摘要：

为了明确大豆中4个参与豆科植物由根瘤菌侵染到根瘤形成和固氮的所有生物过程,并整合结瘤和结瘤自调控信号动态控制根瘤数目的核心转录因子(NIN)基因异同及其在植物生长过程中的作用,利用生物信息学的方法,对大豆4个GmNINs基因进行了系统进化、蛋白序列分析及基因序列和启动子分析,并利用Real-time PCR进行了组织表达模式验证。结果表明,大豆中4个GmNINs蛋白均属于豆类植物中特异的NIN蛋白家族,定位在细胞核,序列相似,且均具有多个磷酸化位点,蛋白核心三级结构类似而在转角处有较大区别。以上结果说明,大豆4个GmNINs蛋白可能在核心功能上是冗余的,而又在具体的表达模式上具有差异性。对GmNINs基因ATG上游-3 kb启动子序列分析表明,GmNINs启动子序列中除NBS、CYC元件外,还含有如ABRE、DRE1等多种与逆境和激素响应相关的元件。转录组分析及Real-time PCR结果显示,4个GmNINs基因均在根瘤中高表达,并受到高氮环境抑制;均可不同程度的响应高盐与干旱胁迫,其中GmNIN2a与GmNIN2b在响应非生物胁迫上较GmNIN1a与GmNIN1b更加敏感与显著,可能在植物响应非生物胁迫的过程中发挥着重要作用。综上所述,结果揭示了GmNINs除了调控大豆结瘤及根瘤数目外,还参与了大豆根系响应非生物胁迫的过程,这一调节机制的发现为选育优质高产大豆新品种提供关键线索。

关键词: 大豆, NIN转录因子, 生物信息学, 非生物胁迫, 表达分析

Abstract:

In order to clarify the similarities, differences and biological functions of Nodule inception(NIN)genes, which expressed core transcription factors NIN involved in all the biological processes from rhizobia infection to root nodule formation and nitrogen fixation in soybean, and integrates nodulation and autoregulation of nodulation signals to dynamically control the number of nodules, methods of bioinformatics were used to analyse the phylogeny, protein sequence, gene sequence and promoter elements of the four GmNINs, and Real-time PCR were carried out to verify the tissue expression pattern.The results showed that the four GmNINs proteins with similar protein sequences belonged to the specific NIN protein family in Legumes, located in the nucleus, had multiple phosphorylation sites and similar core tertiary structure with significant difference in corner. Acorrdingly, the above results indicate that the four GmNINs proteins may be redundant in core functions, but have differences in specific expression patterns.Analysis of the upstream-3 kb promoter sequence of GmNINs showed that the promoter sequence of GmNINs contained many elements such as ABRE, DRE1 and other elements related to stress and hormone response, except NBS and CYC elements.Transcriptome analysis and Real-time PCR results showed that GmNINs were highly expressed in nodules and inhibited in high nitrogen condition; they responded to salt and drought stress of varying degrees, among which GmNIN2a and GmNIN2b were more sensitive and significant in responding to abiotic stress, and GmNIN2a and GmNIN2b may play an important role in the process of plant response to abiotic stress. In short, the results revealed that GmNINs not only regulate the nodule number of soybean and nodulation, but also participate in the process of root response to abiotic stress. The discovery of this regulatory mechanism provides essential clues for breeding new varieties with high quality and high yield.

Key words: Soybean, NIN transcription factor, Bioinformatics, Abiotic stress, Expression analysis