| RACE初步克隆胡麻β-酮脂酰-CoA合酶基因以及胡麻再生体系建立的研究 |
| Alternative Title | Preliminary Cloning the 3-ketoacyl-CoA synthase (KCS) with RACE Technique and Establishment of Regeneration System in Linumusita tissimum
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| 张彦萍 |
| Thesis Advisor | 侯岁稳
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| 2009-06-03
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | 胡麻
β-酮脂酰-CoA合酶
FKCS
RACE技术
再生体系
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| Abstract | 胡麻中超长链脂肪酸具有广泛的生理功能，它参与种子甘油酯、生物膜膜脂及鞘脂的合成，并为表皮蜡质的生物合成提供前体物质。超长链脂肪酸的合成由脂肪酰-CoA 延长酶催化，其合成的第一步反应——缩合反应是限速步骤，由β-酮脂酰-CoA 合酶(KCS)催化。目前，人们对脂肪酸延长酶基因的研究主要集中在KCS基因上。本实验首先利用简并引物获得胡麻β-酮脂酰-CoA 合酶基因的DNA片段，并将预期得到的β-酮脂酰-CoA 合酶基因命名为FKCS。再以此DNA片段为基础，利用3’RACE反应从胡麻叶片中克隆到FKCS的1082bp的cDNA片段，NCBI的ORF分析表明，它含有一个长度为822 bp的开放阅读框和252 bp完整的3’UTR。由于本实验中的5’RACE没有得到相应目的片段，仅通过3’RACE反应获得的cDNA推测出FKCS的273个氨基酸，位于FKCS的C端。序列同源性分析表明，FKCS所编码的蛋白质，其氨基酸序列与拟南芥中五种β-酮脂酰-CoA 合酶有较高的同源性。通过系统进化树分析得知，FKCS与拟南芥KCS家族中亲缘关系最近的是KCS-1。而拟南芥的KCS-1主要与超长链脂肪酸的合成有关，因此推测胡麻中FKCS也与超长链脂肪酸的合成有关，FKCS可能是胡麻中一种新型的β-酮脂酰-CoA 合酶。FKCS基因的克隆和测序为将来研究其在胡麻中的生物学功能奠定了基础。为了便于以后研究FKCS基因的功能，以胡麻的下胚轴为外植体，成功建立了胡麻的快繁再生体系：MS﹢300mg/L水解酪蛋白﹢0.5mg/L 6－BA有利于愈伤组织及不定芽的生成；在促进不定芽生根方面，不添加任何植物激素的1/2MS培养基更有利于根的生长。 |
| Other Abstract | Very long chain fatty acids (VLCFAs) play a comprehensive role in Flax. They are essential biological components of triacylglycerols (TAGs), membrane lipids, and sphingolipids. They also serve as precursors of wax layer compounds. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase. The first step of biosynthesis which is catalyzed by 3-ketoacyl-CoA synthase (KCS) is the rate-limiting step. At present, the study of the fatty acyl-CoA elongase focuses on the KCS. In this study, firstly, a DNA fragment was got by degenerate primer, designated FKCS. A cDNA encoding the 3-ketoacyl-CoA synthase was isolated and sequenced from the leaves tissue of flax (Linum fusitatissimum) for the first time, by 3’ rapid amplification of cDNA ends (RACE) technique based on the DNA fragment. Analysis on the cDNA sequence of FKCS, we obtained the open reading frame (ORF) from NCBI. The ORF contained 822 bp bases, flanked by a complete 3’ untranslated region with 252 bp bases. Because of the failure of 5’ RACE, we only got a putative 273 amino acid of FKCS located at the carboxyl terminal. Sequence analysis indicated that the sequence of amino acids of FKCS is more similar to five members of KCS family in Arabidopsis. The phylogenetic tree based on the amino acid sequences showed that genetic relationship between FKCS and KCS-1 was closest in Arabidopsis. Therefore, FKCS is possibly a novel member of KCS family. The cloning and sequencing of FKCS establishes a good foundation for the future work on the biological functions of FKCS in the flax (Linum usitatissimum). For the future study of FKCS, MS was found to be the best basal medium in regeneration of flax in which hypocotyl was used as explant. 0.5mg/L 6-BA was effective to induce callus and shoot. An optimized protocol was established as following: a MS medium supplemented with 300mg/L casein acid hydrolysates and 0.5mg/L6-BA was beneficial to callus induction and shoot regeneration, then a half-strength MS medium is helpful to shoots elongation and rooting. |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/221873
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| Collection | 生命科学学院
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Recommended Citation
GB/T 7714 |
张彦萍. RACE初步克隆胡麻β-酮脂酰-CoA合酶基因以及胡麻再生体系建立的研究[D]. 兰州. 兰州大学,2009.
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