欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (5): 814-826.doi: 10.3724/SP.J.1006.2021.04140

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花GhMADS7基因正调控棉花花瓣发育

马欢欢(), 方启迪, 丁元昊, 池华斌, 张献龙, 闵玲*()   

  1. 华中农业大学植物科学技术学院/作物遗传改良国家重点实验室, 湖北武汉 430070
  • 收稿日期:2020-06-28 接受日期:2020-11-13 出版日期:2021-05-12 网络出版日期:2020-12-15
  • 通讯作者: 闵玲
  • 作者简介:E-mail: 1106825930@qq.com
  • 基金资助:
    国家重点研发计划项目(2016YFD0101402)

GhMADS7 positively regulates petal development in cotton

MA Huan-Huan(), FANG Qi-Di, DING Yuan-Hao, CHI Hua-Bin, ZHANG Xian-Long, MIN Ling*()   

  1. National Key Laboratory of Crop Genetic Improvement / College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-06-28 Accepted:2020-11-13 Published:2021-05-12 Published online:2020-12-15
  • Contact: MIN Ling
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101402)

RichHTML

34

PDF

348

摘要：

MADS-box基因家族作为一类重要的转录因子, 主要参与植物花器官的生长发育。GhMADS7/98具有保守的MADS-box及K结构域, 属于AG亚家族MIKCC型MADS-box基因。通过同源序列比对发现, GhMADS7/98与拟南芥AtAG (AT4G18960)基因的蛋白序列具有64%的同源性。组织表达分析表明, GhMADS7基因在花瓣、花药、柱头和胚珠等花器官组织中均有表达。为进一步研究该基因的功能, 构建了该基因的RNAi干涉载体并转化棉花, 获得了表达量明显下调的转基因株系。表型观察发现, 在干涉植株长度为5~6 mm和7~8 mm的花蕾中出现花瓣发育延缓的表型; 通过对干涉系转基因植株花瓣进行石蜡切片观察发现, 相较于野生型植株, 干涉系植株花瓣中的维管束存在明显的收缩现象; 通过qRT-PCR检测发现, 转基因株系中控制花瓣发育的A、B类基因的表达量出现异常。因此推测GhMADS7在棉花花瓣发育过程中起着重要的作用。

关键词: 棉花, MADS-box基因, GhMADS7, RNAi, 花瓣发育

Abstract:

The MADS-box gene family, as an important class of transcription factors, is mainly involved in plant floral organs growth and development. GhMADS7/98 has a conserved MADS-box and K domain, belonging to the AG subgroup of MIKCC type MADS-box gene family. Through homology sequence alignment, GhMADS7/98 was 64% homologous to the Arabidopsis AGAMOUS (AG) gene. The GhMADS7 gene was expressed in petals, anthers, stigmas, and ovules, but with different temporal and spatial expression. To study the function of the gene, the RNA interference (RNAi) vector was constructed and transformed into cotton, and the transgenic lines with significantly reduced expression was obtained. These transgenic lines showed delayed petal development in the 5-6 mm and 7-8 mm buds. By observing the paraffin sections of the petals, it was found that vascular bundles in the petals of RNAi lines were shrived, compared with that in the wild type plants. To explore the reason, the expression of class A and B gene of ‘ABC’ model of floral development was detected in the petals by qRT-PCR, the result showed that the expression of most genes were upregulated in the RNAi plants, compared to WT. Therefore, it is speculated that GhMADS7 might cooperative with other petal development genes to regulate the cotton petal development.

Key words: cotton, MADS-box gene, GhMADS7, RNAi, petal development

表1

本试验所用引物及用途"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Purpose
ubiquitin7-F GAAGGCATTCCACCTGACCAAC 实时荧光定量PCR
ubiquitin7-R CTTGACCTTCTTCTTCTTGTGCTTG qRT-PCR
GhAMDS7-S TCATAGTTCAATTCCTTGCCAGC 基因扩增
GhAMDS7-A CTTGAATAGATGGAGGTGGAAGAGAG Gene amplification
OGhMADS7-F CCTTGCCAGCTTTGAACCAT 基因扩增
OGhMADS7-R ATGGAGGTGGAAGAGAGAAGCTAC Gene amplification
qGhMADS7-F AATGATTACTCCAACCAAGACCAA 实时荧光定量PCR
qGhMADS7-R TTGGTAGAACATTAGAGGCTGCTG qRT-PCR
bpGhMADS7-F GGGGACAAGTTTGTACAAAAAAGCAGGCTGGCCTTGCCAGCTTTGAACCAT BP反应
bpGhMADS7-R GGGGACCACTTTGTACAAGAAAGCTGGGTCATGGAGGTGGAAGAGAGAAGCTAC BP reaction
NPTII-F TTGTCACTGAAGCGGGAAGG 探针
NPTII-R CGATACCGTAAAGCACGAGGAA Probe

图1

陆地棉MIKCC型MADS-box基因家族系统进化树 A: 陆地棉MIKCC型MADS-box基因家族进化树; B: 陆地棉MIKCC型MADS-box基因家族AG亚族进化树。"

图2

GhMADS7和GhMADS98基因序列及表达模式分析 A: GhMADS7基因及其同源基因蛋白序列分析; B: GhMADS7基因结构域分析; C: GhMADS7和GhMADS98基因表达模式分析。R: 根; S: 茎; L: 叶; P: 花瓣; A: 花药; S: 柱头; O: 胚珠; F10DPA/20DPA: 10 DPA和20 DPA纤维; S10DPA/20DPA: 10 DPA和20 DPA种子。"

图3

GhMADS7基因组织表达模式分析 F<9: 花蕾长度在9 mm以下; F9-14: 花蕾长度为9~14 mm; F14-19: 花蕾长度为14~19 mm; F>19: 花蕾长度在19 mm以上; F0: 开花当日花蕾。误差线为3次重复的误差。通过ANOVA方法分析A, B, C, D图的差异显著性, 不同的字母代表不同的差异显著性水平。"

图4

GhMADS7转基因株系表达量检测及T0代T-DNA插入拷贝数分析 A: GhMADS7转基因株系表达量检测; B: T0代转基因株系T-DNA插入拷贝数分析; YZ1: 野生型植株; iGhMADS7-3, iGhMADS7-17, iGhMADS7-20, iGhMADS7-27, iGhMADS7-32代表GhMADS7的5个干涉转基因株系。误差线表示3次技术重复的误差。"

图5

GhMADS7-RNAi转基因植株表型考察 1~2 mm、2~3 mm、3~4 mm、4~5 mm、5~6 mm、6~7 mm、7~8 mm代表花蕾的长度; Se: 萼片; Pe: 花瓣; Sti: 柱头; Sta: 雄蕊; Ca: 心皮; Ov: 胚珠。图WT和iGhMADS7-27的标尺均为500 µm。"

图6

石蜡切片比较GhMADS7-RNAi转基因植株和野生型‘YZ1’的花瓣构造 iGhMADS7: 抑制GhMADS7转基因植株; YZ1: 野生型植株。(2~3) mm~(7~8) mm代表花蕾的发育长度。El: 上表皮细胞; Hl: 下表皮细胞; Pt: 薄壁组织; Vb: 维管束。8、10、12代表薄壁组织的细胞层数。图WT和iGhMADS7-27的标尺均为20 µm。"

图7

GhMADS7-RNAi转基因植株中A、B类基因表达量检测 A: A类基因在GhMADS7-RNAi转基因植株中表达量水平; B: B类基因在GhMADS7-RNAi转基因植株中表达量水平。YZ1: 野生型植株; iGhMADS7: 植株GhMADS7转基因植株。F<5: 花蕾长度在5 mm以下; F5-6: 花蕾长度为5~6 mm; F6-7: 花蕾长度为6~7 mm; F7-8: 花蕾长度为7~8 mm; F8-9: 花蕾长度在8~9 mm; F>9: 花蕾长度在9 mm以上。误差线表示3次重复的误差。*, **分别表示在0.05和0.01水平显著差异。"

图8

GhMADS7、GhMADS3与AtAG蛋白序列比对 A: GhMADS7与ATAG蛋白序列比对; B: GhMADS3与ATAG蛋白序列比对。"

附表1

用于进化树分析的78条MADS蛋白序列基因号"

基因号
ID		 基因名称
Name		 基因号
ID		 基因名称
Name		 基因号
ID		 基因名称
Name
Gh_D04G1849 GhMADS1 Gh_A08G1148 GhMADS45 Gh_D03G1493 GhMADS75
Gh_D05G2375 GhMADS2 Gh_A07G1339 GhMADS46 Gh_D10G0308 GhMADS76
Gh_A10G2220 GhMADS3 Gh_D08G1430 GhMADS47 Gh_D13G1226 GhMADS77
Gh_A10G2221 GhMADS4 Gh_A09G2157 GhMADS48 Gh_D11G0882 GhMADS78
Gh_D05G2596 GhMADS5 Gh_A04G1265 GhMADS49 Gh_D12G1000 GhMADS79
Gh_D04G0341 GhMADS7 Gh_A01G1608 GhMADS50 Gh_A04G1491 GhMADS80
Gh_A12G0910 GhMADS9 Gh_D03G0105 GhMADS51 Gh_D11G0883 GhMADS81
Gh_D10G0309 GhMDS10 Gh_D05G2452 GhMADS52 Gh_D12G1027 GhMADS82
Gh_D04G1892 GhMADS11 Gh_A05G2191 GhMADS53 Gh_D06G0245 GhMADS84
Gh_A02G0736 GhMADS12 Gh_D06G0267 GhMADS54 Gh_D11G3150 GhMADS85
Gh_A11G0754 GhMADS13 Gh_D02G2012 GhMADS55 Gh_D11G0082 GhMADS86
Gh_D12G2226 GhMADS14 Gh_A04G0934 GhMADS57 Gh_D02G0779 GhMADS87
Gh_A11G0077 GhMADS15 Gh_A12G0936 GhMADS58 Gh_A03G1085 GhMADS88
Gh_D02G1502 GhMADS16 Gh_A12G0570 GhMADS59 Gh_A07G0605 GhMADS89
Gh_D07G1814 GhMADS17 Gh_A11G0755 GhMADS60 Gh_A03G2004 GhMADS90
Gh_D13G0877 GhMADS20 Gh_D07G0780 GhMADS61 Gh_A12G2048 GhMADS91
Gh_D04G1451 GhMADS27 Gh_A07G1615 GhMADS62 Gh_A04G1264 GhMADS93
Gh_A13G0524 GhMADS28 Gh_A06G0244 GhMADS64 Gh_A13G0981 GhMADS94
Gh_D11G0400 GhMADS36 Gh_D13G0605 GhMADS66 Gh_A03G1563 GhMADS95
Gh_A06G1875 GhMADS37 Gh_A08G1275 GhMADS67 Gh_A12G0150 GhMADS96
Gh_D11G0534 GhMADS38 Gh_D12G0778 GhMADS68 Gh_A13G0425 GhMADS97
Gh_A12G0775 GhMADS40 Gh_D12G0163 GhMADS70 Gh_A05G3267 GhMADS98
Gh_A13G0751 GhMADS41 Gh_D09G2362 GhMADS71 Gh_A05G2136 GhMADS99
Gh_D04G1891 GhMADS42 Gh_D13G0878 GhMADS72 Gh_A05G2334 GhMADS100
Gh_A03G0634 GhMADS43 Gh_D07G0671 GhMADS73 Gh_Sca004768G07 GhMADS101
Gh_D02G1311 GhMADS44 Gh_D03G0922 GhMADS74 Gh_Sca007246G01 GhMADS102
[1] Coen E S, Meyerowitz E M. The war of the whorls: genetic interactions controlling flower development. Nature, 1991,353:31-37.
[2] Theien G. Development of floral organ identity: stories from the MADS house. Curr Opin Plant Biol, 2001,4:75-85.
[3] Alvarez-Buylla E R, Liljegren S J, Pelaz S, Gold S E, Ditta B C, Vergara-Silva F, Yanofsky M F. MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. Plant J, 2000,24:457-466.
[4] Arora R, Agarwal P, Ray S, Singh A K, Singh V P, Tyagi A K, Kapoor S. MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics, 2007,8:242.
[5] Litt A, Irish V F. Duplication and diversification in the APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development. Genetics, 2003,165:821-33.
[6] Svensson M. Evolution of a Family of Plant Genes with Regulatory Functions in Development; Studies on Picea abies and Lycopodium annotinum. PhD Dissertation of Uppsala University, Uppsala, Sweden, 2000.
[7] Smaczniak C, Immink R G, Angenent G C, Kaufmann K. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies. Development, 2012,139:3081-3098.
[8] José D R, Diego L, Martínez-Zapater J M, María José C. Genome-wide analysis of MIKCC-type MADS box genes in grapevine . Plant Physiol, 2009,149:354-369.
[9] Dreni L, Kater M M. MADS reloaded: evolution of the AGAMOUS subfamily genes. New Phytol, 2014,201:717-732.
[10] Gao Z H, Zhang Y M, Wang S, Zhang Z. Research progress in floral organ identity gene AGAMOUS. Acta Bot Boreali-Occident Sin, 2008,28:638-644.
[11] Jack T, Sieburth L, Meyerowitz E. Targeted misexpression of AGAMOUS in whorl 2 of Arabidopsis flowers. Plant J, 1997,11:825-839.
[12] Liu Z, Zhang D, Liu D, Li F, Lu H. Exon skipping of AGAMOUS homolog PrseAG in developing double flowers of Prunus lannesiana (Rosaceae). Plant Cell Rep, 2013,32:227-237.
[13] Hou J H, Gao Z H, Zhang Z, Chen S M, Ando T, Zhang J Y, Wang X W. Isolation and characterization of an AGAMOUS homologue pmAG from the Japanese Apricot ( Prunus mume Sieb. et Zucc.). Plant Mol Biol Rep, 2011,29:473-480.
[14] Junko K, Ko S. Ectopic expression of OsMADS3, a rice ortholog of AGAMOUS, caused a homeotic transformation of lodicules to stamens in transgenic rice plants. Plant Cell Physiol, 2002,43:130-135.
[15] Pnueli L. Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell, 1994,6:163-173.
[16] Helliwell C A, Wesley S V, Wielopolska A J, Waterhouse P M. High-throughput vectors for efficient gene silencing in plants. Funct Plant Biol, 2002,29:1217-1225.
[17] Jin S X, Zhang X L, Liang S G, Nie Y C, Guo X P, Huang C. Factors affecting transformation efficiency of embryogenic callus of Upland cotton ( Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult, 2005,81:229-237.
[18] Min L, Zhu L F, Tu L L, Deng F L, Yuan D J, Zhang X L. Cotton GhCKI disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase. Plant J, 2013,75:823-835.
[19] Ding Y H, Ma Y Z, Liu N, Xu J, Hu Q, Li Y Y, Xie S, Zhu L F, Min L, Zhang X L. microRNAs involved in auxin signalling modulate male sterility under high temperature stress in cotton ( Gossypium hirsutum). Plant J, 2017,91:977-994.
[20] Becker A, Winter K U, Meyer B, Saedler H, Theissen G. MADS-Box gene diversity in seed plants 300 million years ago. Mol Biol Evol, 2000,17:1425-1434.
[21] Zhang T Z, Hu Y, Jiang W K, Fang L, Guan X Y, Chen J D, Zhang J B, Christopher A S, Brian E S, David M S, Amanda M H K, Wan Q, Liu B L, Liu C X, Wang S, Pan M Q, Wang Y K, Wang D W, Ye W X, Chang L J, Zhang W P, Song Q X, Ryan C K, Chen X Y, Elizabeth D, Danny J L, Daniel G P, Peggy T, Don C J, Wang Q, Xu X Y, Zhang H, Wu H T, Zhou L, Mei G F, Chen S Q, Tian Y, Xiang D, Li X H, Ding J, Zuo Q Y, Tao L N, Liu Y C, Li J, Lin Y, Hui Y Y, Cao Z S, Cai C P, Zhu X F, Jiang Z, Zhou B L, Guo W Z, Li R Q, Chen Z J. Sequencing of allotetraploid cotton ( Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol, 2015,33:531-537.
[22] Mizukami Y, Ma H. Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell, 1992,71:119-131.
[23] 靳春梅, 周坤, 张今今. 茶树花发育MADS-box转录因子CsGLO1、CsGLO2与CsAG之间的互作关系研究. 植物科学学报, 2017,35(1):79-86.
Jin C M, Zhou K, Zhang J J. Interactions of MADS-box transcription factors CsGLO1, CsGLO2 and CsAG in Camellia sinensis flower development. Plant Sci J, 2017,35(1):79-86 (in Chinese with English abstract).
[24] 田亚然, 范天刚, 张钢, 李永红. 低温引起月季花朵过度重瓣化关键基因的表达及分析. 热带作物学报, 2016,37:1147-1154.
Tian Y R, Fan T G, Zhang G, Li Y H. Expression and analysis of key genes of excessive double flowers in rose caused by low temperature. Chin J Trop Crops, 2016,37:1147-1154 (in Chinese with English abstract).
[25] 陈之琳, 秦波, 蔡明, 郑唐春, 潘会堂, 王国熙, 潘隆应, 朱嫄, 张启翔. 大花紫薇B类和C类基因克隆及表达模式分析. 见: 张启翔主编. 中国观赏园艺研究进展2017. 北京: 中国林业出版社, 2017. pp 746-756.
Chen Z L, Qin B, Cai M, Zheng T C, Pan H T, Wang G X, Pan L Y, Zhu Y, Zhang Q X. Cloning and expression pattern analysis of B-function and C-function genes in Lagerstroemia speciosa. In: Zhang Q X, eds. Advances in Ornamental Horticulture of China (2017). Beijing: Chinese Forestry Publishing House, 2017. pp 746-756(in Chinese).
[26] 孙迎坤. 山茶花MADS-box家族A类和C类基因克隆及功能分析. 中国林业科学研究院博士学位论文, 北京, 2013.
Sun Y K. Isolation and Function Analysis of Class A and C Genes of MADS-box Family from Camellia japonica. PhD Dissertation of Chinese Academy of Forestry, Beijing, China, 2013 (in Chinese with English abstract).
[27] Narumi T, Aida R, Niki T, Nishijima T, Mitsuda N, Hiratsu K, Ohme-Takagi M, Ohtsubo N. Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application. Jpn Soc Plant Cell Mol Biol, 2008,25:45-53.
[28] Guo Y L, Zhu Q L, Zheng S Y, Li M Y. Cloning of a MADS box gene ( GhMADS3) from cotton and analysis of its homeotic role in transgenic tobacco. J Genet Genomics, 2007,34:527-535.
[29] Yamaguchi T, Lee D Y, Miyao A, Hirochika H, An G, Hirano H Y. Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. Plant Cell, 2006,18:15-28.
[30] Yun D P, Liang W Q, Dreni L, Yin C S, Zhou Z G, Kater M M, Zhang D B. OsMADS16 genetically interacts with OsMADS3 and OsMADS58 in specifying floral patterning in rice. Mol Plant, 2013,6:743-756.
[31] 段晓姗. 毛茛科C类MADS-Box基因的克隆和表达研究. 陕西师范大学硕士学位论文, 陕西西安, 2011.
Duan X S. Study on Cloning and Expression of C-type MADS-Box Gene in Ranunculaceae. MS Thesis of Shaanxi Normal University, Xi’an, Shaanxi, China, 2011 (in Chinese with English abstract).
[32] Kramer E M, Jaramillo M A, Stilio V S D. Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms. Genetics, 2004,166:1011-1023.
[1] 周静远, 孔祥强, 张艳军, 李雪源, 张冬梅, 董合忠. 基于种子萌发出苗过程中弯钩建成和下胚轴生长的棉花出苗壮苗机制与技术[J]. 作物学报, 2022, 48(5): 1051-1058.
[2] 孙思敏, 韩贝, 陈林, 孙伟男, 张献龙, 杨细燕. 棉花苗期根系分型及根系性状的关联分析[J]. 作物学报, 2022, 48(5): 1081-1090.
[3] 闫晓宇, 郭文君, 秦都林, 王双磊, 聂军军, 赵娜, 祁杰, 宋宪亮, 毛丽丽, 孙学振. 滨海盐碱地棉花秸秆还田和深松对棉花干物质积累、养分吸收及产量的影响[J]. 作物学报, 2022, 48(5): 1235-1247.
[4] 郑曙峰, 刘小玲, 王维, 徐道青, 阚画春, 陈敏, 李淑英. 论两熟制棉花绿色化轻简化机械化栽培[J]. 作物学报, 2022, 48(3): 541-552.
[5] 张艳波, 王袁, 冯甘雨, 段慧蓉, 刘海英. 棉籽油分和3种主要脂肪酸含量QTL分析[J]. 作物学报, 2022, 48(2): 380-395.
[6] 张特, 王蜜蜂, 赵强. 滴施缩节胺与氮肥对棉花生长发育及产量的影响[J]. 作物学报, 2022, 48(2): 396-409.
[7] 赵文青, 徐文正, 杨锍琰, 刘玉, 周治国, 王友华. 棉花叶片响应高温的差异与夜间淀粉降解密切相关[J]. 作物学报, 2021, 47(9): 1680-1689.
[8] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[9] 曾紫君, 曾钰, 闫磊, 程锦, 姜存仓. 低硼及高硼胁迫对棉花幼苗生长与脯氨酸代谢的影响[J]. 作物学报, 2021, 47(8): 1616-1623.
[10] 许乃银, 赵素琴, 张芳, 付小琼, 杨晓妮, 乔银桃, 孙世贤. 基于GYT双标图对西北内陆棉区国审棉花品种的分类评价[J]. 作物学报, 2021, 47(4): 660-671.
[11] 周冠彤, 雷建峰, 代培红, 刘超, 李月, 刘晓东. 棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究[J]. 作物学报, 2021, 47(3): 427-437.
[12] 卢合全, 唐薇, 罗振, 孔祥强, 李振怀, 徐士振, 辛承松. 商品有机肥替代部分化肥对连作棉田土壤养分、棉花生长发育及产量的影响[J]. 作物学报, 2021, 47(12): 2511-2521.
[13] 王晔, 刘钊, 肖爽, 李芳军, 吴霞, 王保民, 田晓莉. 转PSAG12-IPT基因对棉花叶片衰老及产量和纤维品质的影响[J]. 作物学报, 2021, 47(11): 2111-2120.
[14] 杨琴莉, 杨多凤, 丁林云, 赵汀, 张军, 梅欢, 黄楚珺, 高阳, 叶莉, 高梦涛, 严孙艺, 张天真, 胡艳. 棉花花器官突变体的鉴定及候选基因的克隆[J]. 作物学报, 2021, 47(10): 1854-1862.
[15] 李晓旭, 王蕊, 张利霞, 宋亚萌, 田晓楠, 葛荣朝. 水稻基因OsATS的克隆及功能鉴定[J]. 作物学报, 2021, 47(10): 2045-2052.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2