| 甘薯S-腺苷甲硫氨酸合成酶基因克隆与表达 |
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| 作者姓名： | 岳昌武 何博文 何明雄 张义正 |
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| 作者单位： | 四川大学生命科学学院资源生物学与生态环境教育部重点实验室四川大学生命科学学院分子生物学与生物技术四川省重点实验室 成都610065 |
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| 基金项目： | 甘薯抗冻基因克隆与鉴定,四川省科技攻关项目（05NG002-002-2）. |
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| 摘 要： | 从实验室建立的甘薯冷胁迫抑制消减文库中分离出一cDNA片段,经NCBI比对后发现该片段与其它植物的S-腺苷甲硫氨酸合成酶基因具有90%的同源性,根据相关物种S-腺苷甲硫氨酸合成酶基因cDNA序列设计引物,以甘薯总RNA为模板,经RT-PCR扩增首次获得全长1182bp的甘薯SAMS完全编码区,NCBI比对分析结果表明:该片段与不同种属植物sams基因的编码区序列的核苷酸相似性达85.48%,所推导的氨基酸序列相似性达93.6%。根据核苷酸序列和氨基酸序列分别构建了SAMS系统进化树,从分子水平阐明了植物种属间的亲缘进化关系,为其种质资源利用提供理论依据。利用该序列与原核表达载体pET32a连接构建融合表达载体pET-SAMS,酶切鉴定后转入大肠杆菌BL21,在不同温度下诱导3h后均表达出一63KDa大小融合蛋白。
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| 关 键 词： | S-腺苷甲硫氨酸合成酶 冷胁迫 RT-PCR 序列分析 原核表达 |
Cloning and Expression of s-adenosylmethionine Synthetase of Sweetpotato |
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| Authors: | Yue Changwu He Bowen He Mingxiong Zhang Yizheng |
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| Abstract: | In order to explore the relationship between cold stress and the change of gene expression. Suppression subtractive hybridization (SSH) method was used to identify novel genes regulated by cold stressed in sweetpotato. A full length cDNA coding region of S-Adenosyl-L-Methionine synthase (SAMS) which contains 1182bp was isolated by this way combined with RT-PCR technology. Sequence analysis revealed that this genes hared 85.48 % similarity in gene sequence and 93.6 % similarity in protein sequence with the other SAMS gene in plant. The differences of nucleotides sequences were higher than those of amino acids. The phylogenetic tree was constructed based on the alignment nucleotides sequences elucidated the relationship among different plant species at nucleotide and amino acids level, which could proved some help for the utilization of plant germplasms. Fused expression plasmid vector pET-SAMS was constructed with sams and pET32a. A protein about 63KDa was expressed after 3h induced by IPTG at different temperature in BL21. |
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| Keywords: | S-adenosylmethionine synthetase Cold stress RT-PCR Sequence analysis Pronucleus expression |
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