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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (12): 2925-2939.doi: 10.3724/SP.J.1006.2024.44029

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

Identification of the CLE gene family in Gossypium hirsutum and functional analysis of the drought resistance of GhCLE13

RONG Yu-Xuan(), HUI Liu-Yang(), WANG Pei-Qi, SUN Si-Min, ZHANG Xian-Long, YUAN Dao-Jun(), YANG Xi-Yan   

  1. National Key Laboratory of Crop Genetic Improvement / Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2024-02-21 Accepted:2024-08-15 Online:2024-12-12 Published:2024-09-02
  • Contact: *E-mail: robert@mail.hzau.edu.cn
  • About author:**Contributed equally to this work
  • Supported by:
    Bingtuan Science and Technology Program(2022DB012);Biological Breeding—Major Projects in National Science and Technology(2023ZD04038)

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Abstract:

The CLAVATA3/Embryo surrounding region-related (CLE) peptide family is the largest family of small peptide hormones in plants. It is widely present in various plant species and participates in multiple important life activities of plants. In this study, the CLE gene family was identified at the genome-wide level in Gossypium hirsutum, and their gene structures, cis-acting elements in the promoter, protein physicochemical properties, and phylogenetic analysis of GhCLE gene family members were analyzed. The expression profiles of GhCLE gene family members in various tissues were constructed using RNA-seq data. Finally, the drought resistance function of the screened GhCLE genes was validated by virus-induced gene silencing (VIGS) technology. The results showed that a total of 40 GhCLE genes were identified in the whole genome of Gossypium hirsutum. The structures of GhCLE genes were relatively simple, with 32 GhCLE genes having no introns, and their protein sequences all contained a 12 aa CLE domain. The GhCLE gene promoter region contains a variety of cis-acting elements related to light response, stress response, hormone response, and development. The gene expression profile showed that the GhCLE gene was expressed in multiple tissues of Gossypium hirsutum, with GhCLE13-D-2 being specifically expressed in root tissue and induced by drought. The function of the GhCLE13-D-2 gene in improving cotton drought resistance was validated through VIGS technology and the measurement and comparison of MDA content. This study provides a new theoretical basis for the in-depth study of small peptides, such as CLE, in plant drought resistance and cotton germplasm innovation.

Key words: Gossypium hirsutum, small peptide gene family CLE, root specific expression, drought resistance, GhCLE13-D-2

Table S1

Perl scripts used to count amino acid sequence information"

stat_protein_fa.pl
die "perl \$0 <in> <out>" unless(@ARGV==2);
use Bio::SeqIO;
use Bio::Seq;
use Bio::Tools::SeqStats;
use Bio::Tools::pICalculator;
use Data::Dumper;

my \$in = Bio::SeqIO->new(
-file => "\$ARGV[0]",
-format => 'Fasta'
);

open OUT,">\$ARGV[1]" or die "\$!";
print OUT "#ID\tlength\tMW(Da)\tpI\n";
my \$calc = Bio::Tools::pICalculator->new(-places => 2,-pKset => 'EMBOSS');

while ( my \$seq = \$in->next_seq() ) {
#my ( \$id, \$sequence, \$desc ) = ( \$seq->id, \$seq->seq, \$seq->desc );
my \$weight = Bio::Tools::SeqStats ->get_mol_wt(\$seq);
\$calc->seq(\$seq);
my \$iep = \$calc->iep;
print OUT sprintf("%s\t%s\t%s\t%s\n",
\$seq->id,
\$seq->length,
"\$weight->[0]",
\$iep);
}
\$in->close();
close(OUT);

Table S2

Login number for transcriptome data downloaded from NCBI database"

NCBI数据编号
Number in NCBI		 棉花组织
Tissue of cotton
SRR1695160
SRR1695175
SRR1695177
SRR1695179
SRR1695173
SRR1695178
SRR1695174
SRR1695176		 种子Seed
叶片Leaf
花瓣Petal
雌蕊Pistil
根Root
雄蕊Stamen
茎Stem
花托Torus

Table S3

Primer sequences used in this study"

基因
Gene		 引物序列
Sequence (5°-3°)		 引物用途
Primer usage
GhCLE13-D-F ATGCTTCCCCTTCAGGCC 基因克隆
Gene clone
GhCLE13-D-R TCAATGATGCAATGGGTTCG
GhCLE13-D-VIGS-F GCGTGAGCTCGGTACCGCTTGGAAAGTATCCCACATGC 病毒诱导基因沉默(VIGS)载体构建
Construction of viral induced gene
silencing (VIGS) vector
GhCLE13-D-VIGS-R GCCTCCATGGGGATCCCAATGATGCAATGGGTTCGGA
GhCLE13-D-qRT-PCR-F ATGCTTCCCCTTCAGGCC qRT-PCR引物
qRT PCR primers
GhCLE13-D-qRT-PCR-R GCCTTGATCTGGTGCTTGA

Table 1

Information of CLE family genes in Gossypium hirsutum"

基因ID
Gene ID		 基因名称
Gene name		 肽链长度
Length/amino acid
(aa)		 小肽相对分子量
Molecular weight
(kD)		 小肽等电点
Isoelectric point
(pI)		 拟南芥同源基因Homologous gene in Arabidopsis
Ghir_A10G002770 GhCLE1-A 82 9.1282 8.22 AtCLE1
Ghir_D05G016000 GhCLE1-D-1 84 9.5808 8.22
Ghir_D10G003580 GhCLE1-D-2 82 9.0922 8.74
Ghir_D11G033760 GhCLE1-D-3 306 32.5428 10.91
Ghir_A09G010680 GhCLV3-A 98 10.4149 8.68 AtCLV3
Ghir_D09G010440 GhCLV3-D 98 10.4270 8.68
Ghir_A01G005030 GhCLE5-A 87 9.7073 7.84 AtCLE5
Ghir_D09G000220 GhCLE5-D 82 9.4078 9.32
Ghir_A09G000230 GhCLE6-A 106 12.0738 10.24 AtCLE6
Ghir_D05G015970 GhCLE6-D-1 81 8.7670 10.38
Ghir_D09G000240 GhCLE6-D-2 107 12.1749 10.24
Ghir_A03G011700 GhCLE9-A 98 11.1668 8.93 AtCLE9
Ghir_D02G013210 GhCLE9-D 98 11.1398 9.46
Ghir_A11G000690 GhCLE13-A 108 12.4310 10.23 AtCLE13
Ghir_D11G000720 GhCLE13-D-1 108 12.4120 9.83
Ghir_D13G007160 GhCLE13-D-2 131 15.0093 10.55
Ghir_A03G002330 GhCLE14-A-1 85 9.6739 8.77 AtCLE14
Ghir_A08G018820 GhCLE14-A-2 85 9.8511 9.79
Ghir_D03G016680 GhCLE14-D 85 9.7860 9.16
Ghir_A07G019890 GhCLE19-A-1 76 8.8853 12.68 AtCLE19
Ghir_A11G016310 GhCLE19-A-2 79 8.8802 12.01
Ghir_D11G016380 GhCLE19-D 79 8.8792 12.22
Ghir_D07G020180 GhCLE20-D-1 76 8.8642 12.42 AtCLE20
Ghir_D08G005670 GhCLE20-D-2 76 8.7749 12.02
Ghir_D05G007670 GhCLE22-D-1 97 10.7140 8.28 AtCLE22
Ghir_D08G001490 GhCLE22-D-2 82 9.2473 6.23
Ghir_D13G000120 GhCLE22-D-3 78 8.6591 10.55
Ghir_A07G023300 GhCLE25-A 106 12.0169 11.82 AtCLE25
Ghir_D07G023370 GhCLE25-D-1 106 11.9448 12.02
Ghir_D11G028800 GhCLE25-D-2 74 8.2654 5.03
Ghir_D02G017600 GhCLE27-D 90 10.3039 9.49 AtCLE27
Ghir_A03G016340 GhCLE27-A-1 90 10.4641 9.37
Ghir_A11G028610 GhCLE27-A-2 74 8.1913 5.02
Ghir_A08G008310 GhCLE40-A 84 9.4238 8.56 AtCLE40
Ghir_D08G008390 GhCLE40-D 84 9.4008 8.56
Ghir_D05G031300 GhCLE45-D-1 96 10.7723 10.66 AtCLE45
Ghir_D12G017800 GhCLE45-D-2 84 9.5651 11.99
Ghir_A05G031450 GhCLE45-A 96 10.8465 11.25
Ghir_D08G021160 GhTDIF-D 126 13.9278 7.62 AtTDIF
Ghir_A10G012570 GhTDIF-A 96 10.1514 7.52

Fig. 1

Gene structure, protein sequence conserved domain, and cis-acting element analysis of GhCLE gene family A: schematic diagram of gene structure analysis and protein domain analysis of GhCLE gene family members; B: schematic diagram of conserved signal peptide domain of GhCLE gene family; C: schematic diagram of conserved CLE motif of GhCLE gene family."

Fig. 2

Schematic diagram of the distribution of cis-acting elements"

Fig. 3

Phylogenetic analysis and clustering of GhCLE gene family A: phylogenetic tree of the GhCLE gene family; B: schematic diagram of the CLEmotif characteristics of each subfamily of the GhCLE gene family."

Fig. 4

Analysis of GhCLE expression patterns A: expression heatmap of GhCLE gene family members in various tissues of cotton. B: expression levels of root-specific genes GhCLE13-D-2, GhCLE14-A-2, and GhCLE25-A-1 under drought treatment and CK treatment (FPKM value). *, **, *** indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

Fig. 5

VIGS experiments verified the drought resistance of GhCLE13 A: the control group and VIGS silent lines under normal treatment; B: leaf phenotypes between control group and VIGS silent lines after 7 days of drought treatment; C: leaf phenotypes of control group and VIGS silent lines after 10 days of drought treatment; D: whitening phenotype of TRV::GhCLA lines; E: relative expression levels of GhCLE13-D-2 gene between VIGS silent lines and control lines; F: MDA content (nmol g-1) of VIGS silent lines and control groups after 10 days of normal treatment and drought. The scale is 3 cm. *, **, *** indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

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