棉花种子特异性启动子的克隆及表达载体构建

Cloning Seed-specific Promoter from Cotton and Construction of Plant Expression Vector

种子特异性表达启动子是植物种子基因工程改良的重要工具。Lea(Late embryogenesis abundant)蛋白是胚胎发育后期种子中大量积累的一系列蛋白质,因此,其调控序列可能提供一个很好来源的种子特异性表达启动子。为研究植物Lea蛋白基因启动子在种子中的特异性表达,本研究通过PCR扩增,从亚洲棉(Gossypi-um arboreum L.)石系亚1号中克隆了Lea蛋白基因家族中D113基因上游1262 bp的调控序列和D34基因上游1445 bp的调控序列。DNA序列分析结果表明,克隆到的两个片段与已报道的Lea蛋白基因同一家族该基因的对应序列的相似性分别达到97.43%和94.27%。分别用限制性内切酶HindⅢ和xbaI双酶切重组质粒pGEMT-D和改造后的表达载体pBI121-T,分别回收pGEMT-D重组质粒中的小片段和pBI121-T植物表达载体中的大片段,经连接、转化和鉴定,获得由D113基因启动子和D34基因启动子驱动报告基因GUS的新型植物表达载体pBI121-D113、pBI121-D34,为外源基因在棉花种子中的定位表达研究奠定基础。

In order to study the expression of late embryogenesis abundant gene in cotton seeds, the 1262 bp 5＇flanking sequence of D113 gene and the 1450 bp 5＇flanking sequence of D34 gene were cloned by PCR from Gossypium arboreum L. The similarity of cloned D113 gene promoter and D34 gene promoter compared with the sequence of Lea protein gene family which had been reported earlier were 97.43% and 94.27%, respectively. Two new plant expression vectors named pBI121-D113 and pBI121-D34 in which the reporter gene GUS is drived by D113 and D34 gene promoter were constructed after cutting two vectors pGEMT-D and pBI121-T with two restriction enzymes HindlIland xbaI, subsequently recovered the small fragment from pGEMT-D recombined vector and the long fragment from pBI121-T plant expression vector, and then ligation, transformation and identification were conducted. Finally, foundation has been set up for further research work in expression and function of this promoter.