甘蓝型油菜LPAT4和LPAT5基因的克隆与功能分析

发布时间：2018-05-29 19:56

本文选题：油菜 + LPAT4/5基因　；参考：《湖南农业大学》2015年硕士论文

【摘要】：油菜(Brassica napus)——全球重要经济作物,食用植物油及优质蛋白的主要来源。数据表明,就单位面积产油量而论,油菜油脂含量每提高1个百分点,相当于增产菜籽2.5个百分点,故而提高菜籽含油量是提升生产效益的有效途径。LPAT为三酰甘油组装过程中的一个关键酶,能影响脂质生物合成。提高LPAT酶活性能够降低合成抑制作用。因此,通过基因工程手段克隆LPAT基因,并分析其功能,对研究菜籽含油量具有一定的实践价值。主要实验结论汇总如下：1.采用同源克隆方法,获得2条LPAT4基因全长CDS序列,长度依次为1143 bp和1140 bp。生物信息学分析显示,它们都包含LPLAT_LCLAT1样结构域,是LPLAT超基因家族中的成员,命名为BnLPAT4-1和BnLPAT4-2。2.同源克隆得到BnLPAT4启动子序列,启动子元件分析显示其可能参与光、缺氧、逆境胁迫、真菌、脱落酸、赤霉素、水杨酸、乙烯等诱导和茎尖特异性表达及昼夜节律调控。pBnLPAT4启动子介导GUSA基因拟南芥异源表达显示,该启动子介导的基因主要表达部位为幼苗、根、茎、叶、花瓣、花萼、花托及果荚,而花药及种子中未检测到启动子激活。3. BnLPAT4-1和BnLPAT4-2为普遍型表达基因,BnPAT4-1和BnPAT4-2的最高表达分别在叶和胚中。逆境分析显示,NaCl、PEG4000、水渍、6-BA和ABA胁迫下,BnLPAT4-1和BnLPAT4-2呈现不同的表达模式。极差分析表明,ABA对BnLPAT4-1影响较大,而BnLPAT4-2却对PEG4000敏感。4. BnLPAT4-1和BnLPAT4-2酵母异源表达显示,该酶具备转移酶活性。转BnLPAT4-1菌株,66.88%为饱和脂肪酸,是不饱和脂肪酸的2.89倍,较YES2.0转化子饱和脂肪酸25.62%,净增长1.61倍。其中硬脂酸比例最高,为50.60%。BnLPAT4-2转化子显著提高了C16脂肪酸(棕榈酸及棕榈油酸)的比例。较YES2.0转化子C16脂肪酸25.62%净增长22.40%,提高70%。5. napin启动子介导BnLPAT4-1在拟南芥T3代种子中表达结果显示,BnLPAT4-1能够降低芥酸、饱和脂肪酸以及长链多不饱和脂肪酸含量,且特异性地提高花生一烯酸含量。6.同源克隆湘油15 LPAT5全长CDs序列,其长度是1041bp,命名BnLPAT5。序列分析表明它拥有LPLAT超基因家族的特征,是此基因家族中的一员。7. BnLPAT5为普遍型表达基因,水渍、NaCl及PEG4000处理下,BnLPAT5呈“骤降缓升”的表达模式,3h时,达到最低表达量。对于ABA和6BA的处理,BnLPAT5呈“缓降缓升”的表达模式,12h时,表达量最低。8. BnLPAT5酵母表达显示其表达产物具备转移酶活性。转BnLPAT5菌株,含不饱和脂肪酸85.73%,是饱和脂肪酸的5.82倍。亚油酸和油酸的比例之和约占总比例的85%,符合健康油标准。BnLPAT5对油酸和亚油酸具有偏好性,底物选择的顺序依次为：亚油酸油酸。9. napin启动子介导BnLPAT5在拟南芥T3代种子中表达表明,BnLPAT5能够抑制拟南芥LPAT对芥酸、花生酸、花生二烯酸及花生三烯酸的选择,且不同株系BnLPAT5选择性呈现出差异,但均能提高长链低不饱和脂肪酸的比例。
[Abstract]:Brassica napusus is the main source of edible vegetable oil and high quality protein in the world. In terms of oil production per unit area, the oil content of rape increased by 1 percentage point, which was equivalent to 2.5 percentage points of rapeseed yield. Therefore, increasing the oil content of rapeseed is an effective way to improve the efficiency of production. LPAT is a key enzyme in triglyceride assembly, which can affect lipid biosynthesis. Increasing the activity of LPAT could reduce the inhibition of synthesis. Therefore, cloning and analyzing the function of LPAT gene by genetic engineering has certain practical value for the study of rapeseed oil content. The main experimental conclusions are summarized as follows: 1: 1. By using homologous cloning method, two full-length CDS sequences of LPAT4 gene were obtained, the length of which was 1143 BP and 1140 BP, respectively. Bioinformatics analysis shows that they all contain LPLAT_LCLAT1 like domains and are members of LPLAT supergene family named BnLPAT4-1 and BnLPAT4-2.2. Homologous cloning of BnLPAT4 promoter sequence showed that the promoter might be involved in light, hypoxia, stress, fungi, abscisic acid, gibberellin and salicylic acid. Ethylene induction and stem tip specific expression and circadian regulation. PBnLPAT4 promoter mediated the heterologous expression of GUSA gene in Arabidopsis thaliana. The main sites of the promoter gene expression were seedlings, roots, stems, leaves, petals, calyx, receptacle and fruit pods. However, no promoter activation. 3 was detected in anthers and seeds. BnLPAT4-1 and BnLPAT4-2 were the highest expression of BnPAT4-1 and BnPAT4-2 in leaves and embryos, respectively. Stress analysis showed that NaClPAT4000. BnLPAT4-1 and BnLPAT4-2 showed different expression patterns under the stress of 6-BA and ABA. Range analysis showed that ABA had a great effect on BnLPAT4-1, while BnLPAT4-2 was sensitive to PEG4000. 4. The heterologous expression of BnLPAT4-1 and BnLPAT4-2 showed that the enzyme had the activity of transferase. 66.88% of BnLPAT4-1 strain was saturated fatty acid, 2.89 times of unsaturated fatty acid, which was 1.61 times higher than that of YES2.0 transformant 25.62%. The ratio of stearic acid was the highest, and the ratio of C 16 fatty acid (palmitic acid and palmitic oil acid) was significantly increased by 50.60%.BnLPAT4-2 transformant. Compared with the YES2.0 transformant C16 fatty acid 25.62%, the net increase was 22.4040%, and increased 70%. 5. The expression of BnLPAT4-1 mediated by napin promoter in T3 generation seeds of Arabidopsis thaliana showed that BnLPAT4-1 could reduce the contents of erucic acid, saturated fatty acid and long chain polyunsaturated fatty acids. The content of arachidonic acid. The homologous clone Xiangyou 15 LPAT5 was named BnLPAT5 with a length of 1041bp. Sequence analysis shows that it has the characteristics of LPLAT supergene family and is a member of this gene family. BnLPAT5 is a universal expression gene. The lowest expression level of BnLPAT5 can be reached at 3 h after treatment with PEG4000 and WBnLPAT5. For the treatment of ABA and 6BA, the expression level of BnLPAT5 was the lowest when the expression pattern of BnLPAT5 was "slowly descending and rising slowly" for 12h. The expression of BnLPAT5 yeast showed that its expression product had the activity of transferase. The BnLPAT5 strain contained 85. 73% unsaturated fatty acids, 5.82 times of saturated fatty acids. The ratio of linoleic acid to oleic acid is about 85% of the total, which meets the healthy oil standard. BnLPAT5 has a preference for oleic acid and linoleic acid. The order of substrate selection was linoleic acid .9.The expression of BnLPAT5 in Arabidopsis thaliana T3 seeds mediated by napin promoter showed that BnLPAT5 could inhibit the selection of erucic acid, arachidonic acid and arachidonic acid by LPAT in Arabidopsis thaliana. The BnLPAT5 selectivity of different lines was different, but all of them could increase the proportion of long chain low unsaturated fatty acids.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S565.4

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