不结球白菜分蘖调控基因BcMAX1的同源克隆及功能分析

Homologous cloning and functional analysis of the tillering regulation gene BcMAX1 in non-heading Chinese cabbage

[目的]本文旨在克隆不结球白菜BcMAX1基因及其启动子,对其进行表达模式分析,并对其在分蘖调控中的功能进行探索。[方法]对BcMAX1基因及其启动子进行克隆,并利用生物信息学方法对该基因和启动子序列、基因编码的蛋白序列以及不同物种间MAX1的进化关系进行分析。比较不结球白菜品种‘马耳头’(分蘖品种)和‘苏州青’(不分蘖品种)不同生长时期以及不同组织(根、茎、叶片和腋芽)中BcMAX1的相对表达量。利用BcMAX1过表达拟南芥转基因植株和不结球白菜沉默植株对BcMAX1基因的功能进行初步研究,并利用实时荧光定量PCR检测BcMAX1及独角金内酯响应基因BcMAX2和BcD14的表达水平。[结果]BcMAX1基因包含1个长度为1593 bp的开放阅读框,编码531个氨基酸。MAX1在不同物种间具有很高的保守性。BcMAX1的启动子含有多个决定转录起始和效率的基序以及响应干旱和光照的基序。BcMAX1在不结球白菜‘马耳头’和‘苏州青’腋芽伸长形成分蘖的过程中发挥作用,主要表达部位为根、叶片和腋芽。BcMAX1过表达转基因拟南芥植株的侧枝减少,BcMAX1基因及独角金内酯响应基因BcMAX2和BcD14的表达量在过表达植株中均升高,在不结球白菜沉默植株中下降。[结论]不结球白菜BcMAX1基因在‘马耳头’和‘苏州青’中的表达存在时空特异性和组织特异性,它参与植物分蘖的负调控,同时正向调控独角金内酯响应基因BcMAX2和BcD14的表达。

[Objectives]The paper aimed to clone BcMAX1 gene and its promoter,analyze the expression pattern of BcMAX1 and explore its function in tillering regulation.[Methods]BcMAX1 gene and its promoter were cloned,and the bioinformatics method was used to analyze the gene and promoter sequence,the protein sequence encoded by the gene,and the evolutionary relationship of MAX1 among different species.The relative expression levels of BcMAX1 in different tissues(root,stem,leaf and axillary)at different growth stages in‘Maertou’(the tillering variety)and‘Suzhouqing’(the non-tillering variety)were also compared.The function of BcMAX1 gene was preliminarily studied in Arabidopsis thaliana transgenic plants and non-heading Chinese cabbage silent plants.The expression levels of BcMAX1 and strigolactones response genes BcMAX2 and BcD14 were detected by RT-qRCR.[Results]The BcMAX1 gene contained an open reading frame of 1593 bp,encoding 531 amino acids.MAX1 was highly conserved among different species.The promoter of BcMAX1 contained the multiple motifs that determine transcription initiation and efficiency,and the motifs that respond to drought and light.BcMAX1 gene played an important role in the process of the axillary buds elongation and tiller formation in‘Maertou’and‘Suzhouqing’,and the main expression sites were root,leaf and axillary bud.BcMAX1 overexpression transgenic A.thaliana plants had fewer lateral branches,and the expression levels of BcMAX1 gene and strigolactones response genes BcMAX2 and BcD14 increased in overexpressed A.thaliana plants,but decreased in non-heading Chinese cabbage which silenced endogenous BcMAX1.[Conclusions]The expression of BcMAX1 gene in‘Maertou’and‘Suzhouqing’was spatiotemporal and tissue-specific.It was involved in the negative regulation of plant tillering and the positive regulation of the expression of strigolactones response genes BcMAX2 and BcD14.