外源SAD基因在蛋白核小球藻中转化和表达及对脂肪酸组成的影响

发布时间：2018-10-16 22:12

【摘要】：为了探究微藻硬脂酰载体蛋白去饱和酶(SAD)基因在蛋白核小球藻不饱和脂肪酸合成调控中的作用,提高藻细胞中不饱和脂肪酸的含量,本文采用基因枪法将若夫小球藻的SAD基因导入到蛋白核小球藻的叶绿体中,利用链霉素抗性筛选,经同质化培养,成功获得了转化藻。以野生蛋白核小球藻为对照,对转化藻进行了PCR验证和荧光定量分析,并且对脂肪酸组成和SAD基因在蛋白核小球藻不饱和脂肪酸合成过程中的调控作用进行了分析。本文系首次用基因枪法对蛋白核小球藻成功进行了叶绿体转化,并探究了SAD基因对微藻不饱和脂肪酸合成的影响,加深了对微藻油脂合成调控机理的认识。本文的主要研究内容及结果如下:(1)确定了蛋白核小球藻基因工程筛选标记及对同源片段chlL基因进行了验证。通过研究蛋白核小球藻对6种抗生素的敏感性,结果表明链霉素浓度为200μg/mL时能在8天左右观察到蛋白核小球藻细胞全部死亡,确定aadA基因可作为蛋白核小球藻基因工程筛选标记;通过PCR扩增验证了chlL基因作为蛋白核小球藻叶绿体转化中同源片段的可行性。(2)成功构建了以chlL基因序列为同源片段、分别以莱茵衣藻叶绿体中的atpA和rbcL作为SAD基因的启动子和终止子、aadA基因作为筛选标记的叶绿体表达载体。(3)利用基因枪法将携带有SAD基因的重组质粒导入到蛋白核小球藻的叶绿体中,利用链霉素抗性对转化后的蛋白核小球藻进行了筛选,在抗性压力下完成了同质化培养。(4)对转化藻的外源基因进行了普通PCR检测,对aadA基因进行了RT-PCR检测及对SAD基因荧光定量PCR检测,结果表明aadA基因、SAD基因均成功整合到蛋白核小球藻的叶绿体基因组中并成功转录,而且SAD基因实现了过量表达。(5)藻的油脂脂肪酸组成分析结果显示,C17:1大幅上升,提示若夫小球藻SAD基因编码的酶可能具有对脂肪酸C17:0去饱和的能力;结果还显示总不饱和脂肪酸含量有所增加,提示外源SAD基因表达促进了不饱和脂肪酸合成。
[Abstract]:In order to investigate the role of (SAD) gene in the synthesis and regulation of unsaturated fatty acids in Chlorella vulgaris, the content of unsaturated fatty acids in algae cells was increased. In this paper, the SAD gene of Chlorella luteus was introduced into chloroplast of Chlorella Proteus by gene gun method. The transformed algae was successfully obtained by streptomycin resistance screening and homogenization culture. The PCR verification and fluorescence quantitative analysis of transforming Chlorella sp. Were carried out with the wild protein Chlorella nucleocephala as control, and the fatty acid composition and the regulation of SAD gene in the synthesis of unsaturated fatty acids of Chlorella protein were analyzed. In this paper, the chloroplast transformation of Chlorella Proteinuclear Chlorella was successfully carried out by means of gene gun method, and the effect of SAD gene on the synthesis of unsaturated fatty acids in microalgae was studied, which deepened the understanding of the regulation mechanism of microalgae oil synthesis. The main contents and results are as follows: (1) the screening markers of Chlorella protein nuclear Chlorella were identified and the homologous chlL gene was verified. The sensitivity of Chlorella Proteinuclear Chlorella to 6 antibiotics was studied. The results showed that when the concentration of streptomycin was 200 渭 g/mL, all the cells could be observed to die at about 8 days. It was confirmed that aadA gene could be used as a screening marker for gene engineering of Chlorella putamen, and the feasibility of using chlL gene as a homologous fragment in chloroplast transformation of Chlorella putamen was verified by PCR amplification. (2) the sequence of chlL gene was successfully constructed as homologous fragment. AtpA and rbcL in chloroplast of Chlamydomonas rhinella were used as promoter and Terminator of SAD gene respectively. AadA gene was used as the chloroplast expression vector of screening marker. (3) Recombinant plasmid carrying SAD gene was introduced into protein nucleus by gene gun method. In the chloroplasts of Chlorella vulgaris, Streptomycin resistance was used to screen the transformed Chlorella Proteinuclear Chlorella, and homogenized culture was completed under the pressure of resistance. (4) Common PCR was used to detect the exogenous genes of the transformed algae. AadA gene was detected by RT-PCR and SAD gene was detected by fluorescence quantitative PCR. The results showed that aadA gene and SAD gene were successfully integrated into chloroplast genome of Chlorella putamen and transcribed successfully. Moreover, the SAD gene was overexpressed. (5) the fatty acid composition analysis of the oil and fat of the algae showed that C17: 1 increased significantly, suggesting that the enzyme encoded by the SAD gene of Chlorella luteum may have the ability to desaturate the fatty acid C17: 0; The results also showed that the content of total unsaturated fatty acids increased, suggesting that exogenous SAD gene expression promoted the synthesis of unsaturated fatty acids.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS201.2

【相似文献】