基于CRISPR/Cas9的棉花GhbHLH71基因编辑突变体的分析

Analysis of mutants developed by CRISPR/Cas9-based GhbHLH71 gene editing in cotton

碱性/螺旋-环-螺旋(basic/helix-loop-helix,bHLH)转录因子在植物的生长发育、次生代谢、信号转导、逆境胁迫等方面起重要的调控作用。棉花GhbHLH71基因c DNA全长996 bp,编码331个氨基酸残基,蛋白序列中含有保守的bHLH结构域,属于bHLH转录因子家族成员。实时荧光定量分析结果表明,GhbHLH71基因在棉花纤维快速伸长期3~12 DPA (days post anthesis,DPA)相对高表达,暗示其主要在棉花纤维发育过程中发挥作用。构建该基因的CRISPR/Cas9基因编辑载体进行了棉花遗传转化,T_0代再生株经过Cas9基因的PCR检测以及靶位点突变情况检测分析,获得6个T_0代基因编辑突变体。T_1代不同突变株系在棉花生长发育期的表型性状与对照相比无明显差异,但成熟纤维的长度与对照相比皆明显变短。T_2代突变株系可以稳定遗传T_1代株系纤维变短的表型。其中4#和8#株系连续2代的纤维长度与对照相比缩短比率皆达20%以上,表明GhbHLH71基因的突变主要影响了棉花纤维细胞的伸长。本研究为深入了解棉花中bHLH转录因子的生物学功能以及棉花纤维发育的分子机制提供了一定的参考。

The basic/helix-loop-helix(bHLH)transcription factors play important regulatory roles in plant growth and development,secondary metabolism,signal transduction,and stress responses.The cDNA of cotton GhbHLH71 gene is 996 bp in length,encoding 331 amino acid residues.Protein sequence of GhbHLH71 contains a conserved bHLH structural domain,which is a member of the bHLH transcription factor family.The qRT-PCR showed that GhbHLH71 gene was relatively highly expressed at 3–12 DPA(days post anthesis,DPA)at rapid elongation stage in cotton fiber,implying that it mainly played a role in cotton fiber development.A CRISPR/Cas9 gene editing vector of GhbHLH71 gene was constructed,and then was transferred into upland cotton(Gossypium hirsutum L.).Six T0 mutant plants were obtained after PCR detection of Cas9 gene and mutation detection of target loci.The phenotypic traits of different T1 GhbHLH71 gene mutant plants were not significantly different from the control during cotton growth and development,but the fiber length of T1 mutants was significantly shorter compared with the control,and T2 generation mutant lines stably inherited the shortened fiber phenotype of T1 generation lines.The shortened fiber lengths of the 4#and 8#mutant lines in two consecutive generations were more than 20%shorter compared with the control,indicating that the mutation of GhbHLH71 gene mainly affected the elongation of cotton fiber cells.This study provides some insight into the biological functions of bHLH transcription factors in cotton and the molecular mechanism of cotton fiber development.