干旱诱导基因GmNF-YA7克隆及植物表达载体构建

Cloning and Plant Expression Vector Construction of Drought-Induced GmNF-YA7

为了鉴定大豆核因子YA(Nuclear Factor YA,NF-YA)与非生物胁迫的关系,本研究检测大豆干旱胁迫下GmNF-YA7和GmNF-YA8的表达量情况,克隆GmNF-YA7基因并构建植物表达载体,同时获得其转基因工程菌株。qRT-PCR结果显示,GmNF-YA7和GmNF-YA8均可以被干旱胁迫诱导表达,且GmNF-YA7对干旱胁迫的应答更明显。从大豆中克隆出GmNF-YA7基因,其位于大豆8号染色体上,编码含有336个氨基酸的蛋白质,预测分子量为37.06 kDa,pI6.11。GmNF-YA7蛋白氨基酸序列中含有1个CBF保守结构域。亚细胞定位预测结果显示,GmNF-YA7定位于细胞核中。蛋白系统进化分析表明GmNF-YA7蛋白与拟南芥AtNF-YA1蛋白的亲缘关系较近。利用限制性内切酶Nde I和Sal I将GmNF-YA7与植物表达载体pRI101连接并转化农杆菌EHA105,获得转基因工程菌株。

In order to identify the relationship between soybean nuclear factor YA(NF-YA)and abiotic stress,we detected the expression levels of GmNF-YA7 and GmNF-YA8 in soybean under drought stress,cloned the GmNF-YA7,constructed the plant expression vector,and obtained genetically engineered strain in this reaserch.qRT-PCR results showed that both expression levels of GmNF-YA7 and GmNF-YA8 could be induced by drought stress,and the response of GmNF-YA7 to drought stress was more obvious.GmNF-YA7 was cloned from soybean,it was located on chromosome 8 of soybean and encoded a protein containing 336 amino acids.The predicted molecular weight of GmNF-YA7 protein was 37.06 kDa and the pI was 6.11.The amino acid sequence of GmNF-YA7 protein contained a conserved CBF domain.Subcellular localization prediction results showed that GmNF-YA7 protein was localized in the nucleus.Phylogenetic analysis showed that GmNF-YA7 protein was closely related to Arabidopsis AtNF-YA1 protein.GmNF-YA7 was constructed into plant expression vector pRI101 with restriction endonucleases Nde I and Sal I and transformed into Agrobacterium EHA105.This study showed that the GmNF-YA7 gene was up regulated by drought,and we constructed plant expression vector with GmNF-YA7 and genetically obtained engineered Agrobacterium strain with this vector.