羽衣甘蓝类蛋白质二硫键异构酶PDIL1-2的基因克隆及蛋白表达分析

以羽衣甘蓝（Brassica oleracea var.acephala）自交不亲和系（S13-bS13-b）为试材，利用RT-PCR和RACE的方法从柱头中分离类蛋白质二硫键异构酶BoPDIL1-2基因。将BoPDIL1-2编码区的序列构建到原核表达载体pET-14b上，并转化到大肠杆菌中进行原核表达与纯化；用纯化后的蛋白免疫小鼠，制备BoPDIL1-2多克隆抗体；通过免疫印迹法检测BoPDIL1-2在不同组织及不同发育阶段柱头的表达情况。氨基酸序列比对分析结果显示，羽衣甘蓝BoPDIL1-2与油菜BnPDIL1-2、拟南芥AtPDIL1-2的一致性分别是97.3%和85.5%。SDS-PAGE结果显示，在分子量58 kD处有BoPDIL1-2蛋白特异性地诱导表达。免疫印迹结果显示BoPDIL1-2在羽衣甘蓝柱头中特异表达，而且在柱头发育早期表达量较低，在开花期柱头表达量较高。

Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var.acephala)

The BoPDIL1-2 gene was isolated from the stigma of ornamental kale(Brassica oleracea var.acephala)S13-bS13-b homozygotes by RT-PCR and RACE.The BoPDIL1-2 coding region sequence was inserted into the prokaryotic expression vector pET-14b and transformed into the E.coli cell for purification.We prepared the polyclonal antibodies against recombinant BoPDIL1-2 through immune mouse.The expression level of BoPDIL1-2 in the various tissue anddifferent developmental stigmas were detected by Western blot.The deduced amino acid sequence of BoPDIL1-2 shared 97.3%and 85.5%identity with Brassica napus BnPDIL1-2 and Arabidopsis thaliana AtPDIL1-2,respectively.SDS-PAGE results showed that the recombinant BoPDIL1-2 fusion protein about 58 kDa was induced by IPTG.Western blot results revealed BoPDIL1-2 wasspecifically expressed inthe stigma,the expression of BoPDIL1-2 was lower in the early stage of stigma,while it was higher in the stigma at theflowering stage.