大豆GmNAC73-like基因的克隆和表达及其对GmIFS2基因的影响  

Cloning and expression of GmNAC73-like gene from Glycine max and its effect on GmIFS2 gene

作　　者：万群[1] 刘晓庆[1] 张大勇[1] 何晓兰[1] 徐照龙[1] 宁丽华[1] 黄益洪[1] 邵宏波[1] WAN Qun LIU Xiaoqing ZHANG Dayong HE Xiaolan XU Zhaolong NING Lihua HUANG Yihong SHAO Hongbo(Provincial Key Laboratory of Agrobiology,Institute of Biotechnology,Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)

机构地区：[1]江苏省农业科学院农业生物技术研究所江苏省农业生物学重点实验室,江苏南京210014

出　　处：《植物资源与环境学报》2016年第3期12-18,27,共8页Journal of Plant Resources and Environment

基　　金：江苏省自然科学基金资助项目(BK20130727);江苏省农业三新工程项目〔SXGC(2016)335〕;江苏省农业科技自主创新资金项目〔Cx(15)1005〕;江苏省"双创计划"人才项目

摘　　要：为研究NAC转录因子对大豆〔Glycine max(Linn.)Merr.〕异黄酮合成的影响,根据大豆基因组序列设计引物,从豆荚中克隆获得Gm NAC73-like基因,并对该基因序列进行生物信息学分析。结果显示:Gm NAC73-like基因包含1个长度981 bp的完整开放阅读框,编码326个氨基酸。Gm NAC73-like蛋白的理论相对分子质量37 000,理论等电点p I 6.4,为亲水性蛋白,无信号肽,并被定位在细胞核上,包含核定位信号"PKRRK"。同源性比对结果显示:Gm NAC73-like蛋白与野大豆(Glycine soja Sieb.et Zucc.)、蒺藜苜蓿(Medicago truncatula Gaertn.)、可可(Theobroma cacao Linn.)、葡萄(Vitis vinifera Linn.)及拟南芥〔Arabidopsis thaliana(Linn.)Heynh.〕的NAC蛋白具有较高的相似性,相似度分别为93%、69%、73%、75%和58%。在NJ系统树上,Gm NAC73-like蛋白与野大豆的Gs NAC8蛋白和木豆〔Cajanus cajan(Linn.)Millsp.〕的Cc NAC8蛋白聚在一起,显示出较近的亲缘关系。半定量RT-PCR分析结果显示:在大豆的三叶期、开花期和结荚期,Gm NAC73-like基因在根中均不表达,在茎和叶中可不同程度表达且茎中表达量较高;而在开花期或结荚期,该基因在花或豆荚中也可表达,且豆荚中表达量较高。酵母单杂交实验结果显示:Gm NAC73-like可与异黄酮生物合成关键酶基因Gm IFS2启动子中的CGTG基序结合;在大豆转基因发状根系中过表达Gm NAC73-like基因后,除查尔酮异构酶基因的表达量无变化外,其他异黄酮生物合成相关基因的表达量均不同程度提高,其中,肉桂酸-4-羟化酶基因和查尔酮合酶基因的表达量明显提高。此外,在Gm NAC73-like基因过表达的大豆转基因发状根系中总异黄酮含量显著降低。综合分析结果表明:Gm NAC73-like可能通过与MYB转录因子的互作调控Gm IFS2基因的表达,并在大豆异黄酮的生物合成过程中起负调控作用。In order to study the effect of NAC transcription factor on isoflavone synthesis of Glycine max （Linn.） Merr., primers were designed according to genome sequence from G. max, GmNAC73-like gene was cloned from pod of G. max, and bioinformatic analysis of this gene sequence was carried out. The results show that GmNAC73-like gene includes a whole open reading frame with length of 981 bp, 326 amino acids are encoded. Theoretical relative molecular mass of GmNAC73 -like protein is 37 000, theoretical isoelectric point is pI 6. 4, which is a hydrophilic protein without signal peptide, it is located in nucleus and contains nuclear localization signal “PKRRK” .The homology alignment result shows that GmNAC73-like protein has a high similarity with NAC protein from Glycine soja Sieb. et Zucc., Medicago truncatula Gaertn., Theobroma cacao Linn., Vitis vinifera Linn. and Arabidopsis thaliana （ Linn.） Heynh.,with a similarity degree of 93% , 6 9% , 7 3% , 75% and 5 8% , respectively. In NJ phylogenetic tree, GmNAC73-like protein, GsNAC8 protein from G. soja and CcNAC8 protein from Cajanus cajan （Linn.） Millsp. are clustered together, showing close phylogenetic relationship. The semi-quantitative RT-PCR analysis result shows that at trefoil,flowering and pod setting stages, GmNAC73-like gene has no expression in root,and has different degrees of expression in stem and leaf,and has a higher expression in stem ; while at flowering or pod setting stages,this gene also has expression in flower or pod,and has a higher expression in pod. The result of yeast one-hybrid experiment shows that GmNAC73-like can bind to CGTG motif in promoter of key enzyme gene GmIFS2 of isoflavone biosynthesis. After over expression of GmNAC73-like gene in transgenic hairy roots of G. max, except expression of chalcone isomerase gene without change, expression of other related genes of isoflavone biosynthesis is enhanced with different degrees,i

关 键 词：大豆 GmNAC73-like基因 蛋白特性 表达特性 GmIFS2基因 总异黄酮含量 

分 类 号：Q786[生物学—分子生物学] S565.1[农业科学—作物学]