茶树脂肪酸去饱和酶家族基因的克隆与表达分析  

Cloning and Expression Analysis of Fatty Acid Desaturase Family Genes in Camellia sinensis

作　　者：王鹏杰[1] 曹红利[1] 陈丹[1] 陈笛 陈桂信[1] 杨江帆[1] 叶乃兴[1] WANG Pengjie;CAO Hongli;CHEN Dan;CHEN Di;CHEN Guixin;YNAG Jiangfan;YE Naixing(College of Horticulture,Fujian Agriculture and Forestry University,Key Laboratory of Tea Science at Universities in Fujian,Fuzhou 350002,China)

机构地区：[1]福建农林大学园艺学院茶学福建省高校重点实验室,福州350002

出　　处：《园艺学报》2020年第6期1141-1152,共12页Acta Horticulturae Sinica

基　　金：福建省“2011协同创新中心”专项(闽教科[2015]75号);福建农林大学园艺学院优秀博士学位论文资助基金项目(2018B02)。

摘　　要：以茶树品种‘铁观音’为试材,利用RT-PCR技术克隆得到5个脂肪酸去饱和酶(fatty acid desaturase,FAD)家族基因的cDNA序列,分别命名为Cs FAD2-2、CsFAD3、Δ7-CsFAD、Δ8-CsFAD和CsFAB2,其编码序列长度1 137~1 320 bp。系统发育与基序分析结果表明,茶树FAD成员来自4个亚家族,即ω3/ω6-FAD亚家族、Δ7/Δ9-FAD亚家族、FrontendFAD亚家族和FAB亚家族。同一亚家族成员的保守基序一致,而不同亚家族的保守基序存在较大不同。荧光定量PCR结果表明,5个CsFAD家族基因都显著响应低温诱导,Δ8-CsFAD对低温胁迫响应最强烈,表达水平上调千倍;外源ABA处理后,除CsFAD3外,其他4个CsFAD基因的表达均上调2倍以上;干旱胁迫下,5个Cs FAD中CsFAD2-2、Δ7-CsFAD和Δ8-CsFAD表达水平都上调1.5倍以上,其中Δ8-CsFAD的表达上调最大,达5.5倍,而CsFAD3和CsFAB2的表达受到干旱处理抑制显著下调。克隆并分析Δ8-CsFAD启动子,发现存在多种与逆境胁迫响应相关的顺式元件。烟草亚细胞定位观察表明Δ8-CsFAD定位于细胞膜。根据上述结果推测5个CsFAD可能参与茶树抗逆响应,尤其是Δ8-CsFAD,可能通过调控茶树细胞膜不饱和脂肪酸的合成进而影响茶树的抗逆性。In this experiment,the cDNA sequences of five fatty acid desaturase(FAD)family genes were cloned from the tea plant cultivar‘Tieguanyin’by RT-PCR,and named as CsFAD2-2,CsFAD3,Δ7-CsFAD,Δ8-CsFAD and CsFAB2,respectively.The length of coding sequences were ranged from 1137 to 1320 bp.Phylogenetic and motif analysis showed that CsFAD members could be divided into four subfamilies,including theω3/ω6-FAD subfamily,theΔ7/Δ9-FAD subfamily,the Front end FAD subfamily,and the FAB subfamily.The conserved motifs of FAD members were identical in the same subfamily,while they were quite different in different subfamilies.The results of quantitative real-time PCR indicated that all of CsFAD genes were significantly induced in response to low temperature treatment,especially the expression level ofΔ8-CsFAD could be strongly up-regulated to thousand folds.After exogenous ABA treatment,the expression level of CsFAD genes was up-regulated to more than two folds except for CsFAD3.Under drought stress,the expression levels of CsFAD2-2,Δ7-CsFAD andΔ8-CsFAD were up-regulated to more than 1.5 folds,and the expression ofΔ8-CsFAD was induced to 5.5 folds,whereas the expression of CsFAD3 and CsFAB2 was significantly repressed by drought treatment.Moreover,we isolated theΔ8-CsFAD promoter sequence and analysis results showed that it contains multiple stress responsive cis-elements.Subcellular localization analysis in tobacco indicated thatΔ8-CsFAD was localized to the cell membrane.Our results showed that CsFAD genes might be involved in the tea plants stress response,especially ofΔ8-CsFAD,reflecting that they could regulate the synthesis of unsaturated fatty acids in the cell membrane to participate in the stress resistance of tea plants.

关 键 词：茶树 脂肪酸去饱和酶 逆境胁迫 启动子 亚细胞定位 

分 类 号：S571.1[农业科学—茶叶生产加工]