紫苏PfSAD3和PfSAD4基因克隆及功能分析  

Gene Cloning and Functional Analysis of PfSAD3 and PfSAD4 in Perilla frutescens

作　　者：尹苗 王尧 王壮琳 胡婷 陈树溦 周雅莉[1,2] 邢志 王计平 李润植[1,2] YIN Miao;WANG Yao;WANG Zhuang-Lin;HU Ting;CHEN Shu-Wei;ZHOU Ya-Li;XING Zhi;WANG Ji-Ping;LI Run-Zhi(College of Agronomy,Shanxi Agricultural University,Taigu 030801,China;Shanxi Engineering Research Center for Genetics and Metabolism of Specific Crops,Taigu 030801,China)

机构地区：[1]山西农业大学农学院,太谷030801 [2]山西省特用作物遗传和代谢工程研究中心,太谷030801

出　　处：《农业生物技术学报》2024年第11期2526-2539,共14页Journal of Agricultural Biotechnology

基　　金：山西省基础研究计划(自由探索类)(20210302123418);山西农业大学农学院育种工程重点培育项目(YZ2021-08);山西农业大学校科技创新提升工程(CXGC2023048)。

摘　　要：9-硬脂酰-ACP脱氢酶(Δ9-stearoyl-ACP dehydrogenase,SAD)是植物体内不饱和脂肪酸合成积累的关键酶。为探究SAD在紫苏(Perilla frutescens)脂肪酸生物合成中的作用,本研究克隆了PfSAD3和PfSAD4基因,并对其进行生物信息学分析及功能验证。以'晋紫苏1号'种子为实验材料,分离得到PfSAD3(GENE_023515)和PfSAD4(GENE_042129)基因的全长序列,其ORF长度分别为1173和1116 bp;使用MEGA 11.0对PfSAD3和PfSAD4蛋白序列、拟南芥(Arabidopsis thaliana)及其他物种SAD序列构建进化树,发现PfSAD3与白头翁(Leucas cephalotes)LcSAD亲缘关系较近,PfSAD4与蓖麻(Ricinus communis)RcSAD亲缘关系较近。进一步构建酵母(Saccharomyces cerevisiae)表达载体pYES2.0-PfSAD3和pYES2.0-PfSAD4,分别转入野生型酵母INVSc1和缺陷型酵母BY4389中,结果表明,PfSAD3和PfSAD4过表达使酵母总脂肪酸及不饱和脂肪酸含量显著升高(P<0.05),并使缺陷型酵母恢复合成棕榈油酸(C16:1)和油酸(C18:1)的能力。通过添加外源脂肪酸分析PfSAD3和PfSAD4蛋白的底物选择特异性,结果表明,与PfSAD3相比,PfSAD4对C18:0更具偏好性,酶活性更强。将PfSAD3和PfSAD4基因在野生烟草(Nicotiana tabacum)中过表达来验证其功能,结果表明,转基因烟草叶片总脂肪酸及不饱和脂肪酸含量显著升高(P<0.05),与酵母实验结果基本一致。本研究为深入解析紫苏等油料作物不饱和脂肪酸合成的分子调控机制及利用基因工程手段培育富含油脂的油料作物新品系提供科学基础和分子靶标。Δ9-stearoyl-ACP dehydrogenase(SAD)is a key enzyme involved in the synthesis and accumulation of unsaturated fatty acids in plants.In this study,to explore the role of the SAD enzyme in the fatty acid biosynthesis of Perilla frutescens,PfSAD3(GENE_023515)and PfSAD4(GENE_042129)genes were cloned and bioinformatic analysis and functional verification on them were conducted.Using the seeds of'Jinzisu 1'as the experiment material,two full-length sequences of PfSAD3 and PfSAD4 were obtained,with the ORF lengths of 1173 and 1116 bp,respectively.MEGA 11.0 was used to construct evolutionary trees for the protein sequences of PfSAD3 and PfSAD4,as well as the SAD sequences of Arabidopsis thaliana and other species,it was found that PfSAD3 was closely related to LcSAD of Leucas cephalotes,and PfSAD4 was closely related to RcSAD of Ricinus communis.After constructing the yeast(Saccharomyces cerevisiae)expression vectors pYES2.0-PfSAD3 and pYES2.0-PfSAD4,PfSAD3 and PfSAD4 were transformed into the wild-type yeast strain INVSc1 and the defective yeast strain BY4389,respectively.The results showed that overexpression of PfSAD3 and PfSAD4 significantly increased the total fatty acid content and unsaturated fatty acid content in yeast(P<0.05),and restored the ability of the defective yeast to synthesize palmitoleic acid(C16⁚1)and oleic acid(C18⁚1).The substrate specificity of the PfSAD3 and PfSAD4 proteins was analyzed by adding exogenous fatty acids.The result showed that compared to PfSAD3,PfSAD4 had a more substantial substrate preference ratio for C18⁚0,and exhibited stronger enzyme activity.The functions of PfSAD3 and PfSAD4 were verified by overexpressing them in wild tobacco(Nicotiana tabacum).The result showed that the total fatty acid content and unsaturated fatty acid content in transgenic tobacco leaves were significantly increased(P<0.05),which was consistent with the results of yeast experiments.This study provides scientific bases and molecular targets for further elucidating the molecular regulatory mechanism

关 键 词：紫苏 Δ9-硬脂酰-ACP脱氢酶(SAD) 基因功能 遗传转化 不饱和脂肪酸 

分 类 号：S565.8[农业科学—作物学]