巴西橡胶树Hb GSK1的基因克隆与表达分析

Molecular Cloning and Expressional Analysis of HbGSK1 from Rubber Tree( Hevea brasiliensis Müll. Arg. )

采用RACE和RT-PCR技术,从橡胶树CATAS7-33-97的胶乳c DNA中扩增到1条全长c DNA,共1506 bp,包含1 230 bp开放阅读框,编码409个氨基酸,含有特征性的STKc_GSK3和S_TKc结构域,命名为Hb GSK1 (Gen Bank:JN835185. 1),其编码蛋白分子量为46. 35 k D,理论等电点为8. 69。q PCR分析结果表明:机械伤害上调巴西橡胶树胶乳中Hb GSK1的基因表达;首次割胶胶乳中Hb GSK1基因的表达水平下调,而第2次割胶上调,然后恢复到未处理的对照水平;排胶对胶乳中Hb GSK1基因表达的效应大于机械伤害的效应;胶乳中Hb GSK1基因的表达受生物钟调节。伤害和割胶过程中,胶乳中Hb GSK1基因转录水平的变化表明,Hb GSK1蛋白参与橡胶树的非生物胁迫响应。

Recent studies have revealed that plant GSK3 proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. The GSK in rubber tree function in stress tolerance is unknown. Rubber trees of clone CATAS7-33-97 were treated with mechanical wounding （without latex flow）, tapping （with latex flow） with the untreated as the control, and then tapped after 2hr, 6 hr and 1d, 3d, 5d, 7d of treatment to extract the total RNA from the latex sampled, and the full length cDNA of HbGSK1 （GenBank： JN835185.1） was amplified from the cDNA library in the latex. It was 1 506 base pairs （bp） in length, containing a 1 230 bp open reading frame （ORF）, encoding 409 amino acid residues with a predicted molecular mass of 46.35 ku and a pI of 8.69. Real-time PCR analysis showed that the expression of HbGSK1 was upregulated by mechanical wounding （without latex flow）. However, the expression of HbGSK1 was downregulated by first tapping, upregulated by second tapping, and then recovered to the level of that of the untreated control. The tapping had higher influence on HbGSK1 expression than the mechanical wounding. Furthermore, the expression patterns of HbGSK1 in the latex was regulated by biological clock. The change of HbGSK1 transcript levels in the latex indicated that the HbGSK1 protein was involved in abiotic stress responses of rubber tree.