杠柳顺式-1,4-聚异戊二烯生物合成关键基因PsIPPI和PsCPT的克隆及功能分析

发布时间：2018-04-30 19:56

本文选题：杠柳 + 天然橡胶　；参考：《宁夏大学》2017年硕士论文

【摘要】：天然橡胶是事关国民经济和国家安全的一种重要战略物资,主要成分为顺式-1,4-聚异戊二烯。巴西橡胶树(Hevea rasiliensis Muell.Arg)是目前天然橡胶原料的主要来源,由于受到该树种热带生长特性限制,我国种植面积有限,产能严重不足,开发新的天然橡胶资源已成为我国迫切需要解决的问题。本研究首先从分析具有天然橡胶生产能力的植物入手,选择了杠柳(Periploca sepium Bunge)作为我国生产天然橡胶替代植物的研究对象;然后通过转录组测序等生物信息学手段,明确了杠柳顺式-1,4-聚异戊二烯生物合成途径的关键酶系及其所编码的基因;利用基因的表达调控技术阐明了部分关键基因在杠柳顺式-1,4-聚异戊二烯生物合成中的作用。本研究的研究结果如下:1.以杜仲聚异戊二烯提取得率以及分子量分布为衡量标准,通过对材料预处理、提取方法进行比较,确定了有效的聚异戊二烯分析测定方法。结果表明采用氯仿作为有机溶剂,在水浴下采用索氏提取杜仲聚异戊二烯的方法最佳,在提取过程中使用超声波辅助处理,可以进一步促进小分子或大分子的聚异戊二烯聚合物的溶出,提取更加充分。2.以三种广泛分布于我国西北、东北和华北等干旱、半干旱地区的野生灌木或草本植物,含乳汁成分的鹅绒藤(Cyanchum chinense R.Br.)、罗布麻(Apocynum venetum L.)以及杠柳为材料,进行聚异戊二烯提取和分析。通过对提取物进行凝胶渗透色谱(GPC)分子量分析以及核磁共振(NMR)结构分析,发现杠柳中含有顺式-1,4-聚异戊二烯成分,可作为生产天然橡胶原料的替代植物加以研究。3.以杠柳的叶片、枝条和树皮为材料,提取RNA进行转录组测序分析,从中筛选出杠柳顺式-1,4-聚异戊二烯生物合成途径的相关酶类及其编码的基因,即杠柳异戊烯基二磷酸异构酶(PsIPPI)、r{牛儿基二磷酸合酶(PsGPPS)、法尼基二磷酸合酶(PsFPPS)、双r{牛儿基二磷酸合酶(PsGGPPS)及顺式-异戊烯基转移酶(PsCPT)及其基因,并进行了生物信息学分析。4.根据转录组测序结果选择了杠柳顺式-聚异戊二烯生物合成途径中的两个关键基因,即PsIPPI和PsCPT,利用PCR方法全长克隆,并以单独或组合的形式构建成三种基因过表达载体,以鉴定这两个基因的功能以及对杠柳顺式-聚异戊二烯生物合成的影响。5.利用农杆菌介导方法将这三种基因过表达载体导入到杠柳植物体内进行过表达。结果发现在杠柳中过表达PsIPPI基因对其顺式-1,4-聚异戊二烯的生物合成没有明显作用;过表达PsCPT基因对顺式-1,4-聚异戊二烯的生物合成具有明显的提升,增加约5倍;而同时过表达PsCPT基因及PsIPPI基因能更进一步促进杠柳顺式-1,4-聚异戊二烯的生物合成,特别是高分子部分。该研究将为培育杠柳成为生产天然橡胶工业原料替代植物,为解决我国天然橡胶贫乏的工业瓶颈、丰富橡胶资源提供技术支撑。
[Abstract]:Natural rubber is an important strategic material related to national economy and national security. Hevea rasiliensis Muell.Arg is the main source of natural rubber raw materials at present. Due to the restriction of the tropical growth characteristics of the tree species, the planting area in China is limited, and the production capacity is seriously insufficient. Developing new natural rubber resources has become an urgent problem to be solved in our country. Based on the analysis of plants with natural rubber production capacity, Periploca sepium Bunge was selected as the research object of producing natural rubber substitute plants in China, and then bioinformatics methods, such as transcriptome sequencing and other bioinformatics methods, were used. The key enzyme system and its encoding genes in the biosynthesis pathway of cis-1 and 4-polyisoprene were clarified, and the role of some key genes in the biosynthesis of cis-1-4-polyisoprene was elucidated by gene expression regulation technique. The results of this study are as follows: 1. The extraction yield and molecular weight distribution of polyisoprene from Eucommia ulmoides ulmoides were taken as the criterion. The effective method for the analysis and determination of polyisoprene was determined by comparing the pretreatment and extraction methods. The results showed that chloroform was used as organic solvent and Soxhlet was used to extract polyisoprene from Eucommia ulmoides in water bath. Can further promote the dissolution of small or macromolecular polyisoprene polymer, extraction is more complete. 2. Three species of wild shrubs or herbs, Cyanchum chinense R. Br., Apocynum venetum L., and Apocynum venetum L., which are widely distributed in arid and semi-arid regions of Northwest, Northeast and North China, are widely distributed in China. The extraction and analysis of polyisoprene were carried out. Through the molecular weight analysis of gel permeation chromatography (GPC) and the structure analysis of nuclear magnetic resonance (NMR), it was found that the extract contained cis-1 + 4-polyisoprene, which could be used as a substitute plant for the production of natural rubber. RNA was extracted from leaves, branches and bark of Salix wilfordii for transcriptional sequencing analysis. The enzymes related to the biosynthesis pathway of Cis-1 and 4-polyisoprene and their coding genes were screened. The results of bioinformatics analysis were as follows: PsIPPIdiphosphatase (PsIPPIJ), PsGPPSN, PsFPPSN, PsGGPPSs and PsCPT, respectively, and their genes were analyzed by bioinformatics, and bioinformatics analysis was carried out on the basis of bioinformatics analysis of PsIPPIK and PsIPPIP-PsCPT, and the results of bioinformatics analysis of PsGPPSN, PsGPPSG, and PsCPT were also discussed in this paper. According to the results of transcriptome sequencing, two key genes in the cis-polyisoprene biosynthesis pathway, PsIPPI and PsCPT, were selected and cloned by PCR method. In order to identify the function of the two genes and the effect on the cis-polyisoprene biosynthesis. By Agrobacterium tumefaciens-mediated method, the three gene overexpression vectors were introduced into the plant for overexpression. The results showed that the overexpression of PsIPPI gene had no obvious effect on the biosynthesis of cis-1m4-polyisoprene, and the overexpression of PsCPT gene had a significant improvement on the biosynthesis of cis-1-butadiene-4-polyisoprene by about 5 times. The overexpression of PsCPT gene and PsIPPI gene can further promote the biosynthesis of cis-1 and 4-polyisoprene, especially in the polymer part. This study will provide technical support for cultivating willow as a substitute plant for natural rubber industry, for solving the industrial bottleneck of natural rubber shortage in China and for enriching rubber resources.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S793.9;Q943.2

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