陆地棉GhSWEET9基因的克隆及抗黄萎病功能分析

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

摘要/Abstract

摘要：

目的研究GhSWEET9基因与棉花抗黄萎病相关性，为探究棉花抗黄萎病的分子机制及选育抗黄萎病棉花新品种提供理论依据。方法利用生物信息学软件分析GhSWEET9基因的序列特征、系统进化关系和亚细胞定位。利用酵母异源互补系统明确GhSWEET9蛋白的糖转运功能。利用病毒诱导的基因沉默技术（virus induced gene silencing, VIGS）分析该基因在抗黄萎病中的功能。结果 GhSWEET9蛋白有7个跨膜结构域，系统进化树分析显示其属于SWEETs Clade2 亚族成员。亚细胞定位结果显示GhSWEET9定位在细胞质膜上。酵母异源互补实验显示GhSWEET9蛋白可以转运半乳糖、甘露糖、葡萄糖和果糖。利用VIGS技术沉默GhSWEET9，葡萄糖含量测定发现沉默植株（pTRV2:GhSWEET9）根系中的葡萄糖含量明显高于空载体对照（pTRV2:00）；在大丽轮枝菌Vd991侵染棉苗2 d和6 d时发现，沉默植株根系中的葡萄糖含量仍明显高于对照植株；侵染14 d后发现，沉默植株叶片黄化和萎蔫程度比对照植株更为严重，维管束褐化更加明显，病情指数更高。结论 GhSWEET9基因沉默有利于葡萄糖在棉花根系中的积累，这可能促进了大丽轮枝菌对棉花根系的侵染，从而减弱棉花对黄萎病菌的抗性，推测该基因在棉花抗黄萎病过程中可能发挥重要作用。

关键词: 陆地棉, 糖转运蛋白, 棉花黄萎病, 病毒诱导的基因沉默, 葡萄糖

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

张勇, 宋盛龙, 李永泰, 张新宇, 李艳军. 陆地棉GhSWEET9基因的克隆及抗黄萎病功能分析[J]. 生物技术通报, 2025, 41(6): 144-154.

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.

图/表 8

表1 试验所用引物

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

图1 黄萎病菌侵染下GhSWEET9基因的表达模式CK：水处理；Vd991：黄萎病菌Vd991侵染。显著性水平（*P<0.05；**P<0.01；***P<0.001，下同）

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)

图2 GhSWEET9的生物信息学分析A：GhSWEET9蛋白跨膜结构域预测；B：陆地棉与拟南芥SWEETs蛋白的系统进化树

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

图3 陆地棉GhSWEET9基因启动子顺式作用元件分析A：陆地棉GhSWEET9基因上游2 000 bp启动子区顺式作用元件分析；B：不同类型顺式作用元件数量统计

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

图4 GhSWEET9蛋白的亚细胞定位

Fig. 4 Subcellular localization of GhSWEET9 protein

图5 GhSWEET9基因在酵母中的功能验证

Fig. 5 Functional validation of yeast transformed with GhSWEET9 gene

图6 GhSWEET9沉默植株抗病性鉴定A：对照植株和沉默植株中GhSWEET9的表达水平；B：接种黄萎病菌14 d后对照植株和沉默植株的发病症状；C：接种黄萎病菌14 d后对照植株和沉默植株的病情指数；D：接种黄萎病菌14 d后对照植株和沉默植株茎秆维管束褐化情况

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

图7 大丽轮枝菌侵染后对照和VIGS沉默植株根系葡萄糖含量测定A：大丽轮枝菌侵染2 d后棉花根部的葡萄糖含量；B：大丽轮枝菌侵染6 d后棉花根部的葡萄糖含量。CK为水处理，Vd991为黄萎病菌Vd991侵染

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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