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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 814-826.doi: 10.3724/SP.J.1006.2021.04140

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2021-05-12 Published:2020-12-15
  • Contact: MIN Ling E-mail:1106825930@qq.com;lingmin@mail.hzau.edu.cn
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101402)

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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

Table 1

Sequence and purpose of the primers used in this study"

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

Fig. 1

Phylogenetic tree analysis of MIKCC MADS-box gene family in Gossypium hirsutum A: phylogenetic tree of MIKCC MADS-box gene family in Gossypium hirsutum; B: phylogenetic tree of AG subfamily of MIKCC MADS-box gene family in Gossypium hirsutum."

Fig. 2

Sequence analysis and tissue expression pattern analysis of GhMADS7 and GhMADS98 in Gossypium hirsutum A: alignment of the protein sequences of GhMADS7 and its homologous genes; B: the domain sequences analysis of GhMADS7; C: expression pattern analysis of GhMADS7 and GhMADS98 in Gossypium hirsutum. R: root; S: stem; L: leaf; P: petal; A: anther; S: stigma; O: ovule; F10 DPA/20 DPA: the fiber of 10 DPA/20 DPA; S10 DPA/20 DPA: the seed of 10 DPA/20 DPA."

Fig. 3

Expression pattern analysis of GhMADS7 in Gossypium hirsutum F<9: the length of flower bud is less than 9 mm; F9-14: the length of flower bud is 9-14 mm; F14-19: the length of flower bud is 14-19 mm; F>19: the length of flower bud is above 19 mm; F0: the flower bud on the day of flowering. The error bars represent deviations of three replicates. Significant differences in A, B, C, D were found by one-way ANOVA (P<0.05). Values with different letters are significantly different at P<0.05."

Fig. 4

Relative expression level of GhMADS7 RNAi in different transgenic lines and copy number of T-DNA in T0 transgenic lines A: relative expression level of GhMADS7 RNAi in different transgenic lines; B: copy number of T-DNA in T0 transgenic lines; YZ1: wild plant; iGhMADS7-3, iGhMADS7-17, iGhMADS7-20, iGhMADS7-27, iGhMADS7-32 represent five transgenic lines of GhMADS7 RNAi. The error bars represent deviations of three technical replicates."

Fig. 5

Phenotypic investigation of GhMADS7-RNAi transgenic plants 1-2 mm, 2-3 mm, 3-4 mm, 4-5 mm, 5-6 mm, 6-7 mm, and 7-8 mm represent the length of flower buds; Se: sepal; Pe: petal; Sti: stigma; Sta: stamen; Ca: carpel; Ov: ovule. Bar=500 µm (in WT and iGhMADS7-27)."

Fig. 6

Comparison of petal structure between GhMADS7-RNAi transgenic plants and wild type YZ1 by paraffin section iGhMADS7: suppression of GhMADS7 transgenic plants; YZ1: wild type plants. 2-3 mm to 7-8 mm represents the length of the bud. E1: epithelium cells; H1: hypodermic cells; Pt: thin-walled tissue; Vb: vascular bundle. 8, 10, 12 represent the cell layer number of the thin-walled tissue. Bar = 20 µm (in WT and iGhMADS7-27)."

Fig. 7

Relative expression levels of A and B type genes in GhMADS7 transgenic plants A: the expression levels of A genes in GhMADS7-RNAi transgenic plants; B: the expression levels of B genes in GhMADS7-RNAi transgenic plants. YZ1: wild type plant; iGhMADS7: suppression of GhMADS7 transgenic plant. F<5: the length of flower bud is less than 5 mm; F5-6: the length of flower bud is 5-6 mm; F6-7: the length of flower bud is 6-7 mm; F7-8: the length of flower bud is 7-8 mm; F8-9: the length of flower bud is 8-9 mm; F>9: the length of flower bud is above 9 mm. The error bars represent deviations of three replicates. *, ** mean significant differences at the 0.05 and 0.01 probability levels."

Fig. 8

Alignment of the protein sequences of GhMADS7, GhMADS3, and AtAG A: alignment of the protein sequences of GhMADS7 and ATAG; B: alignment of the protein sequences of GhMADS3 and AtAG."

Table S1

Gene ID of 76 MADS protein sequence for phylogenetic tree analysis"

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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