利用REF启动子构建橡胶树乳管特异表达载体及GUS基因的瞬时表达

Construction of Hevea brasiliensis Transient Expression Vector with REF Promoter

采用SDS法提取橡胶树基因组DNA,用2套引物进行PCR扩增,得到2条REF条带,它们与NCBI报道的REF序列间的同源性分别是98.9%,98.4%。用克隆得到的REF片段分别替换载体质粒pBI121中的CaMV35S启动子,得到利用橡胶内源性启动子构建的橡胶树REF启动子瞬时表达载体(pBIR1和pBIR2)。用基因枪法转化橡胶树组培苗叶片后,GUS组织化学检测显示叶脉为GUS染色阳性。研究中发现,长度为306bp的REF启动子片段可以实现REF启动子的全部功能。从而成功地构建了橡胶树乳管瞬时表达载体,为外源基因的橡胶树乳管特异表达载体的构建打下了基础。

Hevea brasiliensis genomic DNA was extracted by SDS and amplified by PCR. The sequences of the products shared high homologies with the reported sequence of REF in NCBI at a rate of 98.9 % and 98.4 % respectively. Replacing CaMV35S promoter of pBI121 with the two cloned fragments, we obtained transient expression vectors of REF promoter which were designated as pBIR （pBIR1 and pBIR2）. When bombarded with pBIR plasmids, blue spots occurred on leaves of cultured plants after dyed. Studies showed that pBIR2 including fragments of 306 bp could execute almost all the functions of REF promoter. Thus a transient vector was accomplished, which was a good foundation for foreign gene to express in laticifer system.