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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (1): 30-41.doi: 10.3724/SP.J.1006.2021.04090

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

Function study of cotton GbSTK gene in regulating flowering and Verticillium wilt resistance

CUI Jing(), WANG Zhi-Cheng, ZHANG Xin-Yu, KE Hui-Feng, WU Li-Qiang, WANG Xing-Fen, ZHANG Gui-Yin, MA Zhi-Ying, ZHANG Yan*()   

  1. State Key Laboratory of North China Crop Improvement and Regulation / Co-Innovation Center for Cotton Industry of Hebei Province / Hebei Agricultural University, Baoding 071001, Hebei, China
  • Received:2020-04-11 Accepted:2020-08-19 Online:2021-01-12 Published:2020-09-21
  • Contact: ZHANG Yan E-mail:cuijing1240861235@qq.com;zhangyan7235@126.com
  • Supported by:
    Natural Science Foundation of Hebei Province(C2019204365);China Agriculture Research System(CARS-15-03);Talents Support Program of Hebei Province

Abstract:

Cotton is an important cash crop, and achieving disease resistance as well as early maturity is an important breeding goal. However, the molecular relationship between disease resistance and early maturity of cotton is still a research gap. In previous study, we identified a GbSTK gene response to V. dahliae that could enhance Verticillium wilt resistance in transgenic Arabidopsis. In this study, we found that GbSTK was more highly expressed in resistance and/or tolerant varieties than in susceptible varieties. Knocking down of GbSTK expression in Nongda 601 showed that the silenced plants displayed serious disease symptoms, with disease index from 27.5 (tolerant) to 63.2 (susceptible), suggesting that GbSTK positively regulates cotton Verticillium wilt resistance. More interesting, we found that GbSTK could promote the development of Arabidopsis. At the flowering time point, the average number of rosette leaves in transgenic lines was 7.5, significantly less than the mock plants, and the transgenic plants bloomed five to seven days earlier than the control. The FT and SOC1 gene, regarded as marker genes in Arabidopsis flowering signaling pathway, maintained higher expression level in transgenic lines than in the mock, indicating that GbSTK could accelerate flowering via regulating FT and SOC1 gene expression. We also obtained 30 candidate proteins that could interacted with GbSTK via yeast double hybrid test, which helped to further revealing the molecular regulatory network of GbSTK. Taken together, we first identified a functional gene that could improve disease resistance and early flowering, which would provide a reference for genetic improvement of cotton disease resistant and early maturity.

Key words: cotton, GbSTK, Verticillium wilt resistance, early maturity

Fig. S1

Seedlings were inoculated by V. dahliae via dipping root method in conidial suspension a: cultivation of cotton seedlings under hydroponic conditions. b: the seedlings were inoculated by V. dahliae."

Table 1

Primers used in the study"

引物名称
Primer name
序列
Primer sequence (5°-3°)
用途
Purpose
STK-F1 CCATATGAAGAATTCAAGCAAATCCTC 基因扩增Gene amplification
STK-R1 GCTGCAGTCAAGGGGCCGTAGGTTGT 基因扩增Gene amplification
VF ACTAGTGCTGCTCGAACTTCTATTGGGAAG 基因扩增Gene amplification
VR GGCGCGCCAGGGGCCGTAGGTTGTGTAACTCT 基因扩增Gene amplification
STK-F2
STK-R2
ATGCGGCAACGAACAAGAAT
TAGATTGGCACTGAGCTGGT
实时定量qPCR
实时定量qPCR
GhUBQ14-F CAACGCTCCATCTTGTCCTT 实时定量qPCR
GhUBQ14-R TGATCGTCTTTCCCGTAAGC 实时定量qPCR
BD-F TTTGTAATACGACTCACTATAGGGCG 通用载体Vector
BD-R TTTTCGTTTTAAAACCTAAGAGTCAC 通用载体Vector
T7 TAATACGACTCACTATAGGGCG 通用载体Vector
3°AD AGATGGTGCACGATGCACAG 通用载体Vector
TUB2-F ATCCGTGAAGAGTACCCAGAT 实时定量qPCR
TUB2-R AAGAACCATGCACTCATCAGC 实时定量qPCR
FT-F CTTGGCAGGCAAACAGTGTATGCAC 实时定量qPCR
FT-R GCCACTCTCCCTCTGACAATTGTAGA 实时定量qPCR
SOC1-F AGCTGCAGAAAACGAGAAGCTCTCTG 实时定量qPCR
SOC1-R GGGCTACTCTCTTCATCACCTCTTCC 实时定量qPCR

Fig. 1

Expression levels of GbSTK gene in different resistant cottons"

Fig. 2

Effects of silencing of STK in cotton (ND601) susceptibility to Verticillium dahliae a: silencing of GhCLA1 gene in cotton; b: qPCR analysis of the STK transcripts in control and silencing seedlings; c: disease symptoms inoculated with V. dahliae on TRV::00 and TRV::GbSTK plants at 25 days; d and e: the rate of diseased plants and disease index were measured at 25 days."

Fig. 3

Phenotypic evaluation of overexpression of GbSTK in Arabidopsis and expression analysis of the flowering marker genes a-c: overexpression (OE) of GbSTK in Arabidopsis led to early flowering; d: the transgenic Arabidopsis showed significantly fewer rosette leaves when flowering; e-g: the transgenic Arabidopsis showed significantly higher expression of SOC1 and FT, rather than LFY. Error bars indicate standard error (SE). *, *** indicate significant difference at the 0.05 and 0.001 probability levels, respectively. WT: wild type Arabidopsis; OE: GbSTK transgenic Arabidopsis; OE1 and OE2: different GbSTK transgenic Arabidopsis lines."

Fig. 4

Construction of bait vector of pGBKT7-GbSTK and identification of toxicity M: DNA marker DL5000; 1-2: double digestion of GbSTK-T vector; 3-6: double digestion of pGBKT7 vector; 7: screening pGBKT7- GbSTK positive clones. d: detection of toxicity for the bait vector of pGBKT7-GbSTK."

Fig. 5

Auto-activation for bait vector of pGBKT7-GbSTK a: yeast growth on SD/-Leu/-Trp agar plate; b: yeast growth on SD/-Ade/-His/-Leu/-Trp/X-α-Gal agar plate; c: bait vector of pGBKT7- GbSTK growth on agar plate."

Fig. 6

Screening of positive clones"

Table 2

Candidate proteins interacting with GbSTK"

编号
No.
蛋白描述
Protein description
功能报道
Function reported
功能分类
Functional classification
1 Cytochrome c oxidase subunit 6b-1 Regulate ROS production[22] ROS production
2 Malate dehydrogenase Related to production of ROS and PCD[23]
3 GTPase-activating protein GYP7 Defense response[24] Involved in defense and abiotic/biotic stresses
4 Cytochrome P450 71D8 Defense response[25]
5 Short-chain dehydrogenase reductase 3b Involved in plant defense responses[26]
6 Cysteine proteinase RD21a Involved in plant defense[27]
7 Vacuolar protein sorting-associated protein 55 Involved in plant defense[28]
8 Pathogenesis-related protein STH-2 Defense response[29]
9 (-)-alpha-terpineol synthase Involved in defense and abiotic stresses[30]
10 Glyceraldehyde-3-phosphate dehydrogenase 2 Negatively regulate autophagy and immunity[31]
11 Phosphoglycerate Ubiquitin-conjugating
enzyme E2
Involved in plant immunity[32]
12 Dirigent protein 16 Responsive to biotic stress[33]
13 Major allergen Pruar 1 Novel Pathogenesis-related protein[34]
14 S-Adenosylmethionine synthase Responsive to abiotic stress[35]
15 S-adenosylmethionine synthase 2 Responsive to abiotic stress[36]
16 Probable inorganic phosphate transporter 1-5 Involved in phosphate homeostasis[37]
17 Glycerophosphodiester phosphodiesterase GDPD1 Response to Pi deficiency[38]
18 Delta-cadinene synthase isozyme C2 Gossypol biosynthetic[39] Secondary metabolism and
synthesis
19 UDP-galactose/UDP-glucose transporter 3 Synthesis of cell walls[40]
20 Aldehyde dehydrogenase family 2 member C4 Regulator of the growth-defense trade-off[41] Plant development and
stress response
21 Probable 2-oxoglutarate-dependent dioxygenase Related to vascular disease/involved in
gibberellin synthesis[42]
22 Patellin-5 Function in plant development and stress
response[43]
23 Triphosphate tunel metalloenzyme 3 Catalyze diverse enzymatic reactions[44]
24 Chloroplastic 2,3-bisphosphoglycerate-independent mutase Involved in photosynthesis and chloroplast development[45] Plant growth and development
25 Synaptotagmin-2 Participates in pollen germination and tube growth[46]
26 Mediator of RNA polymerase II transcription subunit
27 Alpha-taxilin
28 Aconitate hydratase, cytoplasmic
29 Putative zinc transporter At3g08650
30 40S ribosomal protein S5

Fig. S2

Homology analysis of SAMS protein and GhSAMS protein"

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