本文關(guān)鍵詞：棉花LPAAT基因的克隆與遺傳轉(zhuǎn)化　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 棉花 LPAAT基因 表達(dá)載體構(gòu)建 遺傳轉(zhuǎn)化 脂肪酸

【摘要】：棉花是世界上最重要的纖維作物和主要的油料作物,棉子是商品棉的主要副產(chǎn)物,約占子棉總產(chǎn)量的60%,棉子含油量占15.0%~48.7%。棉子油不僅能用于食品用油,而且能用于飼料加工等。TAG是棉子油脂的主要儲存體,其合成途徑中關(guān)建的溶血磷脂酸酰基轉(zhuǎn)移酶(Lysophosphatidic Acid Acyltransferase,LPAAT)對棉子內(nèi)油脂的積累有較大的促進(jìn)作用。本研究通過對2個棉花材料棉子發(fā)育的14個時期胚珠樣品進(jìn)行脂肪酸成分的測定,及其中5個時期的胚珠樣品進(jìn)行粗脂肪含量的測定,發(fā)現(xiàn)油酸和亞油酸的積累模式在兩種材料中有一定差異,且突變體材料5個時期的胚珠樣品粗脂肪含量均高于野生型。溶血磷脂酸�；D(zhuǎn)移酶是棉子脂肪酸合成過程中的關(guān)鍵酶,以雷蒙德氏棉(G.raimondii,D5)和亞洲棉(G.arboreum,A2)2個二倍體棉花中LPAAT基因家族的生物信息學(xué)分析及結(jié)合本實(shí)驗(yàn)室的棉花胚珠的RNA-seq數(shù)據(jù),從中分析并選取在胚珠中表達(dá)的一些GhLPAAT基因。以徐州142野生型為材料,從中克隆了棉花中的GhLPAAT5和GhLPAAT2.2并進(jìn)行功能驗(yàn)證。取得的結(jié)果如下:1.通過對徐州142及其無絨無絮突變體14個時期的胚珠進(jìn)行脂肪酸組成含量及5個時期的胚珠進(jìn)行脂肪含量的測定,分析棉子發(fā)育過程中脂肪及脂肪酸組成的積累模式。發(fā)現(xiàn)兩個材料的棉子從開花后20 d到成熟這一階段脂肪積累呈上升趨勢,其中成熟種子含油量達(dá)到最高。2個材料棉子發(fā)育過程中先后有8種脂肪酸成分出現(xiàn),其中成熟棉子中油酸與亞油酸含量占脂肪酸總含量的72%左右,是棉子脂肪酸的重要組分。對棉子發(fā)育過程中各脂肪酸組成含量做相關(guān)分析發(fā)現(xiàn)徐州142中C18:1與C18:2呈一定正相關(guān)但不顯著,而突變體材料中則呈極顯著正相關(guān)。2.溶血磷脂酸酰基轉(zhuǎn)移酶是油脂合成途徑中的一個關(guān)鍵酶,它催化溶血磷脂酸轉(zhuǎn)變?yōu)榱字�。本研究從雷蒙德氏棉和亞洲棉的基因組數(shù)據(jù)中得到17個LPAAT基因家族成員。利用生物信息學(xué)方法對二倍體棉花LPAAT基因進(jìn)行基因結(jié)構(gòu),染色體分布以及系統(tǒng)進(jìn)化分析,結(jié)果表明,LPAAT基因家族根據(jù)親緣關(guān)系的遠(yuǎn)近可以分為不同的亞家族,各亞家族中LPAAT基因具有相似的基因結(jié)構(gòu);LPAAT家族基因編碼的氨基酸序列具有3個保守基序,其中包括了ΦFPEGTR-G結(jié)合位點(diǎn)和Φ-NHQS-ΦDΦΦ催化位點(diǎn)。基于陸地棉(G.hirsutum)材料不同發(fā)育時期的胚珠RNA-seq數(shù)據(jù)庫和qRT-PCR表達(dá)分析,發(fā)現(xiàn)LPAAT基因可能對脂肪積累起到積極作用。本研究結(jié)果有助于了解棉屬植物L(fēng)PAAT基因家族的功能,以期從中選取一些LPAAT基因進(jìn)行進(jìn)一步功能驗(yàn)證。3.從徐州142材料中克隆出2個溶血磷脂酸�；D(zhuǎn)移酶基因GhLPAAT2.2和GhLPAAT5。其CDS序列長度分別為1164 bp和1065 bp,分別編碼387和354個氨基酸。通過對兩個基因所編碼氨基酸序列的同源比對分析發(fā)現(xiàn)它們均含有F*EGT*和NHR***D保守結(jié)構(gòu)域。對這兩個基因進(jìn)行表達(dá)載體構(gòu)建并轉(zhuǎn)化Col-0野生型擬南芥。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S562;Q943.2

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