丹参漆酶基因的克隆表达和功能研究

发布时间：2018-05-07 15:27

本文选题：丹参 + 漆酶　；参考：《福建中医药大学》2017年硕士论文

【摘要】：丹参(Salvia Miltiorrhiza Bunge)为唇形科鼠尾草属植物,其根及根茎入药,主要用于治疗心血管疾病。课题组前期通过同位素示踪发现迷迭香酸(RA)是合成丹参酚酸B(LAB)的重要前体[2],并推测丹参漆酶是催化RA在植物体内转化形成LAB的关键酶。本课题目的在于验证这个推测,并初步探索丹参漆酶基因家族在酚酸类代谢途径中的功能与作用。我们从丹参基因组数据库中获得了丹参漆酶并借助各种手段进行筛选获得了目标漆酶基因,在体外和体内探索了其在丹参酚酸代谢途径中的作用。本文主要从以下几个方面对丹参漆酶的体内、体外功能进行研究:1.以RA为底物,使用商品化的漆树漆酶进行催化反应,在产物中检测到了少量的LAB。该结果初步验证了植物漆酶在体外具有催化RA生成LAB的能力。2.借助生物信息学分析方法,我们对丹参基因组数据进行了充分的挖掘。将NCBI上的已有漆酶基因序列与丹参基因组数据库进行比对,筛选得到29条完整的丹参漆酶(Smlacs)序列,并对它们进行了理化性质、蛋白结构、氨基酸同源性等特征分析。初步筛选得到15条可能具有该催化功能的Smlacs。3.我们考察了 29条Smlacs在植物中的组织特异性和受甲基茉莉酸甲酯(MeJA)诱导情况。结果显示15条Smlacs中同时满足MeJA诱导能够调控其表达量升高和丹参根中表达量相对其他部位较高两个条件的丹参漆酶有三条,它们分别是Smlac7、Smlac20和Smlac28。因此,本课题将这三条基因作为候选基因进行体外功能和体内功能的研究。4.通过合适的引物进行PCR反应,获得了 3条目的漆酶基因;在毕赤酵母表达体系中进行了蛋白表达;已经完成了毕赤酵母表达载体的构建,并成功转入了GS115宿主细胞中,使用抗性筛选获得了 His+菌株。目前蛋白还在进一步诱导表达当中。5.利用RNAi技术获得了三个Smlacs的转基因发根。使用RT-PCR和qPCR技术对发根进行了鉴定,结果表明阳性发根中的目的基因的表达量与空载对照组发根中该基因表达量相比明显降低,说明目的基因的表达成功的被抑制了。转基因发根与空白对照组之间在形态也出现了差异。利用LC-MS/MS技术对转基因发根进行RA和LAB的含量测定。结果显示三种转基因发根中LAB的含量均有不同程度的下降,三种转基因发根RA的含量较空白对照组均有略微的上升。该结果表明三个漆酶均能够影响LAB在体内的合成;6.通过在转基因丹参发根中过量表达Smlac7和Smlac20,对其调控作用进行进一步确证。RT-PCR和qPCR技术表明阳性发根中的目的基因的表达量均有不同程度的提高。同时,检测发根中LAB和RA的含量,结果表明LAB的含量明显高于空白对照组,而RA的含量并未出现明显的下降。该结果表明过表达两个漆酶基因对发根中丹参酚酸B的含量均有明显的提高,可以作为代谢调控丹参迷迭香酸和丹参酚酸B合成的一种新方法。综上所述,本研究在体外验证了漆酶具有催化RA生成LAB功能的前提下,通过生物信息学的方法对丹参漆酶进行筛选,并对具有潜在功能的三条丹参漆酶进行了体内和体外功能的验证,首次探索了漆酶与丹参水溶性有效成分迷迭香酸及丹参酚酸B在植物次生代谢中的内在联系,扩展了人们对漆酶在调控次生代谢产物方面的认识,为阐明丹参体内丹酚酸B合成途径,进一步开展以生物合成途径为基础的遗传改造、培育优良品系提供了理论基础。同时,实验结果也提醒我们对于丹参漆酶家族中其它可能参与酚酸代谢途径的基因还有待于进行全面探索。
[Abstract]:Salvia miltiorrhiza (Salvia Miltiorrhiza Bunge) is a plant of the genus salvia in the lip family. Its roots and rhizomes are used as medicine for the treatment of cardiovascular diseases. In the early stage of the study, we found that rosmarinic acid (RA) was an important precursor to the synthesis of salvianolic acid B (LAB) by isotopic tracers, and that laccase was the key enzyme to catalyze the transformation of RA in plants to form LAB. The subject of this lesson is to verify this speculation, and to explore the function and function of the salvia miltiorrhiza gene family in the phenolic acid metabolism pathway. We obtained the laccase from Danshen genome database and screened the target laccase gene by means of various means. In vitro and in vivo, we explored its phenolic acid pathway in Salvia miltiorrhiza. The function in vivo and in vitro is studied in the following aspects: 1. using RA as the substrate and using commercialized laccase for laccase, a small amount of LAB. has been detected in the product, and the result preliminarily verifies the ability of plant laccase to catalyze RA to produce LAB in vitro.2. with the help of biological letter. We have fully excavated the genomic data of Salvia miltiorrhiza, and compared the existing laccase gene sequences on NCBI with the Danshen genome database, and screened 29 complete Smlacs sequences, and analyzed their physicochemical properties, egg white structure, amino acid homology and so on. 15 Smlacs.3., which may have the function of this catalysis, we examined the tissue specificity of 29 Smlacs in plants and the induction of methyl jasmonate (MeJA). The results showed that 15 Smlacs were able to satisfy the MeJA induction to regulate the increase of its expression and the two conditions higher in the root of the root of Salvia miltiorrhiza than in the other parts. There are three elements of the ginseng laccase, which are Smlac7, Smlac20 and Smlac28.. Therefore, the three genes were used as candidate genes for in vitro function and in vivo function..4. was obtained by PCR reaction with appropriate primers, and 3 laccase genes were obtained. The construction of yeast expression vector has been successfully transferred into GS115 host cells, and His+ strain is obtained by resistance screening. The protein is still further induced by.5. using RNAi technology to obtain three Smlacs transgenic roots. Using RT-PCR and qPCR techniques to identify the roots, and the results indicate the target base in the positive root. The expression of the gene was significantly lower than that in the no-load control group, indicating that the expression of the target gene was suppressed. The morphological differences between the transgenic root and the blank control group were also different. The content of RA and LAB in the transgenic rooting was measured by LC-MS/MS technology. The results showed that three kinds of transgenic hair roots were found. The content of LAB decreased in varying degrees. The content of three transgenic roots RA increased slightly in comparison with that of the blank control group. The results showed that three laccase could affect the synthesis of LAB in the body. 6. through overexpression of Smlac7 and Smlac20 in the root of transgenic Salvia miltiorrhiza, the regulation effect was further confirmed by.RT-PCR and qPCR technology. The expression of the target gene in the positive root was improved in varying degrees. At the same time, the content of LAB and RA in the hair root was detected. The results showed that the content of LAB was obviously higher than that of the blank control group, while the content of RA was not obviously decreased. The results showed that the content of two laccase based on the content of B in the root of salvianolic acid in the root were obvious. It can be used as a new method to regulate the synthesis of Salvia miltiorrhiza and salvianolic acid B by metabolic regulation. To sum up, this study shows that laccase has the function of catalyzing RA to produce LAB in vitro. By bioinformatics method, the laccase of Salvia miltiorrhiza is screened by bioinformatics, and three laccase with potential function have been carried out in vivo. The internal relationship between laccase and Salvia miltiorrhiza and salvianolic acid B in the secondary metabolism of plants was first explored, and the understanding of laccase in the regulation of secondary metabolites was expanded for the first time, and the synthesis pathway of salvianolic acid B in Salvia miltiorrhiza was clarified, and the biosynthesis pathway was further carried out. The basis of genetic modification and breeding of fine lines provides a theoretical basis. At the same time, the experimental results also remind us of other genes in the family of Salvia miltiorrhiza laccase that may be involved in phenolic acid metabolic pathways in a comprehensive way.

【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.53

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