摘要
根据多种已发表的淀粉粒结合淀粉合成酶 (GBSS)基因序列的对比分析 ,以它们保守区的核酸顺序为基础 ,设计一对简并引物。从膜荚黄芪 (Astragalusmembranaceus (Fisch .)Bunge)毛状根提取mRNA ,用RT_PCR的方法扩增出 5 6 0bp的cDNA片段。序列测定表明 ,此片段与发表的豌豆和马铃薯GBSSI基因相应序列同源性分别达 89.6 %和 73.0 %。通过 5′/ 3′RACE(rapidamplificationofcDNAends)的方法 ,分别扩增出 5′和 3′端序列 ,从而获得全长的cDNA。序列分析表明此cDNA编码黄芪的GBSSI。根据推导的蛋白质序列 ,这是一个由 6 0 7个氨基酸组成的、pI为7.5 5、相对分子量为 6 6 5 6 0的前体蛋白。根据同类GBSS的氨基酸的序列比较 ,其氨基端转运肽同源性较低 ,而成熟蛋白氨基酸序列同源性较高。结合已知GBSSI的转运肽切割位点和 11种植物氨基端对准分析的结果 ,确立了此类GBSS的转运肽切割位点识别模式。对黄芪GBSSI转运肽切割位点进行了预测 ,它含有 77个氨基酸的转运肽 ,成熟蛋白pI为 5 .78,相对分子量为 5 82 70。Northernblot分析表明 。
Two degenerate primers were designed according to the conserved region among the known granule_bound starch synthase ( GBSS ) genes. A cDNA fragment of 560 bp with 89.6% and 73.0% homeologous identical to the corresponding part of GBSSI genes of pea and potato, was amplified by RT_PCR (reverse transcription polymerase chain reaction) of mRNA isolated from hairy roots of Astragalus membranaceus (Fisch.) Bunge). A full_length cDNA of the GBSSI gene was obtained by 5′/3′RACE (rapid amplification of cDNA ends). The deduced polypeptide sequence consisted of 607 amino acids with a predicted molecular mass of 66 560 and pI value of 7.55. Alignments with the GBSSI sequences from eleven species of higher plants showed a high degree of homology among the mature proteins of all the species compared along the entire length, but a low one among the transit peptides. Two consensus cleavage_site motifs were identified as I(V/I)C↓(G/K) and SXVVX↓A in GBSSI transit peptides of dicots and monocots, respectively. The transit peptide characteristics of all the species studied have the same feature as those of the chloroplast transit peptides except their cleavage_sites. The predicted transit peptide sequence of A. membranaceus is 77 amino acids long with a calculated molecular mass of 58?270 and pI value of 5.78. Northern blot analysis indicated that GBSS gene transcripts more abundantly in hairy roots and normal roots than in stems and leaves.
基金
国家"九五"攻关资助项目!( 96_C0 2_0 3_0 6)
国家自然科学基金资助项目!( 3 9670 877)
国家中医药管理局资助项目!( 95B0 2 5 )
关键词
黄芪
GBSS
基因克隆
毛状根
遗传转化
中药材
Astragalus membranaceus
granule_bound starch synthase
transit peptides
