棉花LPAAT基因的克隆与遗传转化

发布时间：2018-01-09 08:21

本文关键词：棉花LPAAT基因的克隆与遗传转化　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：棉花是世界上最重要的纤维作物和主要的油料作物,棉子是商品棉的主要副产物,约占子棉总产量的60%,棉子含油量占15.0%~48.7%。棉子油不仅能用于食品用油,而且能用于饲料加工等。TAG是棉子油脂的主要储存体,其合成途径中关建的溶血磷脂酸酰基转移酶(Lysophosphatidic Acid Acyltransferase,LPAAT)对棉子内油脂的积累有较大的促进作用。本研究通过对2个棉花材料棉子发育的14个时期胚珠样品进行脂肪酸成分的测定,及其中5个时期的胚珠样品进行粗脂肪含量的测定,发现油酸和亚油酸的积累模式在两种材料中有一定差异,且突变体材料5个时期的胚珠样品粗脂肪含量均高于野生型。溶血磷脂酸酰基转移酶是棉子脂肪酸合成过程中的关键酶,以雷蒙德氏棉(G.raimondii,D5)和亚洲棉(G.arboreum,A2)2个二倍体棉花中LPAAT基因家族的生物信息学分析及结合本实验室的棉花胚珠的RNA-seq数据,从中分析并选取在胚珠中表达的一些GhLPAAT基因。以徐州142野生型为材料,从中克隆了棉花中的GhLPAAT5和GhLPAAT2.2并进行功能验证。取得的结果如下:1.通过对徐州142及其无绒无絮突变体14个时期的胚珠进行脂肪酸组成含量及5个时期的胚珠进行脂肪含量的测定,分析棉子发育过程中脂肪及脂肪酸组成的积累模式。发现两个材料的棉子从开花后20 d到成熟这一阶段脂肪积累呈上升趋势,其中成熟种子含油量达到最高。2个材料棉子发育过程中先后有8种脂肪酸成分出现,其中成熟棉子中油酸与亚油酸含量占脂肪酸总含量的72%左右,是棉子脂肪酸的重要组分。对棉子发育过程中各脂肪酸组成含量做相关分析发现徐州142中C18:1与C18:2呈一定正相关但不显著,而突变体材料中则呈极显著正相关。2.溶血磷脂酸酰基转移酶是油脂合成途径中的一个关键酶,它催化溶血磷脂酸转变为磷脂酸。本研究从雷蒙德氏棉和亚洲棉的基因组数据中得到17个LPAAT基因家族成员。利用生物信息学方法对二倍体棉花LPAAT基因进行基因结构,染色体分布以及系统进化分析,结果表明,LPAAT基因家族根据亲缘关系的远近可以分为不同的亚家族,各亚家族中LPAAT基因具有相似的基因结构;LPAAT家族基因编码的氨基酸序列具有3个保守基序,其中包括了ΦFPEGTR-G结合位点和Φ-NHQS-ΦDΦΦ催化位点。基于陆地棉(G.hirsutum)材料不同发育时期的胚珠RNA-seq数据库和qRT-PCR表达分析,发现LPAAT基因可能对脂肪积累起到积极作用。本研究结果有助于了解棉属植物LPAAT基因家族的功能,以期从中选取一些LPAAT基因进行进一步功能验证。3.从徐州142材料中克隆出2个溶血磷脂酸酰基转移酶基因GhLPAAT2.2和GhLPAAT5。其CDS序列长度分别为1164 bp和1065 bp,分别编码387和354个氨基酸。通过对两个基因所编码氨基酸序列的同源比对分析发现它们均含有F*EGT*和NHR***D保守结构域。对这两个基因进行表达载体构建并转化Col-0野生型拟南芥。
[Abstract]:Cotton is the most important fiber crop and main oil crop in the world. Cotton is the main by-product of commercial cotton, accounting for about 60% of the total yield of seed cotton. The oil content of cotton seed is 15.0% and 48.7%. Cotton oil can be used not only in food oil, but also in feed processing. Tag is the main storage of cottonseed oil. Lysophosphatidic Acid Acyltransferase was synthesized by lysophosphatidic Acid Acyltransferase. LPAATs can promote the accumulation of lipid in cotton seeds. In this study, the fatty acid composition of ovule samples from 14 stages of cotton seed development was determined. The crude fat content of the ovule samples in 5 periods was determined and it was found that the accumulation patterns of oleic acid and linoleic acid were different between the two materials. The crude fat content of ovule samples in 5 periods of mutant was higher than that of wild type. Lysophosphatidyltransferase was the key enzyme in fatty acid synthesis of cottonseed. G. raimondii D5 and G. arboreum. A2) bioinformatics analysis of LPAAT gene family in two diploid cotton and RNA-seq data of cotton ovule from our laboratory. Some GhLPAAT genes expressed in ovules were analyzed and selected. The wild type of Xuzhou 142 was used as the material. The GhLPAAT5 and GhLPAAT2.2 in cotton were cloned and verified. The results are as follows:. 1. The fatty acid composition of ovules of Xuzhou 142 and its mutants were determined in 14 periods and the fat contents of ovules in 5 periods were determined. The accumulation pattern of fat and fatty acid composition during cotton development was analyzed. It was found that the fat accumulation of cotton seeds from 20 days after anthesis to maturation showed an increasing trend. The oil content of mature seeds reached the highest. Eight fatty acid components appeared successively during the development of cotton seeds, of which oleic acid and linoleic acid content accounted for about 72% of the total fatty acid content in mature cotton seeds. It was found that there was a positive correlation between C18: 1 and C18: 2 in Xuzhou 142, but there was no significant correlation between C18: 1 and C18: 2. However, there was a significant positive correlation among the mutants. 2. Lysophosphatidyltransferase was a key enzyme in the lipid synthesis pathway. It catalyzes the conversion of lysophosphatidic acid to phosphatidic acid. In this study, 17 members of LPAAT gene family were obtained from genomic data of Raymond's cotton and Asian cotton. Bioinformatics method was used to analyze LPA of diploid cotton. The AT gene carries on the gene structure. The results of chromosome distribution and phylogenetic analysis showed that the LPAAT gene family could be divided into different subfamilies according to their genetic relationship, and the LPAAT genes in each subfamily had similar gene structure. The amino acid sequences encoded by LPAAT family genes have three conserved motifs. It includes 桅 FPEGTR-G binding site and 桅 -NHQS- 桅 D 桅桅 catalytic site. RNA-seq database and qRT-PCR expression analysis of ovule at different developmental stages of materials. It was found that LPAAT gene may play a positive role in fat accumulation. This study is helpful to understand the function of LPAAT gene family in cotton plants. 3. Two lysophosphatidyl transferase genes GhLPAAT2.2 and GhLPAA were cloned from Xuzhou 142. T5. The length of its CDS sequence is 1164, respectively. BP and 1065 BP. The amino acids encoding 387 and 354 amino acids were encoded respectively. By homologous alignment analysis of the amino acid sequences of the two genes, it was found that both of them contained FG EGT * and NHR***D conserved domains. Col-0 wild-type Arabidopsis thaliana was constructed and transformed by expression vector.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S562;Q943.2

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