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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 138-148.doi: 10.3724/SP.J.1006.2024.34041

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-01-12 Published: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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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

Fig. 1

Sketch image of gene editing vector in cotton"

Table 1

Primers used in this study"

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

Fig. 2

Amino acid sequence and conserved domain of GhbHLH71 protein A: amino acid sequence of GhbHLH71 protein. The conserve bHLH domain was underlined, putative DNA binding site was marked by red letters. B: protein domain prediction of GhbHLH71 protein."

Fig. 3

Relative expression pattern of GhbHLH71 gene in different cotton tissue DPA: days post anthesis."

Fig. 4

Cas9 PCR amplification assay of different T0 regeneration plants M: DNA molecular marker; +: the plasmid amplification as positive control; 1-11: different T0 regeneration plants."

Fig. 5

CRISPR/Cas9 editing effect of GhbHLH71 gene in different T0 regeneration lines A: the editing results of different T0 regeneration lines. Blue letters indicate sgRNA1 and sgRNA2 sequence, bold and underline letters indicate PAM site, red letters indicate the different base in A and D genome of GHbHLH71 gene, the deletions indicated by blue dash, the insertions and mutants marked by yellow letters. B: the sequencing peaks image of different T0 regeneration lines of GHbHLH71 gene. Underline indicates the position of different sgRNA, arrow indicates the mutant site."

Fig. 6

Fiber phenotype of different GhbHLH71 T1 and T2 gene editing lines A: the fiber phenotype and statistical analysis of fiber length of different T1 gene editing lines. n > 10, **: P < 0.01. B: the fiber phenotype and statistical analysis of fiber length of different T2 gene editing lines. n > 10, **: P < 0.01."

Fig. 7

CRISPR/Cas9 editing effect of GhbHLH71 gene in 4# and 8# T2generation plants The yellow markers in the figure are the unmutated sgRNA1or sgRNA2 sequences, A and D represent the A and D genomes of GhbHLH71 gene, respectively. Plant from 8-1 to 8-5 represent the five different plant of Line 8#, plant from 4-1 to 4-5 represent five different plant of Line 4#, and the following numbers represent the monoclonal assay samples. CRS25-F is the primer for sequencing. Blue markers are insertions or mutated bases, and dashes indicate base deletions."

Table 2

Hi-TOM editing mutations detection in T2 generation lines"

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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