Cloning,Expression and Functional Analysis of HcMS1 Gene from Kenaf

doi:10.7668/hbnxb.20192151

Abstract

Abstract: To explore the male sterile gene of kenaf and explain the molecular mechanism of flower bud abortion, the transcriptome data was analyzed and the homologous male sterile gene of Arabidopsis thaliana MALE STERILITY1(MS1) was cloned from kenaf anther by homologous cloning, named HcMS1. The structure, physicochemical properties and genetic relationship of HcMS1 protein were analyzed by bioinformatics method, and the expression level of HcMS1 gene in kenaf male sterile line and maintainer at different stages was analyzed by qRT-PCR. Through the construction of over-expression vector, the transgenic tobacco was transformed by leaf disc method, and the phenotype of transgenic tobacco was observed. The open reading frame(ORF) of HcMS1 was 1 950 bp, encoding 649 amino acids. Bioinformatics analysis showed that HcMS1 protein was hydrophilic, located in the nucleus and contained typical PHD-finger domain, without signal peptide and transmembrane domain structure. It had the closest relationship with Gossypium hirsutum MS1 protein, followed by Hibiscus syriacus MS1 protein. qRT-PCR results showed that the expression level of HcMS1 gene in maintainer and male sterile line increased first and then decreased, and the highest expression level was found in the tetrad to mononuclear stage of kenaf buds, which was consistent with the expression pattern of MS1 in Arabidopsis thaliana;In addition, the expression level of HcMS1 gene in maintainer was significantly higher than that in male sterile line, suggesting that the kenaf abortion was related to the low expression of HcMS1 gene. Through genetic transformation experiment, it was found that HcMS1 transgenic tobacco had shorter plant type, different calyx size, shorter anther length and could not produce seeds, which indicated that expression of kenaf HcMS1 heterologous gene could affect the normal fertility of tobacco.We successfully cloned HcMS1 gene from kenaf anther, and analyzed its function, which provided a theoretical basis for subsequent kenaf male sterility breeding.

Key words: Kenaf, Male sterility, HcMS1 gene, Bioinformatics analysis, Expression analysis, Tobacco transformation

CLC Number:

Q78
S563.03

CHEN Wentao, PAN Gen, TANG Huijuan, CHANG Li, LI Defang, ZHAO Lining, CHEN Anguo, LI Jianjun. Cloning,Expression and Functional Analysis of HcMS1 Gene from Kenaf[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 64-74. doi: 10.7668/hbnxb.20192151.

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References

[1] 陶爱芬, 张晓琛, 祁建民. 红麻综合利用研究进展与产业化前景[J]. 中国麻业科学, 2007, 29(1):1-5.doi:10.3969/j.issn.1671-3532.2007.01.001.
Tao A F, Zhang X C, Qi J M. Research progress and industrialization prospect of comprehensive utilization on kenaf[J]. Plant Fiber Sciences in China, 2007, 29(1):1-5.
[2] 李文略, 金关荣, 骆霞虹, 安霞, 李苹芳, 朱关林, 陈常理. 不同红麻品种的土壤重金属污染修复潜力对比研究[J]. 农业环境科学学报, 2018, 37(10):2150-2158.doi:10.11654/jaes.2018-0381.
Li W L, Jin G R, Luo X H, An X, Li P F, Zhu G L, Chen C L. Comparative study on the potential of a kenaf(Hibiscus cannabinus) variety for remediating heavy metal con-taminated soils[J]. Journal of Agro-Environment Science, 2018, 37(10):2150-2158.
[3] 祁建民, 陈幼玉, 周瑞阳, 林荔辉, 梁康迳, 吴建梅, 方平平. 红麻产量和纤维品质性状的遗传效应与杂种优势分析[J]. 作物学报, 2005, 31(4):469-475.doi:10.3321/j.issn:0496-3490.2005.04.012.
Qi J M, Chen Y Y, Zhou R Y, Lin L H, Liang K J, Wu J M, Fang P P. Genetic effects and heterosis analysis for yield and quality traits in kenaf(Hibiscus cannabinus L.) [J]. Acta Agronomica Sinica, 2005, 31(4):469-475.
[4] 石子, 宋伟, 赵久然. 雄性不育在作物杂种优势中的应用途径分析[J]. 中国生物工程杂志, 2018, 38(1):126-134.doi:10.13523/j.cb.20180115.
Shi Z, Song W, Zhao J R. Application of male sterility in crop heterosis[J]. China Biotechnology, 2018, 38(1):126-134.
[5] 赵艳红. 红麻atp9、atp6、atpA 基因克隆、功能分析与分子标签发掘[D].南宁:广西大学, 2014:83-99.doi:10.7666/d.D523541.
Zhao Y H. Cloning and function analysis of mitochondrial genes related to CMS and further development of two molecular markers distinctively to male sterile cytoplasm in kenaf(Hibiscus cannabinus L.) [D].Nanning:Guangxi University, 2014:83-99.
[6] 史奇奇. 红麻细胞质雄性不育系和保持系microRNA表达谱及功能研究[D].南宁:广西大学, 2018:4-5.
Shi Q Q. Study on the expression profile and function of microRNA in cytoplasmic male sterile line and maintainer line of kenaf[D].Nanning:Guangxi University, 2018:4-5.
[7] 韦范. 红麻蛋白乙酰化修饰调控细胞质雄性不育及相关功能研究[D].南宁:广西大学, 2020:4-6.
Wei F. Study of the proteinacetylation on regulation of kenaf cytoplasmic male sterility and its related functions[D].Nanning:Guangxi University, 2020:4-6.
[8] 李增强, 史奇奇, 孔祥军, 汤丹峰, 廖小芳, 韦范, 何冰, 莫良玉, 周瑞阳, 陈鹏. 红麻不育系与保持系基因组DNA甲基化比较分析[J]. 中国农业大学学报, 2017, 22(11):17-27.doi:10.11841/j.issn.1007-4333.2017.11.02.
Li Z Q, Shi Q Q, Kong X J, Tang D F, Liao X F, Wei F, He B, Mo L Y, Zhou R Y, Chen P. Comparative analysis on the kenaf (Hibiscus cannabinus L.) genomic DNA methylation of its male sterility line and maintainer line[J]. Journal of China Agricultural University, 2017, 22(11):17-27.
[9] 钱景华, 周步进, 孔祥军, 李增强, 史奇奇, 廖小芳, 周瑞阳, 陈鹏. 红麻MYB21 基因的克隆、表达及载体构建[J]. 生物技术通报, 2016, 32(11):170-179.doi:10.13560/j.cnki.biotech.bull.1985.2016.11.020.
Qian J H, Zhou B J, Kong X J, Li Z Q, Shi Q Q, Liao X F, Zhou R Y, Chen P. Cloning, expression analysis and vector construction of MYB21 gene in kenaf[J]. Biotechnology Bulletin, 2016, 32(11):170-179.
[10] 陈励虹, 周步进, 周瑞阳. 红麻TIR1 基因克隆及其表达载体构建[J]. 南方农业学报, 2017, 48(8):1343-1350.doi:10.3969/j.issn.2095-1191.2017.08.03.
Chen L H, Zhou B J, Zhou R Y. Cloning of TIR1 gene in Hibiscus cannabinus L. and construction of its expression vector[J]. Journal of Southern Agriculture, 2017, 48(8):1343-1350.
[11] Chen P, Ran S M, Li R, Huang Z P, Qian J H, Yu M L, Zhou R Y. Transcriptome de novo assembly and differentially expressed genes related to cytoplasmic male sterility in kenaf(Hibiscus cannabinus L.) [J]. Molecular Breeding, 2014, 34(4):1879-1891.doi:10.1007/s11032-014-0146-8.
[12] 翟晓光, 辛芳, 韩玉翠, 夏雪姣, 朱婷, 菅明阳, 丁勤, 马翎健. K型小麦雄性不育系绒毡层结构变化及相关基因的表达分析[J]. 麦类作物学报, 2018, 38(12):1400-1407.doi:10.7606/j.issn.1009-1041.2018.12.02.
Zhai X G, Xin F, Han Y C, Xia X J, Zhu T, Jian M Y, Ding Q, Ma L J. Change of tapetum structure and related gene expression in K-type male sterile wheat[J]. Journal of Triticeae Crops, 2018, 38(12):1400-1407.
[13] Zhu J, Lou Y, Xu X F, Yang Z N. A genetic pathway for tapetum development and function in Arabidopsis[J]. Journal of Integrative Plant Biology, 2011, 53(11):892-900.doi:10.1111/j.1744-7909.2011.01078.x.
[14] Li D D, Xue J S, Zhu J, Yang Z N. Gene regulatory network for tapetum development in Arabidopsis thaliana[J]. Frontiers in Plant Science, 2017, 8:1559.doi:10.3389/fpls.2017.01559.
[15] Lu J Y, Xiong S X, Yin W Z, Teng X D, Lou Y, Zhu J, Zhang C, Gu J N, Wilson Z A, Yang Z N. MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis[J]. Journal of Experimental Botany, 2020, 71(16):4877-4889.doi:10.1093/jxb/eraa219.
[16] Wilson Z A, Morroll S M, Dawson J, Swarup R, Tighe P J. The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors[J]. The Plant Journal, 2001, 28(1):27-39.doi:10.1046/j.1365-313X.2001.01125.x.
[17] 李建军, 唐慧娟, 陈艳翠, 陈安国, 黄思齐, 李德芳. 质核互作型红麻雄性不育系细胞学形态观察[J]. 中国麻业科学, 2015, 37(5):229-232.doi:10.3969/j.issn.1671-3532.2015.05.002.
Li J J, Tang H J, Chen Y C, Chen A G, Huang S Q, Li D F. Cytomorphological observation on cytoplasm nuclear male sterile line of kenaf[J]. Plant Fiber Sciences in China, 2015, 37(5):229-232.
[18] 王会强. 根癌农杆菌介导的花青素调节基因Lc-NtAn2 转化烟草的研究[D].杨凌:西北农林科技大学, 2013:22-23.
Wang H Q. Study on Agrobacterium- mediated transformation of anthocyanin regulatory gene Lc-NtAn2 into tobacco[D].Yangling:Northwest A&F University, 2013:22-23.
[19] 朱丽梅, 艾素云, 周瑞阳. 红麻细胞质雄性不育系小孢子败育的细胞学观察[J]. 作物学报, 2007, 33(6):999-1003.doi:10.3321/j.issn:0496-3490.2007.06.022.
Zhu L M, Ai S Y, Zhou R Y. A cytological study on microsporogenesis of cytoplasmic male sterile lines in kenaf(Hibiscus cannabinus L.) [J]. Acta Agronomica Sinica, 2007, 33(6):999-1003.
[20] Ito T, Shinozaki K. The MALE STERILITY1 gene of Arabidopsis, encoding a nuclear protein with a PHD-finger motif, is expressed in tapetal cells and is required for pollen maturation[J]. Plant & Cell Physiology, 2002, 43(11):1285-1292.doi:10.1093/pcp/pcf154.
[21] Ito T, Nagata N, Yoshiba Y, Ohme-Takagi M, Ma H, Shinozaki K. Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development[J]. The Plant Cell, 2007, 19(11):3549-3562.doi:10.1105/tpc.107.054536.
[22] Vizcay-Barrena G, Wilson Z A. Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant[J]. Journal of Experimental Botany, 2006, 57(11):2709-2717.doi:10.1093/jxb/erl032.
[23] Li H, Yuan Z, Vizcay-Barrena G, Yang C Y, Liang W Q, Zong J, Wilson Z A, Zhang D B. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice[J]. Plant Physiology, 2011, 156(2):615-630.doi:10.1104/pp.111.175760.
[24] Wang Z, Li J, Chen S X, Heng Y F, Chen Z, Yang J, Zhou K J, Pei J W, He H, Deng X W, Ma L G. Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility in bread wheat[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(47):12614-12619.doi:10.1073/pnas.1715570114.
[25] Tucker E J, Baumann U, Kouidri A, Suchecki R, Baes M, Garcia M, Okada T, Dong C M, Wu Y Z, Sandhu A, Singh M, Langridge P, Wolters P, Albertsen M C, Cigan A M, Whitford R. Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding[J]. Nature Communications, 2017, 8(1):869.doi:10.1038/s41467-017-00945-2.
[26] 唐文武, 吴秀兰, 李桂花. 芸薹属作物花粉发育相关基因MS1 的生物信息学分析[J]. 南方农业学报, 2017, 48(12):2122-2128.doi:10.3969/j.issn.2095-1191.2017.12.02.
Tang W W, Wu X L, Li G H. Bioinformatics analysis for pollen development related gene MS1 of Brassica crops[J]. Journal of Southern Agriculture, 2017, 48(12):2122-2128.
[27] 高嵩, 吕庆雪, 何欢, 张建新, 张志军, 宋广树, 刘伟. 玉米中绒毡层发育调控基因ZmUdt1 克隆的生物信息学分析[J]. 华北农学报, 2018, 33(1):52-59.doi:10.7668/hbnxb.2018.01.009.
Gao S, Lü Q X, He H, Zhang J X, Zhang Z J, Song G S, Liu W. Bioinformatics analysis of ZmUdt1 gene in maize tapetum development[J]. Acta Agriculturae Boreali-Sinica, 2018, 33(1):52-59.
[28] 许明, 林世强, 倪冬昕, 伊恒杰, 刘江洪, 杨志坚, 郑金贵. 藤茶查尔酮合成酶基因AgCHS1 的克隆及功能鉴定[J]. 中国农业科学, 2020, 53(24):5091-5103.doi:10.3864/j.issn.0578-1752.2020.24.012.
Xu M, Lin S Q, Ni D X, Yi H J, Liu J H, Yang Z J, Zheng J G. Cloning and function characterization of chalcone synthase gene AgCHS1 in Ampelopsis grossedentata[J]. Scientia Agricultura Sinica, 2020, 53(24):5091-5103.
[29] Joshi P S, Agarwal P, Agarwal P K. Overexpression of AlNAC1 from recretohalophyte Aeluropus lagopoides alleviates drought stress in transgenic tobacco[J]. Environmental and Experimental Botany, 2021, 181:104277.doi:10.1016/j.envexpbot.2020.104277.

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