gstexo84质粒的构建和exo842酿酒酵母突变菌株的构建-construction of gst exo 84 plasmid and exo 842 saccharomyces cerevisiae mutant strain.docxVIP

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gstexo84质粒的构建和exo842酿酒酵母突变菌株的构建-construction of gst exo 84 plasmid and exo 842 saccharomyces cerevisiae mutant strain.docx

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gstexo84质粒的构建和exo842酿酒酵母突变菌株的构建-construction of gst exo 84 plasmid and exo 842 saccharomyces cerevisiae mutant strain

GST-Exo84 质粒的构建和 Exo84-2 酿酒酵母突变菌株的构建中文摘要目的1.试图构建 Exo84p 质粒表达载体，为体外乙酰化组蛋白是否可以与 Exo84p 结合做准备。2.制成组蛋白敲除/exo84-2 变异菌株以观察 DYN2 基因内含子的剪接状况。方法：1.从酵母基因中，经 PCR 扩增出所需的 Exo84p，经纯化后用 XmaⅠ酶切，琼脂糖凝胶回收后与 pGEX-2T 载体连接，导入感受态细胞 DH5α中，挑选出正确的克隆。2.用 PCR 方法扩增出带 LEU 的基因片段，将扩增产物转入目的酵母 X 中，得到 Exo84-2 变异菌株。用 RT-PCR 技术检测 Exo84-2 突变体对 DYN2 基因第二外显子的保留/切除情况。结果我们构建出 Exo84 质粒与 Exo84-2 变异菌株，并在 Exo84-2 变异菌株的背景下观察 DYN2 基因第二外显子保留/剪接的情况。结论 1 实验成功构建出 Exo84-2 变异菌株。2 在 Exo84-2 变异菌株中，DYN2 基因第二外显子被保留的效率比野生型低。关键词Exo84 基因；DYN2 基因； mRNA 剪接Objective英文摘要We managed to construct the Exo84p expressed plasmid, in order to perform the experiment to conform that if acetylated histone H3 interact with exo84p in vitro.We also made the temperature sensitive exo84-2 mutant for strain of Saccharomyces cerevisiae , and tested the ratio of inclusion of second exon in DYN2 gene(YDR424C) and SCR1. The minigene for DYN2 gene were constructed by us previously.MethodsWe chose the pGEX-2T as a vector and designed a pair of primers for the EXO84 gene which could be amplified from budding yeast genomic DNA. The amplified EXO84 gene DNA were subcloned into pGEX-2T via xmaI site.We designed a pair of primers for amplifying the Leu gene , and transformed the PCR product into budding yeast strain which containing the DNY2 minigene. The transformants were screened by Sc-Trp-Leu. After we got the mutant, The yeast strain were cultured into permisive temperature(30°C )and then put into non permisive temperature(37°C) for 1.5h and 3h. After harvesting the yeast cells we extracted the mRNA and perform the RT-PCR to detecte the ratio of inclusion of second exon of DYN2 gene znd Scr1 mRNA .ResultsWe got the plasmids containing the exo84 gene. We also make the exo84-2 mutant strain and observed the inclusion/exclusion of second exon of DYN2 gene in the exo84-2 mutation background.ConclusionsThe efficiency of mRNA splicing for DYN2 gene becomes low in exo84-2 background.Key words：Exo84 gene;DYN2 gene;mRNA splicing英汉缩略语名词对照英文缩写英文全称中文全称AMPAm