陆地棉纤维发育相关基因GhEXPs的分析及表达研究

doi:10.11963/1002-7807.llmmzy.20210411

摘要/Abstract

摘要：

【目的】扩展蛋白是1种细胞壁蛋白,能够松弛细胞壁从而促进细胞伸长。本研究旨在挖掘棉花纤维发育过程中特异的扩展蛋白基因。【方法】利用转录组测序和定量PCR（Polymerase chain reaction）研究棉花扩展蛋白基因的表达模式,并运用生物信息学的方法分析扩展蛋白进化关系、预测三维结构和关键氨基酸位点。【结果】以陆地棉cDNA（Complementary DNA,互补DNA）为模板,克隆得到4个棉花扩展蛋白基因GhEXPA1d、GhEXPA4b、GhEXPA23b 和GhEXPB3a,开放阅读框长度分别为768 bp、795 bp、798 bp和804 bp。进化树分析表明,GhEXPA1d、GhEXPA4b、GhEXPA23b属于EXPA（α-expansin）亚家族,GhEXPB3a属于EXPB（β-expansin）亚家族。组织特异性表达分析表明它们是纤维发育特异基因;定量PCR验证了这4个GhEXPs都是在纤维发育的起始期和伸长期表达量较高。根据序列比对以及三维结构模型预测了4个棉花扩展蛋白的关键氨基酸位点,分别是苯丙氨酸、色氨酸和酪氨酸,均为芳香族氨基酸,在三维结构模型中处于同一个平面上。【结论】获得的4个棉花扩展蛋白基因在棉花纤维发育起始期和伸长期特异表达,为进一步阐明GhEXPs对棉花纤维发育的分子机制提供了参考。

关键词: GhEXPs; 纤维发育; 三维结构; 陆地棉

Abstract:

[Objective] The expansins (EXPs) are a kind of cell wall proteins, which can loosen the cell wall and promote cell elongation. The purpose of this study is to explore the specific expansin genes in cotton fiber development. [Methods] The expression pattern of four GhEXPs was analyzed in different tissues using the RNA-seq and qRT-PCR (Quantitative real-time polymerase chain reaction) technology, respectively. The phylogenetic tree, three-dimensional structure prediction of GhEXPs, and locations of key amino acid were analyzed by bioinformatics. [Results] The four GhEXPs, GhEXPA1d, GhEXPA4b, GhEXPA23b, and GhEXPB3a, were cloned from the upland cotton cDNA (complementary DNA), the intact ORF (Open read frame) of them are 768 bp, 795 bp, 798 bp and 804 bp, respectively. The evolutionary analysis showed that GhEXPA1d, GhEXPA4b and GhEXPA23b belonged to the EXPA (α-expansin) subfamily, and GhEXPB3a belongs to EXPB (β-expansin) subfamily. Tissue-specific expression analysis indicated that the four genes were specific-expressed genes in fiber development, and predominantly expressed in the early stage of fiber development, and the reliability of the results was verified by qRT-PCR. According to sequence alignment and three-dimensional structure prediction, we have predicted the key amino acid residues of four GhEXPs, including the phenylalanine, tryptophan and tyrosine. The results showed that they were all aromatic amino acids and located the same surface. [Conclusion] The four GhEXPs specifically expressed at the initiation and elongation stages of cotton fiber development which provided a reference for further elucidating the molecular mechanism of GhEXPs during cotton fiber development.

Key words: GhEXPs; genes; fiber development; three-dimensional structure; Gossypium hirsutum

吕丽敏,左东云,王省芬,张友平,程海亮,王巧连,宋国立,马峙英. 陆地棉纤维发育相关基因GhEXPs的分析及表达研究[J]. 棉花学报, 2021, 33(3): 280-290.

Lü Limin,Zuo Dongyun,Wang Xingfen,Zhang Youping,Cheng Hailiang,Wang Qiaolian,Song Guoli,Ma Zhiying. Analysis and expression of GhEXPs related to fiber development in Gossypium hirsutum L.[J]. Cotton Science, 2021, 33(3): 280-290.

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https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2021/V33/I3/280

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参考文献 33

[1]	Jamet E, Dunand C. Plant cell wall proteins and development[J]. International Journal of Molecular Sciences, 2020, 21(8): 2731. DOI: 10.3390/ijms21082731. doi: 10.3390/ijms21082731
[2]	Cosgrove D J. Loosening of plant cell walls by expansins[J]. Nature, 2000, 407(6802): 321-326. DOI: 10.1038/35030000. doi: 10.1038/35030000
[3]	Cosgrove D J. Plant expansins: diversity and interactions with plant cell walls[J]. Current opinion in plant biology, 2015, 25: 162-172. DOI: 10.1016/j.pbi.2015.05.014. doi: 10.1016/j.pbi.2015.05.014 pmid: 26057089
[4]	Choi D, Kim J H, Lee Y. Expansins in plant development[J]. Advances in Botanical Research, 2008, 47(8): 47-97. DOI: 10.1016/S0065-2296(08)00002-5. doi: 10.1016/S0065-2296(08)00002-5
[5]	Cho H T, Cosgrove D J. Regulation of root hair initiation and expansin gene expression in Arabidopsis[J]. The Plant Cell, 2002, 14(12): 3237-3253. DOI: 10.1105/tpc.006437. doi: 10.1105/tpc.006437
[6]	Lin C, Choi H S, Cho H T. Root hair-specific EXPANSIN A7 is required for root hair elongation in Arabidopsis[J]. Molecular Cells, 2011, 31(4): 393-397. DOI: 10.1007/s10059-011-0046-2. doi: 10.1007/s10059-011-0046-2
[7]	McQueen-Mason S, Durachko D M, Cosgrove D J. Two endogenous proteins that induce cell wall extension in plants[J]. The Plant Cell, 1992, 4(11): 1425-1433. DOI: 10.1105/tpc.4.11.1425. doi: 10.1105/tpc.4.11.1425
[8]	Sampedro J, Cosgrove D J. The expansin superfamily[J]. Genome Biology, 2005, 6(12): 242. DOI: 10.1186/gb-2005-6-12-242. doi: 10 pmid: 16356276
[9]	Sampedro J, Lee Y, Carey R E, et al. Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family[J]. The Plant Journal, 2005, 44(3): 409-419. DOI: 10.1111/j.1365-313X.2005.02540.x. doi: 10.1111/j.1365-313X.2005.02540.x.
[10]	Zhu Y, Wu N N, Song W L, et al. Soybean (Glycine max) expansin gene superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies[J]. BMC Plant Biology, 2014, 14: 93. DOI: 10.1186/1471-2229-14-93. doi: 10
[11]	Krishnamurthy P, Hong J K, Kim J A, et al. Genome-wide analysis of the expansin gene superfamily reveals Brassica rapa-specific evolutionary dynamics upon whole genome triplication[J]. Molecular Genetics and Genomics, 2015, 290(2): 521-530. DOI: 10.1007/s00438-014-0935-0. doi: 10.1007/s00438-014-0935-0
[12]	Sampedro J, Carey R E, Cosgrove D J. Genome histories clarify evolution of the expansin superfamily: new insights from the poplar genome and pine ESTs[J]. Journal of Plant Research, 2006, 119(1): 11-21. DOI: 10.1007/s10265-005-0253-z. doi: 10.1007/s10265-005-0253-z pmid: 16411016
[13]	Ding A M, Marowa P, Kong Y Z. Genome-wide identification of the expansin gene family in tobacco (Nicotiana tabacum)[J]. Molecular Genetics and Genomics, 2016, 291(5): 1891-1907. DOI: 10.1007/s00438-016-1226-8. doi: 10.1007/s00438-016-1226-8
[14]	Kim H J, Triplett B A. Cotton fiber growth in planta and in vitro. Models for plant cell elongation and cell wall biogenesis[J]. Plant Physiology, 2001, 127(4): 1361-1366. DOI: 10.1104/pp.127.4.1361. doi: 10.1104/pp.127.4.1361 pmid: 11743074
[15]	Haigler C H, Betancur L, Stiff M R, et al. Cotton fiber: a powerful single-cell model for cell wall and cellulose research[J]. Frontiers in Plant Science, 2012, 3(104): 1-7. DOI: 10.3389/fpls.2012.00104. doi: 10.3389/fpls.2012.00104
[16]	Li Y, Tu L L, Pettolino F A, et al. GbEXPATR, a species-specific expansin, enhances cotton fibre elongation through cell wall restructuring[J]. Plant Biotechnology Journal, 2016, 14(3): 951-963. DOI: 10.1111/pbi.12450. doi: 10.1111/pbi.12450
[17]	Shan C M, Shangguan X X, Zhao B, et al. Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3[J]. Nature Communications, 2014, 5: 5519. DOI: 10.1038/ncomms6519. doi: 10.1038/ncomms6519
[18]	Xu B, Gou J Y, Li F G, et al. A cotton BURP domain protein interacts with alpha-expansin and their co-expression promotes plant growth and fruit production[J]. Molecular Plant, 2013, 6(3): 945-958. DOI: 10.1093/mp/sss112. doi: 10.1093/mp/sss112
[19]	An C, Saha S, Jenkins J N, et al. Transcriptome profiling, sequence characterization, and SNP-based chromosomal assignment of the EXPANSIN genes in cotton[J]. Molecular Genetics and Genomics, 2007, 278(5): 539-553. DOI: 10.1007/s00438-007-0270-9. doi: 10.1007/s00438-007-0270-9
[20]	Ji S J, Lu Y C, Feng J X, et al. Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array[J]. Nucleic Acids Research, 2003, 31(10): 2534-2543. DOI: 10.1093/nar/gkg358. doi: 10.1093/nar/gkg358
[21]	Harmer S E, Orford S J, Timmis J N. Characterisation of six alpha-expansin genes in Gossypium hirsutum (upland cotton)[J]. Molecular Genetics and Genomics, 2002, 268(1): 1-9. DOI: 10.1007/s00438-002-0721-2. doi: 10.1007/s00438-002-0721-2 pmid: 12242493
[22]	Lv L M, Zuo D Y, Wang X F, et al. Genome-wide identification of the expansin gene family reveals that expansin genes are involved in fibre cell growth in cotton[J]. BMC Plant Biology, 2020, 20: 223. DOI: 10.1186/s12870-020-02362-y. doi: 10.1186/s12870-020-02362-y
[23]	Bajwa K S, Shahid A A, Rao A Q, et al. Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton[J]. Frontiers in Plant Science, 2015, 6: 838. DOI: 10.3389/fpls.2015.00838. doi: 10.3389/fpls.2015.00838
[24]	Cosgrove D J. Diffuse growth of plant cell walls[J]. Plant Physiolgy, 2018, 176(1): 16-27. DOI: 10.1104/pp.17.01541. doi: 10.1104/pp.17.01541
[25]	Kerff F, Amoroso A, Herman R, et al. Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization[J]. Proceedings of the National Academy of Sciences, 2008, 105(44): 16876-16881. DOI: 10. 1073pnas.0809382105. doi: 10. 1073pnas.0809382105
[26]	Yennawar N H, Li L C, Dudzinski D M, et al. Crystal structure and activities of EXPB1 (Zea m1), a beta-expansin and group-1 pollen allergen from maize[J]. Proceedings of the National Academy of Sciences, 2006, 103(40): 14664-14671. DOI: 10.1073/pnas.0605979103. doi: 10.1073/pnas.0605979103
[27]	Zhu T, Liang C Z, Meng Z G, et al. CottonFGD: an integrated functional genomics database for cotton[J]. BMC Plant Biology, 2017, 17: 101. DOI: 10.1186/s12870-017-1039-x. doi: 10.1186/s12870-017-1039-x
[28]	Tamura K, Stecher G, Peterson D, et al. MEGA6: Molecular evolutionary genetics analysis version 6.0[J]. Molecular Biology and Evolution, 2013, 30(12): 2725-2729. DOI: 10.1093/molbev/mst197. doi: 10.1093/molbev/mst197
[29]	Kelley L A, Mezulis S, Yates C M, et al. The Phyre² web portal for protein modeling, prediction and analysis[J]. Nature Protocols, 2015, 10(6): 845-858. DOI: 10.1038/nprot.2015.053. doi: 10.1038/nprot.2015.053
[30]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI: 10.1006/meth.2001.1262. doi: 10.1006/meth.2001.1262 pmid: 11846609
[31]	Wang T, Chen Y, Tabuchi A, et al. The target of beta-expansin EXPB1 in maize cell walls from binding and solid-state NMR studies[J]. Plant Physiolgy, 2016, 172(4): 2107-2119. DOI: 10.1104/pp.16.01311. doi: 10.1104/pp.16.01311
[32]	Georgelis N, Tabuchi A, Nikolaidis N, et al. Structure-function analysis of the Bacterial expansin EXLX1[J]. Journal of Biological Chemistry, 2011, 286(19): 16814-16823. DOI: 10.1074/jbc.M111.225037. doi: 10.1074/jbc.M111.225037 pmid: 21454649
[33]	Cosgrove D J. Microbial expansins[J]. Annual Review of Microbiology, 2017, 71: 479-497. DOI: 10.1146/annurev-micro- doi: 10.1146/annurev-micro-090816-093315 pmid: 28886679

基因名称 Gene names	正向引物 Forward primers	反向引物 Reverse primers
GhEXPA4b	ATGGCTGTTGACGCTTCTTCTTCT	TCAAACACGGAAATTCTTTGCG
GhEXPA1d	ATGGCAAAAATCATTGTTGC	TTAGAATTGTAGTTTGCTAGAGAAAG
GhEXPA23b	ATGGCTACCCTGAAGCTTTT	CTAGAAGTTCTTTTTCCCATCAA
GhEXPB3a	ATGCAGCGCCGCGGT	TTAGAGTTTGAAGTTAAGGCGAG

引物名称 Primer names	引物序列 Sequences
QF2_EXPA1d	ACGCTTCTTCTTCTTCCTT
QR2_EXPA1d	CAGATTCCCATACCCACAT
QF2_EXPA4b	ACTGCCTTGTTCAATAATGG
QR2_EXPA4b	TGTAATGGTCACGGATGTT
QF_EXPA23b	ATTACCGTCGTGTCTTGT
QR_EXPA23b	TAACATCCTTAACATCACCAAC
QF_EXPB3a	GGATGAGTGGTTAGAGATGA
QR_EXPB3a	CCTGCTGATAATGTTGTTAGT
GhUBQ7-QF3	GGAATCCACTCTACACCTT
GhUBQ7-QR3	CTTCTTCTTCTTGTGCTTGA