Cloning and Functional Analysis of Gene GhTIFY9 Related to Cotton Verticillium Wilt Resistance

doi:10.13560/j.cnki.biotech.bull.1985.2021-1519

Abstract

Abstract:

The function of TIFY family gene GhTIFY9 in cotton verticillium wilt response was preliminarily explored,which laid a foundation for mining cotton disease resistance related genes and cotton disease resistance breeding. A TIFY family gene GhTIFY9 was obtained by transcriptome screening and cloning. The physicochemical properties of the gene were analyzed by bioinformatics methods. The expression pattern of GhTIFY9 under verticillium wilt induction was analyzed by real-time quantitative polymerase chain reaction（RT-qPCR）. Meanwhile,the VIGS vector of the gene was constructed,and its function in the cotton resistance to verticillium wilt was preliminarily explored by virus-induced gene silencing（VIGS）technology. GhTIFY9 was cloned using the cDNA of upland cotton TM-1 as the template. The open reading frame（ORF）of GhTIFY9 was 594 bp,encoding an alkaline hydrophilic protein containing 197 amino acids. The relative molecular weight was 21.65 kD,it was located in the nucleus and belonged to the TIFY subfamily. RT-qPCR analysis showed that GhTIFY9 responded to verticillium wilt induction,and the sensitivity of silent plants to verticillium wilt was significantly enhanced after inhibiting the expression of this gene. GhTIFY9 is a positive regulator of cotton resistance to verticillium wilt.

Key words: cotton verticillium wilt, GhTIFY9, gene cloning, virus-induced gene silencing（VIGS）

LI Xiu-qing, HU Zi-yao, LEI Jian-feng, DAI Pei-hong, LIU Chao, DENG Jia-hui, LIU Min, SUN Ling, LIU Xiao-dong, LI Yue. Cloning and Functional Analysis of Gene GhTIFY9 Related to Cotton Verticillium Wilt Resistance[J]. Biotechnology Bulletin, 2022, 38(8): 127-134.

Figures/Tables 7

Fig. 1 Cloning of GhTIFY9 gene M：Trans 2K Plus Ⅱ DNA marker

Table 1 Bioinformatics analysis of GhTIFY9

基因 Gene	开放阅读框ORF/bp	氨基酸 Amino acid	相对分子质量Relative molecular weight/kD	理论等电点Theoretical isoelectric point	平均疏水性Average hydrophobicity	信号肽 Signal peptide	亚细胞定位 Subcellular localization	脂肪系数Fat coefficient	跨膜结构域 Transmembrane domain
GhTIFY9	594	197	21.65	9.08	-0.477	无 None	细胞核 Nucleus	76.63	无 None

Fig.2 Protein domain prediction of GhTIFY9

Fig.3 Analysis of the expression patterns of GhTIFY9 under the treatment of Verticillium dahliae *：Significant difference at the 0.05 level. **：Significant difference at the 0.01 level. The same as below

Fig. 4 VIGS vector construction of GhTIFY9 A：PCR amplification of target fragment of GhTIFY9. B：Identification of TRV﹕GhTIFY9 vectors by restriction enzyme digestion. M：Trans 2K Plus II DNA marker

Fig. 5 Silencing efficiency detection of GhTIFY9 gene A：The phenotype of silenced GhCLA1 in cotton. B：Silencing efficiency detection of GhCLA1. C：Silencing efficiency detection of GhTIFY9

Fig. 6 Disease resistance identification of GhTIFY9 gene-silenced plants A：Phenotype analysis of plants after GhTIFY9 gene silencing. B：Disease index statistics. C：Identification by stem anatomy. D：Restoration culture of pathogenic bacteria

References 38

[1]	Li FG, Fan GY, Lu CR, et al. Genome sequence of cultivated Upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution[J]. Nat Biotechnol, 2015, 33(5):524-530. doi: 10.1038/nbt.3208 URL
[2]	Yang CL, Liang S, Wang HY, et al. Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against Verticillium dahliae[J]. Mol Plant, 2015, 8(3):399-411. doi: 10.1016/j.molp.2014.11.023 URL
[3]	Gong Q, Yang ZE, Wang XQ, et al. Salicylic acid-related cotton(Gossypium arboreum)ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae[J]. BMC Plant Biol, 2017, 17(1):59. doi: 10.1186/s12870-017-1007-5 URL
[4]	He X, Kang Y, Li WQ, et al. Genome-wide identification and functional analysis of the TIFY gene family in the response to multiple stresses in Brassica napus L[J]. BMC Genomics, 2020, 21(1):736. doi: 10.1186/s12864-020-07128-2 URL
[5]	Bai YH, Meng YJ, Huang DL, et al. Origin and evolutionary analysis of the plant-specific TIFY transcription factor family[J]. Genomics, 2011, 98(2):128-136. doi: 10.1016/j.ygeno.2011.05.002 URL
[6]	Nishii A, Takemura M, Fujita H, et al. Characterization of a novel gene encoding a putative single zinc-finger protein, ZIM, expressed during the reproductive phase in Arabidopsis thaliana[J]. Biosci Biotechnol Biochem, 2000, 64(7):1402-1409. doi: 10.1271/bbb.64.1402 URL
[7]	杨锐佳, 张中保, 吴忠义. 植物转录因子TIFY家族蛋白结构和功能的研究进展[J]. 生物技术通报, 2020, 36(12):121-128. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0307
	Yang RJ, Zhang ZB, Wu ZY. Progress of the structural and functional analysis of plant transcription factor TIFY protein family[J]. Biotechnol Bull, 2020, 36(12):121-128.
[8]	Meng L, Zhang T, Geng SS, et al. Comparative proteomics and metabolomics of JAZ7-mediated drought tolerance in Arabidopsis[J]. J Proteomics, 2019, 196:81-91. doi: S1874-3919(19)30036-3 pmid: 30731210
[9]	Ebel C, BenFeki A, Hanin M, et al. Characterization of wheat(Tri-ticum aestivum)TIFY family and role of Triticum Durum TdTIFY11a in salt stress tolerance[J]. PLoS One, 2018, 13(7):e0200566. doi: 10.1371/journal.pone.0200566 URL
[10]	Zhu D, Li RT, Liu X, et al. The positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress[J]. PLoS One, 2014, 9(11):e111984. doi: 10.1371/journal.pone.0111984 URL
[11]	Zhao CY, Pan XW, Yu Y, et al. Overexpression of a TIFY family gene, GsJAZ2, exhibits enhanced tolerance to alkaline stress in soybean[J]. Mol Breed, 2020, 40(3):1-13. doi: 10.1007/s11032-019-1080-6 URL
[12]	Seo JS, Joo J, Kim MJ, et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice[J]. Plant J, 2011, 65(6):907-921. doi: 10.1111/j.1365-313X.2010.04477.x URL
[13]	Yang YX, Ahammed GJ, Wan CP, et al. Comprehensive analysis of TIFY transcription factors and their expression profiles under jasmonic acid and abiotic stresses in watermelon[J]. Int J Genomics, 2019, 2019:6813086.
[14]	Zhang ZB, Li XL, Yu R, et al. Isolation, structural analysis, and expression characteristics of the maize TIFY gene family[J]. Mol Genet Genomics, 2015, 290(5):1849-1858. doi: 10.1007/s00438-015-1042-6 URL
[15]	Yu YH, Wan YT, Jiao ZL, et al. Functional characterization of resistance to powdery mildew of VvTIFY9 from Vitis vinifera[J]. Int J Mol Sci, 2019, 20(17):4286. doi: 10.3390/ijms20174286 URL
[16]	刘嘉鹏, 谭秋月, 伍俊为, 等. 香蕉MaTIFY9基因的克隆及表达[J]. 应用与环境生物学报, 2021, 27(6):1609-1615.
	Liu JP, Tan QY, Wu JW, et al. Cloning and expression analysis of the MaTIFY9 gene in banana[J]. Chin J Appl Environ Biol, 2021, 27(6):1609-1615.
[17]	胡子曜, 代培红, 刘超, 等. 陆地棉小GTP结合蛋白基因GhROP3的克隆、表达及VIGS载体的构建[J]. 生物技术通报, 2021, 37(9):106-113. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0287
	Hu ZY, Dai PH, Liu C, et al. Molecular cloning, expression and VIGS construction of a small GTP-binding protein gene GhROP3 in Gossypium hirsutum[J]. Biotechnol Bull, 2021, 37(9):106-113.
[18]	Li Y, Zhou YJ, Dai PH, et al. Cotton Bsr-k1 modulates lignin deposition participating in plant resistance against Verticillium dahliae and Fusarium oxysporum[J]. Plant Growth Regul, 2021, 95(2):283-292. doi: 10.1007/s10725-021-00742-4 URL
[19]	袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4):427-436. doi: 10.3724/SP.J.1259.2012.00427
	Yuan W, Wan HJ, Yang YJ. Characterization and selection of reference genes for real-time quantitative RT-PCR of plants[J]. Chin Bull Bot, 2012, 47(4):427-436.
[20]	Livak KJ, Schmittgen TD. 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 pmid: 11846609
[21]	王钰静. GhROP6在棉花抗黄萎病中的功能研究[D]. 武汉: 华中农业大学, 2017.
	Wang YJ. Functional analysis of GhROP6 in cotton responsive to Verticillium dahliae[D]. Wuhan: Huazhong Agricultural University, 2017.
[22]	Zhang DD, Wang J, Wang D, et al. Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae[J]. New Phytol, 2019, 222(2):1012-1029. doi: 10.1111/nph.15672 URL
[23]	Hrmova M, Hussain SS. Plant transcription factors involved in drought and associated stresses[J]. Int J Mol Sci, 2021, 22(11):5662. doi: 10.3390/ijms22115662 URL
[24]	Yu FF, Huaxia YF, Lu WJ, et al. GhWRKY15, a member of the WR-KY transcription factor family identified from cotton(Gossypium hirsutum L. ), is involved in disease resistance and plant develop-ment[J]. BMC Plant Biol, 2012, 12:144. doi: 10.1186/1471-2229-12-144 URL
[25]	Ma Q, Wang NH, Ma L, et al. The cotton BEL1-like transcription factor GhBLH7-D06 negatively regulates the defense response against Verticillium dahliae[J]. Int J Mol Sci, 2020, 21(19):7126. doi: 10.3390/ijms21197126 URL
[26]	Hu Q, Xiao SH, Wang XR, et al. GhWRKY1-like enhances cotton resistance to Verticillium dahliae via an increase in defense-induced lignification and S monolignol content[J]. Plant Sci, 2021, 305:110833. doi: 10.1016/j.plantsci.2021.110833 URL
[27]	Xiao SH, Hu Q, Shen JL, et al. GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae[J]. Plant Cell Rep, 2021, 40(4):735-751. doi: 10.1007/s00299-021-02672-x URL
[28]	He X, Wang TY, Zhu W, et al. GhHB12, a HD-ZIP I transcription factor, negatively regulates the cotton resistance to Verticillium dahliae[J]. Int J Mol Sci, 2018, 19(12):3997. doi: 10.3390/ijms19123997 URL
[29]	Liu TL, Chen TZ, Kan JL, et al. The GhMYB36 transcription factor confers resistance to biotic and abiotic stress by enhancing PR1 gene expression in plants[J]. Plant Biotechnol J, 2021:2021 Nov 23.
[30]	Xiong XP, Sun SC, Zhang XY, et al. GhWRKY70D13 regulates resistance to Verticillium dahliae in cotton through the ethylene and jasmonic acid signaling pathways[J]. Front Plant Sci, 2020, 11:69. doi: 10.3389/fpls.2020.00069 URL
[31]	He X, Zhu LF, Xu L, et al. GhATAF1, a NAC transcription factor, confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J]. Plant Cell Rep, 2016, 35(10):2167-2179. doi: 10.1007/s00299-016-2027-6 URL
[32]	Cheng HQ, Han LB, Yang CL, et al. The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection[J]. J Exp Bot, 2016, 67(6):1935-1950. doi: 10.1093/jxb/erw016 URL
[33]	Zhang K, Zhao P, Wang HM, et al. Isolation and characterization of the GbVIP1 gene and response to Verticillium wilt in cotton and tobacco[J]. J Cotton Res, 2019, 2:2. doi: 10.1186/s42397-019-0019-0 URL
[34]	Guo WF, Jin L, Miao YH, et al. An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis[J]. Plant Mol Biol, 2016, 91(3):305-318. doi: 10.1007/s11103-016-0467-6 URL
[35]	Li XC, Liu NN, Sun Y, et al. The cotton GhWIN2 gene activates the cuticle biosynthesis pathway and influences the salicylic and jasmonic acid biosynthesis pathways[J]. BMC Plant Biol, 2019, 19(1):379. doi: 10.1186/s12870-019-1888-6 URL
[36]	He X, Zhu LF, Wassan GM, et al. GhJAZ2 attenuates cotton resistance to biotic stresses via the inhibition of the transcriptional activity of GhbHLH171[J]. Mol Plant Pathol, 2018, 19(4):896-908. doi: 10.1111/mpp.12575 URL
[37]	张祥瑞. 棉花抗黄萎病相关基因GhJAZ4和GhbHLH25的功能初探[D]. 开封: 河南大学, 2018.
	Zhang XR. Preliminary study on functions of GhJAZ4 and GhbHLH25 genes related to Verticillium wilt resistance in cotton[D]. Kaifeng: Henan University, 2018.
[38]	Song Y, Zhai YH, Li LX, et al. BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton[J]. Plant Biotechnol J, 2021, 19(10):2097-2112. doi: 10.1111/pbi.13640 URL