本文選題：法尼基焦磷酸合酶 + RT-PCR　； 參考：《吉林農業(yè)大學》2017年碩士論文

【摘要】：灰樹花(Grifola frondosa)屬于擔子菌門,蘑菇綱,多孔菌目,亞灰樹花科,樹花孔菌屬菌類作物,俗稱云蕈、舞茸,栗子蘑。作為一種重要的食藥用菌,灰樹花的功能有:減弱胰島素的抵抗,提高人體對胰島素的敏感度,增強人體控制血糖的功能,同時還有著抑制脂肪細胞堆積,降血壓,增強人體免疫力,抑制艾滋病等效果。三萜類物質則是灰樹花一種重要的次生代謝產物,同時也是衡量灰樹花藥效的重要指標之一。通過分析生物體三萜類物質的合成途徑可知,法尼基焦磷酸合酶在三萜合成途徑中起到十分關鍵的作用,其活性和表達量直接影響著后續(xù)產物的產量。所以,基于對灰樹花法尼基焦磷酸合酶基因及其表達調控機制的探究,可以更進一步的闡述灰樹花三萜合成的分子機制。首先本文NCBI的Genbank中篩選出一些與灰樹花在物種上親緣關系較為接近的法尼基焦磷酸合酶的氨基酸序列,通過使用DNAMAN軟件進行多序列的比對,從而找到灰樹花的法尼基焦磷酸合酶的氨基酸保守區(qū),然后以其為基礎設計簡并引物。再以灰樹花總從cDNA為模板,進行PCR擴增,得到一條長度825bp的灰樹花法尼基焦磷酸合酶的特異性基因片段。再基于獲得的特異性基因片段,利用5’-RACE法,設計特異性引物,擴增得到灰樹花法尼基焦磷酸合酶的5’端序列。再利用3’-RACE方法擴增灰樹花法尼基焦磷酸合酶的3’端序列。再對其3’和5’端的序列進行序列分析,找到其啟動子和終止子并在其附近設計特異性引物,以灰樹花cDNA為模板,擴增得到灰樹花法尼基焦磷酸合酶的cDNA全長,共1086bp。然后將灰樹花法尼基焦磷酸合酶的cDNA序列克隆到真核酵母表達載體pYF1845中,再將其導入到麥角甾醇依賴型的酵母菌株CC25中。導入成功的陽性轉化子在30℃的環(huán)境下可以成功在SC(-ura)的培養(yǎng)基上生長。在灰樹花的自然生長環(huán)境中,某些未知成分可能對于灰樹花的生長及有效物質的積累起到促進作用。所以為了探究在灰樹花三萜合成途徑中,外源的微生物代謝物或者生長環(huán)境對于灰樹花三萜類物質的產量的影響,本文采用灰樹花宿主栗樹的樹皮及栗樹的根際土水煎液,茉莉酸甲酯的稀釋溶液,木麻黃弗蘭克氏菌的發(fā)酵液和根瘤菌發(fā)酵液為誘導劑,以不同的誘導劑誘導灰樹花菌絲生長,最終以法尼基焦磷酸合酶的表達量為參考值,篩選出最適合的誘導劑及其濃度。結果顯示,在法尼基焦磷酸合酶的表達量上來看,以根瘤菌發(fā)酵液為誘導劑的表達量最高,木麻黃弗蘭克氏菌次之,栗樹皮第三,茉莉酸甲酯溶液第四,灰樹花法尼基焦磷酸合酶的表達量在栗樹根際土的水煎液中表現出明顯的被抑制。
[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，

本文編號：1864915