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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)

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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
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