紫苏二酰基甘油酰基转移酶2基因克隆与功能研究

Cloning and function analysis of a type 2 diacylglycerol acyltransferase(DGAT2)from Perilla frutescens

二酰基甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)是植物合成三酰甘油(TAG)最后一步的关键酶,其中DGAT2在某些植物的种子油中能选择性积累更多不饱和脂肪酸。本文成功克隆了紫苏二酰基甘油酰基转移酶2基因(PfDGAT2),并进行生物信息学分析。PfDGAT2实时荧光定量结果表明,不同器官中PfDGAT2基因均有表达,10 d种子的表达量最高,在根中的表达量次之,在种子发育中后期,PfDGAT2表达量逐渐降低。与野生型拟南芥相比,过表达PfDGAT2拟南芥种子含油率提高了21.68%~77.89%,其中种子含油率增加最多的4个株系,其亚麻酸(C18:3)增加4.57%,花生一烯酸(C20:1)增加7.44%,花生二烯酸(C20:2)增加5.4%,二十二一烯酸(C22:1)增加10.37%,而棕榈酸(C16:0)、硬脂酸(C18:0)和亚油酸(C18:2)含量分别降低了3.47%、6.64%和4.83%,油酸(C18:1)和花生酸(C20:0)分别只降低了0.18%和1.91%。本研究结果表明,紫苏PfDGAT2基因不仅能提高种子含油率,还能促进亚麻酸、花生一烯酸等不饱和脂肪酸的积累,这为研究植物不饱和脂肪酸的合成积累提供了参考及理论依据。

Diacylglycerol acyltransferase(DGAT)is a key enzyme in the final step of triacylglycerol(TAG)synthesis in plant.In seed oil of certain plants,DGAT2 can selectively accumulate more unsaturated fatty acids.In this paper,we successful cloned Pf DGAT2 from Perilla frutescens and performed bioinformatics analysis.Real-time fluorescence quantitative analysis showed that Pf DGAT2 was expressed in different organs,with the highest in seeds at 10 d after anthesis,the medium in roots,and gradual decrement in the middle and late stages of seed.Compared with the wild-type,the oil content of seeds in Pf DGAT2 transgenic Arabidopsis was increased by 21.68%–77.89%.The fatty acid components of the four strains with the largest increase in seed oil content were analyzed.Compared with the control,linolenic acid(C18:3),arachidonic acid(C20:1),arachidonic acid(C20:2),docosaenoic acid(C22:1)increased significantly by 4.57%,7.44%,5.40%,and 0.37%,respectively.Palmitic acid(C16:0),stearic acid(C18:0),and linoleic acid(C18:2)were obviously reduced by 3.47%,6.64%,and 4.83%,respectively.Oleic acid(C18:1)only decreased by 0.18%and arachidic acid(C20:0)by 1.91%.In conclusion that Pf DGAT2 gene can not only increase the oil content,but promote the accumulation of unsaturated fatty acids such as linolenic acid and arachidonic acid,which provides a reference and theoretical basis for studying the synthesis and accumulation of unsaturated fatty acid in plants.