Cloning of GhSWEET9 in Upland Cotton and Functional Analysis of Resistance to Verticillium Wilt

doi:10.13560/j.cnki.biotech.bull.1985.2025-0002

Abstract

Abstract:

Objective To investigate the correlation between GhSWEET9 gene and cotton plant’s resistance to Verticillium wilt, and provide theoretical basis for exploring the molecular mechanism of cotton plant’s resistance and breeding a cultivars of cotton plant combating Verticillium wilt. Method Bioinformatics software was utilized to analyze the sequence characteristics, phylogenetic relationships, and subcellular localization of the GhSWEET9 gene. Yeast heterologous complementary system was used to clarify the sugar transport function of GhSWEET9 protein. Virus induced gene silencing (VIGS) technology was applied to study the function of this gene in combating Verticillium wilt. Result The GhSWEET9 protein has 7 transmembrane domains, and phylogenetic tree analysis shows that it belongs to the SWEETs Clade II subfamily. The subcellular localization results show that GhSWEET9 is located on the cytoplasmic membrane. Yeast heterologous complementation experiments show that GhSWEET9 protein can transport galactose, mannose, glucose, and fructose. Using VIGS technology to silence GhSWEET9, glucose content measurement reveals that the glucose content in the root system of silenced plants (pTRV2:GhSWEET9) was significantly higher than that of the empty vector control (pTRV2:00). Using the Verticillium dahliae Vd991 strain to infect control plants and silenced plants, the glucose contents in the roots of the silenced plants were still significantly higher than that in the roots of control plants after 2 and 6 d of infection. After 14 d of infection, compared with the control plants, the leaves of silenced plants show more severe yellowing and wilting, more obvious browning of vascular bundles, and a higher disease index. Conclusion The silencing of GhSWEET9 gene is beneficial for the accumulation of glucose in cotton roots, which may promote the infection of cotton roots by V. dahliae, thereby reducing the resistance of cotton to Verticillium wilt.It is speculated that this gene may play an important role in cotton's resistance to Verticillium wilt.

Key words: upland cotton, SWEETs, Verticillium wilt, VIGS, glucose

ZHANG Yong, SONG Sheng-long, LI Yong-tai, ZHANG Xin-yu, LI Yan-jun. Cloning of GhSWEET9 in Upland Cotton and Functional Analysis of Resistance to Verticillium Wilt[J]. Biotechnology Bulletin, 2025, 41(6): 144-154.

Figures/Tables 8

Table 1 Primers used in this experiment

引物 Primer	序列 Primer sequence （5'-3'）	用途 Application
CDS-GhSWEET9	ATGGTTTCGCACCTTGTCAC	基因克隆
CDS-GhSWEET9 Y-SWEET9-F Y-SWEET9-R	AGGTGCTGTGCAGTTCTTT aacacgggggactttgcaacATGGTTTCGCACCTTGTCACCACC tcctcgcccttcacgatacaAGGTGCTGTGCAGTTCTTTTTGGGATC
VIGS-GhSWEET9-F	aaggttaccgaattGAACAGTTCTCCCCAGCC
VIGS-GhSWEET9-R	ctcggtaccggatcAGCTATAATTCCGACCACCATG
PDR-GhSWEET9-F	ccagcctcgagcgg ATGGTTTCGCACCTTGTCAC
PDR-GhSWEET9-R	agctggatccgcgc AGGTGCTGTGCAGTTCTTT
qPCR-GhSWEET9-F	CCATCAATGGCACAGGGACT	表达量检测
qPCR-GhSWEET9-R	TCGACGTTGAGTGGTGTGAG	表达量检测
UBQ7-F	GAAGGCATTCCACCTGACCAAC	内参基因扩增
UBQ7-R	CTTGACCTTCTTCTTCTTGTGCTTG	内参基因扩增

Fig. 1 Expression pattern of GhSWEET9 gene infected with V. dahliaeCK: Water treatment; Vd991: inoculated with V. dahliae strain Vd991. Significance level (*P<0.05; **P<0.01; ***P<0.001. The same below)

Fig. 2 Bioinformatics analysis of GhSWEET9A: Prediction of the transmembrane domain of GhSWEET9 protein. B: Phylogenetic tree of SWEETs proteins from upland cotton and Arabidopsis

Fig. 3 Analysis of cis-acting elements in the promoter of GhSWEET9 gene in upland cottonA: Analysis of cis-acting elements in the 2 000 bp promoter region upstream of upland cotton GhSWEET9 gene. B: Number statistics of cis-acting elements in different types

Fig. 4 Subcellular localization of GhSWEET9 protein

Fig. 5 Functional validation of yeast transformed with GhSWEET9 gene

Fig. 6 Identification of GhSWEET9-silenced plants’‍s resistances to diseaseA: Relative expression of GhSWEET9 in control plants and silenced plants at 14 d post infection. B: Disease symptoms of control plants and silenced plants at 14 d post infection.C: Disease index of control plants and silenced plants at 14 d post infection. D: Vascular bundles browning in stem of control plants and silenced plants at 14 d post infection

Fig. 7 Glucose contents in the roots of control plants and VIGS-silenced plants infected with V. dahliaeA: The glucose content in the roots of cotton plants infected with V. dahliae after 2 d of infection. B: The glucose content in the roots of cotton plants infected with V. dahliae after 6 d of infection. CK: Water treatment; Vd991: inoculated with V. dahliae strain Vd991

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