大豆RING/U-box蛋白Glyma.13G115900的克隆及其对非生物胁迫的应答

Cloning and Expression Analysis of A RING/U-box Protein of Glyma. 13G115900 from Soybean under Abiotic Stress

课题组前期对干旱胁迫下大豆转录组的数据分析发现大豆Glyma.13G115900基因编码一个RING/U-box蛋白,其表达水平受干旱胁迫影响显著。本研究以垦丰16大豆为试验材料,克隆Glyma.13G115900基因。氨基酸多重序列比对表明其编码的蛋白与其它物种都具有高度保守的RING/U-box结构域。构建原核表达载体pET-29b-Glyma.13G115900转化到大肠杆菌中,Glyma.13G115900蛋白在大肠杆菌中能够表达。荧光定量PCR分析表明Glyma.13G115900基因的表达量受PEG、NaCl和ABA的影响显著,但基本不受冷胁迫的诱导。经PEG和NaCl处理后,该基因表达量与CK相比呈现出显著下降的趋势,PEG处理的表达量变化比NaCl下调的更明显;在ABA诱导下与CK相比该基因的mRNA丰度呈现出先上升后下降的趋势,在4 h表达量出现峰值,推测该基因可能通过依赖于ABA途径参与非生物胁迫应答。以上结果为进一步深入研究该基因的调控途径奠定基础。

Based on the analysis of the transcriptome data of soybean drought stress,it was found that the soybean Glyma.13G115900 gene encodes a RING/U-box protein,and its expression level is affected by drought stress.In this study,Glyma.13G115900 gene was cloned from soybean.Amino acid multiplex sequence alignment indicated that the encoded protein had the same highly conserved RING/U-box domain as the other species.The prokaryotic expression vector pET-29 b-Glyma.13G115900 was constructed and found to express Glyma13G115900 protein in Escherichia coli.Fluorescence quantitative PCR analysis showed that the expression level of Glyma.13G115900 gene was significantly affected by PEG,NaCl and ABA,and was not induced by cold stress.After treatment of PEG and NaCl,the expression level of this gene showed a significant decrease compared with CK,and the change of PEG expression was more obvious than that of NaCl,compared with CK,the mRNA abundance of the gene increased firstly and then decreased after ABA induction,and peaked at 4 h,suggesting that the gene may be involved in the abiotic response relying on the ABA pathway.The above results lay the foundation for further study of the gene.