本文選題：杠柳 + 天然橡膠��； 參考：《寧夏大學(xué)》2017年碩士論文

【摘要】：天然橡膠是事關(guān)國民經(jīng)濟和國家安全的一種重要戰(zhàn)略物資,主要成分為順式-1,4-聚異戊二烯。巴西橡膠樹(Hevea rasiliensis Muell.Arg)是目前天然橡膠原料的主要來源,由于受到該樹種熱帶生長特性限制,我國種植面積有限,產(chǎn)能嚴重不足,開發(fā)新的天然橡膠資源已成為我國迫切需要解決的問題。本研究首先從分析具有天然橡膠生產(chǎn)能力的植物入手,選擇了杠柳(Periploca sepium Bunge)作為我國生產(chǎn)天然橡膠替代植物的研究對象;然后通過轉(zhuǎn)錄組測序等生物信息學(xué)手段,明確了杠柳順式-1,4-聚異戊二烯生物合成途徑的關(guān)鍵酶系及其所編碼的基因;利用基因的表達調(diào)控技術(shù)闡明了部分關(guān)鍵基因在杠柳順式-1,4-聚異戊二烯生物合成中的作用。本研究的研究結(jié)果如下:1.以杜仲聚異戊二烯提取得率以及分子量分布為衡量標準,通過對材料預(yù)處理、提取方法進行比較,確定了有效的聚異戊二烯分析測定方法。結(jié)果表明采用氯仿作為有機溶劑,在水浴下采用索氏提取杜仲聚異戊二烯的方法最佳,在提取過程中使用超聲波輔助處理,可以進一步促進小分子或大分子的聚異戊二烯聚合物的溶出,提取更加充分。2.以三種廣泛分布于我國西北、東北和華北等干旱、半干旱地區(qū)的野生灌木或草本植物,含乳汁成分的鵝絨藤(Cyanchum chinense R.Br.)、羅布麻(Apocynum venetum L.)以及杠柳為材料,進行聚異戊二烯提取和分析。通過對提取物進行凝膠滲透色譜(GPC)分子量分析以及核磁共振(NMR)結(jié)構(gòu)分析,發(fā)現(xiàn)杠柳中含有順式-1,4-聚異戊二烯成分,可作為生產(chǎn)天然橡膠原料的替代植物加以研究。3.以杠柳的葉片、枝條和樹皮為材料,提取RNA進行轉(zhuǎn)錄組測序分析,從中篩選出杠柳順式-1,4-聚異戊二烯生物合成途徑的相關(guān)酶類及其編碼的基因,即杠柳異戊烯基二磷酸異構(gòu)酶(PsIPPI)、r{牛兒基二磷酸合酶(PsGPPS)、法尼基二磷酸合酶(PsFPPS)、雙r{牛兒基二磷酸合酶(PsGGPPS)及順式-異戊烯基轉(zhuǎn)移酶(PsCPT)及其基因,并進行了生物信息學(xué)分析。4.根據(jù)轉(zhuǎn)錄組測序結(jié)果選擇了杠柳順式-聚異戊二烯生物合成途徑中的兩個關(guān)鍵基因,即PsIPPI和PsCPT,利用PCR方法全長克隆,并以單獨或組合的形式構(gòu)建成三種基因過表達載體,以鑒定這兩個基因的功能以及對杠柳順式-聚異戊二烯生物合成的影響。5.利用農(nóng)桿菌介導(dǎo)方法將這三種基因過表達載體導(dǎo)入到杠柳植物體內(nèi)進行過表達。結(jié)果發(fā)現(xiàn)在杠柳中過表達PsIPPI基因?qū)ζ漤樖?1,4-聚異戊二烯的生物合成沒有明顯作用;過表達PsCPT基因?qū)樖?1,4-聚異戊二烯的生物合成具有明顯的提升,增加約5倍;而同時過表達PsCPT基因及PsIPPI基因能更進一步促進杠柳順式-1,4-聚異戊二烯的生物合成,特別是高分子部分。該研究將為培育杠柳成為生產(chǎn)天然橡膠工業(yè)原料替代植物,為解決我國天然橡膠貧乏的工業(yè)瓶頸、豐富橡膠資源提供技術(shù)支撐。
[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.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S793.9;Q943.2

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