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T. Satyanarayana et al. (eds.), Microorganisms in Sustainable Agriculture
and Biotechnology , DOI 10.1007/978-94-007-2214-9_34,
? Springer Science+Business Media B.V . 2012A bstract D uring the last one decade, rapid advances have been made in the ? eld of functional genomics research in fungi using conventional and non-conventional approaches. RNA interference (RNAi), which is a sequence speci? c silencing of gene at the post transcriptional level, is rapidly becoming a powerful reverse genetic tool, and its potential is also being explored in fungi to validate the gene function. Since over 40 fungal genomes have been sequenced and publicly released and some more genomes are being sequenced, the functional genomics is of utmost important to discover a great deal of new information in the coming years. This review dis-cusses the recent progress on the utilization of RNAi technology in examining gene function in fungi.
Keywords R NA interference ? s iRNA ? Q uelling ? P ost transcriptional gene silencing ?F unctional genomics ? F ungi
34.1 I ntroduction
T
he kingdom fungi comprises of diverse range of eukaryotic organisms which are over 1.5 million in number and it is divided into four major groups, that is, ascomy-cetes, basidiomycetes, zygomycetes and chytrids. The physiology and genetics of fungi share a similarity with plants and animals, including multicellular nature, cell cycle, development and differentiation, intercellular signaling, DNA modi? cation and methylation (Galagan et al. 2005).Fungi in? uence different life forms either directly or indirectly in both positive and negative ways. They colonize the roots of N . S ingh ? M . V . R ajam (*)
D epartment of Genetics ,U niversity of Delhi South Campus ,B enito Juarez Road ,N ew Delhi 110021 ,I ndia
e-mail: r ajam.mv@https://www.doczj.com/doc/de14638182.html,
C hapter 34
R
NA Interference and Functional Genomics in Fungi
N eeru S ingh and M anchikatla V enkat R ajam
774N. Singh and M.V. Rajam plants protecting them from diseases and provide nutrients to them by establishing the symbiotic relationship. Trappe (1987)reported that over 90% of fungi form the mycorrhizal associations with roots of plants. They are the dominant microorgan-isms in soil and play a crucial role in nutrient recycling. Some of the fungi are useful in food industry as the fermentation agents and while some are the producers of important secondary metabolites. Apart from these roles some of the fungi are pathogenic and infect crop plants. Fungal infections also impose a threat to human health, especially affecting the immunocompromised or therapeutically immuno-suppressed patients.
A ll these factors emphasize on the need to understand various processes as pathogenesis, growth, development and metabolism in fungi and hence it is impor-tant to identify and functionally characterize the important genes linked to these processes by using ef?cient functional genomics tools. The elucidation of gene functions would make it possible to do important genetic manipulations so as to upgrade the yield of important secondary metabolites produced by them or to study their interaction with the plants and other hosts and devise the ways to control fun-gal diseases. The revolution in fungal genomics has been brought about by advanced genome sequencing technologies available. There are several sequencing projects which are in process of decoding the different fungal genomes. The ? rst fungal genome to be sequenced was of S accaromyces Cerevisiae(Goffeau et al. 1996)fol-lowed by S hizosaccaromyces pombe and N eurospora craassa genome (Wood et al. 2002; Galagan et al. 2003). In the year 2000, a consortium of mycologists launched the fungal genome initiative (FGI) project which aimed to sequence the genomes of fungi. At present approximately 40 fungal genomes are publicly available and over 40 are in the process of being sequenced, these include important human and plant pathogens and model organisms (Bhadauria et al. 2009). When the genome sequence is available, the important thing remains is to apply the right approach to study the functions of genes identi? ed. The commonly used approaches in fungi are targeted gene disruption/replacement (knock out) which depends on homologous recombi-nation. A very recent approach for manipulating fungal gene expression is RNA silencing or RNA interference (RNAi) which is gaining wide popularity as a tool to identify the functions of gene/s with known sequences, especially of genes present in multiple copies or when particular genes knock out leads to lethality in the organ-ism (Kuck and Hoff 2010). RNAi is a RNA dependent gene silencing phenomenon present in all eukaryotes, with primary role of regulation of gene expression at tran-scriptional or post- transcriptional level (Denli and Hannon 2003).RNAi controls the development of an organism and physiological functions of cells and tissues and is also known to play a role in genome defense against the transposons and invading viruses in some organisms. The basic mechanism of RNAi is common to all organ-isms with small interfering RNAs (siRNAs) mediating the silencing mechanism (Bernstein et al. 2001). The siRNAs reduces gene expression by cleaving homolo-gous transcripts or by translational inhibition and also at transcriptional level by chromatin modi? cation and heterochromatin formation. This chapter mainly focuses on the basic RNAi mechanism with special reference to fungi, and its importance as an alternative tool to study functional genomics in fungi.
77534 RNA Interference and Functional Genomics in Fungi 34.2 B asic RNAi Mechanism
A ndrew
F ire and Craig Mello in 2006 received the Noble prize for their contribution to the understanding of RNAi. The antisense, sense and dsRNA (mixture of sense and antisense RNA) speci? c to the endogenous u nc -22 gene which encodes for myo? lament protein was injected separately in a worm C oenorhabditis elegans to silence it and see the phenotypic effect which is visible in the form of twitching and in severe cases lack of motility and they concluded that dsRNA ef? ciently triggers the target mRNA silencing as compared to either sense or antisense strand alone and this signal can also move from tissue to tissue within the injected organism (Fire
et al.
1998 ). It has also been reported that the silencing signal could be transferred to several generations in C . elegans through germ cells which shows the remark-ability of RNAi in terms of its inheritance in the further generations (Grishok et al. 2000 ) . In general, the RNAi pathway involves the three main steps (Fig. 34.1).
1. I nduction by dsRNA,
2. P rocessing of dsRNA into 21–25 nt small RNA,
3. I ncorporation of siRNAs into effector complexes that bind the complementary target RNA and degrade it.
4. Target mRNA recognition guided by siRNA strand
complexedwith RISC
DICER RISC COMPLEX
RISC COMPLEX
dsRNA molecule siRNA molecule
2. Complex of siRNA molecule and RISC
3. Complex of antisense siRNA strand with RISC
5. Target mRNA degradation by Argonaute
protein of the RISC complex
1. Cleavage of double stranded RNA (dsRNA) by the
DICER enzyme activity into small interfering RNA
(siRNA) molecules
F ig. 34.1 M echanism of RNA mediated gene silencing
776N. Singh and M.V. Rajam T he source of dsRNA could be an inverted repeat sequence or convergent t ranscription of transgenes, transposons, viral RNA and can also be synthesized from aberrant mRNA transcripts by RNA dependent DNA polymerases (RdRps). The basic mechanism of RNAi involves the formation of intermediates called siRNAs. The siR-NAs are the dsRNA molecules of 21–25 nucleotides with characteristic two unpaired nucleotide overhangs at the 3 ￠end of each of the strand. The siRNAs are produced by the endonucleolytic cleavage of the exogenous dsRNA by the multidomain ribonu-clease (RNase) III protein known as Dicer (Elbashir et al. 2001; Bernstein et al. 2001). The dicer comprises of three domains- the RNA helicase domain called PAZ domain (Piwi/Argonaute/Zwille), dsRNA binding domain and of RNase III domains. The slicing of dsRNA to siRNAs is the result of the RNase III activity of the dicer which is functionally active as a dimer, catalyzing four breaks in the phosphodiester back-bone of dsRNA leading to the formation of single siRNA (Zamore 2001).The 3 ￠overhangs of dicer formed upon dicing are methylated by methyltransferases so as to protect them from oligouridylation and degradation. The siRNAs are bound by a ribo-nucleoprotein complex called RNA induced silencing complex (RISC) (Dalmay et al. 2000; Tijsterman et al. 2002). The enzyme component of RISC belonging to argo-naute protein family (AGO) is important as it executes the effector functions leading to gene silencing. The AGO protein has two important domains, PAZ and PIWI domain (Carmell et al. 2002). The PAZ domain is involved in transfer of siRNAs to the RISC complex (Lingel et al. 2003)and PIWI domain has the nuclease activity responsible for siRNA guided cleavage of the target mRNA (Song et al. 2004). Selected siRNA strand incorporates one or several RISC that scans the cell for the complementary nucleic acids to execute their function. The siRNA directed activities include- the slicing activity/endonucleolytic cleavage of target RNA upon transporta-tion to the cytoplasm, retention in the nucleus and DNA cytosine and histone modi? -cations leading to methylation of the target gene sequence and thirdly, it can also cause translational repression. The RNAi is an A TP dependent process; the processing of siRNAs from dsRNA by dicer requires the A TP (Hamilton and Baulcombe 1999 and Zamore et al. 2000). Some of the important features of RNAi are its sequence speci? city that the dsRNA sequence must correspond to the target mRNA sequence to be knocked down. Zamore (2001)has reported that even a short stretch of homology of 23 nucleotides between the target gene and the dsRNA can lead to gene silencing in plants and few molecules of dsRNA are suf? cient to achieve the complete silencing due to ampli? cation of the siRNA molecules by the RdRp enzyme present in worms, fungi and plants. Most of the enzymes involved in RNAi pathway are conserved in different organisms, indicating the common ancestral origin of the pathway.
34.3 R NAi in Fungi
I n fungi, RNAi is commonly referred to as ‘quelling’ and was discovered by Romano and Macino (1992), in the model fungus N eurospora crassa. It has also been reported in many other pathogenic and non pathogenic fungi. The non pathogenic fungi
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S hizosaccharomyces pombe and N. crassa are the commonly used model organisms to study genetic and biochemical basis of RNAi in fungi (Arndt et al. 1995;C ogoni 2001). In fungi, two RNAi related phenomena are present, quelling and meiotic silencing by unpaired DNA. Quelling was observed in N. crassa during the vegeta-tive phase of fungal growth, when Romano and Macino tried to express the exoge-nous genes ( a l-1, a l-2) involved in carotenoid biosynthesis in wild type strain with orange phenotype. Their experiment resulted in an albino phenotype in some of the transformants due to the silencing of transgene as well as the homologous endoge-nous gene. This phenomenon was found to be reversible as the transformants could revert back to the wild type phenotype, probably because of reduction in copy num-ber of exogenous gene. Quelling is mediated post- transcriptionally, since only the gene sequence of few nucleotides could result in gene silencing without the require-ment of promoter along with it. It is similar to co-suppression observed in plants, where integration of transgene in the genome leads to simultaneous silencing of both the transgene and the homologous endogenous gene and both the processes require the presence of aberrant RNA. The proteins involved in quelling and post-transcriptional gene silencing (PTGS) in plants are highly conserved suggesting origin from the common ancestral mechanism that defends the genome from foreign molecules and transposons (Fagard et al. 2000). Romano and Macino used the stably quelled a l-1 strains to isolate the 15 different quelling de? cient mutants ( q de) belonging to three distinct genetic loci- q de1,q de2and q de3. The genes corresponding to these loci were cloned and found to encode three important components of the pathway. The ? rst RNAi gene identi? ed was Q DE-1which encodes for an RdRp and has been shown to be involved in PTGS (Fire et al. 1998). RdRP uses aberrant transgenes as templates to produce dsRNAs (Cogoni and Macino 1999).Through i n vitro studies the RdRP activity of QDE-1 was con? rmed and its secondary structure has been revealed (Fulci and Macino 2007; Laurila et al. 2005). There are evidences which suggest that QDE-1 can function both as an RdRP and as a DNA dependent poly-merase. Q DE-2gene was the second gene cloned and identi? ed to encode for the argonaute protein and it is homologous to r de-1gene of C. elegans, involved in dsRNA mediated gene silencing (Catalanotto et al. 2000). The third important gene required for quelling is Q DE-3which encodes for RecQ DNA helicase homologous to human Werner/Blooms syndrome. QDE-3 facilitates the binding of QDE- 1 to the single stranded DNA at the transgenic region by resolving the complex DNA struc-tures. Another protein called RPA-1 is believed to interact with QDE-3 and helps in recruiting QDE-1 to repetitive transgenic regions. The process of siRNA production requires the dicer like enzymes (DCL) and in N. crassa two dicer proteins were iden-ti? ed, DCL-1 and DCL-2. The double mutants lacking both the dicer enzyme activi-ties are shown to be quelling de? cient. The two enzymes are redundant functionally, with presence of any of the two suf? cient to produce siRNAs. DCL-2 is the major dsRNA processing enzyme since D CL-2mutant show reduced accumulation of siR-NAs (Catalanotto et al. 2004). To execute their function siRNAs get associated with the RISC complex which has argonaute protein QDE-2 as the core component. The RISC is in its inactive form when siRNAs are associated with it in the duplex form, it is only when the passenger strand is cleaved that the RISC is activated. It has been
778N. Singh and M.V. Rajam mRNA Active RISC
Inactive RISC QDE-2
QDE-2
QIP
Duplex siRNA
dsRNA DCL-1/2
QDE-1
QDE-1QDE-3
QDE-1
QDE-3Ribosomal DNA
Ribosomal DNA
Aberrant RNA Transgene
Repetitive DNA
Aberrant RNA F ig. 34.2 A model for the quelling and qiRNA pathway in vegetative cells in N . crassa repetitive transgenes (quelling) or the rDNA locus after DNA damage induce the synthesis of aberrant RNAs by the DdRP activity of Q DE-1 facilitated by Q DE-3 . The aberrant RNA is converted into dsRNAs by the RdRP activity of Q DE-1 . The Dicer proteins D CL-1 and D CL-2 cleave the dsRNAs into siRNAs or qiRNAs, which are then loaded onto the RISC containing Q DE-2 and Q IP .Q DE-2and Q IP convert the siRNA duplex into the mature siRNA, resulting in R ISC activation and gene
silencing of homologous RNAs (Adapted from Li et al.
2010 ) reported that QDE-2 is required for both gene s ilencing and generation of single- stranded siRNA from siRNA duplexes i n vivo (Maiti et al. 2007).It interacts with another protein QIP (QDE-2 interacting protein) to bring about passenger strand degradation. Gene disruption in QIP encoding gene results in accumulation of siRNA duplexes and gene silencing impairment. It acts as an exonuclease and removes nicked passenger strands from the siRNA duplex. Thus, RNA silencing pathway in
fungi can be summarized into following main steps (Fig.
34.2). 1. d sRNA processing into siRNA duplexes by Dicer.
2. A ssociation of siRNA duplexes with RISC (inactive form).
3.
Q DE-2 (argonaute protein) mediated cleavage of passenger strands of siRNA duplexes and removal of nicked passenger strands by QIP.
4. S ingle- stranded siRNA mediated cleavage of homologous mRNAs by activated RISC.
34 RNA Interference and Functional Genomics in Fungi
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I t has also been reported that treatment of N. crassa with DNA damage causing agents results in production of another type of small RNAs called qiRNAs. DNA damage induces signi? cantly high levels of QDE-2 expression and this requires both QDE-1 and QDE-3 activity (Lee et al. 2009). This means during DNA damage dsRNAs are produced by QDE-1 and QDE-3 and the dsRNAs induce the expression of QDE-2. The dsRNA produced on DNA damage are cleaved to a novel class of small RNAs of ~21 nt length which have been named as qiRNA because they inter-act with QDE-2. The qiRNAs are mainly derived from the highly repetitive ribo-somal DNA locus, have a strong 5 ￠uridine preference and a 3 ￠preference for adenine and they are produced by QDE-1, QDE-2 and Dicer enzyme activity suggesting that they are not the non-speci?c products of rRNA degradation (Lee et al. 2009). Interestingly, qiRNAs are derived from the aberrant RNA (aRNA) molecules that can be from both the transcribed as well as untranscribed intergenic spacer regions. Both QDE-1 and QDE-3 are required for the synthesis of DNA damage induced aRNA, since in q de-1 and q de-3 mutants aRNAs are not produced. This also proves that apart from its role in conversion of ssRNA to dsRNA QDE-1 is involved in aRNA production through its RNA polymerase activity. Importantly, qiRNA path-way and quelling share the key components, such as QDE-1, QDE-2. QDE-3 and Dicers and both require aRNA and dsRNA production. It could be said that qiRNA may contribute to the DNA damage response by inhibiting protein translation. Another RNAi related mechanism in N. crassa is the meiotic silencing by unpaired DNA (MSUD) which was discovered by Shiu et al. ( 2001). MSUD functions dur-ing meiotic phase in the life cycle of the fungus to silence the copies of unpaired gene during the pairing of homologous chromosomes. The MSUD de? cient mutants led to the identi? cation of important genes like s ad-1 (suppressor of ascus dominance-1) which is a paralog of q de-1,sms-2 (suppressor of meiotic silenc-ing-2) which encodes for protein homologous to argonaute proteins and another important gene is s ad-2, product of which is required for proper localization of SAD-1 to the perinuclear region (Shiu et al. 2006; Bardiya et al. 2008).SMS-3or DCL-1 is the Dicer protein which is also required for MSUD. All these genes were identi? ed by analyzing the loss of function mutants; s ad-1,sad-2and s ms-2are dominant suppressor of meiotic silencing. All these proteins are found to co-local-ize in the perinuclear region, suggesting that it is the active center for MSUD. Quelling and MSUD require different sets of RNA- related proteins suggesting that there are two different types of RNAi pathways present in N. crassa (Nakayashiki et al.2005).
34.4 E volution of RNA Silencing Pathway in Fungi
I n Eukaryotes the RNA silencing gene loss and gain are very much evident during the course of evolution and all the eukaryotic lineages possess the RNA silencing proteins (Cerutti and Casaa-Mollano 2006). It has been reported that four out of six eukaryotic supergroups have members that do not encode any of the three enzymes
780N. Singh and M.V. Rajam of RNA silencing machinery. In the higher eukaryotic organisms, RNA silencing has the role in important biological processes like, genome defense, chromatin modi? cation and gene regulation therefore is crucial for the growth and develop-ment processes while in fungi the importance of this phenomena is not yet clear. N. crassa RNA silencing mutants are sterile in homozygous crosses but they do not show any morphological abnormalities, (Lee et al. 2009; Shiu et al. 2001).In other ?lamentous fungi like M agnaporthe oryzae and M ucor circinelloides, the Dicer mutants show slight morphological abnormalities (Kadotani et al. 2003;Nicolas et al. 2007)and in S. pombe such mutants show abnormal cell cycle regulation (Carmichael et al. 2004)and cause chromosome segregation defect (V olpe et al. 2003). The RNA silencing pathway is well characterized in N. crassa which has two main RNA silencing pathways, Quelling and MSUD and it has been suggested that a single group of ancestral RNA silencing genes duplicated in an early ancestor of ? lamentous ascomycetes, leading to two paralogous groups of RNA silencing genes with evolutionary divergent functions (Borkovich et al. 2004and Galagan et al. 2003). A number of mutants affected in quelling, PTGS and RNAi have been identi-? ed, which has led to the identi? cation of eight genes controlling these phenomena. The comparative genetic analysis of RNA silencing proteins required for Quelling, RNAi and PTGS revealed that these three pathways are mechanistically linked. In N. crassa,Q DE-1,Q DE-2and Q DE-3genes, in nematode C. elegans,E GO-1, R DE-1a nd M UT-7 genes and in A. thaliana, SGS2and S GS3, all of these genes encode for proteins similar to tomato RdRP. SGS2 and EGO-1 provide a molecular link between PTGS and RNAi but they act in different tissues. EGO-1 is required for RNAi in germline tissues while SGS2 is required for PTGS in somatic tissue as it does not occur in meristems from which germline is derived in plants. The A GO-1 ( A. thaliana), Q DE-1 ( N. crassa) and R DE-1 ( D rosophila) are all necessary for gene silencing in somatic tissues and they all have an amino acid leucine in the highly conserved domain. All these ? ndings con?rm the existence of mechanistic link amongst these pathways (Fagard et al. 2000).Nakayashiki and colleagues (2006) did phylogenetic analysis of RNA silencing proteins Argonaute, Dicer and RdRP in a wide range of fungi belonging to three main classes, ascomycetes, basidiomycetes and zygomycetes. The RNA silencing machinery seems to have undergone diversi-? cation during evolution in fungi which is evident from the fact that there are multiple RNA silencing pathways present in most of these fungi while there are some which either entirely lack them or have only some of the components present in them. N. crassa has three paralogues of RdRP, Argonaute and two of Dicer proteins (Galagan et al. 2003), whereas only one copy of each of these three enzymes has been identi? ed in S chizosaccharomyces pombe genome (Wood et al. 2002). Interestingly, there are some fungi in which RNA silencing machinery seem to have been lost during evolution. For example, S accharomyces cerevisiae, C andida lusitaniae and U stilago maydis do not possess any gene with signi? cant homology to any of the three RNAi proteins i.e. RdRP, Argonaute or the dicer protein, similarly A spergillus nidulans possesses fewer RNA silencing proteins as compared to A. fumigates(Hammond and Keller 2005). Dicer and RdRP like proteins have not been reported in some fungi as C andida albicans and C andida tropicalis.
34 RNA Interference and Functional Genomics in Fungi
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One reason for this could be loss of RNA silencing machinery in these fungi in ancestral groups since C andida and S accharomyces are closely related or they might have been lost sporadically. Most of the members of ? lamentous fungi encode for the single set of proteins for quelling and MSUD but there are some exceptions like A. oryzae and A. ?avus each encode three dicers and three Argonautes, might be due to duplication of genes involved in quelling. In A. nidu-lans the loss of RNA silencing gene has occurred, it has retained the genes involved in quelling, while genes d clA(Dicer), p pdB(Argonaute) corresponding to MSUD pathway in N. craasa are truncated at their respective 3 ￠and 5 ￠ends and these genes encode for the truncated proteins but these are not required for experimental RNA silencing, growth or developmental processes (Liande et al. 2010).
I n conclusion, RNA silencing genes are either lost or have increased in number in fungi indicating that they have either evolved a new pathway or eliminated the exist-ing pathways in response to changes in environmental conditions or with newly devel-oped complexities in the life cycle. The role of RNA silencing pathways in the biology of these fungi is not yet very clear. But the genes involved in quelling in fungi, PTGS in plants and RNAi in animals seem to be mechanistically linked. And in plants and animals they have an important role to play in growth and development.
34.5 F unctional Genomics in Fungi
T he development of different transformation systems together with the efforts towards sequencing of genomes of various ? lamentous fungi had facilitated the ris-ing interest in functional genomics research in ? lamentous fungi. Since for any functional genomics study an ef? cient transformation strategy is a prerequisite, in recent years many transformation strategies have been developed for a wide range of ? lamentous fungi. The following are the successfully used methods for fungal transformation:
1. C alcium chloride/polyethylene glycol
2. E lectroporation
3. P article bombardment
4. A grobacterium mediated transformation
O ne of the earliest reports of transformation came in S. cerevisiae and the process involved the isolation of the protoplast from S. cerevisiae by dissolving the cell wall with glucanase preparation and incubating it with naked DNA in the presence of calcium chloride (Beggs 1978; Hinnen et al. 1978).Soon after this use of protoplasts in transformation was extended to members of Ascomycetes, N. crassa(Case et al. 1979)and A. nidulans(Tilburn et al. 1983)and to many other species. The crucial step in this method is the isolation of protoplast which depends on the choice of enzymes used for digesting the cell wall. These enzymatic preparations are the com-plex mixture of hydrolytic enzymes comprising chie? y of the 1, 3-glucanases and chitinases. The examples of some of the commercially available enzymes are
782N. Singh and M.V. Rajam Novozyme 234 obtained from the fungus T richoderma viride and zymolyase which is of microbilogical origin. Electroporation is also used to transform the protoplast, it involves exposing the protoplast to high amplitude of electric current which per-meablizes the cell membrane thereby permitting the uptake of DNA. The method of particle bombardment depends on coating the gold or tungsten beads with the trans-forming DNA and bombarding it to the fungal tissue. And the more recent method is the A grobacterium-m ediated transformation (AMT) which has proved useful in transforming wide range of fungi and fungal tissues with high frequency even those that are recalcitrant to most other systems of transformation. Most of the functional genomics approaches are based on AMT method, since it produces stable transfor-mants and is suitable for both gene replacement by homologous recombination (Khang et al. 2005) and insertional mutagenesis (Li et al. 2005; Combier et al. 2003) by random integration. With ef?cient transformation system available the only problem that remains is of the multinucleate nature of the most of the fungi and more so because techniques of gene replacement and insertional mutagenesis to achieve gene knock outs rely on the homokaryotic transformants derived from a single transformation event to study loss of functional mutants. It is possible to overcome this problem if it is possible to transform uninucleate tissue or cycle transformed tissue through a uninucleate stage (Cvitanich and Judelson 2003).The other solution to overcome this problem is the gene knock down strategy as RNAi involves inactivation of target mRNA rather than gene mutation. We will brie? y discuss the conventional ways to study functional genomics in fungi before elabo-rating on RNAi approach to achieve the same.
34.5.1 D ifferent Methods to Study Gene Function in Fungi
34.5.1.1 R andom Insertional Mutagenesis
R andom tagged mutations can be created by inserting DNA into the genome of the fungi leading to the disruption of genes, tagging of promoters or enhancers or up-regulation of genes. The phenotypic changes of interest are monitored in the trans-formants and the genomic region carrying the inserted genetic element is retrieved by either PCR based methods as inverse PCR and TAIL PCR or by plasmid rescue (Combier et al. 2003). Gene tagging can be achieved by direct DNA transfer in a non homologous manner and a library of thousands of tagged mutants can be obtained which could be correlated with a particular phenotype leading to the discovery of new genes. T-DNA is another form of insertional mutagenesis that relies on AMT to integrate the T-DNA at random sites in the recipient genome. AMT has various advantages over other methods of insertional mutagenesis as it leads to relatively high frequency of transformation and produces more of single copy integrations. The genome regions ? anking the T-DNA could be retrieved based on general lack of major truncation or rearrangement of the T-DNA. The few drawbacks of this method are that sometimes mutations may be caused in the regions unlinked to the
34 RNA Interference and Functional Genomics in Fungi
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site of T-DNA integration and genomic rearrangements are also observed. This necessitates the proper testing of the putatively tagged mutants to con? rm if the T-DNA insertion is linked to the mutant phenotype. The loss of function mutants obtained through random insertion impose various limitations like it may not be possible to recover mutations in essential or redundant genes, loss of function pro-vides not very con? rmatory information about the mutated gene. These limitations could be overcome to some extent by promoter and enhancer trapping wherein certain elements could be added that allow the detection of promoter activity or increase the transcription of the contiguous genes. For enhancer traps a reporter gene with weak promoter is positioned near the end of the transferred DNA. The insertion of DNA near the enhancer would drive the expression of the reporter gene from the weak promoter. Similarly the promoter-less reporter gene could be used to tag the promoters. Transposon tagging provides another way to achieve inser-tional mutagenesis using endogenous transposons or engineered transposons. The major advantages of this method are especially while dealing with fungal systems that are recalcitrant to transformation, genetic mutations created are unlinked to the transferred DNA and there are rare chances of genomic lesions and rearrange-ments (Langin et al. 1995). Also, the plasmid Impala has been designed for gene tagging in fungi, it transposes by cut and paste method and does not require the host proteins.
34.5.1.2 T argeted Gene Disruption/Replacement
W hen the sequence of gene of interest is known, the function could be identi? ed by gene disruption and correlated with the altered phenotype observed. The gene knock out as it is commonly known as is achieved by homologous recombination. The fungus is transformed with gene disruption cassette consisting of a selectable marker gene ? anked by target gene sequences and inserted into the recipient fungal genome by homologous recombination. The ef? ciency of gene targeting is affected by the length of the homlogous sequences, extent of homology, transformation method and the genomic position of the target gene (Bird and Bradshaw 1997).In ? lamentous fungi, at least 1 kb or more of homologous sequence and that too of very high homology is required to achieve the better frequency of gene knock out ( M ichielse et al. 2005a). Van and Hooykaas (2003) reported that AMT is the most suitable method for achieving high frequency of gene targeting which may be because of linear and single- stranded nature of T-DNA. Particle bombardment and electropo-ration are also the preferred methods for gene knock out studies in fungi. The chances of ectopic integration of the gene of interest are always there which could be minimized by positive and negative selection system which leads to lethality in non viable transfomants resulting from ectopic integration. For example the inclu-sion of negative selection gene a mdS in the knock out construct outside the target gene homologous sequences confers sensitivity to ? uoro-acetamide if the non legit-imate integration takes place (Michielse et al. 2005b).Different approaches have also been tried to overcome these limitations such as split marker technology was
784N. Singh and M.V. Rajam developed for S. cerevisiae(Fairhead et al. 1996)and has been successfully applied to many ? lamentous fungus (Kuck and Hoff 2010). This technique requires three crossing over events to generate the functional resistance marker or auxotrophic gene, which substitutes the target gene by homologous recombination. This tech-nique was applied in P. chrysogenum to obtain l ys2 dirupted mutants using u ra3/5 marker gene involved in uracil biosynthesis and the mutants were selected with 5- ? uorotic acid (Casqueiro et al. 1999). Similar method was adopted for inactivating mecB gene that encodes for cytathionine-gamma-lyase, involved in cephalosporin C biosynthesis in A crmonium chyrsogenum and 5% frequency of gene disruption in transformants was observed (Liu et al. 2001).The modi? ed transposon based ver-sion of split marker technique has also been used in some cases like in C olletotrichum graminicola(Venard et al. 2008). The limitations of this method were that frequency of homologous recombination depended on the recipient strain and on the split marker gene used. So another new approach was devised using strains de? cient in non homologous end joining (NHEJ) so that non homologous recombination is eliminated. A multi protein complex is required for NHEJ which comprises of DNA- dependent protein kinase, DNA ligase IV- XRCC4 complex, exonuclease Artemis and the Ku70/Ku80 heterodimer that binds directly to DNA ends and directs the DNA- Protein kinase, therby showing ef? cient activation (Critchlow and Jackson 1998; Hsu et al. 1999; Hefferin and Tomkinson 2005). The disruption of K u genes resulted in increased homologous recombination frequency of transformation in many of the ? lamentous fungi. The one of the important limitations of this approach has been the increased sensitivity of K u disruption mutants to various chemicals as etyl methanesulfonate and bleomycin due to reduced capability of repair systems in them. These conventional methods to study gene functions are limited by the need to prepare constructs with long stretches of homology and low transformation ef? ciency observed in most of the fungi, also they are not suitable for use in high throughput approach. RNAi is easy and ef? cient approach which provides solution to many of the problems observed with conventional approaches (Shafran et al. 2008).
34.5.2 R NAi as the Alternative Tool to Study Gene
Functions in Fungi
R NAi can be used for silencing the expression of gene whose sequence is known so as to elucidate its role. RNAi could be easily induced in most of the organisms including worms, insects, fungi, plants and mammalian cells, by introducing dsRNA into their genomes. This has become the basis for RNAi as the potential reverse genetic tool in these organisms and has also proven to be an ef? cient tool for high throughput functional genomics studies in most of the eukaryotes. The 90% of D rosophila melanogaster and C. elegans genes were successfully targeted by RNAi for loss of function analysis (Boutros et al. 2004; Kamath et al. 2003). RNAi is gain-ing popularity as the upcoming tool for functional analysis in fungi. RNA silencing has also provided with ways to produce improved genetically modi? ed strains
78534 RNA Interference and Functional Genomics in Fungi c
onsidering the biotechnical and pharmaceutical applications of some of the fungi, which are of industrial value being the producers of important primary and secondary metabolites (Yamada et al. 2007).
E arlier many studies were conducted using antisense mRNA which resulted in the down- regulation of transcript levels of various genes in N . crassa but it was only after discovery of RNA silencing pathway by Romano and Macino in 1998 that the reason for the down- regulation of gene expression was known (
d e Backer et al. 2002 ) . Although there are few reports on the use of RNAi to explore gene function in fungi, the applicability of RNAi has been tested in many of the fungi, including N . crassa ,A . nidulans and C olletotrichum lagenarium (Cogoni et al. 1996 ; Liu et al. 2002 ; Fitzgerald et al. 2004 ; McDonald et al. 2005 ; Nakayashiki et al. 2005).T he ef? cient gene silencing has been achieved using inverted repeat transgenes when expressed in these fungi. There are various strategies by which RNAi can be induced in ? lamentous fungi (Nakayashiki and Nguyen 2008)(Fig. 34.3)
.
5’
3’dsRNA
5’
5’3’3’dsRNA
19 nucleotide duplex
2 overlapping nucleotides 3’
5’5’3’
a b
c F ig. 34.3 S chematic representation of different strategies for inducing RNAi in an organism, using vectors with intron so that hairpin RNA transcripts are produce
d ( a ), with opposing dual promoters also dsRNA formation is facilitated ( b ), th
e RNAi can also be induced transiently using chemically synthesized siRNAs provided exogenously in the nutrient medium ( c )
786N. Singh and M.V. Rajam
1. R NAi using hairpin RNA (hpRNA) expression vectors
2. R NAi using vector with opposing dual promoters
3. D irect delivery of siRNA/dsRNA into fungal cells
R NAi using Hairpin RNAi (hpRNA) expression vectors: The ? rst successful appli-cation of RNAi was reported in pathogenic fungus, C ryptoccus neoformans using dsRNA (Liu et al. 2002). They achieved the signi? cant silencing of genes involved in synthesis of polysaccharide capsule formation in the fungus leading to avirulence in the strains. The plasmid constructs expressing hpRNA or intron containing hair-pin RNA are most ef? cient and reliable source of inducing gene silencing in fungi (Kadotani et al. 2003; Namekawa et al. 2005; de Jong et al. 2006; Takeno et al. 2004). Most of the vectors used for expressing dsRNA have the intron or spacer sequences which lie between the two inversely oriented target gene fragments so that hairpin structure is formed. The psilent-1 is one such versatile vector developed to express hairpin RNA transcripts in the Ascomycete fungi by polymerase chain reaction (PCR)- based cloning (Nakayashiki et al. 2005; Moriwaki et al. 2007).This vector has the transcriptional unit comprising of trpC promoter and terminator sequence, multiple cloning site and a cutinase intron from M. grisea, for selection of the transformants hygromycin resistant marker gene is present (Fig. 34.4). The vector was used to silent the M PG1gene, which encodes a fungal hydrophobin involved in surface interactions during infection-related development in the fungus M. Oryzae(Talbot et al. 1996). The 461 bp fragment of the gene was used to develop the RNAi construct and when expressed in the fungi it resulted in varying degree of silencing of M PG1gene in the transformants. The vector psilent1 has been tested and validated in many of the ? lamentous fungus, but for the high throughput func-tional genomics it is necessary to have the vectors as that of Hellsgate vector in plants (Wesley et al. 2001). Therefore a gateway cloning system known as pTroya based on pSilent1 was designed using invitrogen’s gateway technology (Shafran et al. 2008). In pTroya two destination A cassettes of the gateway system were intro-duced into pSilent1 in opposite orientation in to ligation steps (Fig. 34.5). This system is commercially available and can be used for high throughput functional genomics in ? lamentous fungi. It uses the site- speci? c recombination process in bacteriophage lambda that enables the shuttling of sequences between plasmids bearing compatible recombination sites. With pTroya large scale screening could be done especially in non sequenced organisms based on partial data or the medium scale screens based on transcriptome analysis (Shafran et al. 2008). Another example of vector which has gateway technology is pFANTAi4 developed for human pathogenic fungus B lastomyces dermatidis. This vector has additional feature that it has green ? uores-cent protein (GFP) reporter system (Krajaejun et al. 2007).Importantly, the variable degree of silencing is observed with RNAi in individual trasnfromants and in some cases about 90–98% silencing takes place which is equivalent to gene knock out. Therefore, it is necessary to differentiate between the tranformants based on the extent of silencing in each one of them, to achieve this Liu and co-workers (2001) used vectors with reporter gene to drive the dsRNA expression such that the reporter gene is also down-regulated along with the endogenous target gene. A DE2reporter
78734 RNA Interference and Functional Genomics in Fungi gene was used by them which encodes for phosphoribosylaminoimidazole c arboxylase enzyme required for adenine biosynthesis. Silencing of A DE2gene results in pink colonies and so the transformants with silenced genes can be recog-nized by the pink phenotype. Similarly, G FP gene has also been used for rescuing the silenced transformants by observing loss of GFP ? uorescence in many of the cases like in M agnaporthe oryzae (Kadotani et al. 2003);P hytophtora infestans (Whisson et al. 2005)and B lastomyces dermatidis (Krajaejun et al. 2007).The other alternative is the use of d sRed reporter gene which imparts red phenotype to the transformants when expressed. Fig.34.4 S chematic representation of the silencing vector pSilent-1, and the sequences of multi-cloning sites and splice junctions in the vector. ( a ) A map of pSilent-1. A mp r ,ampicillin-resistant gene; H yg r
, hygromycin-resistant gene; I T ,intron 2 of the cutinase pSilent-1
ap.6.9kb
P t r p C P t r p C H y
g '
T t r p C T t r p C
A m
p
'
Spel
IT
xho l , Sna Bl, Hin dlll
Bg /ll, Sph l, Stu l, Kpn l, Apa l a b (TrpC Promoter)
(TrpC Terminator)
CAAGCATCGATACCGTCGACCTCGAGGTACGT ACAAGCTT GCTGGAGGATACAG GT GAGC
(Cutinase intron)CACAC AG CCAGGGAACGGC AGATCTTCGCATGCTAAGGCCTGTGGTACC TGGGCCCCGGATCCACTT
Hin dlll*Bg /ll*Sph l*Stu l*Kpn l*
Cia l xho l*Sna Bl*Sa /l Apa l*Bam Hl
788N. Singh and M.V. Rajam
R NAi using V ector with Opposing Dual Promoters : Another alternative for inducing RNAi in fungi is use of RNAi vectors with an opposing dual promoter system which require just a single non oriented cloning step. In such a system the sense and anti-sense of the target gene which is required to produce dsRNA in the cell, are both transcribed independently under the control of two opposing RNA polymerase II promoters ( N akayashiki and Nguyen 2008 ) . This kind of vector when used to silence eGFP gene in H istoplasma capsulatum induced moderate silencing of 35% on an average (Rappleye et al. 2004 ) . The pSilent- Dual1 (pSD1) vector is based on dual promoter system; it has trpC and gpd promoters and was used in M . oryzae .The silencing ef? ciency of this vector was found to be lower than the ihpRNA expression vectors and small fraction of the transformants exhibited strong gene silencing (Nguyen et al. 2008 ) . The similar results were obtained with dual- p romoter systems in other fungi as well (Kuck and Hoff 2010).Another vector of this kind which might prove more ef? cient than conventional dual- promoter sys-tems is pSuper RNAi one, initially developed for the mammalian systems. This vector has the human H1 polymerase III gene promoter which allows the cloning and expression of user de? ned oligonucleotide sequences to form short self comple-mentary hairpins and was used for GFP silencing in C oprinopsis cinerea , a basidi-omycete with quite a success (Costa et al. 2008).
D irect Delivery of siRNA/dsRNA into Fungal Cells : The direct uptake of synthetic siRNA duplexes from the nutrient medium is common phenomenon in case of mam-malian cultured cells and was not normally tried in fungal systems until recently when such an application was reported in A . nidulans . In this report key polyamine biosynthesis gene ornithine decarboxylase ( O DC ) was speci? cally silenced by attL2attL1KanR Gene X pENTR221 vector AmpR attB1attB1attB2
attB2Intron Gene X
X Gene GeneX RNAi vector AmpR attR1attR2attR2
attR1HYG HYG Intron
pTroya vector
A Cassette A Cassette F ig. 34.5 S chematic representation of RNAi gateway vector pTroya designed for high throughput functional genomics study in fungi. The vector p Troya has two A-cassettes that include the recom-bination sites ( a ttR1 and a ttR2 ), a chlaramphenicol-resistance gene and hygromycin resistance gene. The target gene is ? rst clone into the p ENTR221 vector after PCR ampli? cation using oligos that have sites a ttB1 and a ttB2 , this creates the RNAi entry clone. This clone and pTroya when mixed with LR clonase enzyme results in insertion of target gene in two different orientations to
create the plasmid pTroya-gene X (Adapted from Shafran et al.
2008 )
34 RNA Interference and Functional Genomics in Fungi
789
treating germinating fungal spores with synthetic 23 nucleotide siRNA duplexes, which were added to the growth media. The siRNA treated fungal spores showed signi? cant decrease in spore germination and germ-tube growth due to reduction in O DC mRNA transcript level and cellular polyamine level (Khatri and Rajam 2007). This method of inducing gene silencing is quite rapid and convenient and remains to be tested in other fungal species. Similarly in one of the reports dsRNA was used to induce RNAi in P hytophtora infestans. The dsRNA (150–300 bp) were delivered into the protoplast by lipofectin-mediated transfection and the gene silencing in this case was only transient after 15–17 days the gene expression was recovered (Whisson et al. 2005).
34.6 A dvantages and Disadvantages of RNAi for Functional
Genomics Study in Fungi
1. R NAi is a gene knock- down phenomenon which does not result in a complete
loss of gene expression and therefore results in variation in phenotypes observed making interpretation of data dif? cult. This feature can be advantageous as well when targeting genes which have a crucial role to play in the growth of the organism.
2. I t is quite ef? cient and convenient when used with inducible promoter thereby
allowing silencing of gene expression at a particular developmental stage. It pro-vides the bene? t of simultaneously silencing all the members of redundant g ene family using a single RNAi construct since it suppresses gene expression in a sequence speci? c manner.
3. I t is a valuable gene analysis tool for fungal species which are dif? cult to analyze
owing to their heterokaryotic nature.
4. R NAi could be applied in fungal species with very low ef? cacy of fungal recom-
bination as well.
5. T he sequence speci? city of RNAi can be used to silence selectively the alterna-
tive splice variants.
6. T he major disadvantage of RNAi is the possibility of “off target” effects which
lead to silencing of genes which bear partial complementarity to the sense or antisense strand of the target gene.
34.7 C onclusions
R NAi is rapidly gaining popularity as the novel tool for rapid gene analysis in fungi ever since its discovery in N. crassa. The presence of quelling and meiotic silencing in most of the fungi demonstrates the importance of RNAi phenomenon. Many of the components of RNAi pathway had been identi? ed and characterized in these two pathways and have contributed to our understanding of RNAi mechanism.
790N. Singh and M.V. Rajam RNAi approach enable identi? cation of gene functions of essential genes that too with only limited amount of sequence information available. The sequences of many of the fungi are still unknown so that gene identi? cation and characterization is a dif? cult task in these fungal species but with RNAi gateway system like pTroya RNAi plasmid large scale screening is possible based on partial data. The use of RNAi is also being expanded to achieve novel traits in fungi so as to improve their industrial value as the producers of important metabolites. The one of the major drawbacks of RNAi is the incomplete repression and possibility of getting off target effects. To improve the ef? cacy of RNAi for gene function analysis in fungi it is important to know the extent of off target effects in fungal cells and to develop the suitable RNAi vectors with tightly controlled inducible promoters which could be used in a wide range of ? lamentous fungi. The use of RNAi technique is being further explored to control fungal infections in economically important crop plants as well as in humans. The host induced silencing of vital fungal genes could provide immunity against the invading fungus as the pathogen would probably ingest the siRNAs synthesized by the host plant against its own gene. Similarly the intake of chemically synthesized fungal gene speci? c siRNAs might lead to control of human and animal diseases caused by fungal pathogens.
R eferences
G.M. Arndt, D. Atkins, M. Patrikakis, J.G. Izant, Mol. Gen. Genet. 248, 293–300 (1995)
N. Bardiya, W.G. Alexander, T.D. Perdue, E.G. Barry, R.L. Metzenberg, P.J. Pukkila, P.K. Shiu, Genetics 178, 593–596 (2008)
J.D. Beggs, Nature 275, 104–109 (1978)
E. Bernstein, A.A. Caudy, S.A. Hammond, G.J. Hannon, Nature 409, 363–366 (2001)
V. Bhadauria, S. Banniza, Y. Wei, Y. Peng, Comp. Funct. Gen. 2009, 380–719 (2009)
D. Bird, R. Bradshaw, Mol. Gen. Genet. 255, 219–225 (1997)
K.A. Borkovich, L.A. Alex, O. Yarden, M. Freitag, G.E. Turner et al., Micro. Mol. Biol. Rev. 68, 1–108 (2004)
M. Boutros, A.A. Kiger, S. Armknecht, K. Kerr, M. Hild, B. Koch, S.A. Haas et al., Science 303(5659), 832–835 (2004)
M.A. Carmell, Z. Xuan, M.Q. Zhang, G.J. Hannon, Genes Dev. 16, 2733–2742 (2002)
J.B. Carmichael, P. Provost, K. Ekwall, T.C. Hobman, Mol. Biol. Cell. 15, 1425–1435 (2004) M.E. Case, M. Schweizer, S.R. Kushner, N.H. Giles, Proc. Natl. Acad. Sci. USA 76,5259–5263 (1979)
J. Casqueiro, S. Gutierrez, O. Banuelos, M.J. Hijarrubia, J.F. Martin, J. Bacteriol. 181,1181–1188 (1999)
C. Catalanotto, G. Azzalin, G. Macino, C. Cogoni, Nature 404, 245 (2000)
C. Catalanotto, M. Pallotta, P. ReFalo, M.S. Sachs, L. Vayssie et al., Mol. Cell. Biol. 24,
2536–2545 (2004)
H. Cerutti, J.A. Casaa-Mollano, Trends Biochem. Sci. 25, 481–482 (2006)
C. Cogoni, Annu. Rev. Microbiol. 55, 381–406 (2001)
C. Cogoni, G. Macino, Nature 399, 166–169 (1999)
C. Cogoni, J.T. Irelan, M. Schumacher, T.J. Schmidhauser, E.U. Selker, G. Macino, EMBO J. 15,
3153–3163 (1996)
34 RNA Interference and Functional Genomics in Fungi
791
J.P. Combier, D. Melayah, C. Raf? er, G. Gay, R. Marmeisse, FEMS Microbiol. Lett. 220,141–148 (2003)
A.S.M.
B. Costa, P.R. Mills, A.M. Bailey, G.M. Foster, M.P. Challen, J. Microbiol. Methods 75,
205–208 (2008)
S.E. Critchlow, S.P. Jackson, Trends Biochem. Sci. 23, 394–398 (1998)
C. Cvitanich, H.S. Judelson, Curr. Genet. 42, 228–235 (2003)
T.A. Dalmay, A. Hamilton, S. Rudd, S. Angell, D.C. Baulcombe, Cell 101, 543–553 (2000)
M.D. de Backer, M. Raponi, G.M. Arndt, Curr. Opin. Microbiol. 5, 323–329 (2002)
J.F. de Jong, H.J. Deelstra, H.A. Wosten, L.G. Lugones, Appl. Environ. Microbiol. 72,1267–1269 (2006)
A.D. Denli, G.J. Hannon, Trends Biochem. Sci. 28, 196–201 (2003)
S.M. Elbashir, W. Lendeckel, T. Tuschl, Genes Dev. 15, 188–200 (2001)
M. Fagard, S. Boutet, J.B. Morel, C. Bellini, H. Vaucheret, Proc. Natl. Acad. Sci. USA 97, 11650–11654 (2000)
C. Fairhead, B. Llorente, F. Denis, M. Soler, B. Dujon, Yeast 12, 1439–1457 (1996)
A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Nature 391,806–811
(1998)
A. Fitzgerald, J.A. Van Kan, K.M. Plummer, Fungal Genet. Biol. 41, 963–971 (2004)
V. Fulci, G. Macino, Curr. Opin. Microbiol. 10, 199–203 (2007)
J.E. Galagan, S.E. Calvo, K.A. Borkovich, E.U. Selker, N.D. Read, D. Jaffe et al., Nature 422, 859–868 (2003)
J.E. Galagan, M.R. Henn, L.J. Ma, C.A. Cuomo, B. Birren, Genome Res. 15, 1620–1631 (2005) A. Goffeau, B.G. Barrell, H. Bussey, R.W. Davis, B. Dujon, H. Feldmann, F. Galibert, J.D. Hoheisel,
C. Jacq, M. Johnston, Science 274, 546–567 (1996)
A. Grishok, H.G. Tabara, C.C. Mello, Science 287(5462), 2494–2497 (2000)
A.A. Grishok, A.E. Pasquinelli, D. Conte, N. Li, S. Parrish, I. Ha, D.L. Baillie, A. Fire, G. Ruvkun,
C.C. Mello, Cell 106, 23–24 (2001)
A.J. Hamilton, D.C. Baulcombe, Science 286, 950–952 (1999)
T.M. Hammond, N.P. Keller, Genetics 169, 607–617 (2005)
M.L. Hefferin, A.E. Tomkinson, DNA Repair. 4, 639–648 (2005)
A. Hinnen, S.J.
B. Hick, G.R. Fink, Proc. Natl. Acad. Sci. USA 75, 1929–1933 (1978)
H.L. Hsu, D. Gilley, E.H. Blackburn, D.J. Chen, Proc. Natl. Acad. Sci. USA 96,12454–12458
(1999)
N. Kadotani, H. Nakayashiki, Y. Tosa, S. Mayama, Mol. Plant Microbe Interact. 16,769–776 (2003)
R.S. Kamath, A.G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Botle, S. Moreno, M. Sohrmann, D.P. Welchman, P. Zipperlen, J. Ahringa, Nature. 421 , 231–237 (2003)
C.H. Khang, S.Y. Park, Y.H. Lee, S. Kang, Fungal Genet. Biol. 42, 483–492 (2005)
M. Khatri, M.V. Rajam, Med. Mycol. 45, 211–220 (2007)
T. Krajaejun, G.M. Gauthier, C.A. Rappleye, T.D. Sullivan, B.S. Klein, Eukaryot. Cell. 6,1299–1309 (2007)
U. Kuck, B. Hoff, Appl. Microbiol. Biotechnol. 86, 51–62 (2010)
T. Langin, P. Capy, M.J. Daboussi, Mol. Gen. Genet. 246, 19–28 (1995)
M.R. Laurila, P.S. Salgado, E.V. Makeyev, J. Nettelship, D.I. Stuart et al., Struct. Biol. J. 149, 111–115 (2005)
H.C. Lee, S.S. Chang, S. Choudhary, A.P. Aalto, M. Maiti et al., Nature 459, 274–277 (2009) M. Li, X. Gong, J. Zheng, D. Jiang, Y. Fu, M. Hou, FEMS Microbiol. Lett. 243, 323–329 (2005) L. Li, S. Chang, Y. Liu, Cellul. Mol. Life Sci. 67, 3849–3863 (2010)
A. Lingel,
B. Simon, E. Izaurralde, M. Sattler, Nature 426, 465–469 (2003)
G. Liu, J. Casqueiro, O. Banuelos, R.E. Cardoza, S. Gutierrez, J.F. Martin, J. Bacteriol. 183,
1765–1772 (2001)
H. Liu, T.R. Cottrell, L.M. Pierini, W.E. Goldman, T.L. Doering, Genetics 160, 463–470 (2002) M. Maiti, H.C. Lee, Y. Liu, Genes Dev. 21, 590–600 (2007)
792N. Singh and M.V. Rajam T. McDonald, D. Brown, N.P. Keller, T.M. Hammond, Mol. Plant Microbe Interact. 18,539–545 (2005)
C.B. Michielse, A.E. Paul, J.J. Hooykaas, A.M. Cees, J.J. Van den, A.E. Arthur, F.J. Ram, Curr.
Genet. 48, 1–17 (2005a)
C.B. Michielse, M. Arentshorst, A.F.J. Ram, C.A.M. Hondel, J.J. van Den, Fungal Genet. Biol. 42,
9–19 (2005b)
A. Moriwaki, M. Ueno, S. Arase, J. Kihara, FEMS Microbiol. Lett. 269, 85–89 (2007)
H. Nakayashiki, Q.B. Nguyen, Curr. Opin. Microbiol. 11, 494–502 (2008)
H. Nakayashiki, S. Hanada, B.Q. Nguyen, N. Kadotani, Y. Tosa, S. Mayama, Fungal Genet. Biol.
42, 275–283 (2005)
H. Nakayashiki, N. Kadotani, S. Mayama, J. Mol. Evol. 63, 127–135 (2006)
S.H. Namekawa, K. Iwabata, H. Sugawara, F.N. Hamada, A. Koshiyama, H. Chiku, T. Kamada, K. Sakaguchi, Microbiology 151, 3669–3678 (2005)
Q.B. Nguyen, N. Kadotani, S. Kasahara, Y. Tosa, S. Mayama, H. Nakayashiki, Mol. Microbiol. 68, 1348–1365 (2008)
F.E. Nicolas, J.P. de Haro, S. Torres-Martinez, M. Ruiz-Vazquez, Fungal Genet. Biol. 44,504–516
(2007)
C.A. Rappleye, J.T. Engle, W.E. Goldman, Mol. Microbiol. 53, 153–165 (2004)
N. Romano, G. Macino, Mol. Microbiol. 6, 3343–3353 (1992)
H. Shafran, I. Miyara, R. Eshed, D. Prusky, A. Sherman, Fungal Genet. Biol. 45,1147–1154
(2008)
P.K.T. Shiu, N.B. Raju, D. Zickler, R.L. Metzenberg, Cell 107, 905–916 (2001)
P.K.T. Shiu, D. Zickler, N.B. Raju, G. Ruprich-Robert, R.L. Metzenberg, Proc. Natl. Acad. Sci.
USA 103, 2243–2248 (2006)
J.J. Song, S.K. Smith, G.J. Hannon, L. Joshua-Tor, Science 305, 1434–1437 (2004)
S. Takeno, E. Sakuradani, S. Murata, M. Inohara-Ochiai, H. Kawashima, T. Ashikari, S. Shimizu, Appl. Microbial. Biotechnol. 65, 419–425 (2004)
N.J. Talbot, M.J. Kershaw, G.E. Wakley, O.M.H. de Vries, J.G.H. Wessels, J.E. Hamer, Plant Cell 8, 985–999 (1996)
M. Tijsterman, R.F. Ketting, K.L. Okihara, T. Sijen, R.H. Plasterk, Science 295, 694–697 (2002) J. Tilburn, C. Scazzocchio, G.G. Taylor, J.H. Zabicky-Zissman, R.A. Lockington, R.W. Davies, Gene 26, 205–221 (1983)
J.M. Trappe, in E cophysiology of VA Mycorrhizal Plants, ed. by G.R. Sa? r (CRC, Boca Raton, 1987), pp. 5–25
H. van Attikum, P.J.J. Hooykaas, Nucl. Acids Res. 31 , 826–832 (2003)
C. Venard, S. Kulshrestha, J. Sweigard, E. Nuckles, L. Vaillancourt, Fungal Genet. Biol. 45,
973–983 (2008)
T. V olpe, V. Schramke, G.L. Hamilton, S.A. White, G. Teng, R.A. Martienssen, R.C. Allshire, Chromosome Res. 11, 137–146 (2003)
S.V. Wesley, C. Helliwell, N.A. Smith, M.-B. Wang, D. Rouse, Q. Liu, P. Gooding, S. Singh,
D. Abbott, P. Stoutjesdijk, S. Robinson, A. Gleave, A. Green, P.M. Waterhouse, Plant J. 27,
581–590 (2001)
S.C. Whisson, A.O. Avrova, P.V. West, J.T. Jones, Mol. Plant Pathol. 6, 153–163 (2005)
V.G. Wood, R. William, M.A. Rajandream et al., Nature 415, 871–880 (2002)
O. Yamada, R. Ikeda, Y. Ohkita, R. Hayashi, K. Sakamoto, O. Akita, Biosci. Biotechnol. Biochem.
71, 138–144 (2007)
P.D. Zamore, Nat. Struct. Biol. 8, 746–750 (2001)
P.D. Zamore, T. Tuschl, P.A. Sharp, D.P. Bartel, Cell 101,25–33 (2000)
安装、使用产品前，请阅读安装使用说明书。 请妥善保管好本手册，以便日后能随时查阅。 GST-DJ6000系列可视对讲系统 液晶室外主机 安装使用说明书 目录 一、概述 (1) 二、特点 (2) 三、技术特性 (3) 四、结构特征与工作原理 (3) 五、安装与调试 (5) 六、使用及操作 (10) 七、故障分析与排除 (16) 海湾安全技术有限公司
一概述 GST-DJ6000可视对讲系统是海湾公司开发的集对讲、监视、锁控、呼救、报警等功能于一体的新一代可视对讲产品。产品造型美观，系统配置灵活，是一套技术先进、功能齐全的可视对讲系统。 GST-DJ6100系列液晶室外主机是一置于单元门口的可视对讲设备。本系列产品具有呼叫住户、呼叫管理中心、密码开单元门、刷卡开门和刷卡巡更等功能，并支持胁迫报警。当同一单元具有多个入口时，使用室外主机可以实现多出入口可视对讲模式。 GST-DJ6100系列液晶室外主机分两类（以下简称室外主机），十二种型号产品： 1.1黑白可视室外主机 a)GST-DJ6116可视室外主机（黑白）； b)GST-DJ6118可视室外主机（黑白）； c)GST-DJ6116I IC卡可视室外主机（黑白）； d)GST-DJ6118I IC卡可视室外主机（黑白）； e)GST-DJ6116I（MIFARE）IC卡可视室外主机（黑白）； f)GST-DJ6118I（MIFARE）IC卡可视室外主机（黑白）。 1.2彩色可视液晶室外主机 g)GST-DJ6116C可视室外主机（彩色）； h)GST-DJ6118C可视室外主机（彩色）； i)GST-DJ6116CI IC卡可视室外主机（彩色）； j)GST-DJ6118CI IC卡可视室外主机（彩色）； k)GST-DJ6116CI（MIFARE）IC卡可视室外主机（彩色）； GST-DJ6118CI（MIFARE）IC卡可视室外主机（彩色）。 二特点 2.1 4*4数码式按键，可以实现在1~8999间根据需求选择任意合适的数字来 对室内分机进行地址编码。 2.2每个室外主机通过层间分配器可以挂接最多2500台室内分机。 2.3支持两种密码（住户密码、公用密码）开锁，便于用户使用和管理。 2．4每户可以设置一个住户开门密码。 2.5采用128×64大屏幕液晶屏显示，可显示汉字操作提示。 2.6支持胁迫报警，住户在开门时输入胁迫密码可以产生胁迫报警。 2.7具有防拆报警功能。 2.8支持单元多门系统，每个单元可支持1～9个室外主机。 2.9密码保护功能。当使用者使用密码开门，三次尝试不对时，呼叫管理中 心。 2.10在线设置室外主机和室内分机地址，方便工程调试。 2.11室外主机内置红外线摄像头及红外补光装置，对外界光照要求低。彩色 室外主机需增加可见光照明才能得到好的夜间补偿。 2.12带IC卡室外主机支持住户卡、巡更卡、管理员卡的分类管理，可执行 刷卡开门或刷卡巡更的操作，最多可以管理900张卡片。卡片可以在本机进行注册或删除，也可以通过上位计算机进行主责或删除。
一、数据类型概述 数据:计算机能够处理数值、文字、声音、图形、图像等信息，均称为数据。 数据类型:根据数据描述信息的含义，将数据分为不同的种类，对数据种类的区分规定，称为数据类型。数据类型的不同，则在内存中的存储结构也不同，占用空间也不同 VB的基本数据类型： 数值型数据（主要数据类型）日期型字节型 货币型逻辑型字符串型对象型变体型 二、数值数据类型 数值类型分为整数型和实数型两大类。 1、整数型 整数型是指不带小数点和指数符号的数。 按表示范围整数型分为：整型、长整型 （1）整型（Integer，类型符%） 整型数在内存中占两个字节（16位） 十进制整型数的取值范围：-32768~+32767 例如：15，-345，654%都是整数型。而45678%则会发生溢出错误。 （2）长整型（Long，类型符&） 长整数型在内存中占4个字节（32位）。 十进制长整型数的取值范围： -2147483648~+2147483647 例如：123456，45678&都是长整数型。 2、实数型（浮点数或实型数） 实数型数据是指带有小数部分的数。 注意：数12和数12.0对计算机来说是不同的，前者是整数（占2个字节），后者是浮点数（占4个字节） 实数型数据分为浮点数和定点数。 浮点数由三部分组成：符号，指数和尾数。 在VB中浮点数分为两种： 单精度浮点数（Single） 双精度浮点数（Double） （1）单精度数(Single，类型符！) 在内存中占4个字节（32位），,有效数字：7位十进制数 取值范围：负数-3.402823E+38~-1.401298E-45 正数 1.401298E-45~3.402823E+38 在计算机程序里面不能有上标下标的写法，所以乘幂采用的是一种称为科学计数法的表达方法 这里用E或者e表示10的次方(E/e大小写都可以） 比如：1.401298E-45表示1.401298的10的负45次方
F6门禁管理系统用户手册 目录 1.系统软件 (2) 2.服务器连接 (2) 3.系统管理 (3) 3.1系统登录 (3) 3.2修改密码 (3) 4.联机通讯 (4) 4.1读取记录 (4) 4.2自动下载数据 (5) 4.3手动下载数据 (5) 4.4实时通讯 (6) 4.5主控设置 (6) 5.辅助管理 (8) 5.1服务器设置 (8) 5.2系统功能设置 (9) 5.3读写器设置 (10) 5.4电子地图 (13) 6.查询报表 (14) 6.1开锁查询 (14) 7.帮助 (18) 7.1帮助 (18)
1.系统软件 图1 门禁管理软件主界面 F6版门禁管理系统的软件界面如上图，顶端菜单栏包括“系统管理”、“联机通讯”、“辅助管理”、“查询报表”和“帮助”菜单；左侧快捷按钮包括“系统管理”、“联机通讯”、“辅助管理”、“查询报表”、“状态”等主功能项，每个主功能项包含几个子功能，在主界面上可以不依靠主菜单，就可在主界面中找到每个功能的快捷按钮。以下按照菜单栏的顺序进行介绍。 2.服务器连接 如图2点击设置则进入远程服务器设置，此处的远程服务器IP地址不是指数据库服务器，而是指中间层Fujica Server服务管理器的IP地址。 图2 服务连接
图2 远程服务器设置 3.系统管理 3.1系统登录 系统默认的操作员卡号为“0001”，密码为“admin”，上班人员输入管理卡号和密码后可以进入系统，进行授权给他的一切操作。 图3 系统登录 3.2修改密码 修改密码是指操作员登录成功后，可以修改自己登录的密码。先输入操作员的旧密码，再输入新密码并确认，则密码修改成功。
数据库系统原理实验报告 实验名称：__用户定义数据类型与自定义函数_ 指导教师：_叶晓鸣刘国芳_____ 专业：_计算机科学与技术_ 班级：__2010级计科班_ 姓名：_文科_____学号： 100510107 完成日期：_2012年11月10日_成绩： ___ ___一、实验目的： （1）学习和掌握用户定义数据类型的概念、创建及使用方法。 （2）学习和掌握用户定义函数的概念、创建及使用方法。 二、实验内容及要求： 实验 11.1 创建和使用用户自定义数据类型 内容： （1）用SQL语句创建一个用户定义的数据类型Idnum。 （2）交互式创建一个用户定义的数据类型Nameperson。 要求： （1）掌握创建用户定义数据类型的方法。 （2）掌握用户定义数据类型的使用。 实验 11.2 删除用户定义数据类型 内容： （1）使用系统存储过程删除用户定义的数据类型Namperson。 （2）交互式删除用户定义的数据类型Idnum。 要求： （1）掌握使用系统存储过程删除用户定义的数据类型。 （2）掌握交互式删除用户定义的数据类型。 实验 11.3 创建和使用用户自定义的函数 内容： （1）创建一个标量函数Score_FUN，根据学生姓名和课程名查询成绩。 （2）创建一个内嵌表值函数S_Score_FUN，根据学生姓名查询该生所有选课的成绩。 （3）创建一个多语句表值函数ALL_Score_FUN，根据课程名查询所有选择该课程学生的成绩信息。
要求： （1）掌握创建标量值函数的方法。 （2）掌握创建内嵌表值函数的方法。 （3）掌握创建多语句表值函数的方法。 实验 11.4 修改用户定义的函数 内容： （1）交互式修改函数Score_FUN，将成绩转换为等级输出。 （2）用SQL修改函数S_Score_FUN，要求增加一输出列定义的成绩的等级。要求： （1）掌握交互式修改用户定义函数的方法。 （2）掌握使用SQL修改用户定义函数的方法。 实验 11.5 输出用户定义的函数 内容： （1）交互式删除函数Score_FUN。 （2）用SQL删除函数S_Score_FUN。 要求： （1）掌握交互式删除用户定义函数的方法。 （2）掌握使用SQL删除用户定义函数的方法。
ID一体式/嵌入式门禁管理系统 使用说明书
1 软件使用说明 （1）配置要求 在安装软件之前，请先了解您所使用的管理电脑的配置情况。本软件要求安装在（基本配置）： Windows 2000，windows xp操作系统； 奔腾II600或更高的处理器(CPU)； 10GB以上硬盘； 128MB或更大的内存； 支持分辨率800*600或更高的显示器。 （2）安装说明 在光盘中运行“智能一卡通管理系统”安装程序（ID版），按照安装提示依次操作即可。 安装数据库以后，有两种创建数据库的方式，手动创建和自动创建。手动创建：在数据库SQL Server2000的数据库企业管理器中，建立一个database(数据库)。进入查询分析器/Query Analyzer 运行智能一卡通管理系统的脚本文件，形成门禁数据库表；自动创建：在安装智能一卡通管理软件中自动创建默认门禁数据库，默然数据名：znykt。 上述安装完后，在安装目录下,在first.dsn 文件中设置其参数，计算机server的名字（无服务器时即本机名）和数据库database的名字。 在桌面运行智能一卡通管理系统运行文件，选择卡号888888，密码为123456即可进入系统。 2 人事管理子系统 部门资料设置 首先运行‘智能一卡通管理系统’软件后，进入软件主界面，如下图所示：
然后点击进入“人事管理子系统”，如图所示： 选择<人事管理>菜单下的<部门管理>或点击工具栏内的‘部门管理’按钮，则会出现如下所示界面： 在<部门管理>中可以完成单位内部各个部门及其下属部门的设置。如果公司要成立新的部门，先用鼠标左键单击最上面的部门名，然后按鼠标右键弹出一菜单，在菜单中选择“增加部门”，则光标停留在窗口右边的“部门编号”输入框中，在此输入由用户自己定义的部门编号后，再在“部门名称”输入框中输入部门名称，最后按 <保存>按钮，此时发现窗口左边的结构图中多了一个新增的部门。如果要给部门设置其下属部门，则首选用鼠标左键选中该部门，再按鼠标右键弹出一菜单，在菜单中选择“增加”，最后输入、保存。同时也可以对选中的部门或下属部门进行“修改”或“删除”。特别要注意的是，如果是“删除”,则被选中的部门及其下属部门将被全部删除，所以要特别谨慎。
用户自定义的数据类型------记录 保存多个相同或不同类型数值的结构称为记录（record）。 在VISUAL BASIC 中定义记录，用Type语句，其语法如下： Type varType Variable1 As varType Variable2 As varType … Variablen As varType End Type 例如定义一个名为CheckRecord的记录： Type CheckRecord CheckNumber as Integer CheckDate as Date CheckAmount as Single End Type CheckRecord结构可以像普通变量类型一样使用。要定义这个类型的变量，使用如下语句： Dim check1 As CheckRecord 要对结构的各个字段访问，可使用如下语句： check1. CheckNumber=123 check1. CheckDate=#08/14/1996# check1. CheckAmount=240.00 例： 简单例（自定义类型1.frm） 数组自定义类型1.FRM 用一维数组存放学生年龄。并可通过学生姓名输入或显示该学生的年龄。 Private Type StudentInformation StudentAge As Integer StudentName As String End Type Dim N As Boolean Dim Information(1 To 4) As StudentInformation Dim infIndex As Integer Dim stuName As String Private Sub cmdInputname_Click() For i = 1 To 4 Information(i).StudentName = InputBox("PL input name") Next i End Sub Private Sub cmdInput_Click() infIndex = 1 N = False
《门禁系统使用说明书》
陕西********科技有限公司 单位地址：**************************** 联系电话：**************************** 目录 （ 1.1）软件系统---------------------------------------------------------------------------------------1-135 第一章软件基本操作...................................................................................................................... - 5 - 2.1进入操作软件 (5) 2.4人事管理 (7) 2.4.1 企业信息.................................................................................................................................................................. - 7 - 2.4.2添加/编辑部门信息 ................................................................................................................................................ - 9 - 2.4.2.1添加部门 ............................................................................................................................................................... - 9 - 2.4.2.2修改部门 ............................................................................................................................................................ - 10 - 2.4.2.3 删除部门 ........................................................................................................................................................... - 11 -
-- - XX职业技术学院信息工程学院 门禁管理系统 操作说明书
制作人：X珍海 日期：2014年3月25日 目录 (请打开【帮助H】下的【使用说明书】，这样方便您了解本系统) 第1章软件的基本操作3 1.1 登录和进入操作软件3 1.2 设备参数设置4 1.3 部门和注册卡用户操作4 1.3.1 设置部门4 1.3.2 自动添加注册卡功能（自动发卡）5 1.4 基本操作7 1.4.1 权限管理8 1.4.2 校准系统时间11 1.5 常用工具12 1.5.1 修改登陆用户名和密码12 第2章考勤管理功能模块13 2.1 正常班考勤设置13 2.1.1 设置考勤基本规则13 2.1.2 设置节假日和周休日14 2.1.3 请假出差的设置15 2.2 考勤统计和生成报表17 2.2.1 生成考勤详细报表17 2.2.2 启用远程开门错误!未定义书签。
第1章软件的基本操作 1.1登录和进入操作软件 1．点击【开始】>【程序】>【专业智能门禁管理系统】>【专业智能门禁管理系统】或双击桌面钥匙图标的快捷方式，进入登录界面。 2．输入缺省的用户名：abc 与密码：123（注意：用户名用小写）。该用户名和密码可在软件里更改。 3．登录后显示主操作界面
入门指南。如果您没有经验，您可以在该向导的指引下完成基本的操作和设置。我们建议您熟悉后, 关闭操作入门指南，仔细阅读说明书，熟悉和掌握软件的操作。 “关闭入门指南”后，操作界面如下。 1.2设备参数设置 1.3部门和注册卡用户操作 1.3.1设置部门 点击【设置】>【部门】，进入部门界面。 点击【添加最高级部门】。
门禁系统管理平台详细设计报告 2015年09月20日
目录 一、基本信息 .................................................................................................................. 错误！未定义书签。 二、市场分析 (4) 1．客户需求分析 (4) （1）国际国内市场需求量预测及客户咨询类似产品情况..... 错误！未定义书签。 （2）客户对该产品的功能、安全、使用环境要求等............. 错误！未定义书签。 2．市场现状分析 (4) 三、详细设计 (4) 1. 模块描述 (4) 2. 功能描述 (4) 3. 信息传输过程 (6) 4. 标准符合性分析 (6) 5. 验证（试制/试验/检测）确认方法、手段的分析 (8) 四、资源论证 (8) 1．人力资源需求分析 (8) 2．开发设备资源需求分析 (9) 3．项目开发成本预算 (9) 五、研发时间安排 (9) 六、项目风险评估 (10) 1．技术方面 (10) 2．人员方面 (10) 3．其它资源 (10) 七、评审结论 (11) 八、公司意见 (11)
一、市场分析 1．客户需求分析 1.2014年7月份由三大运营商出资成立了中国通信设施服务股份有限公司，同年9月份 变更名称为中国铁塔股份有限公司。铁塔公司成立后，2015年12月下旬，2000多亿存量铁塔资产基本完成交接。而从2015年1月1日起，三大运营商停止新建铁塔基站，交由中国铁塔进行建设。据统计，2015年1-11月，中国铁塔累计承接三家电信运营企业塔类建设需求53.2万座，已交付41.8万座。针对如此庞大的存量基站及新建基站。 铁塔公司总部急需对基站人员进出做到统一管理，有效管控。提高效率。因此所产生的市场需求量是很大的。 2.随着互联网及物联网技术的快速发展，原有传统门禁管理系统、单一功能的管理软件已 经无法管理众多不同品牌、不同通讯方式、不同厂家的IC/ID读卡设备，因此客户需要一种开放式、分布式的云管理平台，来管理整个基站门禁系统中的所有设备 2．市场现状分析 ●同行业中，各厂家的产品采用传统的门禁方案，既读卡器和控制器及电磁锁或电插锁对 现场的基站门进行管理。造价昂贵，安装复杂。。 ●目前大部分厂家的管理平台架构单一，系统兼容性差，各家的门禁管理平台只能兼容自 家的控制器。开放性不够。 ●目前很多厂商的平台都是针对某一个硬件厂商的设备来运行的，当项目中有多家设备时 平台的控制力明显不足 二、详细设计 1. 模块描述 铁塔基站门禁系统管理平台系统主要包括三部分：BS/CS客户端、云服务器和手机APP。 其中客户端的主要功能包括： 支持对多家基站锁具设备的识别、获取、登录 支持对不同用户进行权限划分。 支持对锁具根据区域进行分组。 支持多家基站锁具设备的设备配置 支持多家设备通过手机APP开锁、获取状态、日志查询。 支持多家设备的设备时间校准 支持设备更新，当设备更新时，可以方便的只更新涉及到的文件，而不需要重装整个系统 支持电子地图
IC一体式/嵌入式门禁管理系统 使用说明书
目录 1．系统简介 (3) 2．功能特点 (3) 3、主要技术参数 (4) 4、系统组成 (4) 5、设备连接 (5) 6、门禁管理系统软件 (6) 6.1 软件的安装 (6) 6.2 人事管理子系统 (7) 6.3 一卡通管理系统 (9) 6.4 门禁管理子系统 (12) 7. 调试操作流程 (28) 8、注意事项 (28)
1．系统简介 在高科技发展的今天，以铁锁和钥匙为代表的传统房门管理方式已经不能满足要求，而集信息管理、计算机控制、Mifare 1 IC智能(射频)卡技术于一体的智能门禁管理系统引领我们走进新的科技生活。 Mifare 1 IC智能(射频)卡上具有先进的数据通信加密并双向验证密码系统，卡片制造时具有唯一的卡片系列号，保证每张卡片都不相同。每个扇区可有多种密码管理方式。卡片上的数据读写可超过10万次以上；数据保存期可达10年以上，且卡片抗静电保护能力达2KV以上。具有良好的安全性，保密性，耐用性。 IC卡嵌入式门禁管理系统以IC卡作为信息载体，利用控制系统对IC卡中的信息作出判断，并给电磁门锁发送控制信号以控制房门的开启。同时将读卡时间和所使用的IC卡的卡号等信息记录、存储在相应的数据库中，方便管理人员随时查询进出记录，为房门的安全管理工作提供了强有力的保证。 IC卡嵌入式门禁管理系统在发行IC卡的过程中对不同人员的进出权限进行限制，在使用卡开门时门禁控制机记录读卡信息，在管理计算机中具有查询、统计和输出报表功能，既方便授权人员的自由出入和管理，又杜绝了外来人员的随意进出，提高了安全防范能力。 IC卡嵌入式门禁管理系统，在线监控IC卡开门信息、门状态，给客户以直观的门锁管理信息。 IC卡嵌入式门禁（简称门禁读卡器，门禁控制机，控制器）是目前同行业产品中体积较小的门禁，可以嵌入到市场上几乎所有的楼宇门禁控制器中，解决了因为楼宇门禁控制器内部空间小所带来的麻烦，是楼宇门禁控制器的最佳配套产品；它绝不仅仅是简单的门锁工具，而是一种快捷方便、安全可靠、一劳永逸的多功能、高效率、高档次的管理系统。它能够让你实实在在享受高科技带来的诸多实惠和方便。 2．功能特点 2．1．IC卡嵌入式门禁具有的功能： 2.1.1使用MIFARE 1 IC卡代替钥匙，开门快捷，安全方便。 2.1.2经过授权，一张IC卡可以开启多个门（255个以内）。 2.1.3可以随时更改、取消有关人员的开门权限。 2.1.4读卡过程多重确认，密钥算法，IC卡不可复制，安全可靠。 2.1.5具有512条黑名单。
门禁管理系统使用说明书
乌石化汽车定量装车系统 门禁管理系统使用说明书 河北珠峰仪器仪表设备有限公司 2013.08.03
目录 一、系统组成 (5) 二、道闸 (5) 1．主要特点 (5) 2. 设备组成 (6) 3. 基本工作原理 (7) 4. 设备使用说明 (7) 三、车辆检测器 (8) 1.车辆检测器的安装 8 2.主要技术参数 8 3.车辆检测器的接线图 8 4.车辆检测器灵敏度设置 9 四、门禁控制器 (9) 五、读卡器 (10) 六、车牌识别 (11) 七、摄像机 (12) 八、门禁控制管理软件 (13) 九、常见问题及解决方法 (14)
一、系统组成 门禁管理系统由行人出入口读卡器、行人大门电磁锁、车辆出入口道闸、摄像机、白光灯、车牌识别仪、控制主机、数据存储服务器等组成。 门禁控制系统组成 二、道闸 车辆出入口道闸采用深圳捷顺生成的JSDZ0203数字式道闸，该道闸采用先进的直流伺服技术和全电路无触点控制技术，使整机运行更加平稳、可靠。而且采用了数字化电路自学习检测功能，有效地杜绝砸车现象，使系统运行更安全可靠。并配备了标准的外接电气接口，可配置车辆检测器以及上位机，实现系统的自动控制。可广泛适用于道路管理、道路收费及停车场管理等系统中。1．主要特点 1)外形美观大方，结构轻巧；部件标准化，可方便更换；箱体铝合金制作，防 水防锈。 2)集光、电、机械控制于一体，操作灵活、方便，使用安全、可靠。
3)系统具有极限位置自锁功能或人为抬杆报警功能(可根据要求设定)。 4)采用先进的直流伺服控制技术，确保系统动作更加准确、平稳。 5)全电路无触点控制，确保系统运行更加安全、可靠。 6)采用PWM调速实现了无极变速，可根据现场需要在速度段内任意调整。 7)按钮滚动菜单设置方式，方便快捷设置闸机运行参数，可根据现场需要进行 设置。 8)采用数字化电路的自学习功能，采集闸杆运行数据并进行计算来判断闸杆是 否碰到障碍物，若检测碰到障碍物，闸杆则会立即自动升起。 9)强、弱电智能控制系统，除具有一般电气控制功能外，既可使用三联按钮、 遥控装置进行手动控制，也可通过车辆检测器进行自动控制，而且系统对外配置标准485电气接口，可通过电脑对其进行远程控制与管理。 10)手动开闸记忆功能：在系统自动运行中，非正常人为开闸数据将被系统自动 记录下来备查询，有效防止人为作弊。 11)开闸次数记忆功能：在系统自动运行中，道闸将会记忆上位机发出的开闸指 令次数，闸杆会保持开状态直到车辆检测器感应车次与开闸指令次数等同时才进行关闸动作。 12)手摇自动升杆功能：在意外断电情况下可用手摇摇柄轻轻摇动使关到位的闸 杆偏离水平方向约30度，闸杆则会自行升起。 13)温度控制功能：闸机可自动检测工作环境温度，并启动温控装置进行温度调 整，保证设备在高低温环境下正常工作。 14)各运动部件均已调整到最佳运动和平衡状态，故本机性能稳定，运行平稳， 噪声小，使用寿命长。 2. 设备组成 JSDZ0203道闸主要由主机、闸杆插头、闸杆等组成，而主机则由机箱、机芯和电控系统等组成 ,见下图。
UDT----用户自定义数据类型（看不懂也要坚持一下，理解了这部分就不是新手了） 在本章中，我们将介绍如何通过用户自定义数据类型和数据范围划定来规划标签数据库。这里将学到 § 了解使用 UDT 的优势 § 学习如何优化 UDT 规划 § 使用数据范围划定帮助简化并加快开发工作 我们现在将重点关注 Logix 控制器中的数据规划。 打开现有控制器文件 1. 在计算机桌面上，双击Lab Files文件夹。 2. 双击名为Conveyor_Program_S 3.ACD的现有项目。 这样将在 RSLogix 5000 中启动该项目。 为传送带创建用户自定义数据类型 您已重新组织了程序规划以更好地利用 Logix，现在已准备好开始对数据规划进行重新组织。可注意到，工程师规划数据的方式仍像使用带有整数、实数和定时器数据表的传统 PLC 一样。问题是，当与设备关联的数据分布到控制器内存中的各处时便很难进行跟踪。您已再次决定充分利用 Logix，使用用户自定义数据类型。 用户自定义数据类型 用户自定义数据类型也称为 UDT 或结构，借此按逻辑方式对数据进行组织或分组，以便所有与设备关联的数据都可组合在一起。 例如，每个传送带都有 8 个整数值、3 个实数值、2 个定时器和 11 个与其关联的布尔值。在传统PLC 中，可能需要 4 个不同的数据表。然后，当您具有多条传送带时，您可能需要详细地将传送带映射到各个数据表中。这样就会变得很难管理。 通过 UDT 能够实现的是将不同的数据类型（整数、实数、定时器、布尔等）组合到一起，共同作为用户自定义数据类型。然后便可创建该 UDT 类型的数组。这可使得编程工作、代码的记录和数据的跟踪都更加轻松。 1. 在控制器项目管理器中，双击"控制器标签"(Controller Tags)。
门禁控制系统 使 用 说 明 书 公司名称： 联系人： 联系电话：
目录 一、系统概况 ..................................................... 错误！未定义书签。 二、系统组成 ..................................................... 错误！未定义书签。 三、系统使用 ..................................................... 错误！未定义书签。 1、IC感应卡 ................................................. 错误！未定义书签。 2、读卡器 ...................................................... 错误！未定义书签。 3、进出门按钮 .............................................. 错误！未定义书签。 4、机箱电源 .................................................. 错误！未定义书签。 5、系统控制器 .............................................. 错误！未定义书签。 6、电脑软件 .................................................. 错误！未定义书签。 6.1 进入和登陆操作软件 .......................... 错误！未定义书签。 6.2 添加IC感应卡的用户 ...................... 错误！未定义书签。 6.3 设置I C卡进出权限 ......................... 错误！未定义书签。 6.4 怎样查询记录 ...................................... 错误！未定义书签。 7、485/232信号转换器 ................................ 错误！未定义书签。 8、电锁........................................................... 错误！未定义书签。 四、简单的维修维护............................................................................. 错误！未定义书签。
用户自定义的数据类型、默认值、规则 一、用户自定义的数据类型 用户自定义数据类型可看做是系统数据类型的别名。 在多表操作的情况下，当多个表中的列要存储相同类型的数据时，往往要确保这些列具有完全相同的数据类型、长度和为空性（数据类型是否允许为空）。例如,对于student数据库中表student、grade和course三张表的xh,kh两个列必须具有相同的数据类型。 创建用户自定义数据类型时首先应考虑如下三个属性： （1）数据类型名称 （2）新数据类型所依据的系统数据类型（又称为基类型） （3）为空性 如果为空性未明确定义，系统将依据数据库或连接的ANSI NULL默认设置进行指派。 1、创建用户自定义数据类型的方法如下： （1）利用企业管理器定义 （2）利用SQL命令定义数据类型 在SQL Server中，通过系统存储过程实现用户数据类型的定义。 语法格式如下： sp_addtype [@typename=] type, /*自定义类型名称*/ [@phystype=] system_data_type /*基类型*/ [,[@nulltype=] null_type /*为空性*/ [,[@owner=] owner_name] /*创建者或所有者*/ 其中： type：用户自定义数据类型的名称。 System_data_type：用户自定义数据类型所依据的基类型。如果参数中嵌入有空格或标点符号，则必须用引号将该参数引起来。 null_type：指明用户自定义数据类型处理空值的方式。取值可为’NULL’、’NOT NULL’、’NONULL’三者之一（注意：必须用单引号引起来）。如果没有用sp_addtype显式定义null_type，则将其设置为当前默认值，系统默认值一般为’NULL’。 例：定义学号字段的数据类型 sp_addtype ’student_xh’,’char(4)’,’not null’ 2、删除用户自定义数据类型 （1）利用企业管理器 （2）利用SQL语句 语法格式如下： sp_droptype [@typename=] type 其中type为用户自定义数据类型的名称，应用单引号括起来。 例：删除student_xh用户自定义数据类型 sp_droptype ’student_xh’ 说明： （1）如果在表定义内使用某个用户定义的数据类型，或者将某个规则或默认值绑定到这种数据类型，则不能删除该类型。 （2）要删除一用户自定义类型，该数据类型必须存在，否则返回一条错误信息。 3、执行权限
欢迎阅读 I 第1章系统简介 1.1系统功能简介 安全管理在近些年的现代企业管理中越来越受到管理者的关注。本系统实现门禁系统管理统一化、流程化， 并帮助客户实现运营安全。 ? 系统特点 ．强大的数据处理能力，能管理30000个人员的门禁数据，能连接100台设备。 ．形象而合理的操作流程融合了多年的门禁经验。 ．自动化的用户名单管理，使得管理更科学、高效。 ．建立在多级管理角色上的权限管理，能保证用户数据的保密性。 服务器硬件配置要求 ：主频2.0G 以上。 内存：1G 及以上。 硬盘：可用空间10G 及以上，推荐使用NTFS 的硬盘分区作为系统安装目录（NTFS 硬盘分区能提供更好的性能和更高的安全性）。 系统运行环境 可支持的操作系统：WindowsXP/Windows2003/WindowsVista/Windows7 可支持的数据库：MSSQLServer2005/MicrosoftAccess 系统功能模块介绍 本系统主要分为四大功能模块： 人事：主要包括两部分，一是部门管理设置，即设置公司的主要架构；二是人员管理设置，为系统录入人员，分配部门，然后进行人员维护管理。 设备：设置连接设备的通信参数，通信参数正确才能够与设备正常通信，包括系统中的设置和设备中的设置。 通信成功后就能查看到已连接设备的信息并能对设备进行远程监控、上传、下载等操作。 ? 备注：指静脉功能在系统的“设备”和“人员”界面显示。 门禁：基于C/S 框架的管理系统，能够实现普通门禁功能，通过计算机对网络门禁控制器进行管理，实现 对人员进出的统一管理。门禁系统是对已经登记用户的开门时间及权限进行设置；即在某个时间段内，在某些门 上，允许某些用户可以验证开锁。 1
门禁系统使用说明 一、硬件设备稳定运行的先决条件 保证系统各组成部分----前段读卡器、磁力锁、门禁控制器有稳定的UPS电源支持，各个相关门自然状态开关闭合良好。 二、门禁管理系统的操作指南 2.1 登录和进入操作软件 点击开始\程序\iCCard\一卡通[门禁考勤]V6.5，或者双击桌面的快捷方式 进入登录界面。 输入缺省的用户名：admin密码：888888 （注意：用户名用小写）。该用户名和密码可在软件里更改。具体操作请参考相关内容。
3登录后显示主操作界面 2.2 怎样更改控制方式和设置开门延时时间 在【总控台】界面中，鼠标右键单击某个门会弹出菜单。可以设置开门延时和控制方式。所谓开门延时时间，是指门打开多长时间后会自动关闭，缺省是 3秒，可设置为 1－6000秒之间的任一时间。 2.3.1 设置部门和班组名称
单击设置\部门信息进入以下界面 单击 [新增] 可添加部门，可设置所属公司、部门级别、上级部门、部门描述。 2.3.2 添加注册卡用户 单击门禁\持卡人进入以下界面
单击然后在文本输入栏中填写您要添加的相应 姓名选定卡号(在ID感应卡表面一般会印刷两组号码， 0013951989 212 58357 前面10位数为内置出厂号不用管他，后面 212 58357 中间的空格不要，这8位数就是真正的卡号。如果卡上没有印刷卡号, 请用实时监控功能来获取卡号)。选择相应的部门和班组名称。除卡 号外所有的信息都可以修改。如果卡遗失，请到（工具――挂失卡）菜单中挂失相应的卡片。一般的软件挂失卡后会用新卡号全部修改以 前的记录设置，我们的软件会进行科学的标注，以前的记录继续可以 保留。 编号可以自动生成无需修改。姓名和卡号是必填项目，工号可以输入 字母和数字的组合，可填可不填。如果该持卡人不需要考勤，请将 的勾去掉。但是如果需要考勤，此处一定要打勾。 单击该按钮后，就已经将该用户加入系统中。
门禁管理系统使用 说明书
乌石化汽车定量装车系统 门禁管理系统使用说明书 河北珠峰仪器仪表设备有限公司 .08.03
目录 一、系统组成 .............................................................. 错误!未定义书签。 二、道闸 ...................................................................... 错误!未定义书签。 1．主要特点 ....................................................... 错误!未定义书签。 2. 设备组成 ......................................................... 错误!未定义书签。 3. 基本工作原理.................................................. 错误!未定义书签。 4. 设备使用说明.................................................. 错误!未定义书签。 三、车辆检测器 .......................................................... 错误!未定义书签。 1. 车辆检测器的安装......................................... 错误!未定义书签。 2. 主要技术参数................................................. 错误!未定义书签。 3. 车辆检测器的接线图 ..................................... 错误!未定义书签。 4. 车辆检测器灵敏度设置 ................................. 错误!未定义书签。 四、门禁控制器 .......................................................... 错误!未定义书签。 五、读卡器 .................................................................. 错误!未定义书签。 六、车牌识别 .............................................................. 错误!未定义书签。 七、摄像机 .................................................................. 错误!未定义书签。 八、门禁控制管理软件............................................... 错误!未定义书签。 九、常见问题及解决方法........................................... 错误!未定义书签。
用户自定义的数据类型复习题 一、选择题 1．下列程序的输出结果是（）。 A) 5B) 6 C) 7 D) 8 struct abc { int a, b, c; }; main() { struct abc s[2]={{1,2,3},{4,5,6}}; int t; t=s[0],a+s[1],b; printf("%d \n",t); } 2．下列程序执行后的输出结果是（）。 A) 6 B) 8C) 10D) 12 #define MA(x) x*(x-1) main() { int a=1,b=2; printf("%d \n",MA(1+a+b));} 3. 有以下结构体说明和变量的定义,则不能把结点b连接到结点a之后的语句是（）。 A) a.next=q; B) p.next=&b; C) p->next=&b; D) (*p).next=q; struct node { char data; struct node *next; } a,b,*p=&a,*q=&b; 4．变量a所占内存字节数是（）。 A) 4B) 5C) 6D) 8 union U { char st[4]; int i; long l; }; struct A { int c; union U u; }a; 5．有如下程序 #define N 2 #define M N+1 #define NUM 2*M+1 #main() { int i; for(i=1;i<=NUM;i++)printf(“%d\n”,i); }
该程序中的for循环执行的次数是（）。 A) 5 B) 6C) 7D) 8 6．以下程序的输出结果是（）。 A) 16 B) 2C) 9D) 1 #define SQR(X) X*X main() { int a=16, k=2, m=1; a/=SQR(k+m)/SQR(k+m); printf(“d\n”,a); } 7．以下程序的输出是（）。 A) 10B) 11 C) 51D) 60 struct st { int x; int *y;} *p; int dt[4]={ 10,20,30,40 }; struct st aa[4]={ 50,&dt[0],60,&dt[0],60,&dt[0],60,&dt[0],}; main() { p=aa; printf(“%d\n”,++(p->x)); } 8．以下程序的输出结果是（）。 struct HAR { int x, y; struct HAR *p;} h[2]; main() { h[0],x=1;h[0];y=2; h[1],x=3;h[1];y=4; h[0],p=&h[1],p=h; printf(“%d %d \n”,(h[0],p)->x,(h[1],p)->y); } A) 12 B) 23C) 14 D) 32 9. 以下程序的输出结果是（）。 union myun { struct { int x, y, z; } u; int k; } a; main() { a.u.x=4; a.u.y=5; a.u.z=6; a.k=0; printf(%d\n”,a.u.x); } A) 4 B) 5C) 6D) 0 10. 以下程序的输出结果是（）。 #define M(x,y,z) x*y+z