大白菜—结球甘蓝易位系外源NAC086基因的鉴定与表达分析  

Identification and Expression Analysis of Foreign NAC086 Gene in Chinese Cabbage(Brassica campestris ssp.pekinensis)-Cabbage(B.oleracea var.capitata)Translocation Line

作　　者：尚金梦 王汝颖 轩淑欣[1] 江丹 费得清 王彦华[1] 冯大领[2] 申书兴[1] SHANG Jin-Meng;WANG Ru-Ying;XUAN Shu-Xin;JIANG Dan;FEI De-Qing;WANG Yan-Hua;FENG Da-Ling;SHEN Shu-Xing(College of Horticulture,Hebei Agricultural University/Key Laboratory for Vegetable Germplasm Enhancement and Utilization of Hebei/Collaborative Innovation Center of Vegetable Industry in Hebei,Baoding 071001,China;College of Life Science,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]河北农业大学园艺学院/河北省蔬菜种质创新与利用重点实验室/河北省蔬菜产业协同创新中心,保定071001 [2]河北农业大学生命科学学院,保定071001

出　　处：《农业生物技术学报》2021年第9期1678-1687,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31930098);河北省自然科学基金(C2018204135,C2020204111);河北省创新能力提升计划(20592901D);河北省“巨人计划”创新团队项目。

摘　　要：NAC(NAM/ATAF/CUC)转录因子是植物中特有且最大的转录因子家族之一,在抵抗逆境胁迫反应、调控植物生长发育、参与器官模式建成以及生长素传导、叶片凋亡等生理过程中具有重要作用。为了鉴定大白菜(Brassica campestris ssp.pekinensis)—结球甘蓝(B.oleracea var.capitata)双单倍体(double haploid,DH)易位系AT1-18中的外源甘蓝NAC086基因,同时了解该基因的功能,以AT1-18及其亲本为研究对象,通过特异性引物设计、PCR扩增片段序列分析、逆转录PCR(reverse transcription-PCR,RT-PCR)、实时定量PCR(real-time quantitative PCR,qRT-PCR)等方法,对大白菜和结球甘蓝NAC086基因进行了鉴定,同时对NAC086基因的组织表达特异性、响应外源生长素或盐水处理后的表达模式进行了研究。结果表明,基于大白菜Bra006392设计的特异性引物仅在大白菜中具有扩增产物,基于结球甘蓝基因Bol034440设计的特异性引物同时在结球甘蓝和易位系中具有扩增产物,且在易位系中扩增产物的序列和Bol034440基因CDS序列的一致性高达99%,证明了在易位系中结球甘蓝的Bol034440基因代换了大白菜的Bra006392基因。RT-PCR分析表明,Bol034440在大白菜不同组织中几乎无表达,在易位系不同组织中具有较高的表达量(依次是根>叶>蕾>花>茎,其中根与花,蕾,茎差异显著)。Bra006392在易位系中除在根中有少量表达外,其他组织几乎无表达;在大白菜叶中表达量显著高于其他组织。Bra008567在大白菜和易位系不同组织中均具有一定表达量,其中在大白菜的根中表达量最高,显著高于其他组织;在易位系的叶中表达量最高,除与根差异不显著,与其他组织均达到显著差异。qRT-PCR分析表明Bol034440和Bra008567基因负向响应外源生长素处理作用,正向响应盐水处理的作用,响应时间均在处理后2 h,其中Bol034440在响应过程中占主导地位。研究结果为开展NAC086基因的功能验证和研究�The NAC(NAM,ATAF,and CUC)transcription factors(TFs),which constitute one of the largest plant-specific transcription factor family,play an important role in resisting stress response,regulating plant growth and development,participating in organ model building,auxin transduction and leaf apoptosis,etc.In order to identify the foreign NAC086 gene of Chinese cabbage(Brassica campestris ssp.pekinensis)-cabbage(B.oleracea var.capitata)double haploid translocation line AT1-18,the cloning and expression of NAC086 genes were carried out from the translocation line AT1-18 and its parents by PCR,RT-PCR(reverse transcriptionPCR)and qRT-PCR(real-time quantitative PCR)methods.The results showed that the gene Bol034440 was amplified with the specific primers in cabbage and translocation line,however,the gene Bra006392 was only obtained in Chinese cabbage.Sequence analysis showed that the identity was as high as 99%between the sequence of PCR product from the translocation line using Bol40-1 primers and CDS sequence of gene Bol034440.It was proved that Bra006392 gene of Chinese cabbage was replaced by Bol034440 gene of cabbage in the translocation line.RT-PCR analysis showed that there was almost no Bol034440 expression in the different tissues of Chinese cabbage,but the high relative expression was in the different tissues of the translocation line,followed by roots>leafs>buds>flowers>stems,with significant difference between roots and flowers,buds,stems.Bra006392 had almost no expression in the tissues(except roots)of the translocation line,but had the highest expression in the leaves of Chinese cabbage,which was significantly higher than that in other tissues.Bra008567 had high expression level in different tissues of Chinese cabbage and translocation lines,and the highest expression was in the root of Chinese cabbage,which was significantly higher than other tissues.Whereas,the highest expression was in the leaves of the translocation line,which was also significantly higher than other tissues except roots.There were signi

关 键 词：大白菜 易位系 NAC086 外源基因鉴定 逆转录PCR(RT-PCR) 基因表达 

分 类 号：S643.1[农业科学—蔬菜学]