竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒，它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域，对CoYMV启动子进行了5′端五种不同长度的缺失分析，用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后，每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中，并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%，870bp比585bp启动子介导的GUS活性略高但差别不明显，缺失到447和232时GUS活性有明显下 降，但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性，GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关，870bp上游可能存在一个负调控序列，所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比， 前者为后者的70%左右，考虑到前者仅在韧皮部细胞表达而后者为组成型表达，所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。

DELETION ANALYSIS AND FUNCTIONAL STUDIES OF THE PROMOTER FROM COMMELINA YELLOW MOTTLE VIRUS

Commelina Yellow Mottle Virus(CoYMV) is a double-stranded, circular DNA virus and its promoter could direct GUS gene specifically expressing in phloem tissue of transgenic tobacco plants. To determine the optimal promoter sequence for pholem-specific gene expression, CoYMV promoter was deleted from its 5 end to form promoter fragments with 5 different lengths. Chimeric GUS genes were constructed using the promoter deletion based on the binary vector pBI121. Transgenic tobacco plants evidenced by PCR analysis were obtained with each kind of chimeric GUS gene structure by Agrobacterium mediated transformation. The results of GUS activity assay and histo-chemical staining showed that most of the chimeric GUS genes were expressed in transgenic plants. The GUS activity with the promoter deleted to -870 bp was about 78% higher than that of the full length promoter(1040 bp) and was a little higher than that of the promoter deleted to -585 bp, but the difference is not significant. The GUS activity reduced significantly when the promoter was deleted to -447 bp or -232 bp, whereas the property of phloem-specific expression pattern was still retained. When the promoter was deleted to -44 bp, just upstream adjacent to the TATA box, its tissue-specificity was lost and the activity was reduced to undetectable level. These results suggest that the region between -870 bp-232 bp and downstream of -232 bp of CoYMV promoter could be responsible for promoter activity and tissue specific expression, respectively. A negative regulation sequence might exist upstream of -870 bp of the CoYMV promoter. Therefore, we recommend that the optional CoYMV promoter sequence for phloem specific expression could be downstream from -870 bp or -585 bp. In comparison with CaMV 35S promoter, the GUS activity when driven by -870 bp CoYMV promoter was about 70% of that when driven by the 35S promoter. Considering the fact that 35S promoter-GUS gene is constitutively expressed, while the CoYMV promoter-GUS gene is expressed only in phloem tissues, the activity of the latter in phloem may be the same with or even higher than that of the 35S promoter.