甜菜树贝壳杉烯合酶基因的克隆与原核表达

发布时间：2018-03-13 11:05

本文选题：甜菜树　切入点：贝壳杉烯合酶　出处：《食品与生物技术学报》2017年05期 　论文类型：期刊论文

【摘要】：本文作者研究了赤霉素合成途径中的关键酶-贝壳杉烯合酶,对其功能的研究可以为以后优化品种提供基因层面的基础。首先通过RACE-PCR技术,从甜菜树叶片中克隆得到贝壳杉烯合酶基因(Ent-kaurene synthase gene,Yl KS)的全长c DNA,共2 512 bp(Gen Bank登录号为KP872698),其ORF长度为2 232 bp,共编码743个氨基酸,预测蛋白质相对分子质量大小为84 987,蛋白质等电点为5.264,表明该蛋白质呈酸性。将该基因构建到表达载体pET32a中,得到重组质粒pET-32a-Yl KS。通过将p GG/An2、p IRS和重组质粒pET-32a-Yl KS 3个质粒共转入菌株BL21(DE3)中,得到重组菌株,并进行发酵。通过SDS-PAGE分析发现,重组蛋白成功得到了表达。最后通过对发酵产物进行萃取,并使用GC-MS进行检测,确定了该基因所编码的酶确实是贝壳杉烯合酶。
[Abstract]:In this paper, we studied the key enzyme of gibberellin synthesis pathway-kainiaxene synthase. The study of its function can provide the basis of gene level for the later optimization of varieties. Firstly, we use RACE-PCR technique to optimize the gene level of gibberellin synthase. The full-length cDNA of Ent-kaurene synthase gene Yl KS) was cloned from leaves of sugarbeet. The total number of bp(Gen Bank was KP872698, and the length of ORF was 2 232 BP, encoding 743 amino acids. The predicted molecular weight and isoelectric point of the protein were 84 987 and 5.264 respectively, which indicated that the protein was acidic. The gene was constructed into the expression vector pET32a. The recombinant plasmid pET-32a-Yl KS. the recombinant strain was obtained by co-transferring the three plasmids of p G / An2P IRS and recombinant plasmid pET-32a-Yl KS into strain BL21DDE3. The recombinant strain was found by SDS-PAGE analysis. The recombinant protein was successfully expressed. Finally, the enzyme encoded by the gene was determined by extracting the fermentation product and detecting by GC-MS.
【作者单位】：林木遗传育种国家重点实验室中国林业科学研究院林业研究所;四川农业大学农学院;中国医学科学院/中国协和医科大学药物研究所;中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室;
【基金】：中国林业科学研究院林业研究所中央级公益性科研院所基本科研业务费专项(RIF2014-01);中国林业科学研究院中央级公益性科研院所基本科研业务费专项(CAFYBB2012042)
【分类号】：Q943.2;S649

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