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本研究采用RACE和RT-PCR方法,从巴西橡胶树无性系热研7-33-97的乳管细胞中克隆到转录复合体关键成员TFIIB的同源基因,命名为HbTFIIB。该基因全长1 472 bp,其中开放阅读框810 bp,编码一个由270个氨基酸残基组成的蛋白质。该蛋白质分子量为29.48 kDa,理论等电点为8.3。实时荧光定量PCR结果表明,HbTFIIB基因受割胶上调表达,而且在后期排出的胶乳中表达量显著高于前期排出的胶乳。Western blot分析表明,开割树胶乳中的HbTFIIB蛋白含量高于未开割树,而且在早期排出的胶乳中的含量明显高于后期排出的胶乳中的含量。本研究表明,割胶促进转录复合体蛋白基因表达和转录蛋白质合成,而且排胶会流失转录复合体蛋白。
In this study, homologous genes of TFIIB, a key member of the transcriptional complex, were cloned from the laryngeal tube cells of Acacia crassipes 7-33-97 of Brazil rubber tree by RACE and RT-PCR methods and named as HbTFIIB. The gene is 1 472 bp in length, including an open reading frame of 810 bp encoding a protein consisting of 270 amino acid residues. The protein has a molecular weight of 29.48 kDa and a theoretical isoelectric point of 8.3. Real-time PCR results showed that HbTFIIB gene was upregulated by tapping, and the expression level of HbTFIIB gene was significantly higher than that of the latex discharged in the early stage. Western blot analysis showed that the content of HbTFIIB protein in open cut latex was significantly higher than that of uncut cored tree, and the content of HbTFIIB in early latex was significantly higher than that in late latex. The present study shows that tapping promotes gene expression of transcriptional complex proteins and transcriptional protein synthesis, and shedding of transcriptional complex proteins by gel exclusion.