十倍体长穗偃麦草雄性育性基因ThMs1的克隆、表达及功能分析

doi:10.3864/j.issn.0578-1752.2020.23.001

摘要/Abstract

摘要：

【目的】从小麦隐性核雄性不育突变体ms1与十倍体长穗偃麦草异附加系中,克隆雄性育性恢复基因ThMs1,解析该基因的表达模式、编码蛋白的生物化学活性和生物学功能,鉴定新的小麦ms1育性恢复基因,从而更好地应用于小麦杂交育种。【方法】通过基因组原位杂交（genomic in situ hybridization,GISH）,鉴定ms1与十倍体长穗偃麦草异附加系中小麦外源的染色体类型;通过同源克隆法在小麦-十倍体长穗偃麦草异附加系中克隆雄性育性基因ThMs1,并稳定转化小麦ms1进行基因功能互补验证;利用RT-PCR及实时荧光定量PCR分析检测基因的表达模式;进一步通过RNA原位杂交分析基因的组织细胞特异性表达特性;利用特异性抗体进行Western blot检测ThMs1蛋白在体内的表达情况;经SignalP软件分析预测蛋白结构;通过蛋白-脂质结合活性分析检测ThMs1蛋白是否具有脂类分子结合活性。【结果】证实小麦ms1与十倍体长穗偃麦草异附加系细胞中含有偃麦草4Ag染色体;从小麦-十倍体长穗偃麦草异附加系中克隆了雄性育性基因ThMs1,遗传转化功能互补试验证实ThMs1能够完全恢复小麦ms1突变体的雄性不育表型,基因的聚类分析表明MS1只存在于禾本科植物中;通过RT-PCR和实时荧光定量PCR检测发现ThMs1在减数分裂期的花药中特异表达,RNA原位杂交分析证实ThMs1在小麦小孢子发生过程中特异表达;Western blot检测发现ThMs1在小麦减数分裂期的花药中表达;对ThMs1蛋白氨基酸序列进行结构预测发现,该蛋白具有推测的脂类结合分子结构域,ThMs1蛋白脂类分子结合试验表明ThMs1蛋白特异结合磷脂酸和磷酸化的磷脂酰肌醇。【结论】克隆了一个新的来自十倍体长穗偃麦草、可恢复小麦隐性核雄性不育突变体ms1雄性育性的ThMs1,该基因与小麦Ms1具有相似的分子结构、时空表达模式和脂结合活性,导致2个蛋白在小麦中也具有相似的生物学功能,ThMs1可以替代普通小麦Ms1的功能调控小麦花粉育性,该结果为利用小麦ms1通过分子设计建立小麦杂交育种体系（新一代杂交育种体系）提供了一个新的育性恢复基因。

关键词: 普通小麦, 小麦-十倍体长穗偃麦草异附加系, 隐性核雄性不育, ThMs1, Ms1, 小麦杂交育种体系, 分子设计育种

Abstract:

【Objective】To clone a new male fertility gene, ThMs1, from the ms1g-4Ag wheat-Thinopyrum ponticum alien addition line, and characterize the expression pattern and biological function of ThMs1 in bread wheat, with the hope that ThMs1 will be useful in the application in the preparation of wheat hybrid breeding system. 【Method】4Ag chromosome from Thinopyrum ponticum in wheat-Thinopyrum ponticum alien addition line was identified by GISH. ThMs1 was cloned from the wheat-Thinopyrum ponticum alien addition line by homology-based cloning, and the analysis of the function of ThMs1 in the control of wheat male fertility was performed through stable genetic transformation. RT-PCR and qRT-PCR were performed to detect the expression pattern of ThMs1, further analyzing its tissue-specific expression by RNA in situ hybridization. The expression of ThMs1 protein in vivo was detected by Western blot using specific anti-Ms1 antibodies. ThMs1 protein structure were predicted by SignalP software. The detection of lipid-binding activity of ThMs1 was performed by dot blot. 【Result】The wheat-Thinopyrum ponticum alien addition line harbored 4Ag chromosome from Thinopyrum ponticum. ThMs1, the homolog of bread wheat male fertility gene Ms1, was cloned from the wheat-Thinopyrum ponticum alien addition line. The functional complementation analysis confirmed that the ThMs1 can completely restore the male fertility phenotype of the wheat ms1. The phylogenetic analysis suggested that the MS1 only exists in the Poaceae family. RT-PCR and qRT-PCR analysis confirmed that ThMs1 gene was expressed in anthers during meiosis. RNA in situ hybridization analysis revealed that ThMs1 was specifically expressed in microspore during wheat microsporogenesis. Western blot showed that ThMs1 was detected in wheat anther. The structure prediction analysis revealed that it has a predicted lipid binding domain, and protein lipid-binding experiments indicated that ThMs1 protein specifically binds PA and several phosphatidylinositols. 【Conclusion】A new male fertility gene, ThMs1, was cloned from wheat-Thinopyrum ponticum alien additiona line. ThMs1 exhibits similar expression pattern to that of Ms1 in bread wheat, and ThMs1 binds to the same phospholipid molecules as Ms1, thus ThMs1 is a structural and functional homolog of wheat Ms1. Therefore, the present work identified a new male fertility gene, and provided a foundation for the establishment of wheat hybrid breeding system through molecular design using wheat ms1.

Key words: bread wheat, wheat-Thinopyrum ponticum alien addition line, recessive nuclear male sterility, ThMs1, Ms1, wheat hybrid breeding system, molecular design

衡燕芳,李健,王峥,陈卓,何航,邓兴旺,马力耕. 十倍体长穗偃麦草雄性育性基因ThMs1的克隆、表达及功能分析[J]. 中国农业科学, 2020, 53(23): 4727-4737.

HENG YanFang,LI Jian,WANG Zheng,CHEN Zhuo,HE Hang,DENG XingWang,MA LiGeng. Cloning, Expression and Functional Analysis of a Male Fertility Gene ThMs1 in Bread Wheat[J]. Scientia Agricultura Sinica, 2020, 53(23): 4727-4737.

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参考文献 36

[1]	TILMAN D, CASSMAN K G, MATSON P A, NAYLOR R, POLASKY S . Agricultural sustainability and intensive production practices. Nature, 2002,418:671-677. doi: 10.1038/nature01014 pmid: 12167873
[2]	FOLEY J A, RAMANKUTTY N, BRAUMAN K A, CASSIDY E S, GERBER J S, JOHNSTON M, MUELLER N D, O’CONNELL C, RAY D K, WEST P C, BALZER C, BENNETT E M, CARPENTER S R, HILL J, MONFREDA C, POLASKY S, ROCKSTRÖM J, SHEEHAN J, SIEBERT S, TILMAN D, ZAKS D P M . Solutions for a cultivated planet. Nature, 2011,478:337-342. doi: 10.1038/nature10452 pmid: 21993620
[3]	IWGSC. A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science, 2014,345:1251788. doi: 10.1126/science.1251788 pmid: 25035500
[4]	CHANG Z Y, CHEN Z F, WANG N, XIE G, LU J W, YAN W, ZHOU J L, TANG X Y, DENG X W . Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proceedings of the National Academy of Sciences of USA, 2016,113:14145-14150. doi: 10.1073/pnas.1613792113
[5]	邓兴旺, 王海洋, 唐晓艳, 周君莉, 陈浩东, 何光明, 陈良碧, 许智宏 . 杂交水稻育种将迎来新时代. 中国科学: 生命科学, 2013,43:864-868. doi: 10.1360/052013-299
	DENG X W, WANG H Y, TANG X Y, ZHOU J L, CHEN H D, HE G M, CHEN L B, XU Z H . Hybrid rice breeding welcomes a new era of molecular crop design. Scientia Sinica Vitae, 2013,43:864-868. (in Chinese) doi: 10.1360/052013-299
[6]	CEOLONI C, FORTE P, KUZMANOVIĆ L, TUNDO S, MOSCETTI I, VITA D P, VIRILI M E, OVIDIO R D . Cytogenetic mapping of a major locus for resistance to Fusarium head blight and crown rot of wheat onThinopyrum elongatum 7EL and its pyramiding with valuable genes from a Th. ponticum homoeologous arm onto bread wheat 7DL. Theoretical and Applied Genetics, 2017,130(10):2005-2024. doi: 10.1007/s00122-017-2939-8 pmid: 28656363
[7]	ZHANG X Y, LI Z S, CHEN S Y . Production and identification of three 4Ag (4D) substitution lines of Triticum aestivum-Agropyron: relative transmission rate of alien chromosomes. Theoretical and Applied Genetics, 1992,83:707-714. doi: 10.1007/BF00226688 pmid: 24202744
[8]	WANG S W, WANG C Y, WANG Y Z, WANG Y J, CHEN C H, JI W Q . Molecular cytogenetic identification of two wheat-Thinopyrum ponticum substitution lines conferring stripe rust resistance. Molecular Breeding, 2019,39:143-153. doi: 10.1007/s11032-019-1053-9
[9]	KLINDWORTH D L, WILLIAMS N D, MAAN S S . Chromosomal location of genetic male sterility genes in four mutants of hexaploid wheat. Crop Science, 2002,42:1447-1450. doi: 10.2135/cropsci2002.1447
[10]	MCINTOSH R A, YAMAZAKI Y, DUBCOVSKY J, ROGERS J, MORRIS C, APPELS R, XIA X C . Catalogue of gene symbols for wheat// Proceedings of the 12th International Wheat Genetics Symposium. Yokohama, Japan, 2013.
[11]	NI F, QI J, HAO Q, LYU B, LUO M, WANG Y, CHEN F, WANG S, ZHANG C, EPSTEIN L, ZHAO X, WANG H, ZHANG X, CHEN C, SUN L, FU D . Wheat Ms2 encodes for an orphan protein that confers male sterility in grass species. Nature Communications, 2017,8:15121-15132. doi: 10.1038/ncomms15121 pmid: 28452349
[12]	XIA C, ZHANG L C, ZOU C, GU Y Q, DUAN J L, ZHAO G Y, WU J J, LIU Y, FANG X H, GAO L F, JIAO Y N, SUN J Q, PAN Y H, LIU X, JIA J Z, KONG X Y . A TRIM insertion in the promoter of Ms2 causes male sterility in wheat. Nature Communications, 2017,8:15407-15416. doi: 10.1038/ncomms15407 pmid: 28497807
[13]	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. Proceedings of the National Academy of Sciences of USA, 2017,114:12614-12619. doi: 10.1073/pnas.1715570114
[14]	TUCKER E J, BAUMANU U, KOUIDRI A, SUCHECKI R, BAES M, GARCIA M, OKADA T, DONG C, WU Y, SANDHU A, CIGAN A M, WHITFORD R . Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding. Nature Communication, 2017,8:869-879. doi: 10.1038/s41467-017-00945-2
[15]	PALLOTTA M A, WARNER P, KOUIDRI A, TUCKER E J, MATHIEU B, SUCHECKI R, WATSON-HAIGH N, OKADA T, GARCIA M, SANDHU A, SINGH M, WOLTERS P, ALBERTSEN M C, CIGANA M, BAUMANN U, WHITFORD R . Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins. The Plant Journal, 2019,99:673-685. doi: 10.1111/tpj.14350 pmid: 31009129
[16]	LIU L, LUO Q, LI H, LI B, LI Z, ZHENG Q . Physical mapping of the blue-grained gene from Thinopyrum ponticum chromosome 4Ag and development of blue-grain-related molecular markers and a FISH probe based on SLAF-seq technology. Theoretical and Applied Genetics, 2018,122:1007-1017.
[17]	刘成, 韩冉, 汪晓璐, 宫文萍, 程敦公, 曹新有, 刘爱峰, 李豪圣, 刘建军 . 小麦远缘杂交现状、抗病基因转移及利用研究进展. 中国农业科学, 2020,53(7):1287-1308. doi: 10.3864/j.issn.0578-1752.2020.07.001
	LIU C, HAN R, WANG X L, GONG W P, CHENG D G, CAO X Y, LIU A F, LI H S, LIU J J . Research progress of wheat wild hybridization, disease resistance genes transfer and utilization. Scientia Agricultura Sinica, 2020,53(7):1287-1308. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.07.001
[18]	ZHANG W, CAO Y P, ZHANG M Y, ZHU X W, REN S F, LONG Y M, GYAWALI Y, CHAO S, XU S, CAI X W . Meiotic homoeologous recombination-based alien gene introgression in the genomics era of wheat. Crop Science, 2017,57:1189-1198. doi: 10.2135/cropsci2016.09.0819
[19]	KUZMANOVIC L, MANDALÀ G, TUNDO S, CIORBA R, FRANGELLA M, RUGGERI R, ROSSINI F, GEVI F, RINALDUCCI S, CEOLONI C . Equipping durum wheat-Thinopyrum ponticum recombinant lines with a Thinopyrum elongatum major QTL for resistance to fusarium diseases through a cytogenetic strategy. Frontiers in Plant Science, 2019,10:1324. doi: 10.3389/fpls.2019.01324 pmid: 31695716
[20]	ZHAO J, HAO W W, TANG C G, YAO H, LI B C, ZHENG Q, LI Z S, ZHANG X Y . Plasticity in Triticeae centromere DNA sequences: a wheat 3 tall wheatgrass (decaploid) model. The Plant Journal, 2019,100:314-327. doi: 10.1111/tpj.14444 pmid: 31259444
[21]	SHITSUKAWA N, TAHIRA C, KASSAI K, HIRABAYASHI K, SHIMIZU T, TAKUMI S, MOCHIDA K, KAWAURA K, OGIHARA Y, MURAIA K . Genetic and epigenetic alteration among three homoeologous genes of a class E MADS Box gene in hexaploid wheat. The Plant Cell, 2007,19:1723-1737. doi: 10.1105/tpc.107.051813 pmid: 17586655
[22]	BENNING C . Mechanisms of lipid transport involved in organelle biogenesis in plant cells. Annual Review of Cell and Developmental Biology, 2009,25:71-91. doi: 10.1146/annurev.cellbio.042308.113414 pmid: 19572810
[23]	DOWLER S, KULAR G, ALESSI D R . Protein lipid overlay assay. Science’s Stke, 2002,129:16-26.
[24]	TESTER M, LANGRIDGE P . Breeding technologies to increase crop production in a changing world. Science, 2010,327:818-822. doi: 10.1126/science.1183700 pmid: 20150489
[25]	LI S, YANG D, ZHU Y . Characterization and use of male sterility in hybrid rice breeding. Journal of Integrative Plant Biology, 2007,49:791-804. doi: 10.1111/j.1744-7909.2007.00513.x
[26]	FREEMAN G F . Heredity of quantitative characters in wheat. Genetics, 1919,4:1-93. pmid: 17245919
[27]	SINGH S K, CHATRATH R, MISHRA B . Perspective of hybrid wheat research: A review. Indian Journal of Agricultural Sciences, 2010,80:1013-1027.
[28]	WHITFORD R, FLEURY D, REIF J C, GARCIA M, OKADA T, KORZUN V, LANGRIDGE P . Hybrid breeding in wheat: Technologies to improve hybrid wheat seed production. Journal of Experimental Botany, 2013,64:5411-5428. doi: 10.1093/jxb/ert333
[29]	SINGH S P, SINGH S P, PANDEY T, SINGH R R, SAWANT S V . A novel male sterility-fertility restoration system in plants for hybrid seed production. Scientific Reports, 2015,5:11274-11288. doi: 10.1038/srep11274 pmid: 26073981
[30]	KIM Y J, ZHANG D . Molecular control of male fertility for crop hybrid breeding. Trends in Plant Science, 2018,23:53-65. doi: 10.1016/j.tplants.2017.10.001 pmid: 29126789
[31]	FRANCKOWIAK J D, MAAN S S, WILLIAMS N D . A proposal for hybrid wheat utilizing Aegilops squarrosa L. cytoplasm. Crop Science, 1976,16:725-728. doi: 10.2135/cropsci1976.0011183X001600050033x
[32]	WILSON J A . Hybrid wheat breeding and commercial seed development. Plant Breeding Reviews, 1984,2:303-319.
[33]	PEREZ-PRAT E, VAN LOOKEREN CAMPAGNE M M . Hybrid seed production and the challenge of propagating male-sterile plants. Trends in Plant Science, 2002,7:199-203. doi: 10.1016/s1360-1385(02)02252-5 pmid: 11992824
[34]	成功海, 龚德平, 郭西陵, 邱文兵, 何庆虎 . C49S温光型两系杂种小麦在江汉平原生产应用中的几个问题. 麦类作物学报, 2005,25:147-148. doi: 10.7606/j.issn.1009-1041.2005.04.168
	CHENG G H, GONG D P, GUO X L, QIU W B, HE Q H . Problems of the hybrid with Chongqing thermo-photo-sensitive male sterility wheat C49S in the plain of Jianghan. Mailei Zuowu Xuebao, 2005,25:147-148. (in Chinese) doi: 10.7606/j.issn.1009-1041.2005.04.168
[35]	SINGH S P, SRIVASTAVA R, KUMAR J . Male sterility systems in wheat and opportunities for hybrid wheat development. Acta Physiologiae Plantarum, 2015,37:1713-1720. doi: 10.1007/s11738-014-1713-7
[36]	SONG S, WANG T, LI Y, HU J, KAN R, QIU M, DENG Y, LIU P, ZHANG L, DONG H, LI C, YU D, LI X, YUAN D, YUAN L, LI L . A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene. Plant Biotechnology Journal, 2020: 1-10. doi: 10.1111/j.1467-7652.2008.00392.x pmid: 19121104

引物 Primer	序列 Sequence (5'→3')	目的 Intention
ThMs1-F	AGATCCCGGCGCCTGCTGCTC	ThMs1扩增引物 PCR primer of ThMs1
ThMs1-R	CGCAGGAGCTGTAGAGCGTGAGGA	ThMs1扩增引物 PCR primer of ThMs1
BF	TAGCCATCTTTGATCAATGAGC	4Ag染色体鉴定 Analysis of 4Ag chromosome
BR	TGATGAAAGAGCTAGGTGATAGTTG	4Ag染色体鉴定 Analysis of 4Ag chromosome
TF1	CGCCAGGGTTTTCCCAGTCACGAC	转基因鉴定 Analysis of transgenic lines
TR1	TACAATGGCTAGTAGAGATTTC
TF2	ATACGTTTCCTGCTACAGATTTGAGG
TR2	AAGCAGTGCCGCCAGAGGATCAACGC
TF3	TTTGCGTTCTGCTGATGATGTG
TR3	CTCAATTGTCCTTTAGACCATGTCTAAC
TaACTIN-QF	TTCCGTTGCCCTGAGGTCC	RT-PCR和实时荧光定量PCR RT-PCR and real time PCR
TaACTIN-QR	TGATCTTCATGCTGCTTGGTGC
Ms1-QF	ACATCATCCTCTGAGTCGCG
Ms1-QR	GACCACGCAAACACGTACG
ThMs1-QF	TCCCGGCGCCTGCTGCTC
ThMs1-QR	CGGCAGAGGCAGGGGACG
Ms1-ISH-F	aagcttGAGCGAGGGAGAGAGAGACC	RNA原位杂交 In situ hybridization
Ms1-ISH-F	gaattcATCACATAGCATCAGTGGTTC
ThMs1-ISH-F	aagcttCTAGCGAGCGAGCGAGAGG
ThMs1-ISH-R	gaattcCAACTGGACGGACCATGGC
ThMs1-SB-F1	TGAGATATACGGAGCGATTTAG	Southern杂交 Southern blot
ThMs1-SB-R1	AGCACGGCAAGCTTTTGCTCTG	Southern杂交 Southern blot
His-F	aattcCATCATCATCATCATCACTGActgca	MBP-His载体构建 Vector construction of MBP-His
His-R	gTCAGTGATGATGATGATGATGg	MBP-His载体构建 Vector construction of MBP-His
Ms1s-His-F	gagggaaggatttcagaattcCAGCCGGGGGCGCCGTGC	MBP-Ms1-His载体构建Vector construction of MBP-Ms1-His
Ms1s-His-R	cagtgccaagcttgcctgcagTCAGTGATGATGATGATGATGGGCCGCCTTGGACGGCG	MBP-Ms1-His载体构建Vector construction of MBP-Ms1-His
ThMs1s-His-F	gagggaaggatttcagaattcGCGTTCGGGCCGCAGC	MBP-ThMs1-His载体构建 Vector construction of MBP-ThMs1-His
ThMs1s-His-R	cagtgccaagcttgcctgcagtcaGTGATGATGATGATGATGGAAGAAGGCCGCCTTGGACG	MBP-ThMs1-His载体构建 Vector construction of MBP-ThMs1-His