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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 138-148.doi: 10.3724/SP.J.1006.2024.34041

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于CRISPR/Cas9的棉花GhbHLH71基因编辑突变体的分析

上官小霞*(), 杨琴莉, 李换丽   

  1. 山西农业大学棉花研究所, 山西运城 044000
  • 收稿日期:2023-03-01 接受日期:2023-05-24 出版日期:2024-01-12 网络出版日期:2023-06-16
  • 通讯作者: *上官小霞, E-mail: sgxx74@126.com
  • 基金资助:
    山西省基础研究计划项目(20210302123409);山西农业大学博士人才引进科研启动项目(2022BQ08)

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

SHANG-GUAN Xiao-Xia*(), YANG Qin-Li, LI Huan-Li   

  1. Cotton Research Institute, Shanxi Agricultural University, Yuncheng 044000, Shanxi, China
  • Received:2023-03-01 Accepted:2023-05-24 Published:2024-01-12 Published online:2023-06-16
  • Contact: *E-mail: sgxx74@126.com
  • Supported by:
    Basic Research Program of Shanxi Province(20210302123409);Doctoral Talent Introduction Scientific Research Initiation Project of Shanxi Agricultural University(2022BQ08)

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摘要：

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cDNA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, T0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T0代基因编辑突变体。T1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。T2代突变株系可以稳定遗传T1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。

关键词: 棉花, bHLH转录因子, GhbHLH71基因, 纤维发育, CRISPR/Cas9基因编辑

Abstract:

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.

Key words: cotton, bHLH transcription factor, GhbHLH71 gene, fiber development, CRIPSR/Cas9 gene editing system

图1

棉花基因编辑载体示意图"

表1

本研究所用引物列表"

引物
Primer name		 引物序列
Primer sequences (5°-3°)		 功能
Function
His3-RT-F GAAGCCTCATCGATACCGTC Real-time RT-PCR检测 Real-time RT-PCR analysis
His3-RT-R CTACCACTACCATCATGG Real-time RT-PCR检测 Real-time RT-PCR analysis
bHLH71-RT-F ATGCTAGAAAGCGGTTTAGTCT Real-time RT-PCR检测 Real-time RT-PCR analysis
bHLH71-RT-R TCCAATGGTGACTCCTCCACA Real-time RT-PCR检测 Real-time RT-PCR analysis
bHLH71-1-F ATGCTAGAAAGCGGTTTAGTCT 基因克隆 Gene cloning
bHLH71-2172-R TCAACATAAGGCAGTGGCATCT 基因克隆 Gene cloning
CRS25-F ATGCTAGAAAGCGGTTTAGTCT 编辑位点突变检测 Edit site mutation detection
CRS25-R CCCTCAATCAGTCCAGTTTTC 编辑位点突变检测 Edit site mutation detection
CRS25-Hi-F ggagtgagtacggtgtgcCTCAATGGAAGAACAATCTACT Hi-TOM编辑位点突变检测
Hi-TOM edit site mutation detection
CRS25-Hi-R gagttggatgctggatggTTGAGTCTCGGCCTCTTCTTT 编辑位点突变检测 Edit site mutation detection
Cas9-787-F AATCTGATCGCCCAGCTGCCC 阳性植株鉴定 Positive plants detection
Cas9-1550-R GAAGTTCCAGGGGGTGATGGT 阳性植株鉴定 Positive plants detection

图2

GhbHLH71蛋白氨基酸序列及保守结构域 A: GhbHLH71蛋白氨基酸序列。bHLH保守结构域用下画线标记, 推测的DNA结合位点用棕色标记。B: GhbHLH71蛋白结构域预测。"

图3

GhbHLH71基因在棉花不同组织的表达特征分析"

图4

不同T0代再生植株的Cas9基因的PCR检测 M: DNA分子量标准; +: 质粒模板阳性对照; 1~11: 不同T0代再生株。"

图5

不同T0株系GhbHLH71基因CRISPR/Cas9编辑效率检测 A: 不同T0株系靶位点基因编辑结果。蓝色标记为sgRNA1和sgRNA2序列, 加粗且下画线标记为protospacer adjacent motif (PAM)序列, 红色标记为GhbHLH71基因A、D基因组中sgRNA1序列差异位点, 蓝色破折号表示碱基缺失, 黄色标记为碱基插入或突变。B: 不同T0株系靶位点测序峰图。下画线表示sgRNA1或sgRNA2位置, 箭头所示为突变位点。"

图6

不同T1、T2代GhbHLH71基因编辑株系纤维表型分析 A: 不同 T1株系棉花纤维长度表型及统计分析, n > 10, **P < 0.01。B: 不同T2株系棉花纤维长度表型及统计分析, n > 10, **P < 0.01。"

图7

GhbHLH71基因4#和8#株系T2代材料CRISPR/Cas9编辑效率检测 图中黄色标记为未突变的sgRNA1或sgRNA2序列, A、D分别代表GhbHLH71基因的A和D基因组, 8-1至8-5代表8#株系的5个单株, 4-1至4-5代表4#株系的5个单株, 后面的数字代表单克隆编号。CRS25-F为测序所用引物, 蓝色标记为插入或突变的碱基, 破折号表示碱基缺失。"

表2

T2代株系的Hi-TOM编辑突变检测"

株系
Line		 突变编号
Mutation number		 Reads读数
Reads
number		 突变比率
Mutation
ratio (%)		 左侧突变类型
Left variation type		 右侧突变类型
Right variation type		 左侧突变信息
Left variation		 右侧突变序列
Right variation
8#-1 1 2434 43.06 SNP SNP C->T, A->G A->G, G->T
2 1656 29.29 SNP SNP G->A G->A
3 718 12.70 1D, SNP 1D, SNP C->T, G, A->G G, A->G, G->T
4 436 7.71 1I, SNP 1I, SNP A, G->A A, G->A
5 409 7.24 1I, SNP 1I, SNP T, G->A T, G->A
8#-2 1 1676 33.58 1I, SNP 1I, SNP A, G->A A, G->A
2 1582 31.70 SNP SNP C->T, A->G A->G, G->T
3 937 18.77 1I, SNP 1I, SNP A, G->A A, G->A, G->T
4 796 15.95 SNP SNP C->T, A->G A->G
8#-3 1 1258 31.53 SNP SNP G->A G->A
2 827 20.73 1D, SNP 1D, SNP C->T, G, A->G G, A->G, G->T
3 735 18.42 SNP SNP G->A G->A, G->T
4 648 16.24 SNP SNP C->T, A->G A->G, G->T
5 522 13.08 1D, SNP 1D, SNP C->T, G, A->G G, A->G
4#-1 1 1352 22.47 1D, SNP 1D, SNP C->T, G, A->G G, A->G, G->T
2 1192 19.81 1I, SNP 1I, SNP A, G->A A, G->A
3 1096 18.22 1I, SNP 1I, SNP C->T, T, G->A T, G->A
4 1081 17.97 3D, SNP 3D, SNP C->T, CAA, A->G AAC, A->G, G->T
5 464 7.71 3D, SNP 3D, SNP C->T, CAA, A->G AAC, A->G
6 426 7.08 1D, SNP 1D, SNP C->T, G, A->G G, A->G
7 406 6.75 1I, SNP 1I, SNP T, G->A T, G->A, G->T
4#-2 1 2493 40.96 1D, SNP 1D, SNP C->T, G, A->G G, A->G, G->T
2 1087 17.86 1I, SNP 1I, SNP C, G->A C, G->A
3 1079 17.73 3D, SNP 3D, SNP CAG, G->A GCA, G->A
4 987 16.22 1D, SNP 1D, SNP C->T, G, A->G G, A->G
5 440 7.23 1I, SNP 1I, SNP C, G->A C, G->A, G->T
4#-3 1 2984 50.10 3D, SNP 3D, SNP CAG, G->A GCA, G->A
2 2162 31.26 1I, SNP 1I, SNP C->T, A, A->G A, A->G, G->T
4 644 10.81 3D, SNP 3D, SNP CAG, G->A GCA, G->A, G->T
5 466 7.82 1I, SNP 1I, SNP C->T, A, A->G A, A->G
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