绿针假单胞菌ZL3全基因组测序及atoB重组菌株对灰葡萄孢的抑菌活性

doi:10.16420/j.issn.0513-353x.2024-0879

摘要/Abstract

摘要： 为明确绿针假单胞菌ZL3对灰葡萄孢的抑菌机理，通过全基因组测序分析，筛选出与萜类和聚酮类代谢途径相关基因atoB，预测其蛋白结构，并构建重组菌株Escherichia coli BL21（DE3）/pET32-atoB。采用双皿对扣法测定重组菌株E. coli BL21（DE3）/pET32-atoB对7种常见病原菌的抑制效果和对樱桃灰霉病的防效，以及对樱桃防御酶活性的影响。结果表明：绿针假单胞菌ZL3全长6 715 612 bp，有1个染色体，无质粒，GC含量63.04%，基因组中包含5 956个编码基因，GO、COG和KEGG数据库中分别注释到2 873、5 032和3 446个基因，发现参与代谢的基因最多；重组菌株E. coli BL21（DE3）/pET32-atoB对7种病原菌具有较好抑菌效果，其中对灰葡萄孢的抑制效果最好，抑制率达到83.21%；重组菌株处理后提高了樱桃过氧化氢酶（CAT）、几丁质酶（CHI）和苯丙氨酸解氨酶（PAL）活性，樱桃灰霉病的发病率下降了43.75%，病斑直径减小了39.01%。

关键词: 樱桃, 绿针假单胞菌, 全基因组测序, atoB基因, 灰霉病, 生物防治, 抑菌活性

Abstract:

In order to clarify the antibacterial mechanism of Pseudomonas chlororaphis ZL3 against Botrytis cinerea，the gene atoB related to metabolic pathways of terpenoids and polyketones was screened by whole genome sequencing analysis. The protein structure of gene atoB was predicted. The recombinant strain Escherichia coli BL21（DE3）/pET32-atoB was constructed. The inhibitory effects of E. coli BL21 （DE3）/pET32-atoB on seven pathogenic fungi were determined by using two-sealed-base-plate method. The control effects of E. coli BL21（DE3）/pET32-atoB against cherry gray mold was determined. The effects of E. coli BL21（DE3）/pET32-atoB on cherry defense enzyme activities were detected by using ultraviolet spectrophotometry. The results showed that the total length of strain ZL3 was 6 715 612 bp，with one chromosome and no plasmid，and the GC content was 63.04%. The genome contained 5 956 coding genes. A total of 2 873，5 032 and 3 446 genes were annotated in GO，COG and KEGG databases，respectively. The recombinant strain E. coli BL21（DE3）/pET32-atoB had good antifungal effects on seven pathogenic fungi，and presented the highest inhibition effects on B. cinerea with an inhibition rate of 83.21%. After treatment with recombinant strain，the catalase（CAT），chitinase（CHI）and phenylalanine aminolyase（PAL）activities in cherry. The incidence of cherry gray mold decreased by 43.75%，while the diameter of the disease spots decreased by 39.01%.

Key words: cherry, Pseudomonas chlororaphis, whole genome sequencing, atoB gene, Botrytis cinerea, biological control, antifungal activity

袁胜楠, 袁佳琪, 王梦茜, 王燕, 王美琴, 王春伟. 绿针假单胞菌ZL3全基因组测序及atoB重组菌株对灰葡萄孢的抑菌活性[J]. 园艺学报, 2025, 52(11): 3044-3056.

YUAN Shengnan, YUAN Jiaqi, WANG Mengxi, WANG Yan, WANG Meiqin, WANG Chunwei. Whole Genome Sequencing of Pseudomonas chlororaphis ZL3 and Antifungal Activity of atoB Gene Recombinant Strain Against Botrytis cinerea[J]. Acta Horticulturae Sinica, 2025, 52(11): 3044-3056.

图/表 10

图1 绿针假单胞菌Pseudomonas chlororaphis ZL3的基因组圈图

Fig. 1 Genome circle map of Pseudomonas chlororaphis ZL3

图2 绿针假单胞菌Pseudomonas chlororaphis ZL3基因组GO功能注释分析

Fig. 2 GO functional annotation analysis of Pseudomonas chlororaphis ZL3 genome

图3 绿针假单胞菌Pseudomonas chlororaphis ZL3基因组COG功能注释分析

Fig. 3 COG functional annotation analysis of Pseudomonas chlororaphis ZL3 genome

图4 绿针假单胞菌Pseudomonas chlororaphis ZL3基因组KEGG功能注释分析

Fig. 4 KEGG functional annotation analysis of Pseudomonas chlororaphis ZL3 genome

图5 atoB编码蛋白二级预测结构蓝色：α螺旋；绿色：β转角；紫色：无规则卷曲；红色：延伸链

Fig. 5 The predicted secondary structure of atoB encoded proteins Blue：α helix；Green：β corner；Purple：Irregular curl；Red：Extension chain

图6 atoB基因（A）及重组菌株E. coli BL21（DE3）/pET32-atoB（B）PCR验证

Fig. 6 PCR validation of the atoB gene（A）and the recombinant strain E. coli BL21（DE3）/pET32-atoB（B）

图7 atoB重组蛋白表达条件

Fig. 7 Conditions for expression of the recombinant protein atoB

图8 重组菌株E. coli BL21（DE3）/pET32-atoB对不同病原菌的抑制效果

Fig. 8 Inhibition effects of E. coli BL21（DE3）/pET32-atoB on different pathogens

图9 重组菌株E. coli BL21（DE3）/pET32-atoB对樱桃灰霉病发病率和病斑直径的影响不同小写字母表示各处理间差异显著（P < 0.05）

Fig. 9 Effects of E. coli BL21（DE3）/pET32-atoB on incidence and spot diameter of cherry Different lowercase letters indicate significant differences among different treatments at 0.05 level

图10 重组菌株E. coli BL21（DE3）/pET32-atoB对樱桃酶活性的影响

Fig. 10 Effects of E. coli BL21（DE3）/pET32-atoB on enzyme activity of cherry * P < 0.05，** P < 0.01

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