玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

发布时间：2018-03-28 12:30

  本文选题：玉米大斑病菌　切入点：STK-EGFP融合基因　出处：《生物工程学报》2017年06期

【摘要】：STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因(EGFP)的毕赤酵母GSS115(Pichia pastoris GS115)表达载体p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌丝c DNA为模板,PCR扩增STK1基因,克隆到p PIC3.5K-EGFP,构建了STK1-EGFP融合基因的GS115表达载体p PIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内,利用MD培养基筛选、PCR鉴定,获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察,发现STK1基因和EGFP基因均可以高效稳定地表达。另外,在试验中我们还发现,在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。
[Abstract]:STK1 gene is the regulation of conidial development by Mycobacterium macrophylla. The important MAPK gene of osmotic stress regulation and pathogenicity. Firstly, the expression vector of Pichia pastoris GSS115(Pichia pastoris GS115 containing enhanced green fluorescent protein gene (GSS115(Pichia pastoris GS115) was constructed, and then the mycelia c DNA of strain 01-23 was constructed. STK1 gene was amplified by PCR. The GS115 expression vector pPIC3.5K-STK1-EGFPof STK1-EGFP fusion gene was cloned into pPIC3.5K-EGFP.The expression vector pPIC3.5K-STK1-EGFPwas transformed into GS115 receptive cells by electroporation. Pichia pastoris transformant of STK1-EGFP fusion gene was obtained. By RT-PCR and fluorescence observation, it was found that both STK1 gene and EGFP gene could be expressed efficiently and stably. The expression intensity of STK1-EGFP fusion gene could be increased by 4.8-fold by adding Kozak sequence before the initiation codon of STK1 gene. These results laid a foundation for subcellular functional localization and antibody preparation of STK1 gene expressed protein.
【作者单位】： 唐山师范学院生命科学系;
【基金】：河北省高等学校科学技术研究项目(No.QN2017415) 河北省自然科学基金(No.C2014105067) 河北省留学人员科技活动择优资助项目(No.C2015005009) 唐山师范学院科学研究基金(Nos.2016C05,2014E04)资助~~
【分类号】：S435.131.4
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本文编号：1676332