油菜Oleosin基因启动子的克隆、分析及瞬时表达

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

以甘蓝型油菜幼苗为材料,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。

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 Oleosi n(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.