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GDA2(G2peadarkaccumulatedgene)是与G2豌豆(Pisumsativum L.)短日照条件下不衰老现象紧密相关的基因之一。实验用cDNA差示分析法(representationaldifferenceanalysis,RDA)得到一个短日照下特异表达的G2豌豆cDNA,并命名为GDA2。核酸序列分析表明,该cDNA全长1120bp,最大的开放阅读框为642bp,编码一个由213个氨基酸构成的分子量约为24kD的蛋白质,与GDA1的同源性为36%。在GDA2的cDNA两端分别引入BglⅡ与XhoⅠ的酶切位点,用PCR方法将其克隆到原核表达载体pGEX-4T-1中,经过酶切筛选和测序鉴定,得到所需的表达载体pGEX-GDA2。将pGEX-GDA2导入E.coli BL21菌株,经IPTG诱导,得到分子量约51kD的GST-GDA2融合蛋白,并利用GlutathioneSepharose4B亲和柱对该融合蛋白加以纯化。GDA2-GST融合蛋白的表达和纯化工作,为深入研究GDA2蛋白的结构与功能以及该基因同衰老的关系打下良好的基础。
GDA2 (G2peadarkaccumulatedgene) is one of the genes closely related to the aging phenomenon of G2 pea (Pisum sativum L.) under short daylight conditions. A representative cDNA of G2 pea, which is specifically expressed in short sunlight, was obtained by representational analysis (RDA) and named as GDA2. Nucleic acid sequence analysis showed that the full-length cDNA was 1120bp and the largest open reading frame was 642bp, encoding a protein with a molecular weight of about 24kD consisting of 213 amino acids with a homology of 36% with GDA1. The BglII and XhoI restriction sites were introduced into both ends of GDA2 cDNA. The recombinant plasmid was cloned into prokaryotic expression vector pGEX-4T-1 by PCR. After digestion and sequencing, the desired expression vector pGEX- GDA2. The pGEX-GDA2 was introduced into E.coli BL21 strain and induced by IPTG to obtain a GST-GDA2 fusion protein with a molecular weight of about 51 kD. The fusion protein was purified by Glutathione Sepharose 4B affinity chromatography. GDA2-GST fusion protein expression and purification, in order to further study the structure and function of GDA2 protein and the relationship between the gene and aging to lay a good foundation.