大豆GmPHD3基因的克隆及逆境表达分析

Cloning and Abiotic Expression Analysis of GmPHD3 Gene in Soybean

为探讨大豆PHD-finger转录因子家族编码基因GmPHD3在抵抗中国南方高温高湿非生物胁迫造成种子劣变过程中的调控作用,分离全长GmPHD3基因并进行生物信息学分析、亚细胞定位和转录激活活性分析,以种子劣变抗性品种湘豆3号和不抗品种宁镇1号叶片及不同组织cDNA为材料,通过RT-PCR,进行组织表达模式分析和高温高湿胁迫下的表达模式分析。生物信息学分析结果表明基因CDS序列长度为738 bp,编码246个氨基酸,包含Alfin和PHD-finger 2个结构域。进化树结果表明该基因与木豆ALFIN-Like 3-like(XM020363358.1)的遗传距离较近。亚细胞定位结果显示该蛋白在细胞核内表达。转录激活试验结果表明基因全长有转录激活活性,激活域为N端Alfin结构域,C端PHD-finger结构域无转录激活活性。组织表达模式分析发现该基因主要在成熟期高表达,且2个品种间存在差异。胁迫下的表达模式分析发现随着胁迫时间的延长,基因的表达量逐渐升高。研究结果为进一步阐明高温高湿胁迫下的调控机制研究奠定一定理论基础。

In order to investigate the regulatory role of soybean PHD-finger transcription factor family coding gene GmPHD3 in resisting high temperature and humidity abiotic stress in seed degradation in southern China, we isolated the full-length GmPHD3 gene and conduct bioinformatics analysis, subcellular localization and transcriptional activation analysis. We used leaves and cDNAs of pre-harvest seed deterioration resistance varieties Xiangdou 3 and deterioration varieties Ningzhen 1 as materials to perform the tissue expression pattern analysis and expression pattern analysis under high temperature and humidity stress with RT-PCR. The results of bioinformatics analysis showed that the length of CDS sequence was 738 bp, encoding 246 amino acids, containing two domains of Alfin and PHD-finger. The results of the phylogenetic tree indicated that the genetic distance of this gene was close to that of pigeon pea ALFIN-Like 3-like(XM020363358.1). Subcellular localization results showed that the protein was expressed in the nucleus. Transcription activation test results showed that the gene had transcription activation activity in the full length, the activation domain was the N-terminal Alfin domain, and the C-terminal PHD-finger domain had no transcription activation activity. Analysis of tissue expression patterns revealed that the gene was mainly expressed at maturity and there was a difference between the two varieties. Analysis of the expression pattern under stress showed that the relative expression of the gene gradually increased with the extension of stress time. The research results lay a theoretical foundation for further elucidating the research on the regulation mechanism under high temperature and humidity stress.