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[目的]将特异存在于豆科植物的II型查尔酮异构酶基因CHI1A构建到目前最有效的食品级乳酸乳球菌NICE诱导表达系统中。[方法]利用RT-PCR技术从大豆总RNA中克隆CHI1A基因,连入pMD18-T克隆载体中并进行测序,然后重组到乳酸乳球菌表达载体PNZ8149-CHI1A,利用电穿孔方法转入乳酸乳球菌NZ3900中。[结果]克隆得到CHI1A完整开放阅读框670bp,与已报逍的大豆查尔酮异构酶基因的核苷酸序列(GenBank AY595413)的同源性达到99%;通过PCR和酶切鉴定,成功地将CHI1A导入到NICE表达系统中。[结论]含有CHI1A基因的乳酸乳球菌高效诱导表达载体的构建为利用微生物发酵产生类黄酮奠定基础。
[Objective] The purpose of this study was to construct CHI1A, a Chalcone-type II chalcone-specific gene, present in the most effective food-grade Lactococcus lactis NICE-induced expression system. [Method] The CHI1A gene was cloned from total RNA of soybean by RT-PCR and cloned into pMD18-T cloning vector and sequenced. Then the recombinant plasmid was transformed into L. lactis vector PNZ8149-CHI1A and transformed into L. lactis NZ3900. [Result] The complete open reading frame of CHI1A was 670bp, which was 99% homologous with the reported nucleotide sequence of soy chalcone isomerase (GenBank AY595413). The PCR products were confirmed by PCR and restriction enzyme digestion Import CHI1A into NICE Expression System. [Conclusion] The construction of efficient expression vector of Lactococcus lactis containing CHI1A gene laid the foundation for the production of flavonoids by microbial fermentation.