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 58270 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.
黄芪毛状根GBSSI 基因cDNA克隆及其结构分析
彭佶松1* 赵淑娟1 吴晓俊1 刘涤1** 胡之璧1 许政日岂2
（1. 上海中医药研究院中药研究所，上海200032；2. 中国科学院上海植物生理研究所植物分子遗传国家重点实验室，上海200032）
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（Astragalus membranaceus (Fisch.) Bunge）毛状根提取mRNA，用RT-PCR 的方法扩增出560bp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%和73.0%。通过5‘/3‘RACE（rapid amplification of cDNA ENDS）的方法，分别扩增出5‘‘和3‘A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607个氨基酸组成的、pI 为7.55、相对分子量为66 560 的前体蛋白。根据类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。结合知GBSSI 的转运肽切割位点和!! 种植物氨基端对准分析的结果，确立了此类!GBSSI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58 270。Northern blot 分析表明，GBSSI基因在黄芪毛状根和正常根中的表达高于茎和叶。
关键词： 黄芪；淀粉粒结合淀粉合成酶；转运肽
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