陆地棉纤维起始期优势表达基因GhCRPK1的克隆及功能研究

doi:10.11963/cs20210061

摘要/Abstract

摘要：

【目的】研究陆地棉类受体激酶GhCRPK1（Cold-responsive protein kinase 1,CRPK1）在棉花纤维发育中的功能。【方法】以陆地棉全基因组关联分析（Genome-wide association study,GWAS）数据及陆地棉纤维发育不同时期的表达数据,筛选到GhCRPK1在棉花纤维起始期优势表达,通过基因克隆、亚细胞定位等方法研究了GhCRPK1的功能,对过表达GhCRPK1的拟南芥及突变体crpk1的表皮毛长度和密度进行观察统计,利用酵母双杂交筛选与GhCRPK1相互作用的蛋白。【结果】GhCRPK1基因全长为4 594 bp,开放阅读框长度为1 047 bp,编码348个氨基酸,在开花当天到开花后3 d持续优势表达,编码产物定位在细胞膜上。GhCRPK1在拟南芥中异源表达后可促进表皮毛的伸长;拟南芥中同源基因突变后,表皮毛变短。酵母双杂交实验发现,GhCRPK1与热激蛋白Gh_D08G0057相互作用。【结论】GhCRPK1在棉花纤维发育起始时期优势表达,定位在细胞膜上,在拟南芥中异源表达后调控拟南芥表皮毛的伸长。

关键词: 类受体激酶; GhCRPK1; 纤维发育; 表皮毛; 信号传导

Abstract:

[Objective] The aim of this article is to study the function of GhCRPK1 (Cold-responsive protein kinase 1, CRPK1) during fiber development in upland cotton. [Method] According to the genome-wide association study (GWAS) data and the transcriptome data of fiber development at different stages in cotton, GhCRPK1 may be involved in fiber initiation by gene clone, subcellular localization to study the GhCRPK1. The full length of GhCRPK1 was amplified and transformed into Arabidopsis, and the length and density of trichomes of the GhCRPK1-overexpressed plants and Arabidopsis mutant crpk1 were observed. At last, yeast two-hybrid assay was performed to screen the interaction proteins. [Result] The full-length of GhCRPK1 is 4 594 bp containing a 1 047 bp open reading frame and encoding 348 amino acids. The expression level of GhCRPK1 peaked at the 0-3 days post anthesis. The GhCRPK1 was localized on the plasma membrane. The Arabidopsis plants overexpressing GhCRPK1 had longer trichomes compared with the wild type. While the trichomes were shorter in the crpk1 mutant. Yeast two-hybrid analysis revealed that GhCRPK1 can interact with a heat shock protein Gh_D08G0057. [Conclusion] GhCRPK1, a gene preferentially expressed during fiber initiation and localized on the plasma membrane, regulated the elongation of trichome in Arabidopsis.

Key words: receptor like kinase; GhCRPK1; fiber development; trichome; signal transduction

王雪慧,陈丽锦,赵若林,程海亮,张友平,王巧连,吕丽敏,宋国立,左东云. 陆地棉纤维起始期优势表达基因GhCRPK1的克隆及功能研究[J]. 棉花学报, 2021, 33(6): 459-468.

Wang Xuehui,Chen Lijin,Zhao Ruolin,Cheng Hailiang,Zhang Youping,Wang Qiaolian,Lü Limin,Song Guoli,Zuo Dongyun. Cloning and functional analysis of GhCRPK1, a gene preferentially expressed during fiber initiation in upland cotton[J]. Cotton Science, 2021, 33(6): 459-468.

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链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20210061

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2021/V33/I6/459

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附表1

本研究中所用到的引物序列"

引物名称	引物序列
Primer names	Primer sequence
Histone3PF	CCGTAAATCTGCCCCAACCA
Histone3PR	GACCCACAAGGTATGCCTCTGC
Gh_A09G0177PF	TTTCAGACGCAGACGCCATTGT
Gh_A09G0177PR	GAATGCAGCCGCCAAGTGAATC
Gh_A09G0186PF	TTCCTTGCCCTTCAACGAAGCA
Gh_A09G0186PR	ACTGCCATAACCACCCATTCCA
Gh_A09G1658PF	GTCTCTGCTCGATGCGGTAACT
Gh_A09G1658PR	GCAAGGATTTCTCAAACTCCCCCA
Gh_A10G1849PF	GAATACGCTGTGAGTGGACGCT
Gh_A10G1849PR	ACGATCCACGTCTTGTTCCCAG
Gh_A10G1850PF	AGGCCTTCCGATTCCAACTGTG
Gh_A10G1850PR	ACTATACTGGCTAGCTGGCACGA
Gh_D02G0129PF	GCTCGGTACATGTTTTTATGGCCG
Gh_D02G0129PR	CAAGCGACAGCCAATCGTTCAC
Gh_D05G3666PF	TCTCCTCAACTTGGCAGGCAAC
Gh_D05G3666PR	AGATTCCCCATCGAGTCAGGCA
Gh_D05G3667PF	GCTCGAGACTGATGATACCGCA
Gh_D05G3667PR	CCAAATGTTCAAGATTGCTGCTCCG
Gh_D08G0480PF	TGGGAAGGAGTTGAGTGCAACG
Gh_D08G0480PR	CCATTCACGGTTATCCCATTGACG
Gh_D08G0482PF	TTCATCGGAAAAGGTGCCTCCC
Gh_D08G0482PR	CGCCAAAGAGGACTTTGAGGTGT
Gh_D08G2087PF	TGGTTCTCTTTGCGGAAGGTCT
Gh_D08G2087PR	AGACATGGTGCCAGCAATGGTT
Gh_D13G2124PF	ATCTGAGGGAGATGGTGGTCCC
Gh_D13G2124PR	CCGCCACTGAAATCCAGCTTCT
Gh_D13G0607PF	GGTTCCAAAGCCAAGGGTGCTT
Gh_D13G0607PR	CACAACTCCCTCCCAATCGCAG
Gh_A01G0382PF	ATCTTGCAGGTGAGATACCATCCA
Gh_A01G0382PR	ACCGGAAAGCTTGTTCCCTTTCA
Gh_A01G0896PF	CATGTTTGACGGGCGATTGTGG
Gh_A01G0896PR	GGGACGTTGTAACCGTCAACCA
Gh_A02G0005PF	CAGCAGCTCCACAACCACATCT
Gh_A02G0005PR	TCCTGGAATCCTCGAAGTCGCT
Gh_A02G0017PF	GCCGAAGTCAATCATGCCCCAT
Gh_A02G0017PR	CCGATTCTAAGAGGAGCGGCAC
Gh_A02G1417PF	ACCAACTTTCCGGGGCTTTACC
Gh_A02G1417PR	AAGGTTCCCAGCTCGGGAGTTA
Gh_A02G1419PF	TCCTACGGCGAATGTGAGTCCT
Gh_A02G1419PR	CCCCAGAATTTTTCCGGCAGGT
Gh_D09G0223PF	TGGATGGAACGAGAAGATCGCC
Gh_D09G0223PR	GCCTGTTAGCCGACAGATCCA
Gh_A03G1520PF	TCACGGAAACCTGAAACCGTCC
Gh_A03G1520PR	CGAGTGCTGGTATTCCGGTGAC
Gh_A06G1067PF	GCGTTTGCTACTAAGTTGGACGA
Gh_A06G1067PR	GGCTGCTTTTAAGTCGCGATGG
Gh_A06G1068PF	GCCGTTTCATTGGCAGTTGTCC
Gh_A06G1068PR	TGTTATGAGCTGCATCCGTGGG
Gh_A08G0777PF	GTCTAAGGGGCTACTGCGTTGG
Gh_A08G0777PR	GCAACCAGCGGTTCGAAGTTTG
Gh_A08G0809PF	AAGAGGAGTGTCTGGAAGGTACGG
Gh_A08G0809PR	GAACCACCATGGGCCGAGAAAA
Gh_A08G2273PF	CGCAGAACTGAAAGCGCTCAAG
Gh_A08G2273PR	GCCATAACCCCCTTCCCCAATC
Gh_A08G2419PF	GGCCGACTGCGTCGAAGATT
Gh_A08G2419PR	GCACAGCCCCTAGCATGTCC
Gh_A08G2425PF	ATGCCATTGCACATTCAGCAGC
Gh_A08G2425PR	TGCTGCCTCCTCAACTGGGATA
Gh_A08G2434PF	TCACGCCGATCTTTGCAGACTT
Gh_A08G2434PR	ACAACCTCAGCTGACTGCAAGC
Gh_A09G0625PF	TGGTGGAATGTCACCCCTAACG
Gh_A09G0625PR	TGCTCTAGCAACATCCCCGATT
Gh_A10G1831PF	AGGGGCACAAAGAATGGGTGAC
Gh_A10G1831PR	CTCCGGTTCGGCATGTATTCGT
Gh_A11G1757PF	GGATCCTGACGAGCCGAAAGAC
Gh_A11G1757PR	TCAGATGCCGACACTCAGGAGA
Gh_A13G0484PF	ACGCAAGATCCAGCGCTATTGA
Gh_A13G0484PR	GCTCCATGCCCTCCATCTCATT
Gh_A13G2056PF	CATCCCGTTGGACAGGTGTGTT
Gh_A13G2056PR	TATCCGCAAGCGAGATAAGCGG
GhCRPK1PF1	ATGGAATTTCATGTCCCCACAG
GhCRPK1PR1	TTATGAAGTTTGAAACTGAGGG
GhCRPK1PF2	TCCGGTACCCCCGGGGTCGACTTATGAAGTTTGAAACTGAATGGTGATGGTGATGATGGGG
GhCRPK1PR2	CTGAACCGCCTCCACCGGATCCTGAAGTTTGAAACTGAGGG
GhCRPK1PF3	TCCGGTACCCCCGGGGTCGACTTATGAAGTTTGAAACTGAATGGTGATGGTGATGATGGGG
GhCRPK1PR3	GAATTCCTAATGATGATGATGATGATGTGAAGTTTGAAACTGAGGG

附表1

参考文献 28

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材料Material	表皮毛长度 Trichome length/μm	25 mm²表皮毛的数量 No. of leaf trichome per 25 mm²
WT	282.40±9.2	96±15
crpk1	263.60±8.4^**	90±11
35∷GhCRPK1	340.60±11.5^**	101±15