【摘要】：丹參(Salvia miltiorrhiza Bunge)是唇形科鼠尾草屬的一種常見藥用植物,入藥部分為其干燥的根和莖,水溶性的酚酸類次生代謝物是其重要的藥用成分。植物中的鈣離子作為胞內(nèi)第二信使參與植物生長發(fā)育、代謝和脅迫應(yīng)答等的信號轉(zhuǎn)導(dǎo)過程。本研究使用的材料為懸浮培養(yǎng)的丹參細(xì)胞,從丹參中克隆獲得一條含有C2-SRC2特異結(jié)合位點的、編碼能夠結(jié)合鈣離子的C2-SRC2-like蛋白的基因,將其命名為丹參C2-SRC2-like蛋白(C2-SRC2-like基因),并利用生物信息學(xué)在線軟件分析其核酸和氨基酸序列。分別將丹參培養(yǎng)細(xì)胞進行水楊酸、氯化鈣和缺鈣處理,利用實時定量熒光PCR(qRT-PCR)分析丹參C2-SRC2-like基因表達(dá)情況。進一步構(gòu)建C2-SRC2-like的過表達(dá)和反義表達(dá)載體,并用葉盤法侵染丹參葉片得到過表達(dá)和反義表達(dá)植株,探究C2-SRC2-like蛋白在丹參酚酸類次生代謝物生物合成過程中的作用。取得了以下主要研究結(jié)果:1、克隆出丹參C2-SRC2-like基因,得到了cDNA全長。丹參C2-SRC2-like基因包括一個109 bp的5’-非翻譯區(qū)、1134 bp的開放閱讀框和273 bp的3’-非翻譯區(qū),全長共1516 bp,能夠編碼377個氨基酸。2、生物信息學(xué)分析預(yù)測C2-SRC2-like蛋白分子量為38.9 kDa,等電點為6.9,是不具信號肽、含有一個跨膜結(jié)構(gòu)的親水性蛋白。該蛋白可能位于細(xì)胞核,具有C2-SRC2特異結(jié)合位點,屬于C2超家族。與芝麻SRC2-like蛋白的氨基酸序列相似性最高,為72%。3、丹參懸浮培養(yǎng)細(xì)胞進行激素(SA)、信號分子(Ca2+)和缺鈣處理發(fā)現(xiàn),水楊酸、過量鈣能使C2-SRC2-like基因在m RNA水平相對表達(dá)量顯著提高,且分別在50 min和40 min達(dá)到峰值,為對照組的1.9倍和2.3倍;缺鈣處理后C2-SRC2-like基因在m RNA水平顯著并持續(xù)降低。4、成功構(gòu)建了C2-SRC2-like基因的過表達(dá)和反義表達(dá)載體,為后期研究C2-SRC2-like蛋白在丹參酚酸類次生代謝物生物合成過程中的作用和其生物學(xué)功能奠定基礎(chǔ)。5、成功將C2-SRC2-like基因的過表達(dá)和反義表達(dá)載體轉(zhuǎn)入農(nóng)桿菌GV3101,并用葉盤法侵染丹參葉片得到過表達(dá)和反義表達(dá)植株,在基因水平上檢測出過表達(dá)和反義表達(dá)載體均已成功轉(zhuǎn)入丹參無菌苗,為研究C2-SRC2-like蛋白在丹參酚酸類次生代謝物生物合成過程中的作用研究提供材料。
[Abstract]:Salvia miltiorrhiza (Salvia miltiorrhiza Bunge) (Salvia miltiorrhiza) is a common medicinal plant in the genus Salvia. Calcium ions in plants are involved in the signal transduction of plant growth and development, metabolism and stress response as the second messenger in the cell. In this study, a C2-SRC2-like protein encoding calcium binding site was cloned from Salvia miltiorrhiza cells cultured in suspension culture, and a specific binding site of C2-SRC2-like was obtained from Salvia miltiorrhiza (Salvia miltiorrhiza). It was named Salvia miltiorrhiza C2-SRC2-like protein (C2-SRC2-like gene) and its nucleic acid and amino acid sequences were analyzed by bioinformatics online software. Salicylic acid, calcium chloride and calcium deficiency were treated with salicylic acid, calcium chloride and calcium deficiency in Salvia miltiorrhiza cultured cells. The expression of C2-SRC2-like gene in Salvia miltiorrhiza was analyzed by real-time quantitative PCR (qRT-PCR). The overexpression and antisense expression vectors of C2-SRC2-like were further constructed, and the overexpression and antisense expression of C2-SRC2-like protein were obtained by leaf disk method. The role of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites was investigated. The main results are as follows: 1. The C2-SRC2-like gene of Salvia miltiorrhiza was cloned and the full length of cDNA was obtained. The C2-SRC2-like gene of Salvia miltiorrhiza contains an untranslated region of 10 9 bp, an open reading frame of 1134 bp and a 3 '-untranslated region of 273 bp. The total length of 1516 bp, can encode 377 amino acids. Bioinformatics analysis predicted that the molecular weight of C2-SRC2-like protein was 38.9 kDa, isoelectric point 6.9, which was not a signal peptide and contained a transmembrane hydrophilic protein. This protein may be located in the nucleus, with C2-SRC2 specific binding site, belonging to the C 2 superfamily. The amino acid sequence of Sesame SRC2-like protein was similar to that of Sesame seed protein (722.3. Salvia miltiorrhiza suspension culture cells were treated with hormone (SA), signaling molecule (Ca2) and calcium deficiency), salicylic acid, salicylic acid, Excess calcium significantly increased the relative expression of C2-SRC2-like gene at m RNA level, and reached the peak at 50 min and 40 min, which was 1.9-fold and 2.3-fold higher than that in the control group. After calcium deficiency treatment, C2-SRC2-like gene was significantly decreased at m RNA level. 4. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully constructed. In order to study the role and biological function of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites. 5. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully transferred into Agrobacterium tumefaciens GV3101,. Overexpression and antisense expression plants were obtained from the leaves of Salvia miltiorrhiza infected with leaf disk method. The overexpression and antisense expression vectors were detected at the gene level and were successfully transferred into the sterile seedlings of Salvia miltiorrhiza. To study the role of C2-SRC2-like protein in Salvianolic acid secondary metabolites biosynthesis.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S567.53
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本文編號：2404322