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碳代谢基因gabT调控生防菌Snea253的γ-氨基丁酸代谢途径影响杀线虫活性

于冬梅1 朱峰2 邢志富1 范海燕1 朱晓峰1 段玉玺1 王媛媛3 刘晓宇4 陈立杰1

(1.沈阳农业大学植物保护学院, 辽宁沈阳 110866)
(2.吉林省农业科学院植物保护研究所, 吉林公主岭 130033)
(3.沈阳农业大学生物科学技术学院, 辽宁沈阳 110866)
(4.沈阳农业大学理学院, 辽宁沈阳 110866)

【摘要】【背景】委内瑞拉链霉菌Snea253是本实验室前期获得的具有杀植物线虫活性的生防放线菌,通过生物信息学分析,γ-氨基丁酸转氨酶基因(gabT)是参与Snea253碳代谢的重要基因之一。【目的】明确gabT基因通过调控Snea253的γ-氨基丁酸(γ-Aminobutyric acid,GABA)代谢通路,从而影响菌株的活性。【方法】以紫外诱变所得弱毒株(Snea253-R)为材料,以南方根结线虫为靶标,在弱毒株中过表达gabT基因,通过酶联免疫法(ELISA)和高效液相色谱法(HPLC)分别检测菌株中GABA和下游代谢产物琥珀酸的含量及杀线虫活性,同时检测在不同碳源培养条件下野生型菌株gabT基因表达水平、产物含量和杀线虫活性。【结果】过表达菌株R-p IB139的gabT基因上调表达,GABA含量降低,琥珀酸含量升高,杀线虫活性提高了39%;在8种不同碳源培养条件下,gabT基因在野生株中相对表达量较高的培养基碳源是可溶性淀粉和玉米淀粉,其发酵液中GABA含量较低,发酵液中下游代谢产物增多,杀线虫活性较高。【结论】通过改变gabT基因的表达,明确GABA支路在调控Snea253代谢以提高杀线虫的过程中发挥重要作用。

【关键词】 γ-氨基丁酸转氨酶基因gabT;委内瑞拉链霉菌;γ-氨基丁酸(GABA);琥珀酸;杀线虫活性;

【DOI】

【基金资助】 国家重点研发计划(2017YFD0201104); 国家自然科学基金(31471748);

Carbon metabolism gene gabT regulates γ-aminobutyric acid metabolism of Streptomyces venezuelae Snea253 on nematicidal activity

YU Dong-Mei1 ZHU Feng2 XING Zhi-Fu1 FAN Hai-Yan1 ZHU Xiao-Feng1 DUAN Yu-Xi1 WANG Yuan-Yuan3 LIU Xiao-Yu4 CHEN Li-Jie1

(1.College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning, China 110866)
(2.Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling, Jilin, China 130033)
(3.College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang, Liaoning, China 110866)
(4.College of Science, Shenyang Agricultural University, Shenyang, Liaoning, China 110866)

【Abstract】[Background] Streptomyces venezuelae Snea253 is an actinomycete with nematicidal activity to plant-parasitic nematode. Bioinformatics analysis showed that γ-aminobutyrate transaminase gene (gabT) is one of the important genes involved in carbon metabolism in Snea253. [Objective] The purpose of this research is to clarify that the gabT gene affects the activity of the strain by regulating the γ-aminobutyric acid metabolic pathway of Snea253. [Methods] First, the mutant strain (Snea253-R) obtained by UV mutation was used to overexpress the gabT gene, and Meloidogyne incognita J2 was used as the target nematode. γ-aminobutyric acid and the metabolite succinic acid were detected by enzyme-linked immunoassay (ELISA) and high performance liquid chromatography (HPLC), and the nematicidal activity was also detected. Meanwhile, the gabT gene expression level, product content and nematicidal activity of wild-type strain with eight carbon sources culture were detected. [Results] The expression of gabT gene in overexpression strain R-pIB139 was up-regulated, γ-aminobutyric acid content was decreased, the content of succinic acid was increased, and the nematicidal activity was increased by 39% in comparison with the control. In eight carbon sources cultures, the carbon source medium with higher relative expression of gabT gene in wild plants was soluble starch and corn starch. Under this condition, γ-aminobutyric acid content in fermentation broth was lower, the other products of metabolism were increased, and the nematicidal activity was higher. [Conclusion] By changing the expression of gabT gene, we confirmed that γ-aminobutyric acid shunt plays an important role in regulating the metabolism of Snea253 to enhance nematicidal activity.

【Keywords】 γ-Aminobutyrate transaminase gene gabT; Streptomyces venezuelae; γ-Aminobutyric acid(GABA); Succinic acid; Nematicidal activity;

【DOI】

【Funds】 National Key Research and Development Program of China (2017YFD0201104); National Natural Science Foundation of China (31471748);

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This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 46, No. 12, Pages 3257-3266

December 2019

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Article Outline

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion and conclusion
  • References