油茶果生刺盘孢液泡分选蛋白CfVps26的功能

doi:10.11707/j.1001-7488.20210809

摘要/Abstract

摘要：

目的: 研究液泡分选蛋白Vps26在油茶果生刺盘孢中的生物学功能，为阐明Vps26的作用机制和防治油茶炭疽病提供理论依据。方法: 采用Over-lap法构建CfVPS26基因敲除载体片段，利用PEG介导的转化法将该片段转入果生刺盘孢原生质体中，对具有潮霉素抗性的转化子进行验证筛选；PCR扩增启动子在内的CfVPS26基因回补片段，构建回补质粒并转入突变体原生质体中，荧光筛选回补菌株；对野生型菌株、突变体和回补菌株进行营养生长、附着胞形成率、细胞壁胁迫、糖原染色及致病力等生物学表型测定。结果: 筛选验证获得CfVPS26基因缺失突变体ΔCfvps26；该突变体生长速率均显著小于野生型菌株和回补菌株，气生菌丝也明显减少；在含有细胞壁抑制剂200 μg·mL^-1荧光增白剂和0.01%十二烷基硫酸钠的PDA培养基中，突变体ΔCfvps26生长抑制率显著高于野生型和回补菌株的抑制率；在含有内质网胁迫剂二硫苏糖醇的PDA培养基中，突变体菌株ΔCfvps26耐受性增强，抑制率显著低于野生型和回补菌株。进一步研究发现，CfVPS26基因缺失突变体分生孢子产量和附着胞形成率显著降低、糖原的代谢受阻，无法在油茶叶片上形成病斑，致病力丧失。结论: 液泡分选蛋白CfVps26参与调控油茶果生刺盘孢生长发育、产孢、附着胞形成、外界胁迫、糖原代谢和致病过程。本研究可为阐明CfVps26参与调控果生刺盘孢致病的分子机制提供参考，并为新型杀菌剂的开发提供潜在靶标位点。

关键词: 油茶, 果生刺盘孢, CfVps26, 致病力

Abstract:

Objective: Colletotrichum fructicola is a major epidemic and causes anthracnose of Camellia oleifera. This paper aims to study the biological functions of the vacuolar protein sorting Vps26 in C. fructicola to provide a theoretical basis for the prevention and control of oil-tea tree anthracnose, and also for further analysis of Vps26 action mechanism. Method: The over-lap method was used to construct the CfVPS26 gene knockout vector fragment which was transformed into the protoplast of C. fructicola by using PEG-mediated transformation method to obtain the mutant strain of ΔCfvps26. The PCR-amplified CfVPS26 gene-containing complement of the promoter was taken to construct a complementary vector. Then the complementary vector was transformed into the mutant protoplasts. The complemented strain was screened by fluorescence. The biological phenotypes, such as vegetative growth, appressorium formation, external environmental pressure, glycogen metabolism and pathogenicity, of the wild-type strain, mutant strain, and the complemented strain were measured. Result: The results showed that the mycelial growth rate of ΔCfvps26 was significantly lower than that of the wild type strain and the complemented strain. And the aerial hyphae were significantly decreased, and more sensitive to 200 μg·mL^-1 calcofluor white and 0.01% sodium dodecyl sulfate. Compared with the wild type and the complemented strain, the tolerance of the mutant strain ΔCfvps26 to dithiothreitol was enhanced. The conidia production and appressorium formation rate of CfVPS26 gene deletion mutant were significantly reduced, and glycogen metabolism was blocked. The mutant strain almost lost pathogenicity on the leaves of C. oleifera. Conclusion: This study reveals that the vacuolar sorting protein CfVps26 plays critical roles in growth and development, condiation, appressorium formation, tolerance to external stresses, glycogen metabolism and pathogenicity in C. fructicola on C. oleifera. These results provide potential target sites for the development of new fungicides against C. fructicola.

Key words: Camellia oleifera, Colletotrichum fructicola, CfVps26, pathogenicity

中图分类号:

S718.46

李茜雅,张盛培,李河. 油茶果生刺盘孢液泡分选蛋白CfVps26的功能[J]. 林业科学, 2021, 57(8): 94-101.

Xiya Li,Shengpei Zhang,He Li. Function of Vacuolar Protein Sorting CfVps26 in Colletotrichum fructicola on Camellia oleifera[J]. Scientia Silvae Sinicae, 2021, 57(8): 94-101.

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引物名称 Primer	序列 Sequence(5’—3’)	目的 Purpose
CfVPS26-1F	GATAGAATAAGCGGATGCGG	扩增CfVPS26 5′侧翼序列 Amplify CfVPS26 5′ flank sequence
CfVPS26-2R	TTGACCTCCACTAGCTCCAGCCAAGCCCTTGGCTTCGGTGGTTACCC	扩增CfVPS26 5′侧翼序列 Amplify CfVPS26 5′ flank sequence
CfVPS26-3F	CAAAGGAATAGAGTAGATGCCGACCGAGCTGTGTTGCAATGCTGCA	扩增CfVPS26 3′侧翼序列 Amplify CfVPS26 3′flank sequence
CfVPS26-4R	GAGATGCTTAGTCGCCAGTT	扩增CfVPS26 3′侧翼序列 Amplify CfVPS26 3′flank sequence
CfVPS26-5F	GCATGCATTCAATTGCCTGC	CfVPS26基因缺失验证 Validation of CfVPS26 gene deletion
H855R	GCTGATCTGACCAGTTGC	CfVPS26基因缺失验证 Validation of CfVPS26 gene deletion
CfVPS26-7F	CGGCAAGCGACTAGAGCATA	扩增CfVPS26基因序列 Amplify CfVPS26 gene sequence
CfVPS26-8R	ATCCCGGAATGTAGGGGTCA	扩增CfVPS26基因序列 Amplify CfVPS26 gene sequence
CfVPS26-9F	ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTACACCGACAACCAGGTTGTT	扩增回补序列 Amplify complemented sequence
CfVPS26-10R	CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCGCTTGCGAGGGCTGTGGCA	扩增回补序列 Amplify complemented sequence
Hyg F	GGCTTGGCTGGAGCTAGTGGAGGTCAA	扩增HPH序列 Amplify HPH sequence
Hyg R	CGGTCGGCATCTACTCTATTCCTTTG	扩增HPH序列 Amplify HPH sequence
GFP-R	GACACGCTGAACTTGTGGCCGTT	验证回补序列 Validation of complemented sequence

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