本文選題：丹參　切入點(diǎn)：SmRboh　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：NADPH 氧化酶(NADPH oxidase,NOX)是植物產(chǎn)生活性氧(reactive oxygen species,ROS)的來源之一,NOX家族成員眾多,功能多樣,在植物生長發(fā)育過程中和生物和非生物脅迫應(yīng)答的信號(hào)轉(zhuǎn)導(dǎo)的過程中發(fā)現(xiàn)NADPH氧化酶有著重要的作用,而且在藥用植物中發(fā)現(xiàn)NADPH氧化酶與植物次生代謝有緊密聯(lián)系。本研究以丹參為實(shí)驗(yàn)材料,利用RACE擴(kuò)增技術(shù),然后從丹參中克隆得到一條完整SmRbohE基因的cDNA序列,并對(duì)其轉(zhuǎn)錄組數(shù)據(jù)庫中NOX基因家族的cDNA和其所翻譯表達(dá)的氨基酸序列進(jìn)行生物信息學(xué)分析。通過實(shí)時(shí)定量PCR(qRT-PCR)分析用水楊酸、茉莉酸甲酯、過氧化氫處理丹參懸浮細(xì)胞后其丹參NOX家族基因的相對(duì)表達(dá)量以及在不同組織部位的表達(dá)特異性。構(gòu)建了SmRbohE過表達(dá)載體和RNAi沉默(RNA interference,RNAi)載體,把表達(dá)載體轉(zhuǎn)入農(nóng)桿菌感受態(tài)細(xì)胞中,并用農(nóng)桿菌侵染丹參無菌苗葉片得到轉(zhuǎn)基因丹參,從而研究在次生代謝中丹酚酸B合成過程中SmRbohE的作用。實(shí)驗(yàn)取得了下列主要研究成果:1.依據(jù)NOX蛋白特有功能域,在現(xiàn)有轉(zhuǎn)錄組數(shù)據(jù)庫基礎(chǔ)上對(duì)丹參NOX同源基因進(jìn)行檢索,鑒定到3個(gè)丹參NOX同源基因;采用cDNA末端快速擴(kuò)增技術(shù)的方式,補(bǔ)充得到SmRbohE基因。綜上,共鑒定到3個(gè)丹參MOX同源基因。2.生物信息學(xué)預(yù)測(cè)丹參SmRbohA、SmRbohC、SmRbohE的蛋白結(jié)構(gòu)域,發(fā)現(xiàn)3個(gè)蛋白都有NADPH氧化酶結(jié)構(gòu)域,而且都有一個(gè)跨膜結(jié)構(gòu)域因此都是跨膜蛋白,丹參SmRbohA蛋白有938個(gè)氨基酸,等電點(diǎn)為9.23,分子量為107303.32,平均疏水性-0.251。丹參SmRbohC蛋白有935個(gè)氨基酸,等電點(diǎn)為9.06,分子量為105232.13,平均疏水性-0.340。丹參SmRbohE蛋白有908個(gè)氨基酸,等電點(diǎn)為8.98,分子量為101983.07,平均疏水性-0.124,丹參 SmRbohA、SmRbohC、SmRbohE 均無信號(hào)肽,不是分泌蛋白。構(gòu)建丹參中NOX家族基因系統(tǒng)發(fā)育樹,SmRbohA與SiRbohA相似度最高為90%,SmRbohA與AtRbohF相似度為78%,SmRbohE與SiRbohE相似度最高為85%,SmRbohE與AtRbohE相似度為64%。SmRbohC與SiRbohC相似度最高為90%,SmRbohC 與 AtRbohC 相似度為 68%。3.克隆出SmRbohE的基因,得到了其cDNA全長,命名為SmRbohE。SmRbohE全長 cDNA 全長為 3020 bp,包含 147bp 的 5'-URT,146 bp 的 3'-URT 和一個(gè) 2727 bp的開放閱讀框(ORF)。4.利用qRT-PCR技術(shù)檢測(cè)NOX基因家族在丹參植株根、莖、花、葉和愈傷組織中的表達(dá)量,SmRbohA在葉和愈傷組織中表達(dá)量最高,在花中也有很高的表達(dá),SmRbohC在葉中表達(dá)量最高,SmRbohE在葉和愈傷組織中表達(dá)量最高。用不同激素、信號(hào)分子處理丹參懸浮培養(yǎng)細(xì)胞,結(jié)果表明,水楊酸、茉莉酸甲酯、過氧化氫均能使SmRbohA、SmRbohC、SmRbohE在mRNA水平顯著提高,其中SA、H202的處理效果最顯著。5.構(gòu)建了SmRboh 基因的過表達(dá)和RNAi沉默表達(dá)載體,并轉(zhuǎn)化過表達(dá)和RNAi沉默表達(dá)植株,為后期研究SmRbohE在丹酚酸B合成積累中的作用提供基礎(chǔ)。
[Abstract]:NADPH oxidase NADPH-oxidase NOX is one of the sources of reactive oxygen species-ROSs produced by plants. NADPH oxidase was found to play an important role in the growth and development of plants and in the signal transduction of biological and abiotic stress response. NADPH oxidase was found to be closely related to plant secondary metabolism in medicinal plants. In this study, a complete cDNA sequence of SmRbohE gene was cloned from Salvia miltiorrhiza by RACE amplification. The cDNA of NOX gene family and the amino acid sequence translated and expressed in its transcriptional database were analyzed by bioinformatics. The real-time quantitative analysis was performed with salicylic acid and methyl jasmonate. After being treated with hydrogen peroxide, the relative expression amount of NOX family genes and the expression specificity in different tissues of Salvia miltiorrhiza suspension cells were obtained. SmRbohE overexpression vector and RNAi silencing RNA interference RNAi vector were constructed. Transgenic salvia miltiorrhiza (Salvia miltiorrhiza) was obtained by infusing Agrobacterium tumefaciens into the competent cells of Agrobacterium tumefaciens. Thus, the role of SmRbohE in the synthesis of Salvianolic acid B in secondary metabolism was studied. The following main research results were obtained: 1. According to the specific functional domain of NOX protein, the homologous gene of Salviae miltiorrhiza NOX was searched on the basis of existing transcriptional database. Three homologous genes of Salvia miltiorrhiza NOX were identified and the SmRbohE gene was supplemented by cDNA terminal rapid amplification technique. In summary, three homologous genes of MOX were identified. Bioinformatics was used to predict the protein domain of SmRbohCnSmRbohCnrbohE of Salvia miltiorrhiza. It was found that all three proteins had NADPH oxidase domain, and all of them had a transmembrane domain. The SmRbohA protein of Salvia miltiorrhiza had 938 amino acids, the isoelectric point was 9.23, the molecular weight was 107303.32, and the average hydrophobicity was -0.251. The SmRbohC protein of Salvia miltiorrhiza had 935 amino acids. The isoelectric point was 9.06, the molecular weight was 105232.13, and the average hydrophobicity was -0.340. The SmRbohE protein had 908 amino acids, the isoelectric point was 8.98, the molecular weight was 101983.07, and the average hydrophobicity was -0.124. The highest similarity between SmRbohA and AtRbohF is 78Sm RbohA and the highest similarity between SmRbohA and SiRbohE is 850.SmRbohE and AtRbohE are 64.SmRbohC and SiRbohC have the highest similarity between SmRbohC and AtRbohC. Clone the SmRbohE gene for 68. 3. The full length of its cDNA, named SmRbohE.SmRbohE, is 3020bp. it contains 147bp cDNA with 147bp 5- URT146bp and an open reading frame of 2727bp. QRT-PCR technique is used to detect the NOX gene family in root, stem and flower of Salvia miltiorrhiza (Salvia miltiorrhiza). The expression of SmRbohA in leaf and callus was the highest, and that of SmRbohC in flower was the highest. SmRbohE was the highest in leaf and callus. The results showed that salicylic acid, methyl jasmonate and hydrogen peroxide could significantly increase the level of SmRbohCon SmRbohE in the suspension culture cells of Salvia miltiorrhiza. 5. The overexpression of SmRboh gene and RNAi silencing expression vector were constructed, and the overexpression and RNAi silencing expression plants were transformed to provide the basis for studying the role of SmRbohE in the synthesis and accumulation of Salvianolic acid B.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.53
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本文編號(hào)：1572176