RNA Interference and Functional Genomics in Fungi

作物学报ACTA AGRONOMICA SINICA 2014, 40(7): 1174−1181/ISSN 0496-3490; CODEN TSHPA9E-mail: xbzw@ DOI: 10.3724/SP.J.1006.2014.01174芥菜锌指蛋白转录因子基因Bj26的克隆与鉴定贾双伟1,∗∗高英1,∗∗赵开军1,∗中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程, 北京 100081摘要: 锌指蛋白是一类重要的转录因子家族, 参与植物基因转录调节、发育及胁迫反应等生理过程。

我们前期研究发现芥菜诱导型启动子BjC-P的W-box-like-4为其真菌诱导的核心元件, 本研究通过酵母单杂交技术从芥菜cDNA文库中筛选到与W-box-like-4序列特异互作的转录因子基因Bj26。

生物信息学分析表明, Bj26含735 bp的开放阅读框, 编码一个新的C2H2型锌指蛋白, 包括2个典型的C2H2型锌指结构及2个植物特有的QALGGH氨基酸保守序列, 等电点pI为9.2, 分子量为26.6 kD。

亚细胞定位显示该蛋白位于细胞核。

本氏烟瞬时表达分析表明Bj26蛋白通过与BjC-P的真菌诱导核心元件序列特异互作, 激活启动子BjC-P。

实时荧光定量PCR结果显示Bj26在真菌诱导下表达量明显增高。

Bj26的CDS序列与拟南芥和水稻中的C2H2型锌指蛋白序列比对及进化树分析表明, Bj26与拟南芥的C2H2型蛋白同源性高于水稻。

以上结果揭示Bj26蛋白可能介导BjC-P真菌诱导响应并参与植物抗真菌病原菌的调控过程。

关键词:酵母单杂交; C2H2锌指蛋白; Bj26基因; 真菌诱导; 瞬时表达; 实时荧光定量PCRCloning and Characterization of Brassica juncea Zinc Finger Protein Tran-scription Factor Gene Bj26JIA Shuang-Wei1,∗∗, GAO Ying1,∗∗, and ZHAO Kai-Jun1,∗National Key Facility for Crop Gene Resources and Genetic Improvement / Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaAbstract: Zinc-finger proteins, forming an important transcriptional factor family, are involved in gene transcriptional regulation, development and stress-responses in plants. The previous studies revealed that BjC-P is a fungus-inducible promoter from Bras-sica juncea and W-box-like-4 is the core element responsive to fungal infection. Here, we report the molecular cloning and char-acterization of a zinc finger protein-encoding gene (designated Bj26). Bj26 was screened out from the Brassica juncea cDNA library by the Yeast One-Hybrid System. Bioinformatic analysis showed that Bj26 contains open reading frame of 735 bp that encodes a new C2H2-type zinc finger protein with an isoelectric point (pI) of 9.2 and a molecular weight of 26.6 kD. The protein consists of two typical zinc-finger domains and contains two conserved QALGGH amino acid sequences. Subcellular localization showed that Bj26 is located in the nuclear. The histochemical and quantitative GUS assays, through transient gene expression in Nicotiana benthamiana leaf, showed that Bj26 can bind to the core element W-box-like-4 and activate the function of BjC-P. qRT-PCR analysis showed that the gene Bj26 expression obviously increased under the induction of the fungal elicitor (Hexa-N-Acetyl-Chitohexaose). CDS alignments and phylogenetic analysis of Bj26 and other C2H2-type proteins from Arabi-dopsis thaliana (At) and Oryza sativa (Os) (MEGA 6) showed Bj26 shares high similarity with that from Arabidopsis thaliana (At). All of the above results suggested that Bj26 protein mediates the process of plant response to fungal pathogen.Keywords: Yeast One-Hybrid System; C2H2-type zinc finger protein; Bj26; Fungal-induction; Transient gene expression; qRT-PCR在植物中, 锌指蛋白(zinc-finger protein, ZFP)形成一类较大的转录因子家族[1]。

东南大学博士学位论文基因密码子使用和蛋白质结构的生物信息学分析姓名：***申请学位级别：博士专业：生物医学工程指导教师：***20040401东南大学博士学位论文摘要论文题目：基因密码子使，ｑＪ和蛋白质结构的生物信息学分析研究生姓名：顾万君导师姓名：陆祖宏（教授）学校名称：东南人学随着人类基冈组计划雨Ｉ模式生物基因组计划的完成，公共数据库中生物数据的增艮速度越来越快。

如何从海量的生物数据中解读、提取和获得有片；｜的生物信息，己成为基因组计划下一步亟待解决的问题。

本文中，我们利用生物信息学的方法对基因的周义密码子使用进行了统计分析，研究了不局物种中蛋白质结构和基因周义密码子使用间可能存在的相关性，提出了一种基于密码子的氨基酸二级结构偏向性参数。

同时，我们还在蛋白质二级结构预测的神经网络方法中引入了蛋白质编码基因的密码子使用信息．在预测大肠杆菌的蛋白质的二级结构时提高了预测的准确率。

最后，我们还提出了一种根据单倍体数据将基冈组划分成若干很少出现重组现象的块结构的新方法。

论文的主要内容如下：１．在基因组中，基因的同义密码子使用并不是随机选择的。

研究不周物种中基因的密码子使用模式以及形成这种密码子使用模式的内在因素，对于了解基因组的特征和物种的分子进化具有重要作崩。

我们对一些物种的基因密码子使用模式进行了分析，并且进一步分析了这些物种中影响密码子使用的内在因素。

●通过ＳＡＲＳ病毒基因组和进化上相近的病毒基因组的密码子使用偏性的分析，我们发现在这些病毒基冈组中尽管基因的同义密码子使用存在着偏向性，但是偏向性程度并不高。

这些病毒基因组中，影响同义密码子使用的最主要因素是进化中的碱基突变压力。

同时，基冈的功能也在一定程度上决定了这些病毒基因中密码子的选择。

但是，基因长度和基因翻译过程中的选择作用在这些病毒中并不影响基因的密码子使用。

另外，在这些病毒基囡组中，密码子使用模式在进化上是保守的。

基于密码子使用模式的进化分析表明，ＳＡＲＳ冠状病毒在进化上与其它己知的冠状病毒都不是很近。

甘蔗梢腐病菌FusariumsacchariCYP51基因克隆及遗传转化作者：周宇明黄振段真珍李慧雪暴怡雪张木清姚伟来源：《热带作物学报》2021年第12期摘要：甾醇14α-去甲基化酶（CYP51）是生物細胞膜合成所需的一个非常重要的酶，在病原菌的耐药性、致病性和生长繁殖等方面发挥着非常重要的作用。

研究表明干扰真菌的CYP51基因表达导致其无法正常生长，显著降低其致病性。

本研究以甘蔗梢腐病菌甘蔗镰刀菌（Fusarium sacchari）为试验材料，根据基因组测序数据设计特异性引物克隆得到了FsCYP51基因全长和CDS全长。

生物信息学分析表明，该基因序列全长1947 bp，编码区由2个内含子和3个外显子组成，CDS全长1554 bp，编码517个氨基酸，编码蛋白理论相对分子量为58.61 kDa。

其编码蛋白的二级结构主要由α-螺旋和无规卷曲构成，具有典型的CYP51保守结构域。

预测其亚细胞定位于细胞膜，且存在2个跨膜区域。

系统进化分析表明，FsCYP51基因属于CYP51C这一类，与串珠镰刀菌（F. verticillioides）的CYP51C基因亲缘关系最近。

同时，根据克隆到的基因全长和CDS全长构建了多价HIGS植物表达载体，并利用基因枪介导的遗传转化方法将干扰片段成功转化至甘蔗受体材料，为研究FsCYP51基因功能和创制抗梢腐病甘蔗种质奠定基础。

关键词：甘蔗镰刀菌;CYP51;基因克隆;HIGS;遗传转化中图分类号：S432.1 文献标识码：AAbstract： CYP51 is a very important enzyme for the biosynthesis of biological cell membrane， and plays a very impor-tant role in drug resistance， pathogenicity， growth and reproduction of pathogens. The interference with expression of CYP51 gene in fungi can prevent its normal growth and significantly reduce its pathogenicity. In this study， the full length of FsCYP51 gene and CDS were cloned by designing specific primers based on the genome sequencing data of Fusarium sacchari. Bioinformatics analysis showed that the total length of the gene was 1947 bp，and the coding region consisted of two introns and three exons. The total length of CDS was 1554 bp， encoding 517 amino acids. The theoretical relative molecular weight of the coding protein was 58.61 kDa. The secondary structure of the encoded protein was mainly co mposed of α-helix and random coils， and it had a typical conserved domain CYP51. Its subcellular localization was predicted in the cell membrane and there were two transmembrane regions. Phylogenetic analysis showed that the FsCYP51 gene belonged to the CYP51C class， and was closely related to theCYP51C gene of F. verticillioides. At the same time， according to the full length of the gene and CDS， the polyvalent HIGS plant expression vector was constructed， and transformed into sugarcane by particle bombardment，which would lay a foundation for the study of function of FsCYP51 gene and the creation of transgenic sugarcane resistant to Pokkah boeng disease.Keywords： Fusarium sacchari; CYP51; gene cloning; HIGS; genetic transformationDOI： 10.3969/j.issn.1000-2561.2021.12.011甘蔗（Saccharum officinarum L.）属禾本科甘蔗属植物，是我国最主要的糖料作物[1]。

第50卷第3期2021年5月福建农林大学学报(自然科学版)Journal of Fujian Agriculture and Forestry University ( Natural Science Edition )稻瘟病菌MoSCJ l 基因的生物学功能分析陈浩，王敏，杨子峰，廖煌，吴欢，凌菡，汤蔚(福建农林大学闽台作物有害生物生态防控国家重点实验室，福建福州350002)摘要：以稻瘟病菌野生型菌株Guyll 为材料，利用基因敲除和回补得到稻瘟病菌的突变体\Moscj 1和互补菌株A Moscjl / MoSCJ l,并以野生型为对照,分析了 MoSCJ l 的生物学功能.结果发现:突变体b Moscj l 在基本培养基上的生长速率显著下 降,同时,MoSCJ l 基因的缺失导致突变体对细胞壁胁迫剂更加敏感;但MoSCJ l 基因的缺失并不影响稻瘟病菌的无性繁殖、 附着胞发育及致病性.这说明MoSCJ l 参与调控稻瘟病菌的营养生长和对细胞壁胁迫剂的应答过程,不参与调控该菌的无性繁殖及致病过程.关键词：稻瘟病菌；MoSCJ l ；营养生长；细胞壁完整性中图分类号：S432.44 文献标识码：A文章编号：l67l-5470(202l)03-0309-08DOI ：l0.l3323/ki.j.fafu(nat.sci.) .202l.03.003开放科学(资源服务)标识码(OS1D )Biological function of MoSCJl in Magnaporthe oryzaeCHEN Hao, WANG Min, YANG Zifeng, L1AO Huang, WU Huan, L1NG Han, TANG Wei (State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops , Fujian Agriculture and Forestry University ,Fuzhou, Fujian 350002, China)Abstract : We obtained the MoSCJ l gene deletion mutant from the wild type Guyll of Magnaporthe oryzae and the complemented strain via gene knock-out and complementation technologies , and then analyzed the biological functions of MoSCJ l in rice blast fun ­gus. We found that the deletion of MoSCJ l significantly impaired the vegetative growth of M. oryzae on basic medium and led to high ­er sensitivity to cell wall stressors. However , the loss of MoSCJ l had no effects on the sporulation , appressorium development and pathogenicity. 1n conclusion , MoSCJ l is likely to be involved in the regulation of vegetative growth and cell wall integrity of M. oryzae , instead of the regulation of asexual propagation and pathogenicity.Key words : Magnaporthe oryzae ； MoSCJ l ； vegetative growth ； cell wall integrity稻瘟病是全球水稻生产上的重要病害之一，对水稻的产量和品质具有巨大的威胁，发生严重时能造成 整片稻田绝收.稻瘟病菌(Magnaporthe oryzae )通过分生抱子传播，分生抱子在水稻叶片表面萌发产生芽管 并形成附着胞，后产生侵染栓穿透叶片的角质层和表皮细胞壁，在寄主细胞内分化产生次生菌丝并侵染邻 近细胞和组织，继而发病.内质网是由囊状、管状和泡状结构组成的一个连续的网膜系统.真核细胞中，在折叠酶、伴侣结合蛋白 等协助下，内质网对分泌性蛋白质合成、转运和加工并运输到相关的细胞部位发挥功能.为了保证蛋白质 能正确折叠和修饰,以达到天然构象从而转运到目标细胞器或分泌到胞外,细胞内部有一套严格的蛋白质 质量控制系统，一旦蛋白质由于转录翻译失败或不同条件压力等原因发生错误折叠并聚集在内质网中，内 质网就会产生压力，影响细胞的正常生理功能［'-2］.真核细胞存在两条途径调控内质网压力：非折叠蛋白响应(unfolded protein response , UPR)和内质网 相关蛋白降解(endoplasmic reticulum-associated protein degradation , ERAD)⑶.其中,ERAD 主要通过泛素 化降解内质网中错误折叠或未折叠的蛋白［4］，其包括底物识别、定位、泛素化、逆向运输和蛋白酶体降解 等步骤.底物识别水平决定底物的降解程度.蛋白质错误折叠是由于其本应在蛋白质内部的疏水区暴露在 外从而引起蛋白质之间的聚集而发生,在底物识别的过程中,热休克蛋白Hsp70家族成员(Bip/Kar2)会收稿日期：2020-07-28 修回日期：2020-l0-22基金项目：福建农林大学科技创新专项基金项目(CXZX20l9007G 、CXZX20200l4A ).作者简介：陈浩(l998-),男.研究方向：植物保护.Email : chenho998@ .通信作者汤蔚( l988-),男，讲师，博士.研究方向：真菌功能基 因 组 学 .Email : tangw@ .-310-福建农林大学学报(自然科学版)第50卷与疏水区结合促进多肽链折叠,从而避免蛋白质分子的聚集[5].与Hsp70长时间作用后的底物会被E3泛素连接酶Hrdl复合物识别⑷.Hsp40/DnaJ是一个可以调节Hsp70分子伴侣活性的蛋白质家族,Hsp40s刺激Hsp70蛋白固有的弱腺苷三磷酸(adenosine triphosphate,ATP)酶活性，使其由ATP状态转变为腺苷二磷酸(adenosine diphosphate,ADP)状态，从而对底物有强亲和性，促进Hsp70与多肽底物的相互作用. Hsp70家族成员通常有多个Hsp40伴侣，其特定的配对控制Hsp70伴侣参与调控特定过程.所有Hsp40均含有高度保守的75个氨基酸J结构域，该结构域与Hsp70的ATP酶结构域相互作用，促进ATP水解[7].研究表 明，酿酒酵母(Saccharomyces cerevisiae)基因组的Hsp40/DnaJ家族中有22种蛋白质，其中包括Scjl蛋白囚.Scjl作为一种蛋白分子伴侣参与蛋白的折叠过程.已有研究发现，缺失Scjl会影响Hsp70的活性，进而影响ERAD途径中底物的识别及降解过程，最终启动细胞的凋亡程序冈.目前，有关Scjl在丝状真菌中的研究较少.本试验利用基因敲除技术获得MoSCJ l基因敲除突变体(^Moscj l)，研究其营养生长、无性繁殖、致病力及对细胞壁胁迫剂的应答等情况，以探讨MoSCJ l基因在稻瘟病菌中的生物学功能，为其他病原真菌中SCJl基因的研究提供一定的参考.1材料与方法1.1供试菌株及其他材料本研究中所用稻瘟病菌野生型菌株Guyll由福建农林大学闽台作物有害生物生态防控国家重点实验室保存,△M oscj l突变体和互补菌株A Moscj l/MoSCJ l均由本试验获得.试验所用的水稻品种CO39、大麦种子均保存于福建农林大学闽台作物有害生物生态防控国家重点实验室的4冷库中.完全培养基(complete medium,CM)：20x硝酸盐50mL•L_l,l000x微量兀素l mL•L_l,l000x维生素溶液l mL-LT,葡萄糖l0g-LT,蛋白胨2g-LT,水解酪蛋白l g-LT,酵母提取物l g-LT，琼脂粉l5g•L_l.基本培养基(minimal medium,MM):NaNO36g,KCl0.52g,MgSO4•7H2O0.l52g,KH2PO4 l.52g,维生素Bl0.0l g,微量元素溶液l mL,葡萄糖l0g,琼脂粉l5g.稻秆培养基(straw decoction and corn,SDC):稻秆l00g-L-l(煮沸20min,两层纱布过滤)，玉米粉40g-，琼脂粉l5g-⑼.TB3培养基:水解酪蛋白3g-LT,酵母提取物3g-LT,蔗糖20%,琼脂粉l2g-L»色氨酸缺陷型合成葡萄糖基础培养基(synthetic dextrose minimal medium without tryptophan,SD-Trp):无氨基酵母氮源6.7g,D-山梨糖醇l82.2g,缺色氨酸氨基酸粉末0.74g,葡萄糖20g,琼脂粉l5g,pH5.8[l0].LB培养基:胰蛋白胨l0g-L'1，酵母提取物5g•L_1,NaCl10g-L_1，琼脂15g-L_1;酵母膏胨葡萄糖琼脂培养基(yeast extract peptone dex­trose medium,YPD):葡萄糖20g-L'1，蛋白胨20g-L'1，酵母提取物10g-L'1，琼脂粉15g•L'1.试剂与仪器:潮霉素B,北京索莱宝科技有限公司；博来霉素，美国Innivogen公司；Southern杂交试剂盒,美国罗氏公司;rTaq酶、RNase,宝日医生物技术(北京)有限公司；刚果红(congo red,CR)，西格玛奥德里奇(上海)贸易有限公司；microcloth,EMD Millipore公司；T100PCR仪，美国Bio-Rad公司;智能培养箱,宁波江南仪器厂;Nikon E200生物显微镜、激光扫描共聚焦显微镜，日本尼康公司；血球计数板，北京索莱宝科技有限公司；Centrifuge5420离心机，德国艾本德股份公司；PowerShot G7X Mark H相机，佳能(中国)有限公司.1.2MoScjl的系统发育分析在酵母基因组数据库中得到酿酒酵母Scjl的蛋白质序列，根据其与本试验稻瘟病菌Guyll的序列同源性比对结果，得到的同源蛋白为MoScjl.在NCBI中利用Blast比对得到一组MoScjl的同源蛋白序列，选取8个不同物种的序列在MEGA5.0软件中采用邻接法(neighbor-joining,NJ)建立系统发育树[11].1.3MoSCJ l突变体的获得1.3.1MoSCJ l基因的敲除以稻瘟病菌野生型菌株Guyll基因组为模板，以MoSCJ1-F1/MoSCJ1-F2和MoSCJ1-F3/MoSCJ1-F4(表1)两对引物对MoSCJ l基因上游和下游各1000bp左右的DNA片段进行PCR 大量扩增.与此同时，将pCX62质粒作为模板，以引物HYH-F/HYH-R(表1)对潮霉素磷酸转移酶基因全长进行PCR扩增,并对扩增结果进行纯化回收.将扩增好的MoSCJ l基因上游和下游各1000bp左右的第3期陈浩等:稻瘟病菌MoSCJ l基因的生物学功能分析-311-DNA片段及潮霉素磷酸转移酶基因全长中的目的片段进行融合PCR,获得长度约为3400bp的重组片段.以引物MoSCJ1-F1/MoSCJ1-F4（表1）对重组片段进行大量扩增,纯化回收所得片段，将回收产物用于原生质体的转化[12].用潮霉素（600愿-mf）筛选转化子.表1试验所用引物Table1Primers used in this study序列（5'f3‘）引物MoSCJl-Fl MoSCJ1-F2 MoSCJ1-F3 MoSCJ1-F4 MOSCJ1-BY-F ACTATAGGGCGAATTGGGTACCGCATCGTCTCCATGGCTGA GCGCCGAATTCGATATCAAGCTTGGCACCTAAGGCTCACTGC TGCCGACCGGGCCGGCCGGATCCGCGAAGGTGCGGTTAGAC GGTGGCGGCCGCTCTAGACGATGTTGTCAACCTCGATG CCTCAGTGAGGTAGCAAGHY-R GTATTGACCGATTCCTTGCGGTCCGAAMoSCJ1-KO-F MoSCJl-KO-R HYH-F CTGAGTTCCAGTGGGAGAGG CCTCCTTCCAGAACAGATCG TGGAGCTAGTGGAGGTCAACAATGAATGHYH-R CGGTCGGCATCTACTCTATTCCTTTGC GFP-R CGGTGGTGCAGATGAACTTCNP-SCJ1-F NP-SCJ1-R ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTGAACTTCTCCTGTCCGTACT CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCAACTCATCATGACCGGAGG1.3.2MoSCJ1突变体的鉴定用灭菌的牙签挑选转化子至CM培养基上，培养4d后，用CTAB法粗提转化子DNA[I3然后以粗提的转化子DNA为模板，用引物MoSCJ1-KO-F/MoSCJ1-KO-R（表1）进行PCR验证，阳性对照以野生型菌株Guy11基因组为模板,阴性对照以无菌水为模板，在阳性对照有条带、阴性对照无条带的前提下,对无条带出现的转化子进行下一步验证;以无条带的粗提转化子DNA为模板，用引物MoSCJ1-BY-F/HY-R（表1）再次进行PCR验证，阳性对照和阴性对照同上，在阳性对照、阴性对照均无条带的前提下，有条带的转化子即为突变体.1.4回补菌株的获得1.4.1回补载体的构建以野生型菌株Guy11基因组为模板，以引物NP-SCJ1-F/NP-SCJ1-R（表1）进行PCR扩增，得到长度为4247bp的片段.将其与pYF11骨架片段共同转入XK-125酵母感受态细胞中，并于SD-Trp培养基上培养24h.以酵母转化子为模板，用引物MoSCJ1-KO-F/GFP-R（表1）进行PCR验证,取有条带的酵母转化子在YPD培养基中摇培18h,提取质粒.将酵母质粒转入大肠杆菌中,再进行PCR验证,并将结果测序，获得回补载体.1.4.2回补菌株的鉴定将回补质粒转入突变体的原生质体中，培养4d后挑出转化子，在激光扫描共聚焦显微镜下用博来霉素（200jig-mL_1）进行筛选.1.5MoSCJl突变体的生物学表型分析1.5.1突变体在不同培养基上的营养生长从保菌袋中取出野生型菌株Guy11、突变体和回补菌株的存菌滤纸片分别接种于CM培养基上，于28T黑暗培养箱中培养5d.然后在各菌落边缘切取3mmX3mm 的菌块并接种于CM、MM、SDC培养基上，每个菌株设3个重复.在28弋黑暗培养箱中培养7d后,测量、记录其直径并拍照.1.5.2突变体对细胞壁胁迫剂的响应从野生型菌株Guy11、突变体和回补菌株的菌落边缘切取3mmX 3mm的菌块，分别接种于含有胁迫剂CR（400ig•mL"1）的CM培养基上，每个菌株设3个重复.在28°C 黑暗培养箱中培养7d后，测量、记录其直径并拍照，同时计算抑制率,抑制率=（空白组菌株直径-处理组菌株直径）/空白组菌株直径X100%.1.6MoSCJ l突变体的产抱量测定及分生抱子梗观察1.6.1产孢量的测定从野生型菌株Guy11、突变体和回补菌株的菌落边缘切取3mmX3mm的菌块，分别接种于SDC培养基上，每个菌株设3个重复，在28C黑暗培养箱中培养5d.刮去表面气生菌丝,置于黑光灯下培养5d后,用无菌水冲洗培养基,2层microcloth过滤于10mL离心管中得到抱子液，并最终定容・3l2・福建农林大学学报(自然科学版)第50卷至2mL.在血球计数板上统计抱子数目，并记录数据.l.6.2分生孢子梗的观察菌株培养方法同l.6.l.切2cmx2cm的菌块于载玻片上，菌丝面贴载玻片，黑光灯下培养2d后，在Nikon E200生物显微镜下观察分生抱子梗产抱情况并拍照.试验重复3次.1.7MoSCJ l突变体的附着胞萌发试验抱子液的制备方法同l.6.l,调整抱子液浓度为8xl05个・mL-l・将疏水性盖玻片置于载玻片上，向盖玻片上滴40|xL抱子液后，将载玻片放入保湿盒中，于28°C黑暗培养箱中培养,分别于4、8、l2、24h在Nikon E200生物显微镜下观察分生抱子的萌发情况并拍照,每个菌株统计l00个抱子，计算抱子萌发率和附着胞形成率.试验重复3次.1.8MoSCJ l突变体的侵染试验l.8.l水稻侵染种植感病水稻品种CO39,于26C温室中培养至三叶一心期.抱子液的制备方法同l.6.l，用血球计数板调整抱子液浓度为8xl05个・mL-1,向抱子液中加入0.2%(体积分数)明胶，混匀，用喷壶均匀地喷洒在水稻叶片上.然后将水稻置于28C黑暗培养箱中培养24h,再放在接种室培养6d,观察水稻的感染情况,统计并记录病斑等级［l4］，对感病水稻叶片进行拍照.试验重复3次.l.8.2离体大麦侵染种植大麦,于温室中培养9d.抱子液的制备方法同l.6.l,用血球计数板调整抱子液浓度为8xl05个・mL-1,向抱子液中加入0.2%(体积分数)明胶.剪取长度一致的大麦叶片，滴20応抱子液于大麦叶片背部，将大麦叶片置于28C黑暗培养箱中保湿培养24h.撕取大麦背部表皮，在Nikon E200生物显微镜下观察菌丝侵染情况并拍照.试验重复3次.1.9数据处理用Excel20l0软件对试验数据进行分析和处理，用DPS数据处理系统进行单因素统计分析.2结果与分析2.1MoScjl系统发育分析系统发育树分为两大分支,MoScjl与酿酒酵母S288C具有高度同源性,且从整体来看与其他7个物种裂殖酵母(Schizosaccharomyces pombe)、拟南芥(Arabidopsis thaliana)、粘菌(Dictyostelium discoideum AX4)、果蝇(Drosophila melanogaster)、利什曼原虫(Leishmania donovani)、秀丽线虫(Caenorhabditis ele-gans)、人类(Homo sapiens)的同源蛋白序列均有相似性.由此推测这些物种的Scjl同源蛋白在功能上可能具有保守性.85---------------------------------------------------酿酒酵母Saccharomyces cerevisiae S288C98--------------------------------------------Magnaporthe oryzae---------------------------------裂殖酵母Schizosaccharomyces pombe-------------------------------拟南芥Arabidopsis thaliana99-------------------------------------------------ttW Dictyostelium discoideum AX4---------------------------------------------果蝇Drosophila melanogaster59------------------------------------------------禾Leishmania donovani88-----------------------------------------Caenorhabditis elegans91-----------------------------人类Homo sapiens图l稻瘟病菌Scjl与其他8个物种同源蛋白序列的系统发育树Fig.l Phylogenetic tree of the homologous amino acid sequences of Scjl in M.oryzae and other8species2.2MoSCJ l突变体的鉴定利用同源重组原理构建了基因敲除载体(图2A)，通过原生质体转化的方法，得到具潮霉素抗性的转化子.粗提转化子基因组DNA,利用基因内部引物、臂外引物进行PCR验证，基因内部引物验证无条带、臂第3期陈浩等:稻瘟病菌MoSCJl 基因的生物学功能分析・313・外引物验证有条带的转化子即为突变体.结果显示,转化子'Mosc/1-4、'Mosc/1-22、'Mosc/1-23和'Mosc/1 - 37为符合条件的MoSCJ l 基因敲除突变体（图2B ）.随机选取'Moscj l -22突变体用于后续试验.2.3 MoSCJ 1突变体的营养生长图3显示,突变体菌株在CM 和SDC 培养基上生长的直径与野生型菌株和回补菌株相比无明显差异, 而在MM 培养基上生长的直径相比于野生型菌株和回补菌株极显著缩小（P <0.01）.可见,MoSCJ l 参与调 控稻瘟病菌在基本培养基上的营养生长过程.A 1 kbB MoSCJl -Guyll -----------------------——---------------------- 寻 gdDsowd 0 旨owd 寸\ hph I\Moscj\2F 2RA.MoSCJl 基因的敲除策略；B.PCR 验证结果（Guyll 为以野生型菌株为模板的阳性对照,Mock 为以无菌水为模板的阴性对照）.图2 MoSCJ1基因的靶向敲除Fig.2 The targeted deletion of MoSCJl geneB ■Guyll □ ^Moscjl**表示同一培养基下不同菌株之间的差异极显著(P <0.01).图3 野生型菌株Guy11、突变体NMoscj1、回补菌株^Moscj1/MoSCJ1在CM 、MM 、SDC 培养基上的营养生长情况(A )和菌落直径(B )Fig.3 Vegetative growth (A) and colony diameters ( B) of Guyl 1, 'Moscjl and 'Moscjl /MoSCJl on CM , MM, SDC plates2.4 MoSCJ 1突变体对细胞壁胁迫剂的响应相比于野生型菌株和回补菌株,突变体对CR 更加敏感（图4）,抑制率呈现极显著差异（P <0. 01）.这 说明MoSCJ l 参与调控稻瘟病菌对细胞壁胁迫剂的应答过程.CK 为不加入胁迫剂的对照组；CR 为加入刚果红的处理组.**表示差异极显著（P <0.01）.图4 野生型菌株Guy11、突变体\Moscj1、回补菌株^Moscj1/MoSCJ1在含有CR 的CM 培养基上的生长情况（A ）和抑制率（B ）Fig.4 The growth （A ） and inhibition rate of Guyll , 'Moscjl and 'Mosc/l/MoSCJl on CM plates containingCR・3l4・福建农林大学学报（自然科学版）第 50 卷2.5 MoSCJ l 突变体的无性繁殖结果显示，野生型菌株、突变体和回补菌株均能产生大量抱子（图5A ）,且三者之间的产抱量无显著 差异（图5B ）.由此推测MoSCJ l 不参与调控稻瘟病菌的无性繁殖过程.AB 二 A n u 204 O 1612Guyl 1 bMoscj\ NMoscj'l MoSCJX 菌株类型旨owaUUSOW 己。

Bio Med CentralBMC BioinformaticsResearch articleThe COG database: an updated version includes eukaryotesRoman L Tatusov*1, Natalie D Fedorova 1, John D Jackson 1, Aviva R Jacobs 1,Boris Kiryutin 1, Eugene V Koonin 1, Dmitri M Krylov 1, Raja Mazumder 2, Sergei L Mekhedov 1, Anastasia N Nikolskaya 2, B Sridhar Rao 1,Sergei Smirnov 1, Alexander V Sverdlov 1, Sona Vasudevan 1, Yuri I Wolf 1, Jodie J Yin 1 and Darren A Natale 2Address: 1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda MD, USA and 2Protein Information Resource, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007, USAEmail: Roman L Tatusov*-tatusov@; Natalie D Fedorova -fedorova@;John D Jackson -jjackson@; Aviva R Jacobs -jacobs@; Boris Kiryutin -kiryutin@; Eugene V Koonin -koonin@; Dmitri M Krylov -krylov@; Raja Mazumder -rm285@;Sergei L Mekhedov -mekhedov@; Anastasia N Nikolskaya -ann2@; B Sridhar Rao -rao@; Sergei Smirnov -smirnov@; Alexander V Sverdlov -asverdlo@;Sona Vasudevan -vasudeva@; Yuri I Wolf -wolf@; Jodie J Yin -yin@; Darren A Natale -dan5@ * Corresponding author AbstractBackground: The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.Results: We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted)proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7eukaryotic genomes: three animals (the nematode Caenorhabditis elegans , the fruit fly Drosophila melanogaster and Homo sapiens ),one plant, Arabidopsis thaliana , two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe ), and the intracellular microsporidian parasite Encephalitozoon cuniculi . The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.Conclusion: The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.Published: 11 September 2003BMC Bioinformatics 2003, 4:41Received: 20 May 2003Accepted: 11 September 2003This article is available from: /1471-2105/4/41© 2003 Tatusov et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.本页已使用福昕阅读器进行编辑。

doi:10.1128/AEM.02309-09.Appl. Environ. Microbiol. 76(1):1-22. .MICROBIOLOGY: 2010 INSTRUCTIONS TO AUTHORS APPLIED AND ENVIRONMENTAL2010. , , , et al.INSTRUCTIONS TO AUTHORSMICROBIOLOGY: 2010APPLIED AND ENVIRONMENTAL/cgi/content/full/76/1/1Updated information and services can be found at:These include:CONTENT ALERTSmore>>cite this article), eTOCs, free email alerts (when new articles RSS Feeds,Receive:/subscriptions/To subscribe to an ASM journal go to: /misc/reprints.dtlInformation about commercial reprint orders: at Shanghai Jiao Tong University July 11, 2010 - DOWNLOADED FROMAPPLIED AND ENVIRONMENTAL MICROBIOLOGY2010INSTRUCTIONS TO AUTHORS*SCOPEApplied and Environmental Microbiology (AEM)pub-lishes descriptions of all aspects of applied microbial research,basic research on microbial ecology,and research of a genetic and molecular nature that fo-cuses on microbial topics of practical value.Research must address salient microbiological principles,fun-damental microbial processes,or basic questions in applied or 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植物病害研究法1.列举五个你熟悉的中英文文献检索数据库。

中文：维普、万方、CNKI三大期刊知识库，超星、书生之家书籍库。

英文：Pubmed、SD、EBSCO、Springer、NPG、Ei Village、CAS、Kluwer、ProQuest、Ovid。

2.谈谈如何做好文献检索工作。

（1）根据题目确定关键词，随着对题目的深入了解，增加关键词，保证文章的查全率。

（2）选择合适的数据库，常用的中文数据库有……英文数据库有……（3）使用相应的检索技术，如布尔逻辑算符、位臵检索、截词、优先算符等，来提高检索的效率，另外，需要注意of、the、in、she、he、to be、as、because、if、when等为禁用词，在检索时自动忽略。

（4）将检索到的文献进行筛选和归类：分精读与速读两类；只保留重要的资料；精细分类，并录入数据库。

（5）科学合理使用文件管理软件，如Endnote和Reference Manage。

（6）学会走进文献、走出文献，从文献中发现Idea。

3.谈谈文献检索工作对科研工作者的重要性。

第一，有助于利用和掌握文献资料，缩短查找文献资料所花费的时间。

在科学研究活动中，搜集、掌握足够的文献资料是研究工作的重要组成部分，往往要耗费较多的时间，而文献数量的成倍增长，各学科的相互渗透导致的文献交叉与分散化，使查找所需文献更加固难。

在这种情况下，文献检索的功能和作用，恰恰能解决众多繁杂的文献与研究者特定需要之间的矛盾，能帮助研究者从浩如烟海的文献中迅速、准确地查找出所需专用文献，达到节省时间和精力的目的。

第二，有助于获取最新知识，及时了解研究课题内容所涉及的专业和相关学科的发展动态，指导课题研究的顺利进行。

第三，有助于开阔视野，扩大知识面，借鉴他人之法，指引治学门径，解决研究过程中的疑难问题。

通过文献检索，不仅能够较快地获得所必须了解和掌握的知识，获得大量的情报信息，而且还能够比较有关文献的异同优劣，明确学术源流，达到正确鉴别、准确选择所需文献资料的目的。