芸薹属植物MYBL2基因的克隆及其在A、B、C基因组中的PCR鉴别  

Cloning of MYBL2 Gene from Brassica and Its PCR Identification in Genomes A,B and C

作　　者：邹婷[1] 刘丽莉[1] 向建华[1] 周定港 吴金锋 李莓[2] 李宝 张大为[1] 严明理[1,2] ZOU Ting;LIU LiLi;XIANG JianHua;ZHOU DingGang;WU JinFeng;LI Mei;LI Bao;ZHANG DaWei;YAN MingLi(College of Life Science and Health,Hunan University of Science and Technology/Hunan Provincial Key Laboratory of Genetic Improvement and Comprehensive Utilization of Cash Crops,Xiangtan 410201,Hunan;Hunan Crop Research Institute,Changsha 410125)

机构地区：[1]湖南科技大学生命科学与健康学院/经济作物遗传改良与综合利用湖南省重点实验室,湖南湘潭410201 [2]湖南省作物研究所,长沙410125

出　　处：《中国农业科学》2023年第3期416-429,I0001-I0020,共34页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31971980,U19A2029)。

摘　　要：【目的】MYBL2负调控拟南芥花青素和原花青素的生物合成。从芸薹属6个物种的不同叶色材料中克隆MYBL2基因,分析其序列和表达模式,探究其在芸薹属植物花青素生物合成途径中的功能,为油菜的品质、抗逆性、观赏性等性状改良提供参考。【方法】以芸薹属6个物种19份供试材料的总DNA为模板,同源克隆MYBL2基因,并进行多序列比对和进化树分析;对白菜、甘蓝型油菜、芥菜型油菜以及埃塞俄比亚芥的紫叶材料进行遮光处理,结合转录组和qRT-PCR分析MYBL2基因表达水平;对甘蓝、甘蓝型油菜、芥菜型油菜以及埃塞俄比亚芥紫、绿叶材料进行qRT-PCR分析MYBL2基因表达水平;根据克隆MYBL2-1和MYBL2-2序列的核苷酸变异位点设计特异性引物,开发能够区分MYBL2基因组来源的PCR标记。【结果】克隆获得MYBL2-1和MYBL2-2各9个同源基因共56个拷贝。其中,BcaMYBL2-1为首次获得,BcaMYBL2-1编码区序列全长为867 bp,包含2个内含子,分别为168和102 bp,编码198个氨基酸,分子量为22.69 kD,等电点(pI)为8.72。序列比对和进化分析表明,BcaMYBL2-1来源于B基因组。芸薹属6个物种MYBL2-1和MYBL2-2同源基因中,仅BraA07.MYBL2-1、BolC06.MYBL2-1和BcaMYBL2-1在不同叶色材料中存在序列差异。经遮光处理后,紫叶材料叶色变浅,在白菜紫宝5号中,BraA07.MYBL2-1和BraA02.MYBL2-2表达量分别为未遮光部分的0.7和0.4倍;在紫叶白花甘蓝型油菜中,BnaA07.MYBL2-1、BnaC06.MYBL2-1、BnaA02.MYBL2-2和BnaC02.MYBL2-2表达量分别为未遮光部分的0.4、0.5、0.4和0.4倍;在紫叶芥中,BjuA07.MYBL2-1、BjuB03.MYBL2-1、BjuA02.MYBL2-2和BjuB05.MYBL2-2表达量分别为未遮光部分的0.4、0.3、0.4和0.2倍;在紫秆埃芥中,BcaMYBL2-1、BcaB03.MYBL2-1和BcaC03.MYBL2-2表达量分别为未遮光部分的0.3、0.4和0.5倍,而BcaB05.MYBL2-2表达量为未遮光部分的2.4倍。对比芸薹属不同叶色材料MYBL2基因表达情况,结果表明,除了羽衣甘�【Objective】In Arabidopsis,MYBL2 negatively regulates the biosynthesis of anthocyanins and proanthocyanidins.The MYBL2 genes from six Brassica species with different leaf colors were cloned.By analyzing the sequence and expression pattern of MYBL2,the function of MYBL2 in the biosynthesis of anthocyanins in Brassica species was explored.【Method】The sequences of MYBL2 from the six Brassica species with different leaf colors were obtained using homology-based cloning method and multi sequence alignment and phylogenetic tree analysis were performed.The purple leaf materials of B.rapa,B.napus,B.juncea and B.carinata were treated with shading,and the expression level of MYBL2 gene was analyzed by transcriptome sequencing and qRT-PCR.The qRT-PCR in B.oleracea,B.napus,B.juncea and B.carinata with purple and green leaves were also performed to evaluate the expression level of MYBL2.Based on the nucleotide variation sites of the cloned MYBL2-1 and MYBL2-2 sequences,PCR markers which could distinguish the genomic origin of alleles were developed.【Result】A total of 56 copies of 9 homologs of MYBL2-1 and MYBL2-2 were cloned from 19 samples of six species of Brassica.The BcaMYBL2-1 gene was obtained for the first time.The total length of BcaMYBL2-1 sequence was 867 bp,including two introns of 168 bp and 102 bp respectively,encoding 198 amino acids,with a molecular weight of 22.69 kD and an isoelectric point(pI)of 8.72.Sequence alignment and evolutionary analysis showed that BcaMYBL2-1 was derived from B genome.Among the MYBL2-1 and MYBL2-2 copies of six species in Brassica,only BraA07.MYBL2-1,BolC06.MYBL2-1 and BcaMYBL2-1 exhibited sequence differences in different leaf color materials.After shading treatment,the leaf color of purple leaf material becomes lighter than that of the unshaded part.In Chinese cabbage Zibao 5,the expression of BraA07.MYBL2-1 and BraA02.MYBL2-2 in the shaded part were 0.7 and 0.4 times of that in the unshaded part,respectively.In Brassica napus with purple leaves and white flowers,the exp

关 键 词：芸薹属植物 MYBL2基因 同源克隆 基因表达 基因组PCR鉴别 

分 类 号：Q943.2[生物学—植物学]