Characterization of six novel NAC genes and their responses to abiotic stresses in Gossypium

WRKY家族基因功能的研究进展鹿宏丽⑺，3付嘉智⑺，3武鹏雨1'2'3龙国辉张锐!-2-3（1.新疆生产建设兵团塔里木盆地生物资源保护利用重点实验室，新疆阿拉尔843300；2.塔里木大学植物科学学院，新疆阿拉尔843300；3.南疆特色果树高效优质栽培与深加工技术国家地方联合工程实验室，新疆阿拉尔843300）摘要：WRKY转录基因对于植物生长发育以及植物抗逆性具有重要的作用。

本文分析了WRKY转录基因的结构，综述了WRKY基因在植物细胞器构成、木质素合成、果实成熟、抗逆等方面的功能，为后期验证WRKY转录基因家族成员在植物生长发育过程的功能研究提供借鉴，同时也为WRKY基因家族功能的明晰提供思路。

关键词：WRKY转录因子；基因调控；功能研究中图分类号：S-3文献标识码：A刖言植物的生长离不开转录因子对基因的调控，WRKY 转录因子存在于高等植物各个生长发育过程中，因整个WRKY基因家族都具有保守的由WRKYCQK氨基酸残基形成的序列而得名。

WRKY基因对植物生长发育、休眠、调控木质素生物合成以及抗逆等过程起到重要调控作用[l]o随着分子技术水平的不断提升，越来越多该基因家族成员被鉴定并进行了克隆和表达。

第1个被克隆的WRKY家族的成员是甘薯中的SPF1[2]o多数植物鉴定得到的WRKY基因家族成员的数量一般在50~90左右，但如苹果、杨树WRKY基因家族成员相对较多，超过100个[3-12]o WRKY转录因子具有特殊的DNA结合域，含有1个或2个由60个氨基酸组成的WRKY保守序列，该序列N端含有由色氨酸、精氨酸等7个氨基酸残基组成的WRKY七肽，C端是具有C2 H2型或C2HC型锌指结构[13]o虽然绝大多数植物中WRKY基因家族成员该序列是绝对保守的，但近年来发现有些植物WRKY基因家族部分成员的WRKY保守序列中出现了N端保守序列氨基酸残基的缺失、替换或C端锌质结构的缺失[4]o除此之外，WRKY转录因子作为转录因子还含有核信号定位区（NLS）、转DOI：10.19754/j.nyyjs.20210615003录调控区、亮氨酸拉链以及各种氨基酸富集区等结构域[14]o以WRKY保守序列作为依据，可将植物WRKY基因家族分为3种类型。

植物NAC转录因子的研究进展邢国芳;张雁明;长魏斌;马新耀;韩渊怀【摘要】近年来,新发现的NAC转录因子是具有多种生物功能的植物特异性转录因子,其N端为150个左右保守的氨基酸组成的NAC结构域.NAC转录因子在植物生长发育、激素调节和抵抗逆境胁迫等方面发挥着重要的作用.就植物NAC转录因子的基本结构特征、生物学功能及其在植物细胞次生壁生物合成过程中的作用进行了综述.%NAC transcription factors were new type transcription regulatory factors that possessed multiple biological functions in plants. They contained a conserved NAC domain about 150 ammo acids in N-terminal ends. The NAC transcription factors play very important roles in regulation of plant growth and development, hormone level and response to various kinds of stresses. This article reviews the basic structure, biology function and functions as master transcriptional in the biosynthesis of secondary walls in vascular plants.【期刊名称】《山西农业科学》【年(卷),期】2012(040)004【总页数】4页(P409-411,423)【关键词】NAC转录因子;生物学功能;纤维素合成【作者】邢国芳;张雁明;长魏斌;马新耀;韩渊怀【作者单位】山西农业大学农学院,山西太谷030801;山西农业大学生物工程研究所,山西太谷030801;山西农业大学农学院,山西太谷030801;山西农业大学农学院,山西太谷030801;山西农业大学农学院,山西太谷030801;山西农业大学农学院,山西太谷030801;山西农业大学生物工程研究所,山西太谷030801【正文语种】中文【中图分类】Q786NAC类转录因子是高等植物所特有的一类转录调控因子，其最初命名来源于矮牵牛NAM基因、拟南芥ATAF1/2和CUC1/2基因首字母的缩写，因为这些基因编码蛋白的N端均包含有一段保守的氨基酸序列与NAM蛋白高度同源，所以，将其称为NAC蛋白的结构域[1]。

ORIGINAL ARTICLEPreparation and Characterization of a NovelExtracellular Polysaccharide with Antioxidant Activity,from the Mangrove-Associated Fungus Fusarium oxysporumYan-Li Chen &Wen-Jun Mao &Hong-Wen Tao &Wei-Ming Zhu &Meng-Xia Yan &Xue Liu &Tian-Tian Guo &Tao GuoReceived:1August 2013/Accepted:7January 2015/Published online:28January 2015#Springer Science+Business Media New York 2015Abstract Marine fungi are recognized as an abundant source of extracellular polysaccharides with novel structures.Mangrove fungi constitute the second largest ecological group of the marine fungi,and many of them are new or inadequate-ly described species and may produce extracellular polysac-charides with novel functions and structures that could be explored as a source of useful polymers.The mangrove-associated fungus Fusarium oxysporum produces an extracel-lular polysaccharide,Fw-1,when grown in potato dextrose-agar medium.The homogeneous Fw-1was isolated from the fermented broth by a combination of ethanol precipitation,ion-exchange,and gel filtration chromatography.Chemical and spectroscopic analyses,including one-and two-dimensional nuclear magnetic resonance spectroscopies showed that Fw-1consisted of galactose,glucose,and man-nose in a molar ratio of 1.33:1.33:1.00,and its molecular weight was about 61.2kDa.The structure of Fw-1contains a backbone of (1→6)-linked β-D -galactofuranose residues with multiple side chains.The branches consist of terminal α-D -glucopyranose residues,or short chains containing (1→2)-linked α-D -glucopyranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.The side chains are connected to C-2of galactofuranose res-idues of backbone.The antioxidant activity of Fw-1was eval-uated with the scavenging abilities on hydroxyl,superoxide,and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro,and the results indicated that Fw-1possessed good antioxidant activ-ity,especially the scavenging ability on hydroxyl radicals.Theinvestigation demonstrated that Fw-1is a novel galactofuranose-containing polysaccharide with different structural characteristics from extracellular polysaccharides from other marine microorganisms and could be a potential source of antioxidant.Keywords Mangrove-associated fungus .Fusarium oxysporum .Extracellular polysaccharide .Preparation .Characterization .Antioxidant activityIntroductionMangroves grow in saline coastal sediment habitats in the tropics and subtropics harboring a great diversity of marine fungi (Shearer et al.2007).Mangrove fungi constitute the second largest ecological group of the marine fungi and may produce chemicals with novel functions and structures (Kobayashi and Tsuda 2004).Fungi often produce extracellu-lar polysaccharides that are secreted into the growth media or remain tightly attached to the cell surface (Seviour et al.1992).The research on extracellular polysaccharides from marine fungi is attempted for providing polysaccharide with novel functions and structures (Chen et al.2012;Sun et al.2011).The extracellular polysaccharides produced by marine fungi become an important research area in new drug discovery and show enormous development prospects (Kanekiyo et al.2005).Polysaccharides with hexofuranose units are of interest be-cause of their unique structures and specific properties (Leal et al.2010).The investigations showed that galactose is the most widespread hexose in furanose form in naturally occur-ring polysaccharides (Pedersen and Turco 2003;Peltier et al.2008).The galactofuranose-containing extracellularY .<L.Chen :W.<J.Mao (*):H.<W.Tao :W.<M.Zhu :M.<X.Yan :X.Liu :T.<T.Guo :T.GuoKey Laboratory of Marine Drugs,Ministry of Education,Institute of Marine Drugs and Foods,Ocean University of China,5Yushan Road,Qingdao 266003,People ’s Republic of China e-mail:wenjunmqd@Mar Biotechnol (2015)17:219–228DOI 10.1007/s10126-015-9611-6polysaccharides with novel structural characteristics have been isolated from the fermented broth or cell walls of some microorganisms(Gander et al.1974;Ikuta et al.1997;Latgéet al.1994;Unkefer and Gander1990).With today’s interest in new renewable sources of polymers,the galactofuranose-containing extracellular polysaccharides represent potential source to be explored.However,the galactofuranose-containing extracellular polysaccharides from marine fungi have not yet been fully studied.In the current study,a novel galactofuranose-containing extracellular polysaccharide was isolated from the fermented broth of the mangrove-associated fungus Fusarium oxysporum by a combination of ethanol precipitation,ion-exchange,and gel filtration chroma-tography,and its structural characterization was investigated using chemical and spectroscopic methods,including one-and two-dimensional nuclear magnetic resonance(1D and 2D NMR)spectroscopic analyses.The antioxidant activity of the extracellular polysaccharide was also evaluated by scavenging assays involving hydroxyl,superoxide,and1,1-diphenyl-2-picrylhydrazyl(DPPH)radicals.Materials and MethodsMaterialsMonosaccharides(D-glucose,L-rhamnose,D-xylose,L-arabi-nose,D-mannose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonic acid,D-mannuronic acid,N-acetyl-β-D-glucos-amine),1,1-diphenyl-2-picrylhydrazyl,trifluoroacetic acid, thiobarbituric acid,trichloroacetic acid,and1-phenyl-3-meth-yl-5-pyrazolone were from Sigma-Aldrich(St.Louis,MO, USA).Pullulan standards(Mw=344,200,107,47.1,21.2, and9.6kDa)were from the Showa Denko K.K.(Tokyo, Japan).Q Sepharose Fast Flow and Sephacryl S-100were from GE healthcare(Piscataway,NJ,USA).Dialysis mem-branes(flat width,44mm;molecular weight cut-off,3500) were from Lvniao(Yantai,China).Microbial Strain and Culture ConditionsThe marine fungus F.oxysporum was isolated from fresh leaves of Ipomoea pes-caprae(Linn.)collected from South Sea,China.It was identified according to its morphological characteristics and18S rRNA sequences,and the accession number of Genbank was JN604549.Briefly,the fungus was cultivated in the liquid medium containing yeast extract(3g/ L),peptone(5g/L),glucose(20g/L),malt extract(3g/L),sea salt(24.4g/L),KH2PO4(0.5g/L),NH4Cl(0.5g/L),pH6.0–6.5,at25°C for40days,and50L of fermented broth was obtained.Preparation of the Extracellular PolysaccharideThe fermented broth was filtered through cheese cloth,the filtrate was concentrated to1/15of its original volume under reduced pressure at40°C,and a threefold of the volume of 95%(v/v)ethanol was added.The resulting precipitate was recovered by centrifugation at3600×g for10min,dialyzed in cellulose membrane tubing against distilled water for72h. The retained fraction was dried,and the protein in the fraction was removed as described by Matthaei et al.(1962).The crude polysaccharide was fractionated by anion-exchange chroma-tography using a Q Sepharose Fast Flow column(30×3cm) coupled to an AKTA FPLC system and elution with a step-wise gradient of0,0.2,and1.0M NaCl.The fractions were assayed for carbohydrate content by the phenol–sulfuric acid method.The fractions eluted with distilled water were pooled, dialyzed,and further purified on a Sephacryl S-100column (70×2cm)eluted with0.2M NH4HCO3at a flow rate of 0.3mL/min.The major polysaccharide fractions were pooled, freeze–dried,and designated as Fw-1.Determination of Purity and Molecular WeightPurity and molecular weight were determined by high-performance gel permeation chromatography(HPGPC)with a Shodex Ohpak SB804(7.8×300mm,Tokyo,Japan)column and a refractive index detector(Agilent RID-10A Series),and elution with0.1M Na2SO4at a flow rate of0.5mL/min(Li et al.2012).Of1%sample solutions in0.2M Na2SO4,20μL was injected.The molecular weight was estimated by refer-ence to a calibration curve made by pullulan standards.General AnalysisTotal sugar content was measured by the phenol–sulfuric acid method using galactose as the standard(Dubois et al.1956). Protein content was assayed according to the modified Lowry method(Bensadoun and Weinstein1976).Sulfate content was measured according to Silvestri et al.(1982).Uronic acid con-tent was determined by the carbazole–sulfuric acid method (Bitter and Muir1962).Analysis of Monosaccharide CompositionFive milligrams of polysaccharide was hydrolyzed with2M trifluoroacetic acid at100°C for6h.Excess acid was re-moved by co-distillation with methanol after the hydrolysis was completed.Sample was subjected to reversed-phase high-performance liquid chromatography(HPLC)after pre-column derivatization and UV detection(Li et al.2011). Sugar identification was done by comparison with reference sugars(D-glucose,L-rhamnose,D-xylose,L-arabinose,D-man-nose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonicacid,D-mannuronic acid,N-acetyl-β-D-glucosamine). Calculation of the molar ratio of the monosaccharide was car-ried out on the basis of the peak area of the monosaccharide. Methylation AnalysisMethylation analysis was performed by the method of Hakomori(1964)with some modifications.In brief, 2mg of polysaccharide in dimethyl sulfoxide was meth-ylated using NaH and iodomethane,and the completion of methylation was confirmed by Fourier transform infrared (FTIR)spectroscopy by the disappearance of OH bands. After hydrolysis with2M trifluoroacetic acid at105°C for6h,the methylated sugar residues were converted to partially methylated alditol acetates by reduction with NaBH4,followed by acetylation with acetic anhydride. The derivatised sugar residues were extracted into dichlo-romethane and evaporated to dryness,and dissolved again in100μL of dichloromethane.The products were ana-lyzed by gas chromatography–mass spectrometry(GC-MS)on a DB225using a temperature gradient of100–220°C with heating at a rate of5°C/min and mainte-nance of a temperature at220°C for15min.GC-MS was performed on an HP6890II instrument.Identification of partially methylated alditol acetates was carried out on the basis of retention time and mass fragmentation patterns.IR Spectroscopy AnalysisFTIR spectra were measured on a Nicolet Nexus470spec-trometer.The polysaccharide was mixed with KBr powder, ground up,and then pressed into1-mm pellets for FTIR mea-surements in the frequency range of4000–500cm−1with a resolution of4.0cm−1and320scans co-addition.NMR Spectroscopy Analysis1H nuclear magnetic resonance(NMR)and13C NMR spectra were measured at23°C using a JEOL JNM-ECP600MHz spectrometer.60mg of polysaccharide was deuterium ex-changed by two successive freeze–drying steps in99%D2O and then dissolved in0.5mL of99.98%D2O.1H–1H corre-lated spectroscopy(COSY),1H–1H total correlation spectros-copy(TOCSY),1H–1H nuclear overhauser effect spectrosco-py(NOESY),1H–13C heteronuclear multiple quantum coher-ence spectroscopy(HMQC)and1H–13C heteronuclear multi-ple bond correlation spectroscopy(HMBC)experiments were also carried out.Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.Analysis of Antioxidant ActivityScavenging ability of hydroxyl radicals was determined ac-cording to the method of Smirnoff and Cumbes(1989). Scavenging ability of superoxide radicals was assessed ac-cording to the method reported by Marklund and Marklund (1974).Scavenging ability of DPPH radicals was measured according to the method described by Shimada et al.(1992). The scavenging ability was calculated according to the equa-tion below:scavenging ability(%)=(1–A sample/A control)×100, where A control is the absorbance of control without the tested samples,and A sample is the absorbance in the presence of the tested samples.The EC50value(mg/mL)was the effective concentration at which the tested radicals were scavenged by 50%.Ascorbic acid was used as positive control in all anti-oxidant assays.All bioassay results were expressed as means ±standard deviation(SD).The experimental data were sub-jected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute.ResultsPreparation and Chemical Composition of the Extracellular PolysaccharideProcedures used for the preparation of the extracellular poly-saccharides from the fermented broth of the mangrove-associated fungus F.oxysporum were shown in Fig.1.Crude extracellular polysaccharide(0.59g/L)was obtained from the fermented broth,and fractionated using a Q Sepharose Fast Flow column(Fig.2a).The polysaccharide fraction,eluted with distilled water,was a major component of the crude polysaccharides.The fraction was further purified by a Sephacryl S-100column(Fig.2b),and a polysaccharide frac-tion Fw-1was obtained.The yield of Fw-1from crude polysaccharide was about 42.86%.Fw-1gave a single and symmetrical peak in the HPGPC chromatogram(Fig.2c),thus Fw-1could be a homo-geneous polysaccharide.The linear relationship between the logarithm of molecular weight of pullulan standards and re-tention time was obtained.The retention time in HPGPC chro-matogram of Fw-1was used to calculate its molecular weight by the obtained regression equation.Thus,the molecular weight of Fw-1was estimated to be about61.2kDa.Fw-1 contained91.3%total carbohydrate and minor amounts of protein(0.79%)and did not have any sulfate ester. Monosaccharide composition analysis by reversed-phase HPLC showed that Fw-1consisted of galactose,glucose, and mannose with a molar ratio of1.33:1.33:1.00.No acidic sugar and amino sugar were detected in Fw-1.Thepolysaccharide fraction Fs,eluted at 0.2M NaCl,was not further investigated due to the limit of sample amount.It is possible that fraction Fs contains an acidic polysaccharide,such as a polysaccharide with phosphate ester (Chen et al.2013).IR SpectroscopyFrom the FTIR spectrum of Fw-1,the broad and intense band at 3416cm −1was the result of valent vibrations OH groups.The signal at 2931cm −1was attributed to the stretch vibration of the C –H bond.The band at 1649cm −1was assigned to the bending vibrations of HOH,and the band at 1416cm −1originated from the bend-ing vibrations of O –H bond.The band at 1241cm −1was due to the stretch vibration of C –O –C linkages.The signal at 1032cm −1was assigned to the stretch vibration of C –O and change angle vibration of O –H.The characteristic ab-sorption bands at 876and 809cm −1suggested the pres-ences of furan ring and mannopyranose units,respectively (Ahrazem et al.2000;Mathlouthi and Koenig 1986).Methylation AnalysisIn order to determine the linkage pattern of the sugar residues,Fw-1was subjected to methylation analysis (Table 1).A large amount of 1,2,4,6-tetra-O -acetyl-3,5-di-O -methyl-galactitol,which originated from the (1→2,6)-linked galactofuranoseresidue,was detected in Fw-1,suggesting that Fw-1was a highly branched polysaccharide.1,5-di-O -acetyl-2,3,4,6-tet-ra-O -methyl-glucitol,1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-mannitol,and 1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-glucitol were also detected,indicating the presence of (1→)-linked glucopyranose,(1→2)-linked mannopyranose and (1→2)-linked glucopyranose residues.In addition,1,5-di-O -acetyl-2,3,4,6-tetra-O -methyl-mannitol,which originated from the (1→)-linked mannopyranose residue,was also found in Fw-1.The results suggested that the structure of Fw-1is com-posed of (1→2,6)-linked galactofuranose,(1→2)-linked glu-copyranose,(1→2)-linked mannopyranose,terminal gluco-pyranose,and mannopyranose residues.NMR SpectroscopyThe 1H NMR spectrum (Fig.3a )of Fw-1showed anomeric proton signals at 5.20,5.10,5.09,4.91,4.75,and 4.65ppm,which were labeled A –F from low to high field.The anomeric signals B and C almost overlapped.The anomeric proton sig-nals A –F had relative integrals of 1.0:0.5:0.5:0.25:0.25:0.25.A might be signal of β-galactofuranose residue.B and C were attributed to the signals of α-configuration pyranose units,and D –F were likely the signals of β-configuration pyranose units.The chemical shifts from 3.42to 4.26ppm were assigned to H2–H6of glycosidic ring.In the anomeric region of the 13C NMR spectrum (Fig.3b )of Fw-1,there were six main anomeric carbon signals that occurred at 107.8,102.4,101.8,101.3,99.6,and 99.5ppm.The anomeric carbon signal at 107.8ppm was due to signal of β-galactofuranose residue because of extremely low field shifts (Ahrazem et al.2006).As shown in the DEPT spectrum,the signal at 70.8ppm was assigned to the substituted C-6of β-galactofuranose units.The result confirmed the presence of the substituted C-6linkage patterns,which was in accordance to the methylation results.The 1H NMR spin systems chemical shifts of the polysac-charide were assigned by means of the 1H –1H COSY spec-trum (Fig.3c )and the 1H –1H TOCSY spectrum (Fig.3d ).Combined with the analysis of the 1H –13C HMQC spectrum of Fw-1(Fig.3e ),the observed 1H and 13C chemical shifts and the assignment of the sugar residues were given (Table 2).A was assigned to →2,6)-β-D -Gal f (1→because of the down-field chemical shifts of the C-2(88.1ppm)and C-6(70.8ppm).B and C were suggested to be Glc p because of the high field chemical shift of H-2(3.59and 3.69ppm).In the 1H –1H TOCSY spectrum,H-1of B and C showed the correlation peaks with H-2,H-3,H-4,and H-5,which con-firmed this speculation.The 1H –13C HMQC spectrum re-vealed the substitution of C at C-2due to the downfield chem-ical shift (77.0ppm)of C-2compared with the parent α-D -Glc p .Thus,B was attributed to α-D -Glc p (1→,and C was due to →2)-α-D -Glc p (1→.Combined with methylationanalysisFig.1Scheme for the preparation of the extracellular polysaccharide produced by the mangrove-associated fungus F .oxysporumand NMR spectra data (Takegawa et al.1997),E was assigned to →2)-β-D -Man p (1→because of C-2(78.0ppm)of E had a relative downfield chemical shifts.D and F were assigned to be β-D -Man p (1→,the different glycosidic bond and sugar rings,which linked with D and F,had different chemical en-vironments and chemical shifts.The sequence of glycosyl residues was determined from the 1H –1H NOESY spectrum,followed by confirmation with 1H –13C correlations obtained from the 1H –13C HMBC spec-trum.In the 1H –1H NOESY spectrum (Fig.3f )of Fw-1,A had a strong NOE contact of its H-1with the H-2of C,indicating C linked to the C-2position of A.B and C had a strongcontactFig.2Isolation and HPGPC chromatogram of the extracellular polysaccharide from the fermented broth of the mangrove-associated fun-gus F .oxysporum .a The crude polysaccharides were fractionated using a Q Sepharose Fast Flow column.The fraction eluted with distill water was pooled and named as Fw.b Fw was purified on a Sephacryl S-100column and eluted with 0.2M NH 4HCO 3.The peak fractions containing the polysaccharides were pooled and named as Fw-1.c HPGPC chro-matogram of Fw-1on a Shodex Ohpak SB-804column and the standard curve of molecular weightof its H-1with the H-2of A,suggesting B and C linked to theC-2position of A.D had a strong inter-residue contact be-tween its H-1and the H-2of E,indicating D linked to theC-2position of E.From the1H–13C HMBC spectrum ofFw-1(Fig.3g),the presence of strong cross peak H-1/C-4,C-6of A confirmed that A wasβ-galactofuranose configura-tion and→6)-β-D-Gal f(1→was the main pattern of linkage.The cross-peak H-1B,C/C-2A,and H-2A/C-1B,C indicatedthat B and C linked to the C-2of→6)-β-D-Gal f(1→.The 1H–13C HMBC spectrum of Fw-1also showed H-1F/C-2 C,H-1E/C-2C,H-1D/C-2E,H-2E/C-1D,B H-1/C-5crosspeaks,which further proved the existence ofβ-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The results also revealed both the furanoid char-acter of A and the pyranoid structure of B–F.The NMR resultswere thus in agreement with methylation results.These anal-yses allowed the identification of most of the1H and13Csignals of the sugar residues.Thus,structure of Fw-1couldbe characterized to consist of the backbone of(1→6)-linked β-D-galactofuranose residues,with multiple branches at C-2 consisting of theα-D-Glc p(1→,β-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The hypothetical structure of Fw-1was shown in Fig.4.Analysis of Antioxidant ActivityAs shown in Table3,the scavenging abilities of Fw-1on hydroxyl,DPPH,and superoxide radicals were in a concentration-dependent manner.Less scavenging of hydrox-yl radicals was observed with Fw-1at2mg/mL,but the scav-enging ability of Fw-1on hydroxyl radicals at10.0mg/mL was up to90.2%.Fw-1showed strong scavenging ability on hydroxyl radicals as evidenced by its low EC50value(1.1mg/ mL).The scavenging ability of Fw-1on superoxide radicals was50.2%at2.0mg/mL,and the scavenging ability of Fw-1 was up to89.2%at10.0mg/mL.The EC50value of scaveng-ing ability of Fw-1on superoxide radicals was2.0mg/mL. The scavenging ability of Fw-1on DPPH radicals was up to 88.2%at10.0mg/mL,and its EC50value was2.1mg/mL, indicating that Fw-1was also good effectiveness in the anti-oxidant attribute.The scavenging abilities of Fw-1on hydroxyl,superoxide and DPPH radicals were all relativelylower than that of ascorbic acid at the same concentrations. DiscussionA novel extracellular polysaccharide Fw-1is successfullyobtained from the mangrove-associated fungus F.oxysporum.Fw-1is an extracellular polysaccharidewith different structural characteristics from other extra-cellular polysaccharides produced by Fusarium sp.Thecell wall polysaccharides from F.oxysporum are com-posed of glucosamine and N-acetylglucosamine(Fukamizo et al.1992,1996),and the polysaccharidefrom Fusarium sp.M7-1consists of mannose,glucose,galactose,and glucuronic acid(Iwahara et al.1992).However,a small amount of→2)-β-D-Gal f(1→and→6)-α-D-Glc p(1→residues present in the cell wall polysac-charide of Fusarium sp.M7-1(Iwahara et al.1996).Somealkali-extractable and water-soluble extracellular polysac-charides from Fusarium species contain a backbone of β-(1→6)-linked galactofuranose residues almost fully branched at O-2by single residues of glucopyranose oracidic chains containing glucuronic acid and mannose.The extracellular polysaccharide from F.oxysporumY24-2is composed of→2)-β-D-Gal f(1→6)-α-D-Glc p(1→units(Guo et al.2013).The structure of Fw1also differs from othergalactofuranose-containing extracellular polysaccharides re-ported previously(Gómez-Miranda et al.2003;Leal et al.2010).The galactofuranans from Aspergillus niger, A.fumigatus,Trichophyton species and Penicillium charlesii,have been characterized as linear chains of(1→5)-linkedβ-D-galactofuranose units(Gander et al.1974;Latgéet al.1994; Unkefer and Gander1990;Ikuta et al.1997).For the extracel-lular polysaccharide from the deep-sea fungus P.griseofulvum,its galactofuranan chain consists of(1→5)-linkedβ-D-galactofuranose,with additional branches at C-6 consisting of(1→)-linkedβ-D-galactofuranose residues and phosphate esters(Chen et al.2013).Fw-1contains a backbone of(1→6)-linkedβ-D-galactofuranose residues with multipleTable1Results of methylation analysis of Fw-1Methylated sugar Primary mass fragments(m/z)Molar ratio Deduced linkage1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-mannitol101,117,129,145,161,205 2.0Man p(→1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol101,117,129,145,161,205 2.0Glc p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-mannitol87,101,129,161,189 1.0→2)Man p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-glucitol101,117,129,161,201,233,277 2.0→2)Glc p(1→1,2,4,6-Tetra-O-acetyl-3,5-di-O-methyl-galactitol87,101,117,129,173,189,201,233 4.0→2,6)Gal f(1→Fig.3NMR spectra of Fw-1.Spectra were performed at23°C on a JEOL ECP600MHz spectrometer Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.a1H NMR spectrum.b13C NMR and DEPT spectra.c1H–1H COSY spectrum.d1H–1H TOCOSY spectrum.e 1H–13C HMQC spectrum.f1H–1H NOESY spectrum.g1H–13C HMBC spectrum.A→2,6)-β-D-Gal f(1→.Bα-D-Glc p(1→.C→2)-α-D-Glc p(1→.Dβ-D-Man p(1→,linked to→2)-β-D-Man p(l→.E→2)-β-D-Man p(l→.Fβ-D-Man p(1→,linked to→2)-α-D-Glc p(l→.Glcpglucopyranose,Manp mannopyranose,Galf galactofuranosebranches at C-2consisting of terminal α-glucopyranose resi-dues,or short chains containing (1→2)-linked α-D -glucopy-ranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.To the best of our knowledge,this is the first report of such kind of galactofuranose-containing mannoglucogalactan isolated from fermented broth of micro-organism.The present result suggested that mangrove-associated fungi could be a potential source of extracellular polysaccharides with unique structures to be worth being fur-ther studied.In order to investigate the antioxidant activity of Fw-1,the assays based on scavenging abilities of hydroxyl,superoxide,and DPPH radicals were carried out and compared with that of ascorbic acid,one standard antioxidant.Hydroxyl radical is considered to be a highly potent oxidant,which can react with most biomacromolecules functioning in living cells and in-duce severe damage to the adjacent biomolecules.In cellular oxidation reactions,superoxide radical is normally formed first,and its effects can be magnified because it produces hydrogen peroxide and hydroxyl radical through dismutationTable 21H and 13C chemical shifts for the extracellular polysaccharide Fw-1Sugar residuesChemical shifts (ppm)a H1/C1H2/C2H3/C3H4/C4H5/C5H6/C6A b 5.20/107.8 4.21/88.1 4.26/76.9 4.05/83.9 4.02/71.0 3.94,3.69/70.8B c 5.10/99.5 3.59/72.6 3.77/73.1 3.47/71.0 3.79/73.8 3.91,3.73/62.1C d 5.09/99.6 3.69/77.0 3.81/73.1 3.45/71.0 3.76/72.6 4.12,3.79/62.3D e 4.91/102.4 4.18/72.6 3.73/72.4 3.61/72.6 3.45/71.9 3.79,3.90/62.6E f 4.75/101.3 4.24/78.0 3.68/68.3 3.95/71.2 3.76/73.5 3.96,3.45/62.4F g4.65/101.84.02/71.93.73/72.43.96/71.13.80/73.63.47,3.86/62.3Glcp glucopyranose,Manp mannopyranose,Galf galactofuranoseaThe spectra were recorded using a JEOL JNM-ECP 600MHz spectrometer.Chemical shifts are referenced to internal acetone at 2.225ppm for 1H and 31.07ppm for 13C b →2,6)-β-D -Gal f (→c α-D -Glc p (1→d →2)-α-D -Glc p (1→e β-D -Man p (1→,linked to →2)-β-D -Man p (l →f →2)-β-D -Man p (l →gβ-D -Man p (1→,linked to →2)-α-D -Glc p (l→Fig.4One of the possible structures of Fw-1(Glcp gluco-pyranose,Manp ,mannopyranose,Galf ,galactofuranose,n ≈16)and other types of reaction and was the source of free radicals formed in vivo.DPPH is a useful reagent to evaluate the free radical scavenging ability of the hy-drogen donating antioxidant,which can transfer hydro-gen atoms or electrons to DPPH radicals.It was found that Fw-1had a more noticeable scavenging ability on hydroxyl radicals than the extracellular polysaccharide AVP produced by coral-associated fungus Aspergillus versicolor LCJ-5-4,and the EC50value of AVP was 4.0mg/mL(Chen et al.2012).Moreover,the scaveng-ing ability of Fw-1on superoxide radicals appears to be higher than that of the extracellular polysaccharide As1-1produced by marine fungi Aspergillus sp.Y16,and the EC50value of As1-1was 3.4mg/mL(Chen et al. 2011).Scavenging ability of Fw-1on DPPH radicals was similar to that of extracellular polysaccharide AVP produced by coral-associated fungus,A.versicolor LCJ-5-4,and its EC50value was2.05mg/mL(Chen et al. 2012).Fw-1had a higher scavenging ability on DPPH radicals than the extracellular polysaccharides PS2-1, PS1-2,and PS1-1isolated from marine fungus Penicillium sp.F23-2(EC50value 2.53–6.81mg/mL) (Sun et al.2009).The present result suggested that the extracellular polysaccharide Fw-1could be a potential antioxidant.The antioxidant activity of Fw-1may be attributed to the extracellular polysaccharide can connect with radicals,and terminate the radical chain reaction. However,the antioxidant mechanisms of polysaccha-rides are complex.Further study on antioxidant property of extracellular polysaccharides with different structural characterization will play an important role in the un-derstanding of the mechanism of antioxidant activity.In conclusion,the extracellular polysaccharide Fw-1pro-duced by the mangrove-associated fungus F.oxysporum is a galactofuranose-containing mannoglucogalactan differing from previously described extracellular polysaccharides.Fw-1exhibits good antioxidant activity in vitro.An in-depth investigation of the antioxidant activity of Fw-1will be re-quired to determine if the extracellular polysaccharide will be useful in the food and pharmaceutical industry. Acknowledgments This work was supported by the Science and Tech-nology Development Program of Shandong Province,China (2014GHY115015),NSFC-Shandong Joint Fund for Marine Science Re-search Centers(U1406402),and the National Oceanographic Center of Qingdao of China.ReferencesAhrazem O,Gómez-Miranda B,Prieto A,Barasoaín I,BernabéM,Leal JA(2000)An acidic water-soluble cell wall polysaccharide:a che-motaxonomic marker for Fusarium and Gibberella.Microbiol Res 104:603–610Ahrazem O,Prieto A,Giménez-Abián MI,Leal JA,Jiménez-Barberoa J, Bernabe M(2006)Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp.Carbohydr Res341:246–252Bensadoun A,Weinstein D(1976)Assay of proteins in presence of in-terfering materials.Anal Chem70:241–256Bitter T,Muir HM(1962)A modified uronic acid carbazole reaction.Anal Biochem4:330–334Chen Y,Mao WJ,Tao HW,Zhu WM,Qi XH,Chen YL,Li HY,Zhao CQ, Yang YP,Hou YJ,Wang CY,Li N(2011)Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp.Y16.Bioresour Technol102:8179–8184Chen Y,Mao WJ,Yang YP,Teng XC,Zhu WM,Qi XH,Chen YL,Zhao CQ,Hou YJ,Wang CY,Li N(2012)Structure and antioxidant activity of an extracellular polysaccharide from coral-associated fun-gus,Aspergillus versicolor LCJ-5-4.Carbohydr Polym87:218–226 Chen Y,Mao WJ,Wang BF,Zhou LN,Gu QQ,Chen YL,Zhao CQ,Li N, Wang CY,Shan JM,Yan MX,Lin C(2013)Preparation and char-acterization of an extracellular polysaccharide produced by the deep-sea fungus Penicillium griseofulvum.Bioresour Technol132: 178–181Dubois C,Gilles KA,Hamilton JK,Rebers PA,Smith F(1956) Colorimetric method for determination of sugars and related sub-stances.Anal Chem28:350–356Table3Antioxidant activity of the extracellular polysaccharide Fw-1in vitroa The results were expressed as means±standard deviation(SD). The experimental data were subjected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute Sample Concentration(mg/mL)a0 2.0 4.0 6.08.010.0Scavenging ability on hydroxyl radicals(%)Fw-10.059.5±1.482.5±2.885.6±2.486.8±3.590.2±2.3 Ascorbic acid0.097.2±2.497.2±2.697.4±2.697.5±1.997.7±2.1 Scavenging ability on superoxide radicals(%)Fw-10.050.2±1.868.3±3.179.1±2.385.7±3.289.2±2.8 Ascorbic acid0.097.2±1.997.3±2.297.4±2.797.5±2.897.8±2.4 Scavenging ability on DPPH radicals(%)Fw-10.049.1±1.766.9±2.475.0±2.585.2±2.388.2±2.6 Ascorbic acid0.097.2±2.297.3±1.797.4±2.097.5±2.597.7±2.8。

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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《齐齐哈#$学学&》('学社会科学版). Journal of Qiqihar University^Phi&Soc Sci)2020年12月ec.2020论刘慈欣小说《三体》中的“人性”复杂性宋睿雪晴（云南民族大学文学与传媒学院，云南昆明650500）摘要：一般认为，科幻小说普遍存在人物形象扁平化倾向，而刘慈欣的小说《三体》深蕴着"给岁月以文明，而不是给文明以岁月”的哲学思辨，"硬科幻”的内核与“软科幻”的叙事相得益彰，呈现出纷繁复杂的人性美。

《三体》的人物刻：与环境烘托聚焦于生存困境的抉择,道德伦理拷问、乌托邦的理想憧憬等问题，引发读者对于道德价值观及人性的思考。

关键词:《三体》;人性复杂性;人性困境;反乌托邦；科学性中图分类号:1207.42文献标识码:A文章编号：1008-2638（2020）12-0107-04On the Complexity of Human Nature in Three-Body Problem by Liu CixinSONG Rui-Xue-qing(School of Literature and Media,YunnanMinzu University,KunmingYunnan650500,China) Abstract:It is generally believed that science fiction generally has a tendency to flatten and individualize characters,while Liu Cixin's novel Three-Body Problem contains a philosophical speculation of:To civilization by the years,but not for the years to civiliza­tion".The core of hard science fiction and the narration of soft science fiction complement well,presenting the beauty of complex hu-an nature.The characterization and environ ent of the novel focus on the choice of survival dile a，oral and ethical torture，uto­pian idealand other issues，hich arouse readers，thinking about oral values and hu an nature.:-;co plexity of hu an nature;dile a of hu an nature;dystopia;scientific nature随着（三体》的持续走红,越来越多的评论家开始研究《三体》，其“人性”的复杂表现及形成原因、生存的困境下如何平衡道德与功利、反乌托邦与科技发展的关系等等问题值得不断的反思和挖掘”《三体》为读者提供了另一种理解故事情节和解读人性的思路，由此引发的关于道德、科技与人性的讨论日趋激烈，这也将引起读者和批评家的广泛关注”一、生存与人性的困境《三体》是刘慈欣关于“人性”的一场文学实验,在这里,作者无数次向读者抛出灵魂拷问：“你会把你妈卖到妓院吗？”“大自然真的是自然吗?”“失去人性，失去很多”失去兽性，失去一切”吗？……在极端的末日的背景下，人类所面临着“道德”与“功利”的抉择,这决定的不再是几个人的命运，而是人类乃至多个文明的延续与否”不同的角色，或对这个世界爱的发狂，或恨之入骨，或爱恨交织，虽然性格与经 历不同，但这其中都暗藏着复杂的人性”刘慈欣曾多次在采 访中提到:其塑造的人物具有很强的“工具性”，他们更像是一种“符号”，其意图在于引发读者对于道德价值观及人性的思考”因此，由人物角色引发的人性讨论比对于人物单纯的评判更为重要”（一）叶文洁：悲观中的背叛叶文洁是《三体》第一部的主角之一，是人性迷失以及悲观主义的代表”他领导了地球文明的三体运动，为了寻求人类问题的解决方法，背叛人类,擅自联系三体，导致地球坐标暴露，诱发后续的三体文明、地球文明的搏杀”叶文洁的迷失与悲观是导致地球一步步走向毁灭的背后推手”叶文洁的悲观主义以及对整个人类的背叛，多来源于当时的社会环境与家庭环境”这是对知识分子精神创伤的一种揭露，主流文学作品中也对知识分子的精神创伤进行过描述,但刘慈欣的创作则更加大胆,在《三体》中将创伤后果推向至高点一一人类乃至整个太阳系和其他文明的毁灭”他的所作所为,是人类文明开始毁灭起点，是知识分子希望的收稿日期：2020-10-07作者简介：宋睿雪晴（1996-）,女，在读硕士。

㊃综述㊃基金项目:国家自然科学基金资助项目(81360114,81660130)通信作者:高霞,E m a i l :ga o x i a l z @163.c o m 血管生成素样蛋白4在肾病综合征中的研究进展刘华杰1,高 霞2(1.甘肃中医药大学研究生院,甘肃兰州730000;2.甘肃省人民医院儿科,甘肃兰州730000) 摘 要:血管生成素样蛋白4(A N G P T L 4)是血管生成素样蛋白家族的重要成员之一,其与脂质代谢㊁糖代谢㊁血管生发及肿瘤的发生密切相关㊂A N G P T L 4在肾脏疾病的发生与发展中有重要作用㊂本文就A N G P T L 4的来源及其生物学功能进行了总结,重点介绍了A N G P T L 4在肾病综合征蛋白尿与高甘油三酯血症中的关联机制,以及基于A N G P T L 4的肾病治疗等方面的进展㊂关键词:血管生成素样蛋白4;肾病综合征;关联机制;治疗中图分类号:R 692 文献标志码:A 文章编号:1004-583X (2018)12-1087-04d o i :10.3969/j.i s s n .1004-583X.2018.12.019 血管生成素样蛋白4(a n g i o po i e t i nl i k e p r o t e i n 4,A N G P T L 4)又称禁食诱导脂肪因子[1](f a s t i n gi n d u c e d a d i po s e f a c t o r ,F I A F )㊁过氧化物酶体增殖物激活受体[2](p e r o x i s o m e p r o l i f e r a t o r -a c t i v a t e d r e c e p t o r s ,P P A R )等,是血管生成素样蛋白家族中主要成员之一㊂2000年由3个独立的研究团队先后发现并进行报道,并在2002年被国际人类基因组组织命名为血管生成素样蛋白4㊂K a d d a t z 等[3]研究显示,生理状态下的A N G P T L 4在脂肪细胞㊁肝细胞㊁心肌细胞㊁内皮细胞㊁巨噬细胞等多种细胞中呈高表达,在垂体㊁下丘脑㊁肺及肾脏等则微量表达㊂研究表明,A N G P T L 4在脂质代谢㊁糖代谢㊁血管生发㊁肿瘤的发生[4-7]等方面发挥重要作用㊂此外,2011年,C l e m e n t 等[8]证实了肾小球足细胞可分泌A N G P T L 4,且A N G P T L 4参与肾小球的足细胞损伤,并与肾病综合征(n e p h r o t i c s y n d r o m e ,N S )蛋白尿的发生有密切关系㊂鉴于A N G P T L 4与N S 之间的关联性,本文以此为主题综述其研究进展㊂1 A N G P T L 4的来源及生物学功能1.1 A N G P T L 4的来源及其调控因素 A N G P T L 4是相对分子质量约为45000~65000的一种分泌性糖蛋白㊂在人类其基因位于19号染色体长臂,包含7个外显子和6个内含子,可编码406个氨基酸㊂与家族中的其他成员类似,A N G P T L 4包含两大功能结构域,分别为氨基末端的螺旋结构域(N -端)和羧基末端的纤维蛋白原同源结构域(C -端),这两个结构域结合并不紧密,常被前蛋白转化酶水解为c A N G P T L 4和n A N G P T L 4,其中c A N G P T L 4通过与整合素β1㊁β5结合发挥作用[9-10],而n A N G P T L 4具有抑制脂蛋白脂肪酶(l i p o p r o t e i n l i pa s e ,L P L )的作用[4]㊂A N G P T L 4有3个能被唾液酸化的潜在N -糖基化位点[2]㊂在生理状态下,A N G P T L 4在脂肪细胞㊁肝细胞㊁心肌细胞㊁内皮细胞㊁巨噬细胞等多种细胞中广泛表达,而在下丘脑㊁垂体㊁肾脏等组织则是微量表达[3]㊂A N G P T L 4在各种组织包括脂肪组织和肝脏的表达受脂质感应过氧化物酶体增殖物激活受体(P P A R )α㊁β和γ的控制,并且被游离脂肪酸(f r e e f a t t y ac id ,F F A )刺激㊂因此,通过膳食调控增加血浆F F A 水平,包括长期禁食㊁极低热量饮食和高脂肪㊁高能量的饮食,可以提高循环A N G P T L 4水平[2,11]㊂研究显示,炎症状态亦可增加循环A N G P T L 4水平[3];糖皮质激素能够抑制A N G P T L 4m R N A 表达和蛋白质分泌,而胰岛素与其作用相反[12];缺氧亦可诱导A N G P T L 4的表达[13]㊂1.2 A N G P T L 4的生物学功能 白色和棕色脂肪组织及肝脏均能分泌较多的A N G P T L 4㊂A N G P T L 4的表达可以受长期禁食㊁极低热量饮食㊁高脂肪㊁高能量饮食的调控,A N G P T L 4与能量代谢有关㊂D i jk 等[4]研究发现,循环A N G P T L 4升高,L P L 活性降低,从而抑制甘油三酯(t r i g l y c e r i d e ,T G )的水解,导致高甘油三酯血症㊂H a t a 等[14]研究表明,A N G P T L 4可以与内皮细胞表面的糖基磷脂酰肌醇锚定的高密度脂蛋白结合蛋白1(G P I H B P 1)与L P L 的复合体结合并使其失活;该研究还发现,无论L P L 是否与G P I H B P 1结合,单独的A N G P T L 4的N -端结构域是比较完整的,且A N G P T L 4能够更有效地抑制L P L ㊂A N G P T L 4的C -端结构域能够刺激脂肪组织中脂肪的分解并促进能量消耗,而当脂㊃7801㊃‘临床荟萃“ 2018年12月5日第33卷第12期 C l i n i c a l F o c u s ,D e c e m b e r 5,2018,V o l 33,N o .12Copyright ©博看网. All Rights Reserved.肪组织中不存在A N G P T L4时能够增强机体对血浆T G清除和组织对脂质摄取[5,15]㊂除了在脂质代谢中发挥重要作用,A N G P T L4对糖代谢亦有影响㊂动物实验表明,循环A N G P T L4水平升高可以抑制肝糖原的水解,使血糖水平降低,并改善糖耐量[16]㊂在一项研究中,K i m等[17]认为A N G P T L4可能在调节胰岛素分泌和胰岛形态发生中起关键作用,既可能增加肝脏的胰岛素敏感性,亦可能提高外周组织的葡萄糖利用率㊂此外,近年来的多项研究表明, A N G P T L4在肿瘤侵袭[7]㊁肠道微生态调节[18]㊁骨折的愈合[19]㊁肌腱细胞增殖[20]以及黏附和迁移等方面也发挥重要作用㊂2A N G P T L4与肾病综合征2.1A N G P T L4与足细胞损伤足细胞分泌的A N G P T L4不能进入血液循环,且此种形式的A N G P T L4与存在于循环中的A N G P T L4并非同一种形式[21]㊂脂肪组织㊁心脏㊁骨骼肌分泌的A N G P T L4为a P2-A N G P T L4,该形式的A N G P T L4等于或低于等电点㊁带负电荷㊁正常唾液酸化,且大部分存在于循环中;而足细胞特异性分泌的A N G P T L4是一种高等电点㊁带正电荷㊁低唾液酸化的N P H S2-A N G P T L4㊂该研究亦显示,在不同病理类型的N S大鼠中,微小病变(m i n i m a lc h a n g e d i s e a s e,M C D)大鼠足细胞A N G P T L4表达水平较其他病理类型大鼠明显增高,据此可推测,此种形式的A N G P T L4可能是导致M C D的"元凶"㊂C l e m e n t等[8]克隆出足细胞特异性转基因过表达N P H S2-A N G P T L4和脂肪组织特异性转基因表达a P2-A N G P T L4的两种大鼠,研究发现:足细胞特异性转基因表达的N P H S2-A N G P T L4可透过肾小球毛细血管袢使大鼠蛋白尿增加约500倍,且为肾病范围的选择性蛋白尿,并从电子显微镜下可观察到肾小球基底膜(g l o m e r u l a rb a s e m e n t m e m b r a n e, GB M)电荷减少和足细胞足突融合,而脂肪组织特异性转基因表达a P2-A N G P T L4则仅导致循环A N G P T L4水平增高,并没有蛋白尿㊂事实上,在此前已有研究显示,带正电荷的N P H S2-A N G P T L4可能与肾小球基底膜上带负电荷的硫酸乙酰肝素蛋白聚糖结合而使其电荷屏障发生改变[22]㊂由此不难推测,足细胞特异性分泌的低唾液酸化N P H S2-A N G P T L4使肾小球基底膜的电荷屏障受损是参与MC D蛋白尿发生的重要分子机制㊂2.2 A N G P T L4与N S蛋白尿和高脂血症的发生蛋白尿与高脂血症是N S的两个重要特征,其中高脂血症包括高胆固醇血症和高甘油三酯血症㊂过去的研究一直将高胆固醇血症归因于蛋白尿和低白蛋白血症引起的肝脏脂蛋白合成增加㊂然而,蛋白尿与肝脏脂蛋白合成增加之间的确切分子机制仍未有明确阐述㊂而且,相对于高胆固醇血症,有关高甘油三酯血症的研究亦报道较少㊂由于L P L能水解T G释放F F A,因此可以认为血浆T G的水平主要取决于内皮细胞结合L P L的活性,而循环A N G P T L4能使L P L活性降低㊂基于T G代谢的理论基础,C h u g h 等[23]把一种合成的突变A N G P T L4静脉注入肾病大鼠体内,结果显示肾病大鼠的蛋白尿减轻,且血浆T G没有明显升高㊂循环A N G P T L4与N S的蛋白尿和高甘油三酯血症之间存在直接的分子联系[24]㊂此项研究指出,N S大鼠在出现严重的蛋白尿之后,循环A N G P T L4水平增高,而A N G P T L4-/-肾病大鼠血T G并未升高;进一步分析发现,循环A N G P T L4介导N S中蛋白尿和高甘油三酯血症的原因在于N S中存在两个负反馈环㊂其一是系统反馈环,当机体出现肾病范围蛋白尿时,血浆白蛋白降低,F F A/白蛋白比例升高引起组织分泌的A N G P T L4增多,而循环A N G P T L4与肾小球内皮a vβ5整合,降低蛋白尿;其二是局部反馈环,该循环只发生在脂肪组织㊁心脏㊁骨骼肌等这些既能分泌A N G P T L4又能分泌L P L的组织与器官,机制是血浆中F F A/白蛋白比例升高促进这些组织与器官对L P L摄取,同时诱导A N G P T L4合成与分泌增多,从而使L P L的活性降低,减少T G分解为F F A,进而使循环T G水平增高,F F A含量减少㊂由此可知,高甘油三酯血症是由局部反馈环导致的,而局部反馈环使F F A含量减少,会使循环A N G P T L4的升高程度受限,因此通过提高循环A N G P T L4水平以减轻蛋白尿的能力有限㊂3基于A N G P T L4的肾病治疗3.1重组突变的A N G P T L4治疗剂基于以往的基础研究,有研究者尝试开发重组型A N G P T L4作为肾病蛋白尿的生物治疗剂㊂目前,已有研究显示, A N G P T L4的L P L抑制位点位于N-端部分,而具有抗蛋白尿作用的位点尚在研究中[25];在遗传学领域, Y i n等[26]研究表明,对于具有基因突变E40K的人, A N G P T L4由于L P L的抑制,血T G水平较低㊂C h u g h等[23]证实,A N G P T L4在氨基酸40处或氨基酸161和164之间产生突变会降低L P L的抑制能力,并延长完整蛋白质在循环中的半衰期,接着其开发了几种形式的突变A N G P T L4,进而将突变A N G P T L4静脉注射到肾病大鼠体内,发现大鼠的蛋白尿显著降低而T G水平未受影响㊂这些重组型㊃8801㊃‘临床荟萃“2018年12月5日第33卷第12期 C l i n i c a l F o c u s,D e c e m b e r5,2018,V o l33,N o.12Copyright©博看网. All Rights Reserved.A N G P T L4多为天然糖基化单体,相对分子质量为65000~700000,大于最大的血浆蛋白,降低了胃肠道给药后从尿液损失的可能性㊂此外,C h u g h等[23]在糖尿病肾病大鼠中也观察到类似的现象,即在静脉注射突变A N G P T L4后,大鼠蛋白尿减少近50%㊂另外,A b i d等[27]的研究指出,A N G P T L4的E40K 和T266M突变能够降低空腹T G的水平,并且可预测2型糖尿病人群的心血管疾病风险㊂3.2基于唾液酸化的治疗剂唾液酸是人体利用葡萄糖合成的一种碳水化合物㊂在M C D大鼠模型中足细胞能分泌N P H S2-A N G P T L4形式A N G P T L4,而这种形式的A N G P T L4似乎与肾小球疾病发展过程中细胞的供需失衡有关;同时, N P H S2-A N G P T L4形式的A N G P T L4缺乏唾液酸残基,并在给予N-乙酰基d-甘露糖胺(M a n N A c)(一种生物可用的口服唾液酸前体)补充唾液酸后,唾液酸化得以改善,且M C D大鼠模型中的蛋白尿得到一定程度的改善[8,21]㊂M a n N A c可以降低M C D大鼠蛋白尿,并且在用糖皮质激素第一次治疗M C D大鼠后,用M a n N A c作为维持药物可以降低M C D复发的频率[28]㊂C h u g h等[27]证实,M a n N A c对治疗糖尿病肾病有部分疗效㊂由此可见,M a n N A c对于M C D 患者是一种很有前景的治疗剂㊂然而,在膜性肾病(m e m b r a n o u s n e p h r o p a t h y,MN)中,足细胞A N G P T L4的轻度上调与膜淀粉样形式的产生无关,因此在这种情况下唾液酸或其前体的效能不明显;而钙调神经磷酸酶抑制剂他克莫司能够促进MN 足细胞的修复,使足细胞分泌A N G P T L4减少,并伴有蛋白尿的降低[29-30]㊂不过,M a n N A c在人类中的功效目前尚未有报道,可能由于人类和啮齿类动物中足细胞的唾液酸合成途径不同,而人类唾液酸的合成途径复杂㊂基于分子途径的基础研究,或许不久的将来研究者能开发出对减轻人类肾病蛋白尿有效的糖衍生物㊂综上所述,作为血管生成素家族的重要成员之一,A N G P T L4在体内多种组织中广泛表达,在脂质代谢㊁糖代谢及肿瘤发生中具有重要作用㊂近年来,多项研究发现,A N G P T L4参与一些肾脏疾病的发生发展,并与N S蛋白尿和高甘油三酯血症之间存在重要联系㊂而且,通过对A N G P T L4的深入研究,研究者揭示了N S蛋白尿㊁高甘油三酯血症及低白蛋白血症之间的关联机制㊂突变的A N G P T L4治疗剂能够降低循环T G水平,而唾液酸化治疗剂能够减少N S蛋白尿的程度,随着A N G P T L4的肾病治疗手段的不断发展,或许不久的将来将两者结合可达到最佳的治疗剂效果㊂因此,A N G P T L4在N S中的作用机制仍需进一步研究,以补充和完善其发病机制,从而为N S乃至其他肾脏疾病的临床诊疗提供新思路㊂参考文献:[1] K e r s t e nS,M a n d a r dS,T a nN S,e t a l.C h a r a c t e r i z a t i o n o f t h ef a s t i n g-i n d u c e d a d i p o s e f a c t o r F I A F,a n o v e l p e r o x i s o m ep r o l i f e r a t o r-a c t i v a t e dr e c e p t o rt a r g e t g e n e[J].JB i o lC h e m, 2000,275(37):28488-28493.[2] K a d d a t zK,A d h i k a r y T,F i n k e r n a g e l F,e t a l.T r a n s c r i p t i o n a lp r o f i l i n g i d e n t i f i e s f u n c t i o n a l i n t e r a c t i o n s o fT G Fβa n dP P A Rβ/δs i g n a l i n g:s y n e r g i s t i c i n d u c t i o n o fA N G P T L4t r a n s c r i p t i o n[J].JB i o l C h e m,2010,285(38):29469-29479.[3] T j e 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