紫苏溶血磷脂酰转移酶基因PfLPAAT的克隆及功能研究  

Cloning and functional study of lysophosphatidic acid acyltransferase gene in Perilla frutescens

作　　者：徐华祥 鲁庚 郭曦 李圆圆 张涛[1] XU Hua-Xiang;LU Geng;GUO Xi;LI Yuan-Yuan;ZHANG Tao(College of Life Sciences,Chongqing Normal University,Chongqing 401331,China)

机构地区：[1]重庆师范大学生命科学学院,重庆401331

出　　处：《作物学报》2022年第10期2494-2504,共11页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31171588);重庆师范大学研究生科研创新项目(YKC21033)资助。

摘　　要：溶血磷脂酰转移酶(Lysophosphatidic acid acyltransferase,LPAAT)是植物甘油三酯生物合成的关键酶,通过研究紫苏LPAAT基因(PfLPAAT)在油脂积累过程中的功能,为揭示植物油脂积累的分子机制提供参考。本研究利用RT-PCR法获得PfLPAAT,采用生物信息学方法分析PfLPAAT的推定蛋白的基本理化性质、跨膜结构域和亚细胞定位等,并进行同源蛋白的系统发育分析。使用实时荧光定量PCR(qRT-PCR)方法对PfLPAAT在紫苏不同组织及种子不同发育时期的表达水平进行了分析。构建植物表达载体pCAMBIA1303-PfLPAAT,通过花序侵染法转化拟南芥,并对转基因拟南芥种子的含油量及脂肪酸组成进行分析。结果表明PfLPAAT序列长度为1149 bp,编码382个氨基酸,推定蛋白的理论等电点为9.60,分子质量为43.02 kD。生物信息学分析表明,PfLPAAT蛋白在内质网行使功能,属于PLN02380超家族。qRT-PCR表明,PfLPAAT在紫苏的各组织中均有表达,其中在叶片和开花后15 d的种子中达到最高水平。与野生型拟南芥相比,转基因拟南芥种子含油量增加2.04%~13.40%。脂肪酸组成分析结果表明,油酸、亚油酸、亚麻酸和花生一烯酸的含量显著增加,棕榈酸、硬脂酸、花生酸、花生二烯酸和芥酸含量降低。紫苏PfLPAAT的过表达不仅可以提高拟南芥种子含油量,还可以不同程度地提高油脂中不饱和脂肪酸的含量。本研究结果为油料作物紫苏的种质资源挖掘和创新利用提供了新颖候选基因。Lysophosphatidic acid acyltransferase(LPAAT)is a key enzyme in plant triacylglycerol biosynthesis.Investigating the function of Perilla frutescens LPAAT gene(PfLPAAT)in oil biosynthesis can help to reveal the molecular mechanism of plant oil accumulation.In this study,RT-PCR was used to obtain PfLPAAT,and the basic physical and chemical properties,transmembrane domain,and subcellular localization of the putative protein of PfLPAAT was analyzed by bioinformatics methods,and phylogenetic analysis of homologous proteins was conducted.The qRT-PCR was used to analyze the relative expression level of PfLPAAT in different tissues and developmental stages of seeds in Perilla.The expression vector pCAMBIA1303-PfLPAAT was constructed and transformed into Arabidopsis thaliana by floral dip method,and the oil content and fatty acid composition of transgenic Arabidopsis seeds were analyzed.The results showed that the PfLPAAT sequence was 1149 bp in length and encoded 382 amino acids.The theoretical isoelectric point of the putative protein was 9.60,and the molecular mass was 43.02 kD.Bioinformatics indicated that PfLPAAT functioned in endoplasmic reticulum and belonged to the PLN02380 superfamily.The qRT-PCR revealed that PfLPAAT expressed in all tissues of Perilla,and the highest level was observed in leaves and seeds of 15 days after flowering.Compared with wild-type Arabidopsis,the overexpression of PfLPAAT in Arabidopsis improved seed oil content significantly,with a range of 2.04%to 13.40%.Fatty acid composition analysis demonstrated that the content of oleic acid,linoleic acid,linolenic acid,and arachidic acid increased significantly,while the content of palmitic acid,stearic acid,arachidic acid,arachidonic acid,and docosaenoic acid decreased.In short,the overexpression of the PfLPAAT in Arabidopsis could not only increase the oil content of transgenic Arabidopsis seeds,but also increase the content of unsaturated fatty acids with varying degrees.The results provide novel candidate genes for the mining and innovative ut

关 键 词：紫苏 溶血磷脂酰转移酶 含油量 不饱和脂肪酸 

分 类 号：S565.9[农业科学—作物学]