甘蓝型油菜长链烷烃合成相关基因的克隆及其与BnCER1-2的互作  

Cloning of genes involved in cuticular very-long-chain alkane synthesis and its interaction with BnCER1-2 in Brassica napus

作　　者：柏成成 姚小尧 王雨璐 王赛玉 李金莹 蒋有为 靳舒荣 陈春杰 刘渔 魏星玥 徐新福[1] 李加纳[1] 倪郁[1] BAI Cheng-Cheng;YAO Xiao-Yao;WANG Yu-Lu;WANG Sai-Yu;LI Jin-Ying;JIANG You-Wei;JIN Shu-Rong;CHEN Chun-Jie;LIU Yu;WEI Xing-Yue;XU Xin-Fu;LI Jia-Na;NI Yu(College of Agronomy and Biotechnology/Academy of Agricultural Sciences,Southwest University,Chongqing 400716,China)

机构地区：[1]西南大学农学与生物科技学院/西南大学农业科学研究院,重庆400716

出　　处：《作物学报》2023年第4期1016-1027,共12页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(32171938,31771694);重庆市自然科学基金项目(cstc2021jcyj-msxmX0332);财政部和农村农业部国家现代农业产业技术体系建设专项(CARS-12)资助。

摘　　要：长链烷烃是甘蓝型油菜角质层蜡质的优势组分,在阻止植株的非气孔性水分散失中起主要作用。BnCER1-2催化甘蓝型油菜长链烷烃的生物合成,但BnCER1-2是否通过与其他蛋白互作调控长链烷烃合成还不清楚。前期通过甘蓝型油菜蜡质差异材料转录组筛选获得4个长链烷烃合成相关基因BnCER3.a10、BnCER3.c02、BnCYTB5B.c09、BnCER1-L2.a05。本研究克隆了这4个基因的编码序列,序列分析表明BnCER3.a10/c02和BnCER1-L2.a05前体蛋白具有典型的脂肪酸羟化酶与WAX2C末端结构域,而BnCYTB5B.c09具有Cyt_B5蛋白家族保守结构域。亚细胞定位结果表明,BnCER3.a10/c02、BnCYTB5B.c09和BnCER1-L2.a05均定位于细胞内质网,与BnCER1-2共定位。双分子荧光互补(bimolecular fluorescent complementation,BiFC)与萤火素酶互补试验(luciferase complementation assay,LCA)检测结果表明,BnCER3.a10、BnCYTB5B.c09、BnCER1-L2.a05与BnCER1-2蛋白存在相互作用,而BnCER3.c02与BnCER1-2蛋白不互作。实时荧光定量PCR结果显示,与BnCER1-2的表达模式一致,BnCER3.a10和BnCYTB5B.c09主要在甘蓝型油菜茎/叶中表达,并受干旱胁迫诱导显著上调。BnCER3.a10在NaCl与低温胁迫下表达量显著减少,其中BnCER3.a10受MeJA、ACC诱导显著下调,BnCYTB5B.c09表达受ABA诱导上调。BnCER1-L2.a05在花中的表达量最高,在茎和叶片中的表达量最低,在干旱、低温及NaCl胁迫下转录水平均显著下降,其中SA诱导BnCER1-L2.a05表达上调,而MeJA诱导其表达下调。蜡质差异材料荧光定量PCR结果证实,BnCER3.a10与BnCYTB5B.c09在高蜡(烷)油菜中的表达量显著高于低蜡(烷)油菜,而BnCER1-L2.a05则呈相反变化。综合分析认为,BnCER3.a10和BnCYTB5B.c09可能通过与BnCER1-2互作而促进甘蓝型油菜长链烷烃的生物合成,BnCER1-L2.a05可能通过与BnCER1-2互作负调控长链烷烃的合成。Very-long-chain(VLC)alkanes are the main components of cuticular wax in Brassica napus,which play a key role in preventing non-stomatal water loss.BnCER1-2 is the core enzyme that catalyzes the synthesis of VLC alkanes in B.napus.However,it is unclear whether BnCER1-2 protein regulates VLC alkane synthesis by interacting with other proteins.Four genes potentially involved in VLC alkane synthesis,BnCER3.a10,BnCER3.c02,BnCYTB5B.c09,and BnCER1-L2.a05,were screened previously by the transcriptome in B.napus with differential wax load.In this study,their coding sequences were cloned from B.napus.A typical fatty acid hydroxylase domain and a wax2 C-terminal domain were detected in the predicted BnCER3.a10/c02and BnCER1-L2.a05 proteins,while cyt_b5 protein family conserved domain was in the predicted BnCYTB5B.c09.Subcellular localization showed that BnCER3.a10/c02,BnCYTB5B.c09,and BnCER1-L2.a05 were all located in the endoplasmic reticulum,which were co-located with BnCER1-2.Bimolecular fluorescence complementary(BiFC)and luciferase complementation assay(LCA)revealed that BnCER3.a10,BnCYTB5B.c09,and BnCER1-L2.a05 interacted with BnCER1-2 protein,while BnCER3.c02did not interact with BnCER1-2.The RT-qPCR indicated that BnCER3.a10 and BnCYTB5B.c09 were mainly expressed in the stems or leaves of B.napus,and the relative expression was significantly up-regulated under drought stress,which were consistent with the expression pattern of BnCER1-2.The relative expression levels of BnCER3.a10 decreased significantly under NaCl and low temperature stresses.Further,the relative expression level of BnCER3.a10 was significantly down-regulated by MeJA and ACC,while the relative expression level of Bn CYTB5B.c09 was up-regulated by ABA.The highest relative expression of BnCER1-L2.a05 was in flowers and the lowest in stems and leaves,and its expression was significantly down-regulated under drought,cold,and NaCl stresses.Further,the relative expression level of BnCER1-L2.a05 was up-regulated by SA,while down-regulated by MeJA.The relat

关 键 词：甘蓝型油菜 角质层蜡质 蛋白互作 长链烷烃 

分 类 号：S565.4[农业科学—作物学]