大豆GmNAC73-like基因的克隆和表达及其对GmIFS2基因的影响

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GmNAC73-like基因,并对该基因序列进行生物信息学分析。结果显示：GmNAC73-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。 GmNAC73-like蛋白的理论相对分子质量37000,理论等电点pI 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：GmNAC73-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,GmNAC73-like蛋白与野大豆的GsNAC8蛋白和木豆﹝Cajanus cajan ( Linn.) Millsp.〕的CcNAC8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,GmNAC73-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：GmNAC73-like可与异黄酮生物合成关键酶基因GmIFS2启动子中的CGTG基序结合；在大豆转基因发状根系中过表达GmNAC73-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GmNAC73-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：GmNAC73-like可能通过与MYB转录因子的互作调控GmIFS2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%, 69%, 73%, 75% and 58%, 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, in which, that of cinnamic acid-4-hydroxylase gene and chalcone synthase gene is enhanced obviously. In addition, total isoflavone content in transgenic hairy roots of G. max with over expression of GmNAC73-like gene decreases significantly. The comprehensive analysis result shows that GmNAC73-like may regulate expression of GmIFS2 gene by interaction with MYB transcription factor, and plays a negative regulation in isoflavone biosynthesis process of G. max.