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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (12): 2925-2939.doi: 10.3724/SP.J.1006.2024.44029

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

陆地棉CLE基因家族的鉴定及GhCLE13参与调控棉花抗旱性的功能分析

戎宇轩(), 惠留洋(), 王沛琦, 孙思敏, 张献龙, 袁道军(), 杨细燕   

  1. 华中农业大学作物遗传改良全国重点实验室 / 湖北洪山实验室, 湖北武汉 430070
  • 收稿日期:2024-02-21 接受日期:2024-08-15 出版日期:2024-12-12 网络出版日期:2024-09-02
  • 通讯作者: *袁道军, E-mail: robert@mail.hzau.edu.cn
  • 作者简介:戎宇轩, E-mail: rong19990831@webmail.hzau.edu.cn;
    惠留洋, E-mail: hly111@webmail.hzau.edu.cn**同等贡献
  • 基金资助:
    兵团财政科技计划项目(2022DB012);农业生物育种国家科技重大专项(2023ZD04038)

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 Published:2024-12-12 Published online: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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摘要：

CLAVATA3/Embryo surrounding region-related (CLE)小肽家族是植物中最大的小肽激素家族, 在植物中广泛存在, 且参与植物多个重要的生命活动。本研究对陆地棉CLE基因家族进行了鉴定, 并对GhCLE基因家族成员进行基因结构、启动子顺式作用元件、蛋白质理化性质、系统发育的分析; 通过RNA-seq数据构建了GhCLE基因家族成员在陆地棉各组织的表达谱及干旱处理下的表达模式, 并筛选在棉花根系特异表达且受干旱诱导的CLE基因; 最后通过VIGS技术对筛选出的GhCLE基因进行了抗旱功能的验证。结果显示,在陆地棉全基因组共鉴定出40个GhCLE基因, GhCLE基因结构较为简单, 32个GhCLE基因没有内含子, 所有基因编码的蛋白序列均包含12 aa的CLE结构域; GhCLE基因启动子区域包含多种光诱导响应、胁迫响应、激素响应和发育相关的顺式作用元件; GhCLE基因在陆地棉多个组织均有表达, 筛选出了1个根系特异表达且受干旱诱导的GhCLE13-D-2基因。通过VIGS技术和MDA含量的测定和比较验证了GhCLE13-D-2基因提高棉花的抗旱性的功能。本研究为CLE小肽在植物抗逆方面的深入研究以及棉花种质创新提供了新的理论依据。

关键词: 陆地棉, CLE小肽基因家族, 根系特异表达, 抗旱, GhCLE13-D-2

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

附表1

统计氨基酸序列信息所用perl脚本"

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);

附表2

NCBI数据库下载的转录组数据登录号"

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

附表3

本试验所用到的引物序列"

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

表1

陆地棉CLE家族基因的相关信息"

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

图1

GhCLE基因家族基因结构、氨基酸列保守结构域以及顺式作用元件分析 A: GhCLE基因家族成员基因结构和结构域示意图; B: GhCLE基因家族保守信号肽结构域示意图; C: GhCLE基因家族保守CLE motif示意图。"

图2

顺式作用元件分布示意图"

图3

GhCLE基因家族系统发育分析及聚类 A: GhCLE基因家族系统进化树; B: GhCLE基因家族各亚家族CLEmotif特征示意图。"

图4

GhCLE表达模式分析 A: GhCLE基因家族成员在棉花各个组织的表达量热图。B: 根系特异表达基因GhCLE13-D-2、GhCLE14-A-2和GhCLE25-A-1在干旱处理和CK处理下的表达量(FPKM值)。*、**、***分别表示在0.05、0.01、0.001概率水平差异显著。"

图5

VIGS试验验证GhCLE13抗旱功能 A: 正常处理下对照组和VIGS沉默株系; B: 干旱处理7 d后对照组和VIGS沉默株系叶片表型; C: 干旱处理10 d后对照组和VIGS沉默株系叶片表型; D: TRV::GhCLA株系白化表型; E: VIGS沉默株系和对照株系中GhCLE13-D-2基因的相对表达量; F: 正常处理和干旱10 d后VIGS沉默株系和对照组MDA含量(nmol g-1)。标尺为3 cm。*、**、***分别表示在0.05、0.01、0.001概率水平差异显著。"

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