灰树花法尼基焦磷酸合酶基因序列克隆及表达分析

发布时间：2018-05-09 06:14

  本文选题：法尼基焦磷酸合酶 + RT-PCR　； 参考：《吉林农业大学》2017年硕士论文

【摘要】：灰树花(Grifola frondosa)属于担子菌门,蘑菇纲,多孔菌目,亚灰树花科,树花孔菌属菌类作物,俗称云蕈、舞茸,栗子蘑。作为一种重要的食药用菌,灰树花的功能有:减弱胰岛素的抵抗,提高人体对胰岛素的敏感度,增强人体控制血糖的功能,同时还有着抑制脂肪细胞堆积,降血压,增强人体免疫力,抑制艾滋病等效果。三萜类物质则是灰树花一种重要的次生代谢产物,同时也是衡量灰树花药效的重要指标之一。通过分析生物体三萜类物质的合成途径可知,法尼基焦磷酸合酶在三萜合成途径中起到十分关键的作用,其活性和表达量直接影响着后续产物的产量。所以,基于对灰树花法尼基焦磷酸合酶基因及其表达调控机制的探究,可以更进一步的阐述灰树花三萜合成的分子机制。首先本文NCBI的Genbank中筛选出一些与灰树花在物种上亲缘关系较为接近的法尼基焦磷酸合酶的氨基酸序列,通过使用DNAMAN软件进行多序列的比对,从而找到灰树花的法尼基焦磷酸合酶的氨基酸保守区,然后以其为基础设计简并引物。再以灰树花总从cDNA为模板,进行PCR扩增,得到一条长度825bp的灰树花法尼基焦磷酸合酶的特异性基因片段。再基于获得的特异性基因片段,利用5’-RACE法,设计特异性引物,扩增得到灰树花法尼基焦磷酸合酶的5’端序列。再利用3’-RACE方法扩增灰树花法尼基焦磷酸合酶的3’端序列。再对其3’和5’端的序列进行序列分析,找到其启动子和终止子并在其附近设计特异性引物,以灰树花cDNA为模板,扩增得到灰树花法尼基焦磷酸合酶的cDNA全长,共1086bp。然后将灰树花法尼基焦磷酸合酶的cDNA序列克隆到真核酵母表达载体pYF1845中,再将其导入到麦角甾醇依赖型的酵母菌株CC25中。导入成功的阳性转化子在30℃的环境下可以成功在SC(-ura)的培养基上生长。在灰树花的自然生长环境中,某些未知成分可能对于灰树花的生长及有效物质的积累起到促进作用。所以为了探究在灰树花三萜合成途径中,外源的微生物代谢物或者生长环境对于灰树花三萜类物质的产量的影响,本文采用灰树花宿主栗树的树皮及栗树的根际土水煎液,茉莉酸甲酯的稀释溶液,木麻黄弗兰克氏菌的发酵液和根瘤菌发酵液为诱导剂,以不同的诱导剂诱导灰树花菌丝生长,最终以法尼基焦磷酸合酶的表达量为参考值,筛选出最适合的诱导剂及其浓度。结果显示,在法尼基焦磷酸合酶的表达量上来看,以根瘤菌发酵液为诱导剂的表达量最高,木麻黄弗兰克氏菌次之,栗树皮第三,茉莉酸甲酯溶液第四,灰树花法尼基焦磷酸合酶的表达量在栗树根际土的水煎液中表现出明显的被抑制。
[Abstract]:Grifola frondosa) belongs to basidiomycetes, Agaricus, Agaricidae, subGrifolaceae, Tremella, commonly known as mushroom, fluffy mushroom, chestnut mushroom. As an important edible medicine bacteria, the functions of Grifola frondosa are to reduce insulin resistance, to improve the sensitivity of the body to insulin, to enhance the ability of the body to control blood sugar, and to inhibit the accumulation of fat cells and lower blood pressure. Enhance human immunity, AIDS suppression and other effects. Triterpenoids are an important secondary metabolite of Grifola frondosa and one of the important indexes to evaluate the efficacy of Grifola frondosa. By analyzing the synthesis pathway of triterpenoids, we can see that farinyl pyrophosphate synthase plays a very important role in triterpene synthesis, and its activity and expression directly affect the yield of subsequent products. Therefore, the molecular mechanism of triterpene synthesis in Grifola frondosa can be further elucidated based on the study of the gene expression and regulation mechanism of farinyl pyrophosphatase in Grifola frondosa. First of all, some amino acid sequences of farnityl pyrophosphatase were screened from the Genbank of NCBI, which were closely related to the species of Grifola frondosa. The sequences were compared by using DNAMAN software. The amino acid conserved region of farnityl pyrophosphatase in Grifola frondosa was found and degenerate primers were designed based on it. The specific gene fragment of farinyl pyrophosphatase (FKP) of Grifola frondosa was obtained by PCR amplification from the template of cDNA in Grifola frondosa. Based on the specific gene fragment obtained, the specific primers were designed by 5'-RACE method, and the 5'terminal sequence of Grifola frondosa farinopyrophosphatase was amplified. The 3 'terminal sequence of farnesyl pyrophosphatase from Grifola frondosa was amplified by 3'-RACE. The 3 'and 5' terminal sequences were sequenced. The promoter and Terminator were found and specific primers were designed nearby. The total length of cDNA was amplified by using Grifola frondosa cDNA as template. The total length of cDNA was 1086 BP. Then the cDNA sequence of Grifola frondosa farnityl pyrophosphatase was cloned into eukaryotic yeast expression vector pYF1845 and then introduced into ergosterol dependent yeast strain CC25. The successful positive transformants could grow on SC-ura medium at 30 鈩，

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