巴西橡胶树43kD橡胶粒子膜蛋白基因的cDNA克隆及表达

Expression and Cloning of cDNA Encoding 43 kD Rubber Particle Membrane Protein of Hevea brasiliensis

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3’RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA。该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸。在终止密码子下游,包含有一个239 bp的3’非编码区。该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体。编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达。

A rubber particle protein with apparent molecular mass of 43 kD as determined by SDS-PAGE was purified. A degenerate oligo-nucleotide primer based on the N-terminal amino acid sequence of this purified protein was used to amplify a 1 385 bp cDNA by 3' rapid amplification of cDNA ends(3'RACE). The cDNA contains five repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extraphenylanaline residue at the carboxyl-ter-minal end(Fig.l). The structure of the cDNA is consistent with the structure of other known polyubiquitin genes. Western blot(Fig.2) dem- onstrated that 43 kD rubber particle protein might be a polyubiquitin. Southern blot analysis revealed that there were multiple copies of gene encoding 43 kD rubber particle protein in Hevea brasiliensis(Fig.3). The results of Northern blot analysis indicated that the gene was expressed in latex, young leaves and bark tissue(Fig.4).