王伟权　李树俊　朱欣琪　丘志慧　施力汛　张哲

摘要 以甘藍型油菜幼苗为材料，根据油脂蛋白Oleosin（O20）基因的启动子序列设计引物，PCR扩增了长度为935 bp的片段，把该片段连接到pBI101载体，GUS瞬时染色结果表明，该片段可以驱动GUS基因在油菜幼苗根部特异性表达。油菜油脂蛋白是一个蛋白质家族，为进一步鉴别扩增到的935 bp油脂蛋白启动子，该研究利用油菜基因组信息进行序列对比，结果表明935 bp的启动子和Oleosin（O20）的启动子序列之间有较多的变异，但和油菜1号染色体上的序列完全一致;1号染色体上紧跟的编码框序列和油脂蛋白Oleosin（O20）的编码框序列也完全一致，但油脂蛋白Oleosin（O20）的基因组序列要比油菜1号染色体的基因组序列短。通过PLACE和PlantCARE软件对935 bp启动子进行了扫描预测，结果表明该启动子中含有许多顺式作用元件，尤其是脱落酸、茉莉酸甲酯和水杨酸这3个与激素有关的元件;将该研究扩增到的另一个928 bp长度的启动子与935 bp的启动子相比，前者启动子区域水杨酸顺式作用元件缺少了1个碱基，同时也缺失了1个碱基的片段，这些序列的变化可能是该启动子丧失活力的原因。

关键词 启动子;油菜;瞬时表达;Oleosin

中图分类号 S188文献标识码 A

文章编号 0517-6611（2020）22-0115-05

doi：10.3969/j.issn.0517-6611.2020.22.030

Cloning，Analysing and Transient Expressing Oleosin Gene Promoter in Brassica napus

WANG Wei-quan，LI Shu-jun，ZHU Xin-qi et al （College of Agriculture and Biology，Zhongkai University of Agricultural and Engineering，Guangzhou，Guangdong 510225）

Abstract According to the young seedlings of Brassica napus as materials，Oleosin promoters designed by the oil protein Oleosin （O20） sequence with length of 935 bp and 928 bp were amplified by PCR.GUS staining showed that 935 bp lipid protein Oleosin promoter can drive the specific expression of GUS gene in B.napus seedlings root.Comparing 935 bp with Oleosin （O20） promoter sequences，we found that there is a lot of variation between them.Because B.napus oil protein is a protein family，when utilizing B.napus genome sequencing information to be compared to confirm the oil protein promoter，we found that the promoter sequence amplified with length of 935 bp and the sequence of B.napus chromosome 1 were completely consistent.Then comparing coding sequences predicted behind chromosome 1，we found that the coding sequences behind the 1 chromosome is exactly the same with that of the Oleosin（O20） oil protein.While there are differences in their genome sequence，the 935 bp promoter sequence on chromosome 1 is much larger than that of the Oleosin （O20）.There is no comparison of their genome sequence in this paper.Studying thoroughly the promoter amplified of 935 bp through the PLACE and the PlantCARE website，we have found many cis-acting elements.Three components related to abscisic acid，methyl jasmonate and salicylic acid are notable.Because we also amplified a promoter with length of 928 bp，the promoter can not drive the expression of the GUS gene.Analysing and comparing the sequence of 935 bp and 928 bp，we found that a base lacked in the promoter TCA motif of 928 bp.The loss of these sequential changes may cause the loss of the vitality of the promoter.

In summary，we have successfully obtained a tissue specific promoter of B.napus that can be used for scientific research and production.

Key words  Promoter;Brassica napus;Transient expression;Oleosin

作者简介 王伟权（1968—），男，江苏宜兴人，副教授，博士，从事分子生物学研究。

李树俊（1998—），男，广东广州人，从事分子生物学研究。王伟权与李树俊为共同第一作者。

收稿日期 2019-09-26;修回日期 2020-04-13

植物作为生产药用蛋白或其他具有重要价值蛋白的生物反应器，为人类提供一个更加安全和廉价的生产系统，是植物基因工程的发展方向，从而使传统农业生产和现代生物技术紧密结合，大幅度地提高农产品对人类的服务功效及市场竞争力，增加农民收入，促進农业的可持续发展。……