大豆核因子GmNF-YA13基因的克隆及功能分析

Cloning and function analysis of nuclear factor GmNF-YA13 gene in soybean

【目的】探究大豆核因子GmNF-YA13基因在大豆应对非生物胁迫中的生物学功能,为GmNF-YA13基因在抗逆育种中的应用奠定基础。【方法】以大豆北豆9号和烟草NC89的种子为供试材料,将大豆培养至第一片三出复叶展开后,分别进行干旱、高盐、低温和脱落酸(ABA)处理,通过实时荧光定量RT-PCR(qRT-PCR)对GmNF-YA13在以上4种非生物胁迫下的相对表达量进行检测,并对GmNF-YA13进行克隆及生物信息学分析,将GmNF-YA13转化烟草,并对转基因烟草中抗逆相关基因(NtABA2、NtOsmotin、NtAPX2、NtSOD、NtCAT1和NtCaMK3)的表达量进行qRT-PCR检测。【结果】干旱、高盐、低温和ABA处理均能不同程度诱导GmNF-YA13的表达,其中干旱胁迫和ABA处理对GmNF-YA13的诱导效果尤为显著。GmNF-YA13位于大豆基因组13号染色体,开放阅读框915 bp,编码含有304个氨基酸的蛋白质,该蛋白质分子量75.14 ku,等电点5.10。GmNF-YA13蛋白序列包含一个保守的DNA结合结构域。蛋白系统进化分析结果表明,GmNF-YA13与GmNF-YA7和拟南芥AtNF-YA1亲缘关系较近。启动子顺式作用元件预测分析结果表明,GmNF-YA13启动子区含有3个ABA响应元件ABRE、1个厌氧诱导元件ARE、1个防御和胁迫反应元件TC-rich repeats及1个干旱诱导的MYB转录因子结合位点MBS。GmNF-YA13在大豆叶片中的相对表达量最高。同时构建GmNF-YA13植物表达载体,并将GmNF-YA13转化烟草,获得3株转基因烟草植株。转基因烟草中抗逆相关基因表达量检测结果显示,转基因烟草中的NtABA2、NtOsmotin和NtAPX2相对表达量均显著高于野生型烟草,转基因烟草中NtSOD、NtCAT1和NtCaMK3的相对表达量与野生型烟草差异不显著。【结论】GmNF-YA13与大豆干旱、高盐、低温等非生物胁迫之间关系密切,GmNF-YA13可能通过促进抗逆相关基因表达量的升高以提高转基因烟草的抗逆性。

【Objective】This study explored the biological function of nuclear factor GmNF-YA13 gene in soybean responding to abiotic stress to provide basis for the application of GmNF-YA13 in resistance breeding.【Method】The seeds of soybean varieties Beidou 9 and tobacco NC89 were selected for drought,salt,cold and ABA treatments after the first terately compound leaf unfolded.The relative expression of GmNF-YA13 was detected by real-time fluorescence quantitative RT-PCR(qRT-PCR).GmNF-YA13 was cloned and analyzed by bioinformatics.GmNF-YA13 was transformed into tobacco,and the expression levels of stress-related genes(NtABA2,NtOsmotin,NtAPX2,NtSOD,NtCAT1 and NtCaMK3)in transgenic tobacco were detected by qRT-PCR.【Result】Drought,salt,cold and ABA treatments could induce the expression of GmNF-YA13 at different degrees,with especially significant effects in drought and ABA treatments.GmNF-YA13 was located on chromosome 13 of soybean genome with 915 bp open reading frame and it encoded a protein containing 304 amino acids with a molecular weight of 75.14 ku and an isoelectric point of 5.10.GmNF-YA13 protein contained a conserved DNA binding domain.The protein phylogenetic analysis showed that GmNF-YA13 was closely related to GmNF-YA7 and Arabidopsis AtNF-YA1.The promoter cis-elements prediction analysis showed that the promoter region of GmNF-YA13 contained three ABA responsive elements(ABRE),one anaerobic induction element(ARE),one defense and stress responsive element(TC-rich repeats)and one MYB binding site involved in drought-inducibility(MBS).The relative expression of GmNF-YA13 was the highest in leaves.The GmNF-YA13 plant expression vector was constructed and transformed into tobacco to obtain three transgenic tobacco plants.The relative expression levels of NtABA2,NtOsmotin and NtAPX2 in transgenic tobacco were significantly higher than those in wild-type tobacco,while those of NtSOD,NtCAT1 and NtCaMK3 were not significantly different.【Conclusion】GmNF-YA13 was closely related to abiotic stress such as drought,salt and cold in soybean.GmNF-YA13 may enhance the stress resistance of transgenic tobacco by promoting the expression of stress-related genes.