[目的]Tasselseed3 (Ts3)是玉米雄穗性别变异突变体，表现典型的雄穗结实（tasselseed）现象，说明Ts3基因是玉米雄穗性别决定的关键基因。本研究通过图位克隆方法对Ts3进行了精细定位，并对定位区间Ts3候选基因进行了表达分析，为最终克隆到Ts3基因、为解析玉米雄穗性别决定的遗传控制机制奠定了坚实基础。[结果]通过对野生型和突变体株高、节间长度、叶长叶宽等表型的统计分析，结果显示Ts3突变体明显的表现出矮化、节间长度缩短、叶短而宽的特点，暗示赤霉素信号可能与Ts3介导的玉米性别决定过程有关。经过本实验的前期研究，将控制玉米性别决定的关键基因Ts3初步定位在玉米1号染色体尾端一个6.9Mb的范围内，该范围内有145个已注释的基因。通过本次Ts3精细定位的研究，我们将定位区间缩小至1.8Mb的范围内，该定位区间内还有29个已注释基因，我们对这些已注释的基因在叶片、根尖、茎尖中的表达做了RT-PCR的检测。结果显示：叶片中，有22个基因检测到有表达，7个基因检测不到表达，在这22个基因中，1个基因的表达水平上升，3个基因的表达水平下降，18个基因的表达水平没有明显变化；在根尖中，有22个基因被检测到有表达，7个基因检测不到表达，在这22个基因里，2个基因的表达水平下降，20个基因的表达水平没有明显变化；在茎尖中，共检测到23个基因的表达，其中有6个基因的表达水平发生了变化，17个基因的表达水平没有发生明显的变化。接着，我们对WT材料进行了GA,JA处理，检测了定位区间的基因对GA,JA处理的反应，结果显示，赤霉素处理后，有21个基因有表达，8个基因没有检测到表达，在这些有表达的基因中，gene132、gene133、gene135，gene148的表达水平先提高再下降，其余的17个基因的表达没有发生明显的变化；茉莉酸处理后检测到，21个基因有表达，8个基因检测不到有表达，在这些有表达的基因中，gene120、gene159、gene164的表达水平先下降再升高，gene132、gene133、gene135三个基因的表达水平先升高后降低，其余的15个基因的表达水平没有发生明显的变化。综合以上结果，我们最终认为gene118、gene120、gene164为Ts3的最终候选基因，它们分别为ZM00001d033637、ZM00001d033635、ZM00001d033680。ZM00001d03367编码一种吡哆醛磷酸酶，是维生素B6代谢的主要酶类；ZM00001d033635编码了一种胞苷/脱氧胞苷脱氨酶，参与了细胞内嘧啶核苷的代谢与维持，其功能与逆境胁迫反向有关；ZM00001d033680为D8基因，编码赤霉素信号的负调控因子DELLA蛋白。说明Ts3可能是参与了赤霉素的信号通路途径从而影响雄穗的性别决定发育。[结论]通过对Ts3精细定位的研究，我们将Ts3基因定位在一号染色体G-273到G305标记之间的一个1.8Mb的范围内，通过该区间内基因表达分析和突变体的表型分析，我们认为gene118、gene120、gene164为Ts3的最终候选基因，它们分别为ZM00001d033637、ZM00001d033635、ZM00001d033680。通过对这三个基因的同源性分析，我们推测了他们可能的功能，ZM00001d033637编码一种吡哆醛磷酸酶，是维生素B6代谢的主要酶类；ZM00001d033635编码了一种胞苷/脱氧胞苷脱氨酶，参与了细胞内嘧啶核苷的代谢与维持，其功能与逆境胁迫反向有关；ZM00001d033680为D8基因，编码赤霉素信号的负调控因子DELLA蛋白。推测Ts3可能是通过参与赤霉素信号传导调控玉米茎尖的性别发育。

[Objective] Tasselseed3 (Ts3) is a tasselseed mutant of maize tassel, showing the typical tasselseed phenomenon, indicated that the Ts3 gene is a critical gene for determining the sex of maize tassel. In this study, Ts3 was finely mapped by map-based cloning, and the expression analysis of Ts3 candidate genes in the mapping interval was carried out, which laid a solid foundation for the final cloning of Ts3 gene and the analysis of the genetic control mechanism of maize tassel sex determination. [Result]Statistical analysis of phenotypes such as plant height, internode length, leaf length and leaf width of wild-type and mutants, showed that Ts3 mutants is dwarf mutant with shorten internode and shorten leaves. It indicates that gibberellin signal may be related to the process of maize sex determination mediated by Ts3.After the preliminary mapping the key gene Ts3, which controls the sex determination of maize, was initially located within a range of 6.9 Mb at the end of the chromosome 1 of maize through BSA. In this study,we has done fine mapping of Ts3 and narrowed the localization interval to 1.8Mb, and there were 29 candidate genes in this the expression interval. RT-PCR results of 29 genes comparing WT with mutant showed that 1 genes was upregulated,3 genes were downregulated, 18 genes has no significant difference. In the root tip, 22 genes were expressed and 7 genes were not expressed. Among these 22 genes, the expression level of 2 genes downregulated, and the expression level of 20 genes did not change significantly; In the stem tip, a total of 23 genes were expressed. Among them, the expression level of 6 genes were changed, and the expression level of 17 genes were not significantly changed. The results of RT-PCR of gibberellin-treated plants showed that 21 genes were expressed and 8 genes were not expressed. Among these expressed genes, the expression level of gene132, gene133, gene135, and gene148 upregulated firstly and then downregulated. The expression of the remaining 17 genes did not change significantly; Among these expressed genes, gene120 and gene159, the expression level of gene164 first downregulated and then upregulated. The expression level of gene132, gene133, and gene135 upregulated firstly and then downregulated. The expression level of the remaining 15 genes did not change significantly. Based on the mentioned results, finally we think that gene118, gene120, and gene164 are the candidate genes of Ts3, which are ZM00001d033633, ZM00001d033635 and ZM00001d033680, respectively. ZM00001d033633 encodes a pyridoxal phosphatase, which belongs to enzymes of vitamin B6 metabolism; ZM00001d033635 encodes a cytidine / deoxycytidine deaminase, involving in the metabolism of pyrimidine nucleosides in cells with maintenance, its function is reversely related to stress; ZM00001d033680 is the D8 gene, which encodes negative regulator of the gibberellin signal, DELLA protein. This indicates that Ts3 may be involved in the gibberellin signaling pathway to participate in the development of tassel sex.

[Conclusion]According to fine mapping of Ts3, we rivet the Ts3 gene in a region of 1.8Mb between the G1-273 and G1-305 markers on chromosome 1, through this gene expression analysis and phenotype analysis of mutants, we believe that gene118, gene120, gene164 are the final candidate genes of Ts3, they are ZM00001d033633, ZM00001d033635, ZM00001d033680, respectively. By analyzing the homology of these three genes, we speculated their possible functions. ZM00001d033633 encodes a pyridoxal phosphatase, which is the main enzyme of vitamin B6 metabolism; ZM00001d033635 encodes a cytidine/deoxycytidine deaminase is involved in the metabolism and maintenance of pyrimidine nucleosides in cells, and its function is reversely related to adversity stress; ZM00001d033680 is the D8 gene, which encodes the negative regulator of the gibberellin signal, DELLA protein. It is speculated that Ts3 may be involved in gibberellin signaling  to regulate the sex development of maize stem tips.

蔡昆争,董桃杏,徐涛.茉莉酸类物质(JAs)的生理特性及其在逆境胁迫中的抗性作用[J].生态环境,2006(02):397-404.

陈伟.我国玉米种植产业发展现状及对策研究[J].中国高新科技,2018(20):6-8.

陈儒钢,巩振辉,逯明辉,李大伟,黄炜.植物基因克隆技术的发展与展望[J].长江蔬菜,2009(20):13-18.

柴紫菲,谢道生,盛文涛.浅谈植物的性别[J].园艺与种苗,2018(06):54-56.

国内外玉米贸易格局变化与对策分析[J].北方牧业,2016(20):15.

黄世全,王棚涛,郭思义,宋纯鹏.玉米核基因的图位克隆方法[J].河南大学学报(自然科学版),2018,48(03):318-327.

洪林,程昌凤,魏文辉,陈霞,谭平.高等植物基因克隆的策略[J].浙江农业学报, 2013, 25(04) :914-920.

胡凯,张保军.我国玉米生产现状及策略研究[J].农业与技术,2019,39(01):65-66.

贾伟,杨艳涛,秦富.世界主要国家玉米贸易的现状及特征[J].世界农业,2012(04):35-41.

李金亭,齐婉桢,郭晓双,王灿,张元昊,王迪,苏换换.茉莉酸甲酯对牛膝生长及主要药用成分积累的影响[J].广西植物,2015,35(06):875-879+929.

李浩戈, 崔迪. 茉莉素生理作用及其信号传导研究进展[D]. , 2010.

李想,张剑波.我国玉米贸易格局变化与粮食安全的关系探讨[J].农业现代化研究, 2012, 33 (05):513-517.

路立平,赵化春,赵娜,刘志全,沈海波.世界玉米产业现状及发展前景[J].玉米科学,2006(05):149-151+156.

路立平,赵化春,赵娜,刘志全,沈海波.世界玉米产业现状及发展前景[J].麦类文摘(种业导报),2007(03):31-33.

吕洪坤,郑军.图位克隆技术在玉米基因分离中的应用[J].分子植物育种,2013,11(03):460-468.

李清清, 李大鹏, 李德全. 茉莉酸和茉莉酸甲酯生物合成及其调控机制[J]. 生物技术通报, 2010, 1: 53-57.

任径幽.植物基因克隆技术的研究进展[J].科技创新与应用,2013(27):20.

孙程, 周晓今, 陈茹梅, 等. 植物 JAZ 蛋白的功能概述[J]. 生物技术通报, 2014, 32(6): 1-8.

孙际薇. 茉莉酸甲酯对曼陀罗毛状根的生长及次生代谢产物产生的影响[D].西南大学,2014.

童继平,刘学军,韩傲男,马忠友,刘敏.水稻功能基因图位克隆研究进展[J].中国生物工程杂志,2012,32(03):115-124.

温小杰,张学勇,郝晨阳,蒲文,刘旭.植物激素信号传导途径研究进展[J].中国农业科技导报,2010,12(06):10-17.

王宏亮,郭建飞,李祖亮,郭思义.适于玉米基因图位克隆的SSR标记开发与应用[J].分子植物育种,2017,15(12):5000-5005.

王丽媛,徐明怡,倪红伟,张玉,冷海南.植物基因克隆的方法[J].国土与自然资源研究,2015(03):91-93.

王永春,刘洪霞,赵伟,司陟智.世界玉米产业发展形势分析[J].经济研究导刊,2011(33):207-208.

王雪娇.近年中国玉米进出口贸易格局转变及原因分析[J].农业经济与管理,2014(03):90-+96.

王学勇,崔光红,黄璐琦,邱德有.茉莉酸甲酯对丹参毛状根中丹参酮类成分积累和释放的影响[J].中国中药杂志,2007(04):300-302.

谢阳姣,何志鹏,林伟.茉莉酸甲酯对苦玄参生长及苦玄参苷积累的影响研究[J].作物杂志,2013(02):80-84.

徐艳霞,李旭业,王晓春,李晓波.建国以来我国玉米育种技术的发展与成就[J].黑龙江农业科学,2009(06):165-168.

朱蔚云,谢天炽,李佩琼,申本昌.生物性别决定的复杂性[J].生物学杂志,2015,32(03):84-87.

朱家红, 彭世清. 茉莉酸及其信号传导研究进展[J]. 西北植物學報, 2006, 26(10): 2166-2172.

张伟锋,刘新娇,李秋枫,卢小雨,郑耘,余道坚.世界玉米生产与贸易概况分析[J].世界农业,2014(03):111-114.

张艳霞,史玲玲,戴向辰,Neo Christophine Mokgolodi,刘玉军.茉莉酸及其衍生物:一类重要的植物次生代谢诱导子[J].海南师范大学学报(自然科学版),2008(03):323-327.

Acosta I F, Laparra H, Romero S P, et al. tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize[J]. Science, 2009, 323(5911): 262-265.

Acosta I F, Farmer E E. Jasmonates[J]. The Arabidopsis book/American Society of Plant Biologists, 2010, 8.

Arondel V, Lemieux B, Hwang I, et al. Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis[J]. Science, 1992, 258(5086): 1353-1355.

Beavis W D, Grant D, Albertsen M, et al. Quantitative trait loci for plant height in four maize populations and their associations with qualitative genetic loci[J]. Theoretical and Applied Genetics, 1991, 83(2): 141-145.

Bensen R J, Johal G S, Crane V C, et al. Cloning and characterization of the maize An1 gene[J]. The Plant Cell, 1995, 7(1): 75-84.

Botella M A, Parker J E, Frost L N, et al. Three genes of the Arabidopsis RPP1 complex resistance locus recognize distinct Peronospora parasitica avirulence determinants[J]. The Plant Cell, 1998, 10(11): 1847-1860.

Bortiri E, Hake S. Flowering and determinacy in maize[J]. Journal of experimental botany, 2007, 58(5): 909-916.

Browse J. Jasmonate passes muster: a receptor and targets for the defense hormone[J]. Annual Review of Plant Biology, 2009, 60: 183-205.

Browse J. Jasmonate: preventing the maize tassel from getting in touch with his feminine side[J]. Science signaling, 2009, 2(59): pe9.

Browse J. The power of mutants for investigating jasmonate biosynthesis and signaling[J]. Phytochemistry, 2009, 70(13-14): 1539-1546.

Calderon-Urrea A, Dellaporta S L. Cell death and cell protection genes determine the fate of pistils in maize[J]. Development, 1999, 126(3): 435-441.

Chen Y, Hou M, Liu L, et al. The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol[J]. Plant physiology, 2014, 166(4): 2028-2039.

Cheng P C, Greyson R I, Walden D B. Organ initiation and the development of unisexual flowers in the tassel and ear of Zea mays[J]. American Journal of Botany, 1983, 70(3): 450-462.

Chuck G. Molecular mechanisms of sex determination in monoecious and dioecious plants [M]//Advances in botanical research. Academic Press, 2010, 54: 53-83.

Chuck G, Meeley R, Irish E, et al. The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1[J]. Nature genetics, 2007, 39(12): 1517.

Chuck G, Meeley R B, Hake S. The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet1[J]. Genes & Development, 1998, 12(8): 1145-1154.

Chung H S, Howe G A. A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis[J]. The Plant Cell, 2009, 21(1): 131-145.

Camus-Kulandaivelu L, Veyrieras J B, Madur D, et al. Maize adaptation to temperate climate: relationship between population structure and polymorphism in the Dwarf8 gene[J]. Genetics, 2006, 172(4): 2449-2463.

DeLong A, Calderon-Urrea A, Dellaporta S L. Sex determination gene TASSELSEED2 of maize encodes a short-chain alcohol dehydrogenase required for stage-specific floral organ abortion[J]. Cell, 1993, 74(4): 757-768.

Doebley J, Stec A, Hubbard L. The evolution of apical dominance in maize[J]. Nature, 1997, 386(6624): 485.

Emerson R A. Heritable Characters of Maize II.-Pistillate Flowered Maize Plants[J]. 1920.

Fujioka S, Yamane H, Spray C R, et al. Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 Seedlings of Zea mays L[J]. Plant physiology, 1988, 88(4): 1367-1372.

Giraudat J, Hauge B M, Valon C, et al. Isolation of the Arabidopsis ABI3 gene by positional cloning[J]. The Plant Cell, 1992, 4(10): 1251-1261.

Hayward A P, Moreno M A, Howard T P, et al. Control of sexuality by the sk1-encoded UDP-glycosyltransferase of maize[J]. Science advances, 2016, 2(10): e1600991.

Hedden P, Phillips A L. Gibberellin metabolism: new insights revealed by the genes[J]. Trends in plant science, 2000, 5(12): 523-530.

Heslop-Harrison J. 4. The experimental control of sexuality and inflorescence structure in zea mays L[C]//Proceedings of the Linnean Society of London. Oxford University Press, 1961, 172(1): 108-123.

Jin J, Huang W, Gao J P, et al. Genetic control of rice plant architecture under domestication[J]. Nature genetics, 2008, 40(11): 1365.

Kimberlin A, Leiboff S, Koo A J, et al. Tasselseed5 overexpresses a wound-inducible enzyme, ZmCYP94B1, that affects jasmonate catabolism, sex determination, and plant architecture in maize[J]. Communications Biology, 2019, 2(1): 114.

Li Q, Liu B. Genetic regulation of maize flower development and sex determination[J]. Planta, 2017, 245(1): 1-14.

Lorenzo O, Chico M, Sánchez-Serrano J, et al. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis[J]. The Plant Cell, 2004, 16(7): 1938-1950.

Leung J, Bouvier-Durand M, Morris P C, et al. Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase[J]. Science, 1994, 264(5164): 1448-1452.

Mandaokar A, Thines B, Shin B, et al. Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling[J]. The Plant Journal, 2006, 46(6): 984-1008.

Phinney B O. Gibberellin A 1 dwarfism and shoot elongation in higher plants[J]. Biologia plantarum, 1985, 27(2-3): 172.

Phinney B O. Growth response of single-gene dwarf mutants in maize to gibberellic acid[J]. Proceedings of the National Academy of Sciences of the United States of America, 1956, 42(4): 185.

Putterill J, Robson F, Lee K, et al. The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors[J]. Cell, 1995, 80(6): 847-857.

Rood S B, Pharis R P, Major D J. Changes of endogenous gibberellin-like substances with sex reversal of the apical inflorescence of corn[J]. Plant Physiology, 1980, 66(5): 793-796.

Ross J J. Recent advances in the study of gibberellin mutants[J]. Plant growth regulation, 1994, 15(3): 193-206.

Saniewski M, Miszczak A, Kawa-Miszczak L, et al. Effects of methyl jasmonate on anthocyanin accumulation, ethylene production, and CO 2 evolution in uncooled and cooled tulip bulbs[J]. Journal of Plant Growth Regulation, 1998, 17(1): 33-37.

Smith A R, Zhao D. Sterility caused by floral organ degeneration and abiotic stresses in Arabidopsis and cereal grains[J]. Frontiers in plant science, 2016, 7: 1503.

Song W Y, Wang G L, Chen L L, et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[J]. science, 1995, 270(5243): 1804-1806.

Spray C R, Kobayashi M, Suzuki Y, et al. The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway[J]. Proceedings of the National Academy of Sciences, 1996, 93(19): 10515-10518.

Insertion in the maize domestication gene tb1[J]. Nature genetics, 2011, 43(11): 1160.

Staswick P E, Tiryaki I. The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis[J]. The Plant Cell, 2004, 16(8): 2117-2127.

Tan L, Li X, Liu F, et al. Control of a key transition from prostrate to erect growth in rice domestication[J]. Nature genetics, 2008, 40(11): 1360.

Tanaka W, Pautler M, Jackson D, et al. Grass meristems II: inflorescence architecture, flower development and meristem fate[J]. Plant and cell physiology, 2013, 54(3): 313-324.

Thines B, Katsir L, Melotto M, et al. JAZ repressor proteins are targets of the SCF COI1 complex during jasmonate signalling[J]. Nature, 2007, 448(7154): 661.

Ueguchi-Tanaka M, Ashikari M, Nakajima M, et al. GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin[J]. Nature, 2005, 437(7059): 693.

Winkler R G, Helentjaris T. The maize Dwarf3 gene encodes a cytochrome P450-mediated early step in Gibberellin biosynthesis[J]. The Plant Cell, 1995, 7(8): 1307-1317.

Winkler R G, Freeling M. Physiological genetics of the dominant gibberellin-nonresponsive maize dwarfs, Dwarf8 and Dwarf9[J]. Planta, 1994, 193(3): 341-348.

Wang H, Nussbaum-Wagler T, Li B, et al. The origin of the naked grains of maize[J]. Nature, 2005, 436(7051): 714.

Warren R F, Henk A, Mowery P, et al. A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes[J]. The Plant Cell, 1998, 10(9): 1439-1452.

Xie D X, Feys B F, James S, et al. COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility[J]. Science, 1998, 280(5366): 1091-1094.

Yan Y, Christensen S, Isakeit T, et al. Disruption of OPR7 and OPR8 reveals the versatile functions of jasmonic acid in maize development and defense[J]. The Plant Cell, 2012, 24(4): 1420-1436.

Yan Y, Borrego E, Kolomiets M V. Jasmonate biosynthesis, perception and function in plant development and stress responses[M]. Lipid metabolism. IntechOpen, 2013.

Yamasaki S, Fujii N, Takahashi H. Hormonal regulation of sex expression in plants[J]. Vitamins & Hormones, 2005, 72: 79-110.

Zhao Y, Zhang Y, Wang L, et al. Mapping and functional analysis of a maize silkless mutant sk-A7110[J]. Frontiers in plant science, 2018, 9.