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MeCWINV6是木薯6个细胞壁转化酶基因之一。本研究中应用酵母单杂交技术筛选MeCWINV6启动子的上游转录调节因子,为进一步研究该基因的表达调控提供基础。由MeCWINV6的启动子构建诱饵融合载体,转化酵母细胞构建诱饵菌株。运用SMART技术构建木薯酵母单杂交c DNA文库,再共转化诱饵菌株,经同源重组筛选MeCWINV6启动子的上游转录调节因子。构建的c DNA文库库容为7.08×106,插入片段大小在250~2 000 bp间。筛选大于1 000 bp的35个阳性克隆,经测序和Blast同源性分析筛选出锌指蛋白、组氨酸蛋白H1.2和AT-hook核定位蛋白三个转录因子以及一个线粒体受体TOM5。为进一步研究MeCWINV6基因的表达调控机制提供了候选转录因子。
MeCWINV6 is one of the six cell wall invertase genes in cassava. In this study, yeast single hybridization was used to screen the upstream transcriptional regulatory factor of MeCWINV6 promoter, which provided a basis for further study on the expression regulation of this gene. The bait fusion vector was constructed from the promoter of MeCWINV6, and the yeast cells were transformed to construct the bait strain. SMART technique was used to construct single-hybrid cDNA library of cassava yeast, and co-transformed into bait strain. The upstream transcription regulatory factor of MeCWINV6 promoter was screened by homologous recombination. The constructed c DNA library has a capacity of 7.08 × 106 and an insert size of 250 ~ 2 000 bp. Thirty-five positive clones of more than 1 000 bp were screened, and three transcription factors of zinc finger protein, histidine protein H1.2 and AT-hook nuclear localization protein and one mitochondrial receptor TOM5 were screened by Blast homology analysis and sequencing. It provided candidate transcription factors for further study on the regulatory mechanism of MeCWINV6 expression.