高山被孢霉多不饱和脂肪酸合成相关基因克隆及转化甘蓝型油菜研究
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摘要
多不饱和脂肪酸是对人体有重要生理活性的物质,有较高的药用价值。利用油料作物生产多不饱和脂肪酸是一种极具潜力的途径,将不饱和脂肪酸生物合成途径中的相关基因转入植物生产长链多不饱和脂肪酸成为近几年油脂类国际会议的热点。油菜是十字花科(Cruiferae)芸薹属(Brassica genus)植物,甘蓝型油菜(Brassica napus L.)在世界上是继大豆和棕榈之后的第三大油料作物,我国油菜种植面积和产量均占世界总量的30%左右,在农业生产和粮油安全上占有重要的战略地位。本研究目的是从高山被孢霉中分离花生四烯酸(Arachidonic acid, AA)和二十碳五烯酸(Eicosapentaenoic acid, EPA)合成途径中的相关基因,并将其连接到带有种子特异表达启动子的植物表达载体上,通过农杆菌介导的方法转化甘蓝型油菜品种,从而在甘蓝型油菜中构筑AA和EPA生物合成的途径,为AA和EPA来源开辟新的方向。本论文主要研究内容和结果如下：
(1)克隆了高山被孢霉的△6去饱和酶基因、△6延长酶基因和△5去饱和酶基因,利用生物信息学手段分析了三个基因编码的氨基酸序列结构,从推导的△6去饱和酶基因和△5去饱和酶基因编码的氨基酸序列中发现均存在一个细胞色素b5结构域和3个组氨酸保守区Ⅰ区(HX3H)、Ⅱ区(HX2HH)和Ⅲ区(QIEHHLFP);△6延长酶基因编码的氨基酸序列中,在183-188和221-225氨基酸残基位置分别有一个组氨酸的富集区和一个酪氨酸的富集区,它们对基因功能的正常表达是必需的。实验中对三个基因的进化也进行了分析。
(2)把三个基因分别构建酵母表达载体,分别转入毕赤酵母构建重组基因工程菌,用于对三个基因的功能验证。提取转△6去饱和酶基因重组毕赤酵母的总蛋白,SDS-PAGE电泳后有目标蛋白条带的出现,大小为51.8 kDa,证明△6去饱和酶在酵母菌体内能进行正常的转录和翻译,脂肪酸分析表明,△6去饱和酶能把酵母自身能合成的LA转化为GLA,合成的GLA含量占总脂肪酸含量的6.3%,实验中没有检测到OTA的生成,说明转入酵母中的△6去饱和酶基因不能利用ALA合成新的产物或合成的产物量太少而不能检测出来,据此推断△6去饱和酶基因对LA底物具有偏好性。在添加GLA底物时,△6延长酶在重组酵母中能催化合成占总脂肪酸含量0.31%的DHGLA。添加底物DHGLA,转入△5去饱和酶基因的重组酵母脂肪酸中有2.85%的AA生成。根据实验结果推测△6延长酶催化的反应在多不饱和脂肪酸合成中是限速步骤。
(3)克隆了甘蓝型油菜种子特异表达的napin启动子,通过napin启动子DNA序列分析,该启动子富含AT碱基,含有3个TATA-box,7个CAAT-box,1个GATA-box,2个G-box和3个(CA)n-motif等调控元件,这些调控元件是高等植物启动子序列的特征。采用酶切连接介导PCR的方法构建△6延长酶基因的表达框和△5去饱和酶基因的表达框,利用napin启动子完成三价植物表达载体pCAMBIA1303-D6ELD5 (napin启动子)的构建。
(4)转化根癌农杆菌LBA4404后,通过愈伤组织转化法、真空渗透法和蘸花法对甘蓝型油菜7633进行了转化,其中愈伤组织转化法的转化效率为1.2%,另外两种转化方法的转化效率最高分别为1.8%和2.5%。利用Hyg对转化的植株和种子进行筛选,获得500多株转基因油菜。对筛选到的阳性植株进行PCR检测和Southern blot分析,证明外源基因已经整合到了油菜的基因组中。
Polyunsaturated fatty acids were one kind of important physiological activity substance to human and had high officinal value. It was a great potential method to produce polyunsaturated fatty acids through oil crops, and to produce long-chain polyunsaturated fatty acids by transferring the related genes in the biosythesis of polyunsaturated fatty acids into plants has turned out to be the hot spot of international lipids conferences recently. Rape is one plant of the Brassica genus of the family Cruiferae. Brassica napus L. has become the third important oil crop following soybean and palm in the world at present. The planting area and output of rape of our country accounted for about thirty percent of the world's total amount, which had an important strategic position in the agriculture production and grain safety. In our research, the related genes in the synthesis of AA and EPA obtained from Mortierella alpina was linked to the plant expression vector with seed-specific promoter, and were transformed to B. napus by Agobacterium-mediated transformation method to build biosynthesis pathway for AA and EPA, which provided one new source for AA and EPA production. The main contents and results of this research work are as follows:
(1) A6 desaturase gene,△6 elongase gene and△5 desaturase gene were cloned from M. alpina. Then the amino acid sequences encoded by the three genes were analyzed through bioinformatics methods. A domain of cytochrome b5 and three histidine conserved domains namely domainⅠ(HX3H) domainⅡ(HX2HH) and domainⅢ(QIEHHLFP) were found in the amino acid sequences encoded by both△6 desaturase gene and△5 desaturase gene. Meanwhile, in the amino acid sequence encoded by△6 elongase gene, a histidine enrichment domain and a tyrosine enrichment domain were found in the amino acid residues position of 183-188 and 221-225, respectively. The evolution of the three genes was analyzed in the research as well.
(2) Yeast expression vectors for the three genes were constructed and transferred into Pichia pastoris GS115 to build recombined engineering strains for the function identification of the three genes, respectively. Total protein of the recombined yeast containing△6 desaturase gene was extracted. The target protein band with the size of 51.8 kDa was found after SDS-PAGE electrophoresis, demonstrating that the△6 desaturase could be transcribed and translated normally in yeast. The fatty acid analysis showed that△6 desaturase could transform LA yielded by yeast itself into GLA, and the output of GLA accounted for 6.3% of the total fatty acids. There was no detection of OTA during the experiment which demonstrated that the A6 desaturase gene could not synthesize new product utilizing ALA or the amount of synthesized product could not be detected. In view of these results, we concluded that△6 desaturase gene had preference to the LA substrate. After the addition of GLA substrate,△6 elongase could catalyze the synthesis of DHGLA, which accounted for 0.31% of the total fatty acids in recombined yeast. While after the addition of DHGLA substrate,2.85% AA was detected in the yeast transformed with△5 desaturase gene. According to the experimental results we suggested that the pathway of△6 elongase catalysis was the rate-limiting step in the synthesis of polyunsaturated fatty acid.
(3) The napin promoter of seed-specific expression in B. napus was cloned. The DNA sequence analysis of the promoter showed that there were rich AT nucleotides and had much regulatory elements, including three TATA-box, seven CAAT-box, one GATA-box, two G-box, and three (CA)n-motif, which was the character of the higher plants promoter. Furthermore, the expression cassettes of△6 elongase gene and△5 desaturase gene were constructed through the method of enzyme cleavage connection mediated PCR, and the trivalent plant expression vector pCAMBIA1303-D6ELD5 (napin promoter) was built with the napin promoter.
(4) B. napus 7633 was transformed with Agrobacterium tumefaciens LBA4404 carrying the pCAMBIA1303-D6ELD5(napin promoter) vector through the methods of callus transformation, vacuum infiltration, and floral dip. The transformation efficiency of callus transformation achieved 1.2%, and the maximum transformation efficiency reached 1.2%,1.8%, and 2.5%, respectively. Hygromycin B was applied to screen the transformed explants and seeds, and more than 500 transgenetic plants were obtained during the reseach. PCR and Southern blot were utilized to analyze the screened positive transgenic plants. The results showed that the exogenous genes have been integrated into the genome of rape.
(1)克隆了高山被孢霉的△6去饱和酶基因、△6延长酶基因和△5去饱和酶基因,利用生物信息学手段分析了三个基因编码的氨基酸序列结构,从推导的△6去饱和酶基因和△5去饱和酶基因编码的氨基酸序列中发现均存在一个细胞色素b5结构域和3个组氨酸保守区Ⅰ区(HX3H)、Ⅱ区(HX2HH)和Ⅲ区(QIEHHLFP);△6延长酶基因编码的氨基酸序列中,在183-188和221-225氨基酸残基位置分别有一个组氨酸的富集区和一个酪氨酸的富集区,它们对基因功能的正常表达是必需的。实验中对三个基因的进化也进行了分析。
(2)把三个基因分别构建酵母表达载体,分别转入毕赤酵母构建重组基因工程菌,用于对三个基因的功能验证。提取转△6去饱和酶基因重组毕赤酵母的总蛋白,SDS-PAGE电泳后有目标蛋白条带的出现,大小为51.8 kDa,证明△6去饱和酶在酵母菌体内能进行正常的转录和翻译,脂肪酸分析表明,△6去饱和酶能把酵母自身能合成的LA转化为GLA,合成的GLA含量占总脂肪酸含量的6.3%,实验中没有检测到OTA的生成,说明转入酵母中的△6去饱和酶基因不能利用ALA合成新的产物或合成的产物量太少而不能检测出来,据此推断△6去饱和酶基因对LA底物具有偏好性。在添加GLA底物时,△6延长酶在重组酵母中能催化合成占总脂肪酸含量0.31%的DHGLA。添加底物DHGLA,转入△5去饱和酶基因的重组酵母脂肪酸中有2.85%的AA生成。根据实验结果推测△6延长酶催化的反应在多不饱和脂肪酸合成中是限速步骤。
(3)克隆了甘蓝型油菜种子特异表达的napin启动子,通过napin启动子DNA序列分析,该启动子富含AT碱基,含有3个TATA-box,7个CAAT-box,1个GATA-box,2个G-box和3个(CA)n-motif等调控元件,这些调控元件是高等植物启动子序列的特征。采用酶切连接介导PCR的方法构建△6延长酶基因的表达框和△5去饱和酶基因的表达框,利用napin启动子完成三价植物表达载体pCAMBIA1303-D6ELD5 (napin启动子)的构建。
(4)转化根癌农杆菌LBA4404后,通过愈伤组织转化法、真空渗透法和蘸花法对甘蓝型油菜7633进行了转化,其中愈伤组织转化法的转化效率为1.2%,另外两种转化方法的转化效率最高分别为1.8%和2.5%。利用Hyg对转化的植株和种子进行筛选,获得500多株转基因油菜。对筛选到的阳性植株进行PCR检测和Southern blot分析,证明外源基因已经整合到了油菜的基因组中。
Polyunsaturated fatty acids were one kind of important physiological activity substance to human and had high officinal value. It was a great potential method to produce polyunsaturated fatty acids through oil crops, and to produce long-chain polyunsaturated fatty acids by transferring the related genes in the biosythesis of polyunsaturated fatty acids into plants has turned out to be the hot spot of international lipids conferences recently. Rape is one plant of the Brassica genus of the family Cruiferae. Brassica napus L. has become the third important oil crop following soybean and palm in the world at present. The planting area and output of rape of our country accounted for about thirty percent of the world's total amount, which had an important strategic position in the agriculture production and grain safety. In our research, the related genes in the synthesis of AA and EPA obtained from Mortierella alpina was linked to the plant expression vector with seed-specific promoter, and were transformed to B. napus by Agobacterium-mediated transformation method to build biosynthesis pathway for AA and EPA, which provided one new source for AA and EPA production. The main contents and results of this research work are as follows:
(1) A6 desaturase gene,△6 elongase gene and△5 desaturase gene were cloned from M. alpina. Then the amino acid sequences encoded by the three genes were analyzed through bioinformatics methods. A domain of cytochrome b5 and three histidine conserved domains namely domainⅠ(HX3H) domainⅡ(HX2HH) and domainⅢ(QIEHHLFP) were found in the amino acid sequences encoded by both△6 desaturase gene and△5 desaturase gene. Meanwhile, in the amino acid sequence encoded by△6 elongase gene, a histidine enrichment domain and a tyrosine enrichment domain were found in the amino acid residues position of 183-188 and 221-225, respectively. The evolution of the three genes was analyzed in the research as well.
(2) Yeast expression vectors for the three genes were constructed and transferred into Pichia pastoris GS115 to build recombined engineering strains for the function identification of the three genes, respectively. Total protein of the recombined yeast containing△6 desaturase gene was extracted. The target protein band with the size of 51.8 kDa was found after SDS-PAGE electrophoresis, demonstrating that the△6 desaturase could be transcribed and translated normally in yeast. The fatty acid analysis showed that△6 desaturase could transform LA yielded by yeast itself into GLA, and the output of GLA accounted for 6.3% of the total fatty acids. There was no detection of OTA during the experiment which demonstrated that the A6 desaturase gene could not synthesize new product utilizing ALA or the amount of synthesized product could not be detected. In view of these results, we concluded that△6 desaturase gene had preference to the LA substrate. After the addition of GLA substrate,△6 elongase could catalyze the synthesis of DHGLA, which accounted for 0.31% of the total fatty acids in recombined yeast. While after the addition of DHGLA substrate,2.85% AA was detected in the yeast transformed with△5 desaturase gene. According to the experimental results we suggested that the pathway of△6 elongase catalysis was the rate-limiting step in the synthesis of polyunsaturated fatty acid.
(3) The napin promoter of seed-specific expression in B. napus was cloned. The DNA sequence analysis of the promoter showed that there were rich AT nucleotides and had much regulatory elements, including three TATA-box, seven CAAT-box, one GATA-box, two G-box, and three (CA)n-motif, which was the character of the higher plants promoter. Furthermore, the expression cassettes of△6 elongase gene and△5 desaturase gene were constructed through the method of enzyme cleavage connection mediated PCR, and the trivalent plant expression vector pCAMBIA1303-D6ELD5 (napin promoter) was built with the napin promoter.
(4) B. napus 7633 was transformed with Agrobacterium tumefaciens LBA4404 carrying the pCAMBIA1303-D6ELD5(napin promoter) vector through the methods of callus transformation, vacuum infiltration, and floral dip. The transformation efficiency of callus transformation achieved 1.2%, and the maximum transformation efficiency reached 1.2%,1.8%, and 2.5%, respectively. Hygromycin B was applied to screen the transformed explants and seeds, and more than 500 transgenetic plants were obtained during the reseach. PCR and Southern blot were utilized to analyze the screened positive transgenic plants. The results showed that the exogenous genes have been integrated into the genome of rape.
引文
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