Cloning and Prokaryotic Expression of the GhMYB42 Gene in Cotton

doi:10.7668/hbnxb.20194454

Abstract

Abstract:

MYB transcription factors play an important role in the growth and development of cotton,and GhMYB42 is one of the transcription factors of the MYB family,which also has certain research value,so this study cloned the coding sequence of GhMYB42 gene from cotton and constructed a prokaryotic expression vector.The nucleotide sequence and amino acid sequence of GhMYB42 were analysed using bioinformatics methods, and the coding sequence of the GhMYB42 gene was constructed into the prokaryotic expression vector pGEX-4T-1 by the Gataway BP and LR reactions, and the optimal conditions for the induction of the protein by IPTG were determined by setting up different IPTG-inducing conditions, and finally the recombinant protein was identified by Western Blot.The results showed that the full-length sequence of GhMYB42(XP_016732693.1) was 1 508 bp,the coding region length was 792 bp,the coding area was 263 amino acids,the predicted molecular weight was about 29.534 ku,and the isoelectric point was 5.18.Amino acid sequence comparison analysis revealed that the sequence similarity of the MYB transcription factors was 80.62%, and that the N-terminus of the GhMYB42 protein contained two tandem SANT structural domains, making it an R2R3 transcription factor.Phylogenetic tree analysis showed that the MYB42 protein in Gossypium hirsutum had the highest homology with another MYB protein (XP_012439547.1) in Gossypium hirsutum and was on one branch.Since the results of each experimental gradient were not significantly different during protein induction,it chose the conditions for IPTG to be 0.2 mmol/L at a final concentration of 0.2 mmol/L,a temperature of 37 ℃ for 3 h,and a temperature and time for protein solubility to be induced at 37 ℃ for 3 h.The Western Blot results showed that the size of the recombinant protein was correct,and finally the GhMYB42 recombinant protein with a size of 55.54 ku was successfully obtained,and it will purify the recombinant protein and further study the function of the transcription factor GhMYB42.

Key words: Gossypium hirsutum, GhMYB42, Clone, Sequence analysis, Prokaryotic expression

LI Chenyu, ZUMUREMU Tolson, LI Xiaorong, YANG Yang, LI Bo, YU Yuehua. Cloning and Prokaryotic Expression of the GhMYB42 Gene in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 48-54. doi: 10.7668/hbnxb.20194454.

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Figures/Tables 8

Tab.1 Primer sequence required for construction of expression vector pGEX-4T-1-MYB42

引物名称 Primer name	引物序列(5'—3' ) Primer sequence(5'—3' )
BP-GhMYB42-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGGTAGGCAGCCTTGTTG
BP-GhMYB42-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGCTGTTTTGTGTACGTGTTGG
M13-F	CGCCAGGGTTTTCCCAGTCACGAC
M13-R	CACACAGGAAACAGCTATGAC
pGEX-4T-1-F	CACGTTTGGTGGTGGCGACC

Fig.1 Schematic diagram of the structure of the GhMYB42 gene

Fig.2 Multiple sequence alignment of MYB protein Gh.Gossypium hirsutum;Gr.Gossypium raimondii;Ga.Gossypium arboreum;Hs.Hibiscus syriacus;Tc.Theobroma cacao;Ptr.Populus trichocarpa; Pto.Populus tomentosa;Ss.Salix suchowensis;Qr.Quercus robur;Gm.Glycine max;Black box.SANT conserved domain of MYB protein.

Fig.3 Phylogenetic tree analysis

Fig.4 Construction and identification of pGEX-4T-1-GhMYB42 prokaryotic expression vector M.DL2000 Marker;A.Amplification of the coding sequence of the GhMYB42 gene in cotton:1.Gene of interest;B.Bacterial PCR identification of BP-GhMYB42 intermediate vectors:1.Positive control,2.Negative control,3—8.Positive bands;C.PCR identification of pGEX-4T-1-GhMYB42 prokaryotic expression vectors:1.Positive control,2.Negative control,3—8.Positive bands.

Fig.5 pGEX-4T-1-GhMYB42 prokaryotic expression vector Red.T7 promoter;Blue.GST label;Green.Coding area of GhMYB42;Purple.Stop codons; Orange.Ampicillin resistance gene promoter;Brown.Ampicillin resistance gene;Black.Nos terminator.

Fig.6 Effects of different conditions induced on expression of GhMYB42 recombinant protein M.Protein ladder;1.pGEX-4T-1 empty vector 0.2 mmol/L IPTG induction for 3 h;2.Recombinant proteins before IPTG induction.A.Effect of different IPTG final concentrations on the expression of GhMYB42 recombinant protein:3—7.Recombinant protein after IPTG induction,the final concentrations induced by IPTG were 0.2,0.5,1.0,1.5,2.0 mmol/L,respectively.B.Effect of different IPTG induction times on the expression of GhMYB42 recombinant protein:3—7.Recombinant protein after IPTG induction,IPTG induction time of 1,3,5,7,9 h.C.Effect of different IPTG induction temperatures on the expression of GhMYB42 recombinant protein:3—5.Recombinant protein after IPTG induction,IPTG induction temperature was 16,28,37 ℃.

Fig.7 pGEX-4T-GhMYB42 recombinant protein solubility detection and Western Blot detection A.Solubility analysis of recombinant proteins;B.Western Blot detection of recombinant proteins. M.Protein ladder;1.pGEX-4T-1 empty vector 0.2 mmol/L IPTG induction for 3 h;2.Recombinant proteins before IPTG induction; 3—5.Total protein, supernatant, and precipitation of recombinant proteins after IPTG-induced.

References 20

[1]	蔺焘, 郭文静, 高黎, 常亮, 王正. 棉秆的解剖特性及化学成分研究[J]. 西北林学院学报, 2012, 27(5):201-206.doi:10.3969/j.issn.1001-7461.2012.05.39.
	Lin T, Guo W J, Gao L, Chang L, Wang Z. Anatomical characteristics and chemical components of cotton stalk[J]. Journal of Northwest Forestry University, 2012, 27(5):201-206.
[2]	邵玉涛, 陈永吉, 葛陈勇. 棉花质量检验员职业素质探讨[J]. 山西农经, 2019(21):118-118, 120.doi:10.16675/j.cnki.cn14-1065/f.2019.21.065.
	Shao Y T, Chen Y J, Ge C Y. Discussion on the professional quality of cotton quality inspectors[J]. Shanxi Agricultural Economy, 2019(21):118-118,120.
[3]	Mansoor S, Paterson A H. Genomes for jeans:cotton genomics for engineering superior fiber[J]. Trends in Biotechnology, 2012, 30(10):521-527.doi:10.1016/j.tibtech.2012.06.003. pmid: 22831638
[4]	Ashraf J, Zuo D Y, Wang Q L, Malik W, Zhang Y P, Abid M A, Cheng H L, Yang Q H, Song G L. Recent insights into cotton functional genomics:progress and future perspectives[J]. Plant Biotechnology Journal, 2018, 16(3):699-713.doi:10.1111/pbi.12856. URL
[5]	杜静静, 田岳, 冯昊, 郭梦兰, 杨琴莉, 丁林云, 李洁, 胡艳, 张天真. 陆地棉基因GhMYB52的克隆及特征分析[J]. 棉花学报, 2019, 31(6):505-514.doi:10.11963/1002-7807.djjhy.20191106.
	Du J J, Tian Y, Feng H, Guo M L, Yang Q L, Ding L Y, Li J, Hu Y, Zhang T Z. Cloning and characterization of the transcription factor gene GhMYB52 in Gossypium hirsutum L.[J]. Cotton Science, 2019, 31(6):505-514.
[6]	Pu L, Li Q, Fan X P, Yang W C, Xue Y B. The R2R3 MYB transcription factor GhMYB109 is required for cotton fiber development[J]. Genetics, 2008, 180(2):811-820.doi:10.1534/genetics.108.093070. pmid: 18780729
[7]	邱迎风. 三个棉花MYB转录因子的表达特性及GhMYB4a的功能验证[D]. 乌鲁木齐: 新疆农业大学, 2016.doi:10.7666/d.Y3101497.
	Qiu Y F. Expression characteristics of three cotton MYB transcription factors and functional verification of GhMYB4a[D]. Urumqi: Xinjiang Agricultural University, 2016.
[8]	黄俊锋. 三个R2R3—MYB转录因子在棉花(Gossypium hirsutum)纤维细胞次生壁发育中的功能研究[D]. 武汉: 华中师范大学, 2019.
	Huang J F. Study on the roles of three R2R3-MYB transcription factors during cotton (Gossypium hirsutum) fiber secondary cell wall thickening stage[D]. Wuhan: Central China Normal University, 2019.
[9]	Sun W J, Gao Z Y, Wang J, Huang Y Q, Chen Y, Li J F, Lü M L, Wang J, Luo M, Zuo K J. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers[J]. The New Phytologist, 2019, 222(2):864-881.doi:10.1111/nph.15620. URL
[10]	Machado A, Wu Y R, Yang Y M, Llewellyn D J, Dennis E S. The MYB transcription factor GhMYB25 regulates early fibre and trichome development[J]. The Plant Journal, 2009, 59(1):52-62.doi:10.1111/j.1365-313x.2009.03847.x. pmid: 19309462
[11]	Hu H Y, He X, Tu L L, Zhu L F, Zhu S T, Ge Z H, Zhang X L. GhJAZ2 negatively regulates cotton fiber initiation by interacting with the R2R3-MYB transcription factor GhMYB25-like[J]. The Plant Journal, 2016, 88(6):921-935.doi:10.1111/tpj.13273. pmid: 27419658
[12]	吴珊珊, 朱芸, 陈珊珊, 何冰芳. 融合标签在蛋白质可溶性表达中的应用进展[J]. 化工进展, 2014, 33(4):993-998.doi:10.3969/j.issn.1000-6613.2014.04.035.
	Wu S S, Zhu Y, Chen S S, He B F. Progress in fusion tags and its applications in protein soluble expression[J]. Chemical Industry and Engineering Progress, 2014, 33(4):993-998.
[13]	张磊, 唐永凯, 李红霞, 李建林, 徐逾鑫, 李迎宾, 俞菊华. 促进原核表达蛋白可溶性的研究进展[J]. 中国生物工程杂志, 2021, 41(S1):138-149.doi:10.13523/j.cb.2006016.
	Zhang L, Tang Y K, Li H X, Li J L, Xu Y X, Li Y B, Yu J H. Advances in promoting solubility of prokaryotic expressed proteins[J]. China Biotechnology, 2021, 41(S1):138-149.
[14]	邱淑彬, 荆韧威, 郭正隆, 谢晓东. 两种不同的原核表达载体蛋白表达及纯化的比较[J]. 基因组学与应用生物学, 2020, 39(9):4136-4144.doi:10.13417/j.gab.039.004136.
	Qiu S B, Jing R W, Guo Z L, Xie X D. Comparison of two different prokaryotic expression vectors in protein expression and purification[J]. Genomics and Applied Biology, 2020, 39(9):4136-4144.
[15]	Vasina J A, Baneyx F. Expression of aggregation-prone recombinant proteins at low temperatures:a comparative study of the Escherichia coli cspA and tac promoter systems[J]. Protein Expression and Purification, 1997, 9(2):211-218.doi:10.1006/prep.1996.0678. pmid: 9056486
[16]	柴政斌, 张更林, 王学政, 韩金祥. 融合蛋白GST-PADI4可溶性表达条件的优化及纯化[J]. 中国生物制品学杂志, 2014, 27(3):404-408,411.doi:10.13200/j.cnki.cjb.000201.
	Chai Z B, Zhang G L, Wang X Z, Han J X. Optimization of condition for soluble expression of GST-PADI4 fusion protein and purification of expressed product[J]. Chinese Journal of Biologicals, 2014, 27(3):404-408,411.
[17]	Kohl T, Schmidt C, Wiemann S, Poustka A, Korf U. Automated production of recombinant human proteins as resource for proteome research[J]. Proteome Science, 2008, 6:4.doi:10.1186/1477-5956-6-4. pmid: 18226205
[18]	郁硕, 陈锋, 刘英富, 霍景瑞, 李光宗, 张益, 丁辉, 樊毫军. 可溶性GST-CRH蛋白原核表达条件的优化及纯化[J]. 天津医药, 2017, 45(2):146-150.doi:10.11958/20161302.
	Yu S, Chen F, Liu Y F, Huo J R, Li G Z, Zhang Y, Ding H, Fan H J. Optimization of prokaryotic expression condition and purification of soluble GST-CRH protein[J]. Tianjin Medical Journal, 2017, 45(2):146-150.
[19]	万超, 张月, 胡莉, 伍炳华, 袁媛. 茉莉花JsMYB305转录因子的原核表达及蛋白纯化[J]. 福建农业学报, 2022, 37(2):164-169.doi:10.19303/j.issn.1008-0384.2022.002.005.
	Wan C, Zhang Y, Hu L, Wu B H, Yuan Y. Prokaryotic expression and purification of JsMYB305 transcription factor in Jasminum sambac[J]. Fujian Journal of Agricultural Sciences, 2022, 37(2):164-169.
[20]	Harper S, Speicher D W. Purification of proteins fused to glutathione S-transferase[J]. Methods in Molecular Biology, 2011, 681:259-280.doi:10.1007/978-1-60761-913-0_14. pmid: 20978970

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