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分离了金华中棉(Gossypiun arboreum var.jinhua)光诱导基因cab 5′上游的调控序列1 009 bp,并对其功能进行了分析,证明获得的这一DNA片段具有驱动光诱导表达的功能。为了进一步分离具有最大转录活性的最小光诱导启动子,根据光诱导表达调控元件所在的位置,构建了Gacab P和197 bp、504 bp、779 bp的5′端缺失体,并将这些缺失体分别与gus(uid A)基因融合,构建植物表达载体。用农杆菌介导法转化烟草,获得转基因烟草。GUS组织化学分析表明,转基因烟草的T1代种子在光下培养时,只有Gacab P驱动gus基因在转基因烟草的叶片表达,其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达;当转基因烟草的T1代种子在暗中萌发及培养时,GacabP驱动gus基因在转基因烟草中无表达,其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达。GUS定量分析表明,-504～-1 bp的启动子缺失体启动活性最高,比CaMV35S启动子高0.6倍。上述结果表明只有全长的Gacab启动子具有光诱导和绿色组织特异表达特性,且?504～?1 bp的启动子缺失体启动活性最高。
The 1 009 bp regulatory sequence upstream of the light-induced gene cab of Gossypiun arboreum var. Jinhua was isolated and its function was analyzed. The obtained DNA fragment showed the function of driving light-induced expression. In order to further isolate the minimal light-induced promoter with the highest transcriptional activity, Gacab P and 5 ’deletion of 197 bp, 504 bp and 779 bp were constructed according to the location of light-induced expression regulatory elements, respectively. Fusion with gus (uid A) gene to construct plant expression vector. Agrobacterium-mediated transformation of tobacco to obtain transgenic tobacco. GUS histochemical analysis showed that only the Gacab P-driven gus gene was expressed in the leaves of transgenic tobacco when T1 generation seeds of transgenic tobacco were cultured under light, and the other three promoters showed that gus gene was expressed in the whole plant of transgenic tobacco. When T1 seed of transgenic tobacco germinated and cultured in the dark, gus gene driven by GacabP was not expressed in transgenic tobacco. The other three promoters driven the expression of gus gene in the whole plant of transgenic tobacco. GUS quantitative analysis showed that the promoter activity of -504 ~ -1 bp promoter was the highest, which was 0.6 times higher than that of CaMV35S promoter. The above results showed that only the full-length Gacab promoter has the characteristics of light-induced and green tissue-specific expression, and the promoter activity of 504 bp to 1 bp promoter is the highest.