【作者】 付瑜华；

【导师】 王文泉；

【作者基本信息】 海南大学 ， 作物遗传育种， 2012， 博士

【摘要】 麻疯树{Jatropha curcas L.)是大戟科能源作物,主要分布于全球热带和亚热带地区,种仁中脂肪类物质含量高,约21%的饱和脂肪酸和70%以上的不饱和脂肪酸,种子油流动性好,被认为是最有可能成为替代化石能源的植物油之一。为深入开展麻疯树基因组学研究,图位克隆功能基因、构建高密度物理图谱和基因组大规模测序,基因组大片段文库是必不可少的基础工具和平台。基因工程技术是改变植物油成分、培育新品种的有效方法之一,植物微粒体co-3脂肪酸去饱和酶基因(FAD3)是脂肪酸去饱和酶基因家族一员,主要催化细胞中亚油酸到亚麻酸的反应,研究表明在非光合作用的组织中,FAD3蛋白主要为ω-3去饱和酶的功能,催化组织中约80%三烯脂肪酸的合成。因此,对麻疯树微粒体ω-3脂肪酸去饱和酶基因的研究,不仅可以加深对植物种子油脂合成途径的理解,也为利用基因工程改善麻疯树种子油成分提供了理论依据。麻疯树BIBAC文库,用BamHⅠ限制性内切酶对基因组DNA进行部分酶切,两次酶切片段选择后,连接载体pCLD04541,转化E.coliDH1OB感受态细胞,蓝白斑筛选阳性克隆。该文库含有30,720个克隆,随机挑选214个克隆进行插入片段的评估,发现片段大小主要集中在141-160kb范围,平均插入片段大小为131.9kb,80%的克隆都具有100kb以上的插入片段,文库空载率为7.9%,所以该文库实际含有插入片段的BIBAC克隆数大约为28,293个。麻疯树基因组约为416Mbp,因此该文库约覆盖麻疯树二倍体基因组8.9倍,理论上筛选出任意基因的几率在99%以上。对随机挑取的5个BIBAC克隆进行稳定性检测表明,插入片段可以在宿主大肠杆菌中稳定保存。将麻疯树BIBAC文库中27,648个克隆(8.0×)以4×4点阵形式制备了高密度杂交膜。每张膜(22.5cm×11.25cm)上含有9,216个BIBAC克隆,每个克隆含有两个拷贝,高密度杂交膜包含整个文库90%以上的克隆,覆盖麻疯树二倍体基因组8.0倍。从NCBI上已经公布的与麻疯树脂肪酸代谢相关基因中选取9个基因(FAD3, FAD6, FAD7, ACCase, KAS Ⅰ, KASⅡ, KASⅢ acyl-ACP thioesterase和chloroplast acyl-ACP thioesterase),根据序列信息共设计15对探针同麻疯树高密度杂交膜杂交进行筛选,除acetyl-CoA carboxylase基因外,其他8个基因都检测到了阳性克隆,平均每个基因可以得到5.3个阳性克隆,该结果说明所构建的麻疯树BIBAC文库能够满足基因克隆研究的需要,并将是构建物理图谱及大规模基因组测序的有力工具。对选取的9个麻疯树脂肪酸代谢相关基因进行Southern分析,结果表明这9个基因在麻疯树基因组中均为单拷贝。将Southern结果中含有FAD3基因的克隆22G19和含有FAD7基因的克隆64D4分别测序并进行数据拼接,同NCBI数据库进行比对后结果表明,麻疯树叶绿体ω-3脂肪酸去饱和酶基因(FAD7)长度约为2655bp,含有8个外显子和7个内含子,氨基酸525个,转录起始位点位于上游-424～--374bp区域,起始碱基为位于-385bp的G,启动子区域为-1730-385bp。顺式作用元件分析表明FAD7基因含有较多的光响应元件,其次为对低温和高温的响应元件,干旱响应元件和胚乳特异表达的顺式作用元件。麻疯树微粒体ω-3脂肪酸去饱和酶基因(FAD3)长度约为1797bp,含有8个外显子和7个内含子,氨基酸377个,转录起始位点位于上游-93-23bp区域,起始碱基为位于-54bp的A。顺式作用元件主要为光响应元件、抗逆响应元件、厌氧诱导响应元件和玉米蛋白代谢响应元件。从麻疯树叶片中克隆FAD3基因,该基因ORF为1134bp,编码377个氨基酸,分子量约为41.1kD,命名为JcFAD3。同源序列比对发现JcFAD3同油桐、白毛杨、蓖麻、大豆和亚麻的氨基酸序列相似性均较高。进化树分析表明,JcFAD3与油桐FAD3的亲缘关系最近。结构分析表明,JcFAD3氨基酸序列含有完整的Deltal2-FADS结构域和Membrane-FADS结构域,三个保守的组氨酸富集区分别位于94位(HDCGH),130位(HRTHH)和297位(HVIHH)。对JcFAD3基因在麻疯树根、茎、叶和叶柄的组织中进行表达分析,结果表明JcFAD3为组成型表达,根中的表达量最高,叶中的表达量最低。麻疯树种子形成过程中,JcFAD3在种子形成的初期和中期表达量逐渐降低,后期表达量上升。植物细胞中三烯酸的含量同植物对温度的敏感性有直接关系。本研究中将麻疯树分别进行4℃和40℃处理,结果发现JcFAD3在不同温度下均有表达,低温情况下,随着处理时间的增加,表达量呈上升趋势；高温情况下,表达量下降,且在整个高温处理阶段,表达量均较低。利用INVScl-pYes2.0酿酒酵母转化体系对JcFAD3基因进行酵母表达研究,气象色谱分析转基因酵母和对照酵母中的脂肪酸含量,结果表明,同对照酵母相比,转JcFAD3基因的酵母菌中多出一个亚麻酸的色谱峰,说明JcFAD3蛋白在酵母中得到了正确的表达,表达产物可以将底物亚油酸转化为亚麻酸。根据植物表达载体pVKH-35S-GUS的多克隆位点特征和麻疯树FAD3基因序列特征,构建了pVKH-JcFAD3过表达载体,采用农杆菌介导法转化野生型拟南芥,PCR鉴定表明获得5株阳性植株。对转JcFAD3基因的拟南芥和野生型拟南芥叶片分别提取脂肪酸,气相色谱分析结果表明,野生型拟南芥和转JcFAD3基因的拟南芥叶片中亚麻酸含量分别为21%和37.5%,说明JcFAD3在拟南芥中得到了正确表达,并提高了转基因拟南芥中亚麻酸的含量。更多还原

【Abstract】 Jatropha curcas L., a member of the Euphorbiaceae, is bio fuel crop and widely distributed in tropical and subtropical areas all over the world. The Jatropha kernel contains high amounts of lipids which with21%saturated fatty acid and over70%unsaturated fatty acid and its seed oil with low viscosity, that it has been considered as one of the substitutes for fossil fuels. In order to investigate Jatropha curcus genome, gene cloning and characterization, physical mapping and large-scale genome sequencing of Jatropha L., Large-insert DNA libraries are fundamental and useful tools and platforms. Genetic engineering technology is one of the effective ways to alter seed oil composition and breed new varieties. Microsomal ω-3fatty acid desaturase (FAD3) of plant is a member of fatty acid desaturase family and catalyze the reaction of linoleic acid to linolenic acid, studies shown that FAD3protein is the major ω-3desaturase in non-photosynthetic tissues and it accounts for nearly80%of C18:3syntheses. Therefore, the study of Jatropha microsomal ω-3fatty acid desaturase will not only lead to a better understanding of seed oil syntheses but also provide the thesis for altering the Jatropha seed oil composition through genetic engineering.The BIB AC library of Jatropha L. which constructed by BamH I with the vector pCLD04541, consisting of30,720clones.214clones in the library have been random sampled and analyzed. Results showed that the library has an average insert size of131.9kbp and more than80%of the clones had inserts larger than100kb, which ranged from141to160kbp. As7.9%of its clones are estimated to none inserts, the BIBAC library had approximately28,293clones containing Jatropha L. genomic DNA inserts. Since the haploid Jatropha L. genome is around416Mbp, the library has a genome coverage equivalent to approximately8.9x haploid genomes and provides more than99%probability of identifying any single-copy gene. Five BIBAC clones were randomly picked and analyzed their stability. Results revealed that the insert DNA was stable in the vector for long terms of cultivation. A subset (8.0x) of the Jatropha L. BIBAC library double-spotted onto three high-density nylon filters, containing27,648clones in total with9,216clones on each filter, representing90%of the whole library. Then the filters were screened by overgos designed nine fatty acid metabolism related genes in Jatropha (FAD3, FAD6, FAD7, ACCase, KASⅠ, KASⅡ, KASⅢ, acyl-ACP thioesterase and chloroplast acyl-ACP thioesterase). The filters Hybridization results showed that from one to fourteen positive clones were identified for each gene, except acetyl-CoA carboxylase. A total of forty-two positive clones were identified, with an average of5.3positive clones per gene. This result indicates that the Jatropha BIBAC library is suitable for gene cloning and a powerful tool for physical map construction and genome sequencing.The Southern result of nine fatty acid metabolism related genes in Jatropha shows that all of the nine genes are single copy in Jatropha genome.Positive clones of clone22G19which contains FAD3gene and clone64D4which contains FAD7gene in Southern hybridization had been sequenced and data spliced, and NCBI blast shows that FAD7is2655bp, containing8exons and7introns and encoding525amino acids. The transcriptional start site is base G locating at-385bp and promoter area is-1730--385bp. Analysis of cis-acting element of FAD7promoter shows that FAD7has many light responsiveness elements, others were heat stress and low-temperature stress resposiveness, drought-inducibility and endosperm expression cis-acting elements. FAD3is1797bp, containing8exons and7introns and encoding377amino acids. The transcriptional start area is-93～-23and the start site is base A locating at-54bp. Analysis of cis-acting element of FAD3promoter shows that cis-acting elements in FAD3are mainly involved in light responsiveness and, defense and stress responsiveness, anaerobic induction, zein metabolism regulation and heat stress responsiveness.Jatropha FAD3gene was cloned from Jatropha leaves, it was named JcFAD3with an open reading fame of1134bp which encoding377amino acid residues with the predicted molecular mass of41.1kD. The deduced amino acid sequence showed high identities with FAD3from Vernicia fordii, Populus tomentosa, Ricinus communis, Glycine max and Linum usitatissimum by homologous sequences blast. The phylogenetic analysis of various FAD3indicated that JcFAD3was close to FAD3from Vernicia fordii. The structural analysis of FAD3protein shows that FAD3has Deltall2-like domain, Membrane-FADS domain and three conserved His motifs which are located at94(HDCGH),130(HRTHH) and297(HVIHH), respectively.The expression patterns of JcFAD3in different tissue of Jatropha were investigated by real-time PCR which showed that JcFAD3was expressed in all the organs of Jatropha and the expression level was the highest in the root and the lowest in the leaves. During the development of Jatropha seed, JcFAD3in the seed was expressed decreasingly in the early and middle stage and increasingly in the late stage. The amount of trienoic fatty acids in the cell are the direct result response to temperature in plant. After exposing Jatropha at4℃and40℃, respectively, it was found that JcFAD3expressed at different temperature and its expression was increased at low temperature while decreased at high temperature.INVScl-pYes2.0yeast transformation system was used to study JcFAD3gene expression in yeast cell and then the fatty acid contents in transformed yeast cell with empty vector and JcFAD3were also investigated by GC analysis, the result showed that a novel fatty acid methyl ester peak corresponding to linolenic acid methyl ester standard was detected in the yeast cell transformed with JcFAD3, which was absent in the yeast cell containing empty vector. This indicated that JcFAD3gene was expressed correctly and JcFAD3protein catalyzed linoleic acid to linolenic acid in yeast cell.The expression vector pVKH-JcFAD3was constructed based on the character of pVH-35S-GUS and JcFAD3gene and then transformed into wild-type Arabidopsis by Agrobacterium-mediated method.5transgenic plants with JcFAD3were obtained by PCR analysis. The fatty acid was isolated from wild Arabidopsis leaves and transgenic Arabidopsis showed there proportions were21%and37.5%in total fatty acids respectively which detected by GC analysis.更多还原