Analog-sensitive蛋白激酶研究系统在植物病原真菌中的建立

doi:10.13560/j.cnki.biotech.bull.1985.2021-1602

摘要/Abstract

摘要：

常用的基因敲除技术无法应用于某些特殊激酶的研究，旨为在植物病原真菌中建立基于蛋白激酶人工改造的analog-sensitive蛋白激酶研究系统，为蛋白激酶的研究提供新的思路和方法。采用网站预测蛋白激酶的“守门员”残基，用载体回补法对其进行突变获得相应的类似物敏感型（analog-sensitive，as）突变体gpmk1-as与pmk1-as，并检测类似物敏感型蛋白激酶的功能及其对ATP类似物抑制剂1-NM-PP1的敏感性。结果显示，Gpmk1与Pmk1的守门员残基分别为Q103和Q104，此位点在多个代表性真菌中均十分保守；类似物敏感型突变体gpmk1-as的菌落生长速度与野生型PH-1一致，均快于Gpmk1敲除突变体，Pmk1-as能够产生与野生型Guy11一样成熟的黑化附着孢，而Pmk1敲除突变体无法产生；在5 μmol/L的1-NM-PP1条件下，gpmk1-as的菌落生长速度下降，但PH-1正常生长，在10 μmol/L的1-NM-PP条件下，pmk1-as无法形成附着孢，但Guy11可以形成成熟的黑化附着孢。结果表明，Gpmk1和Pmk1的类似物敏感型蛋白激酶均能行使正常的蛋白功能，却对ATP类似物抑制剂1-NM-PP1十分敏感。

关键词: 植物病原真菌, 蛋白激酶, 人工改造, 化学遗传学技术, 类似物敏感型

Abstract:

Common gene knockout techniques cannot be applied to the study of some specific kinases. In order to provide new ideas and methods for the study of protein kinases，an analog-sensitive protein kinase system based on artificial modification of protein kinases was established in plant pathogenic fungi. The “gatekeeper” residues of protein kinase were predicted via web technology，and the analog-sensitive（as）mutants gpmk1-as and pmk1-as were obtained by vector complement method. The function of analog-sensitive protein kinase and its sensitivity to ATP analogue inhibitor 1-NM-PP1 were detected. The results showed that the “gatekeeper” residues of Gpmk1 and Pmk1 were Q103 and Q104，respectively，which were very conserved in multiple representative fungi. The colony growth rate of the analog-sensitive mutant gpmk1-as was consistent with that of the wild type PH-1，both faster than that of the Gpmk1 knockout mutant. pmk1-as produced appressorium being same with that of the wild type Guy11，but the Pmk1 knockout mutant could not do so. With 5 μmol/L 1-NM-PP1，the colony growth rate of Gpmk1-as decreased，but the growth of PH-1 was not affected. With 10 μmol/L 1-NM-PP，pmk1-as could not form appressorium，but Guy11 formed mature black appressorium. The results showed that both Gpmk1 and Pmk1 analog-sensitive protein kinases could perform normal protein functions，but were very sensitive to ATP analogue inhibitor 1-NM-PP1.

Key words: plant pathogenic fungi, protein kinase, artificial modification, chemo-genetical technique, analog-sensitive

孙忠娟, 刘倩倩, 郭雨纤, 王光辉, 王晨芳. Analog-sensitive蛋白激酶研究系统在植物病原真菌中的建立[J]. 生物技术通报, 2022, 38(11): 49-57.

SUN Zhong-juan, LIU Qian-qian, GUO Yu-qian, WANG Guang-hui, WANG Chen-fang. Establishment of Analog-sensitive Protein Kinase Research System in Plant Pathogenic Fungi[J]. Biotechnology Bulletin, 2022, 38(11): 49-57.

图/表 9

表1 本实验所用的引物序列

Table 1 Primer sequences used in this study

引物名称Primer	引物序列Sequence（5'-3'）
GPMK1-as/1F	AGGGAACAAAAGCTGGGTACCGGGGTATGGT- GGTCAAGGTTATG
GPMK1-as/2R	ACAGTGGCGTTTCGTACTCCGATCAGATAGAC
GPMK1-as/3F	GTCTATCTGATCGGAGTACGAAACGCCACTGT
GPMK1-as/4R	GATTTCAGTAACGTTAAGTGGATCCATGGTGG- CAAGTAAAGGAATGAG
GPMK1-as/5F	CTGTATCCGTAGATGGGAGGTA
GPMK1-as/6F	GACCGTGCTTGCTGTATCCT
GPMK1-as/7F	GACCGAACGACCGATTGACT
GPMK1-as/8F	CAACCTCCTCCTCAACGCCAACT
GPMK1-as/9F	ACCCATACCTTGAGCCTTACC
PMK1-as/1F	AGGGAACAAAAGCTGGGTACCGCGTCAAGCC- ATCGCGTTATCT
PMK1-as/2R	GAGTGTTCAGCAGCAACGGACCCCGATCAGG- TACACCTCGTT
PMK1-as/3F	TTCAACGAGGTGTACCTGATCGGGGTCCGTTG- CTGCTGAACACTCC
PMK1-as/4R	GATTTCAGTAACGTTAAGTGGATCCGGCACAG- CCAATCCACGAATACACA
PMK1-as/5F	ATTCTCGTTGGTCCATCCGTC
PMK1-as/6F	TCCACCAAGCAACTCTTCGGG
PMK1-as/7F	TCAAGGCAATGCACTCGGCAAAC
PMK1-as/8F	GTACCATGACCCCGATGATGAACC
PMK1-as/9F	CGGCACTCACAATTAAGCCGTTG
GPMK1-5F	CAACTGTGATCTCAAGGTCTGCG
GPMK1-6R	TGCTTCAGAGCTTCCTCCACAG
PMK1-5F	CGAAACGCCGTCTACTGTACTTG
PMK1-6R	CAGTACTGGGGAGAACATCGTG

图1 pKNT-GPMK1-as载体的构建

Fig. 1 Construction of pKNT-GPMK1-as vector

图2 代表性真菌中Gpmk1的序列比对（A）及Gpmk1三维结构展示（B）

Fig.2 Multiple sequence alignments of Gpmk1 in representative fungi（A）and 3D structure of Gpmk1（B）

图3 重要蛋白激酶的“守门员”残基

Fig. 3 “Gatekeeper” residues of some essential protein kinases

图4 gpmk1-as（A）及pmk1-as（B）转化子的PCR检测 M：DNA marker；P：阳性对照；N：阴性对照

Fig.4 PCR detection of gpmk1-as（A）and pmk1-as（B）transformants M：DNA marker；P：positive control；N：negative control

图5 菌株PH-1、gpmk1和gpmk1-as在PDA平板上生长2 d的菌落（A）及菌落直径统计分析（B）经单因素方差分析及Duncan多极差检验，不同字母表示差异显著（P=0.05）

Fig.5 Cultures of the strains PH-1，gpmk1 and gpmk1-as grown on PDA plate for 2 d（A）and colony dia-meters of the indicated strains（B） Different letters indicate significant differences based on ANOVA analysis followed by Duncan’s multiple range test（P=0.05）

图6 菌株Guy11、pmk1和pmk1-as在疏水玻片上的附着孢形成（Bar=50 μm）

Fig. 6 Appressorium formation of strains Guy11，pmk1 and pmk1-as on hydrophobic slides（Bar=50 μm）

图7 1-NM-PP1处理后菌株PH-1、gpmk1-as和gpmk1在PDA平板上生长2 d的菌落（A）及直径统计分析（B）经t检验分析（P=0.05）

Fig.7 Cultures of the strain PH-1，gpmk1 and gpmk1-as grown on PDA plate for 2 d after 1-NM-PP1 treatm-ent（A）and colony diameters of the indicated str-ains（B） Significant differences based on t test（P=0.05）

图8 1-NM-PP1处理后菌株Guy11、pmk1和pmk1-as在疏水玻片上的附着孢形成（Bar=50 μm）

Fig.8 Appressorium formation of strain Guy11，pmk1 and pmk1-as on hydrophobic slides after 1-NM-PP1 treatment（Bar=50 μm）

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