核桃油酸脱氢酶基因JrFAD2的克隆及表达分析

Cloning and expression analysis of oleate dehydrogenase gene JrFAD2 in walnut

【目的】测定并分析不同核桃品种核仁脂肪酸成分,克隆核桃FAD2基因,探究其在核桃不饱和脂肪酸合成中的作用。【方法】采用气相色谱-质谱联用的方法对20份核桃种质资源核仁的脂肪酸组分进行测定并对各组分间的相对含量进行计算及分析;基于核桃全基因组注释信息,通过实时荧光定量PCR技术对核桃油酸脱氢酶FAD2基因在高、低亚油酸品种间的表达模式进行分析;通过RT-PCR技术扩增JrFAD2基因的CDS序列,对其进行结构分析,并利用农杆菌介导的瞬时表达系统选用带有GFP标签的pBI121载体构建35S::JrFAD2重组表达载体,利用激光共聚焦显微镜观察核桃FAD2在本氏烟草叶表皮细胞中的表达。【结果】20份核桃资源核仁脂肪酸主要组分为亚油酸、油酸、硬脂酸和棕榈酸,其中不饱和脂肪酸高达90%。实时荧光定量PCR结果显示,高亚油酸品种及低亚油酸品种的油酸脱氢酶FAD2基因的表达模式具有差异。其中WALNUT_00011902-RA在3个高亚油酸品种的表达量均显著高于低亚油酸品种。选择WALNUT_00011902-RA作为目标基因进行克隆,得到长度为1 152 bp的JrFAD2基因的CDS序列,编码383个氨基酸,含有ω-6脂肪酸去饱和酶的功能结构域,属于Membrane-FADS-like超家族。系统进化分析发现,核桃FAD2与大豆、油桐FAD2的亲缘关系较近。亚细胞定位结果显示,核桃FAD2定位于细胞质。【结论】JrFAD2基因可能在核桃不饱和脂肪酸合成过程中对亚油酸的合成及油酸和亚油酸的比例具有促进和调控作用。

【Objective】Walnut(Juglans regia L.)is an economically important nut tree in China with a long history of cultivation and a wide range of distribution.It is rich in fatty acids of which about 90%are unsaturated fatty acids which are essential nutrients for the people.The degree of unsaturated fatty acids is one of the important indexes to determine the quality of the oil in walnut for the characteristics of lowering cholesterol in the body of human The research on the mechanism of unsaturated fatty acid formation in walnut has also become a hotspot.The composition of the fatty acids in walnut and key genes related to high unsaturated fatty acid synthesis are of great significance for studying the synthesis of unsaturated fatty acids and optimizing the distribution ratio of fatty acids.As a critical enzyme in the lipid biosynthetic pathway,the fatty acid desaturase 2(FAD2)takes part in the desaturated conversion of oleic acid(18∶1)to linoleic acid(18∶2).Currently,the FAD2 gene has been deeply studied in other plants,but it has not been reported in walnut.In this study,we cloned the walnut FAD2 gene to explore its role in the synthesis of unsaturated fatty acids in walnut in order to provide a basis for the analysis of the synthesis mechanism of high unsaturated fatty acid in walnut.【Methods】The components and relative contents of fatty acids of the 20 walnut cultivars were determined by gas chromatography-mass spectrometry(GC-MS).High linoleic acid varieties and low linoleic acid varieties were used as materials,and the expression patterns of the fatty acid desaturase 2(FAD2)in high and low linoleic acid varieties were analyzed by real-time fluorescence quantitative PCR(qRT-PCR)based on the whole genome annotation information of walnut.The CDS sequence of the JrFAD2 gene was amplified by RT-PCR and its structure was analyzed by NCBI.MEGA7 was employed to build the phylogenetic tree of the FAD2.The expression vector 35 S::JrFAD2 was constructed by p BI121 vector with GFP tag through Agrobacterium-mediated transient expression system,and the expression of the walnut FAD2 in the epidermal cells of tobacco leaves was observed by using laser con-focal microscope.【Results】The fatty acids of the 20 walnut varieties were mainly composed of linoleic acid,oleic acid,stearic acid and palmitic acid,of which unsaturated fatty acids were up to 90%.The content of linoleic acid was the highest,its average value was 58.63%,and its coefficient of variation was the smallest,10.4%.The variation range of oleic acid was 24.17%-48.27%,and the coefficient of variation was the largest,21.18%.There were some differences in the expression levels of the FAD2 in the high linoleic acid varieties and the low linoleic acid varieties,the expression level of the WALNUT00002632-RA in high linoleic acid varieties’Daihui’and’Zhengduan 15’was significantly higher than those in the three low linoleic acid varieties(p≤0.05),but there was no significant difference between’Zha 343’and low linoleic acid varieties.The difference of the WALNUT00027620-RA between’Daihui’and the low linoleic acid varieties was significant.The expression level of the WALNUT00011902-RA in the three high linoleic acid varieties was all significantly higher than those of the low linoleic acid varieties.The WALNUT00011902-RA was selected as the gene to clone,and the CDS sequence of the JrFAD2 gene with a length of 1152 bp was obtained,which codes for 383 amino acids,contains the functional domain of omega-6 fatty acid desaturase,and belongs to Membrane-FADS-like Super family.The Jr FAD2 in walnut had higher similarity with the FAD2 in the other plants,and it was speculated that they had similar structure and function.Phylogenetic analysis showed that the walnut JrFAD2 was closely related to the Vernicia fordii FAD2 and the soybean FAD2.The results of subcellular localization showed that the walnut FAD2 emitted fluorescence signal in the cytoplasm,which was presumed to be mainly located in the cytoplasm.【Conclusion】The fatty acids of walnut were mainly composed of linoleic acid,oleic acid,stearic acid and palmitic acid.The critical gene JrFAD2 in walnut may promote and regulate the synthesis of linoleic acid and the ratio of oleic acid and linoleic acid during the synthesis of the unsaturated fatty acids.