油菜O-acetylserine(thiol)lyase isoform A(OASTL)基因的克隆及序列分析

Cloning and Sequence Analysis of O-acetylserine(thiol)lyase isoform A(OASTL)Gene from Rapeseed(Brassica napus L.)

植物OASTL蛋白是半胱氨酸生物合成过程中的关键酶,催化半胱氨酸合成的最后一步。克隆低镉积累基因型油菜中OASTL基因,为研究该基因在镉积累过程中的功能提供参考。本研究以镉低积累品种美国‘油王88’幼苗为材料,采用同源克隆法PCR扩增OASTL基因,并对其进行生物信息学分析。克隆得到美国‘油王88’OASTL基因cDNA编码区长为969 bp,编码322个氨基酸;编码蛋白的理论分子量为33.9 kD,理论等电点为5.50,溶液中的不稳定指数为27.45,为稳定蛋白,属于磷酸吡哆醛(PLP)依赖型的β-取代丙氨酸合成酶超家族成员。氨基酸序列聚类分析表明,其与花椰菜和小白菜的相似性最高。亚细胞定位预测分析表明,OASTL蛋白定位于细胞质中,推测OASTL蛋白主要在细胞质中发挥重要作用。OASTL基因的克隆及其结构、性质与功能的初步分析,为油菜抗重金属育种提供理论基础和基因资源。

O-Acetylserine(thiol)lyase(OASTL)is a key enzyme of cysteine biosynthesis metabolism pathway in plants and catalyzes the final step in cysteine biosynthesis.The present study was conducted to clone and analyze OASTL gene from Rapeseed of low cadmium(Cd)accumulation genotypes in order to provide references for analyzing OASTL function in cadmium accumulation of rapeseed.In this study,the OASTL gene was amplified by PCR using homologous cloning method from the seedlings of the American cultivar’Youwang88’of low Cd accumulation.Sequence analysis revealed that the coding region cDNA of OASTL gene was 969 bp,which encoding 322 amino acids.The encoded protein with a predicted molecular mass of 33.9 kD,a theoretical isoelectric point(pI)of 5.50,is a stable protein of which the instability index in the solution was 27.45,belonging to the pyridoxal phosphate(PLP)-dependentβ-substituted alanine synthase superfamily.Phylogenetic tree analysis showed that the protein was most closely related to Brassica cretica and Brassica rapa.Predictive analysis of subcellular location indicated that OASTL protein was located in the cytoplasm,so it is speculated that OASTL protein mainly plays an important role in the cytoplasm.The cloning of OASTL gene and the preliminary analysis of its structure,properties and functions provide theoretical basis and genetic resources for rapeseed breeding for heavy metal resistance.