北京欧文氏菌4个糖基转移酶活性测定及蛋白相互作用分析

doi:10.13304/j.nykjdb.2022.0736

摘要/Abstract

摘要：

北京欧文氏菌（Erwinia beijingensis）可引起刺芹侧耳细菌性软腐病，为明确该病原菌中糖基转移酶的功能，以糖基转移酶基因为研究对象，构建重组表达载体进行表达纯化；并测定蛋白活性，分析蛋白间相互作用。结果表明，成功构建了原核表达载体，获得4个可溶性蛋白。经亲和层析柱纯化获得MshA、WbnH2、EpsH及TuaG蛋白，活性测定显示4个蛋白均可以利用UDP-糖，并优先利用UDP-半乳糖。GST pull down证实WbnH2及TuaG蛋白分别与MshA及EpsH蛋白在体外具有相互作用。以上研究结果为进一步研究北京欧文氏菌糖基转移酶基因功能奠定了基础。

关键词: 北京欧文氏菌, 软腐病, 糖基转移酶, 原核表达

Abstract:

Erwinia beijingensis can cause the bacterial soft rot disease of Pleurotus eryngii. In order to clarify the function of glycosyltransferase in E. beijingensis， a recombinant expression vector was constructed to express and purify the glycosyltransferase in this pathogen. The protein activity was measured， and the interactions between proteins were analyzed. The results showed that the prokaryotic expression vector was successfully constructed， 4 soluble proteins were obtained， and MshA， WbnH2， EpsH and TuaG proteins were purified by affinity chromatography column. The activity assay showed that four proteins preferentially used UDP galactose. GST pull down results confirmed that WbnH2 and TuaG proteins interacted with MshA and EpsH proteins in vitro， respectively. Above results laid a foundation for the subsequent study on the function of the glycosyltransferase gene in E. beijingensis.

Key words: Erwinia beijingensis, soft rot, glycosyltransferase, prokaryotic expression

中图分类号:

Q933

常晓宁, 郭金英, 荣成博, 谷彤彤, 刘宇. 北京欧文氏菌4个糖基转移酶活性测定及蛋白相互作用分析[J]. 中国农业科技导报, 2024, 26(1): 125-132.

Xiaoning CHANG, Jinying GUO, Chengbo RONG, Tongtong GU, Yu LIU. Activity Determination of 4 Glycosyltransferases and Protein Interaction Analysis of Erwinia beijingensis[J]. Journal of Agricultural Science and Technology, 2024, 26(1): 125-132.

图/表 6

表1 试验引物

Table 1 Primers in this experiment

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp
mshA-F	GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCGAATAGTAACTTTGTTGTATGTTTGC	1 058
mshA-R	TCTCAGTGGTGGTGGTGGTGGTGCTCGAGGATGTTACCGCCCTCTAAAAGATACGATCTAAAATATTCTT
wbnH1 F	GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAATGCCGGAAGGAAAATAATTTATAT	1 044
wbnH1 R	CAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTAATGGAATCAACTCAAGATCTTTTTGGGCTTC
wbnH2 F	CCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCACTTGATCGTGATCCATAATGGTGTTCCTCCTCATGC	642
wbnH2 R	CGTCAGTCAGTCACGATGAATTAAGCTTGAGCTCGAGCTACCGAAGAAATATTTCCTGATACAATTTATTC
EpsH F	AAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAATGAGAAGATCGCATCCCCAAAACTCTCT	936
EpsH R	CAGTGGTGGTGGTGGTGGTGCTCGAGTTTTATAGTTAGCACTCTAGTTTTACGCAACAAAAAACGTAAAA
tuaG F	CTGGAAGTTCTGTTCCAGGGGCCCCTGGGATCCATGAAAATAACAATAGTTACTGCTACATATAATTCTG	685
tuaG R	AGATCGTCAGTCAGTCACGATGCGGCCGCTCGAGTCATCCCCTTAGAATAGCTCTTAGAGCATAATTCAT
His1422 F	AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGACGCCTGTTTATAATAGAGCAGACTTAC	732
His1422 R	AGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCATCTTTCTAATTCCAGAACCGACTCTAATTATG
GST1422 F	GGAAGTTCTGTTCCAGGGGCCCCTGGGATCCATGACGCCTGTTTATAATAGAGCAGACTTACTTAAAAAT	714
GST1422 R	TCGTCAGTCAGTCACGATGCGGCCGCTCGAGTTACATCTTTCTAATTCCAGAACCGACTCTAATTATGTT

图1 小量诱导表达可溶性蛋白的筛选结果A：E. coli BL21/pET-30a-mshA；B：E. coli BL21/pET-30a-EpsH；C：E. coli BL21/pGEX-6p-tuaG；D：E. coli BL21/pGEX-wbnH2。1~4分别为0.1 mmol·L-1 IPTG诱导的诱导前全菌、诱导后全菌、诱导后破碎上清及诱导后破碎沉淀；M—marker；5~8分别为1.0 mmol·L-1 IPTG诱导的诱导前全菌、诱导后全菌、诱导后破碎上清及诱导后破碎沉淀

Fig. 1 Screening results of soluble protein induced by a small amount of induced expression.A： E. coli BL21/ pET-30a-mshA； B： E. coli BL21/pET-30a-EpsH；C： E. coli BL21/pGEX-6p-tuaG；D： E. coli BL21/pGEX-wbnH2. 1~4 are whole bacteria before induction， whole bacteria after induction， broken supernatant after induction and broken sediment after induction with 0.1 mmol·L-1 IPTG induced， respectively； M is marker； 5~8 are the whole bacteria before induction， whole bacteria after induction， broken supernatant after induction and broken sediment after induction induced with 1.0 mmol·L-1 IPTG， respectively

图2 蛋白纯化结果A：MshA蛋白纯化，其中M为marker；1为流穿液，2为缓冲液A洗脱，3~9分别为20、40、60、80、100、150、200 mmol·L-1咪唑浓度洗脱液；B：EpsH蛋白纯化，其中1为流穿液，2~6分别为60、80、100、150、200、300 mmol·L-1咪唑浓度洗脱液，M为marker；C：WbnH2蛋白纯化，其中M为marker，1为流穿液，2~6分别为20 mmol·L-1 GSH洗脱；D：TuaG蛋白纯化，其中M为marker，1为流穿液，2~4为10 mmol·L-1 GSH洗脱，5~6为10 mmol·L-1 GSH+5 mmol·L-1 DTT洗脱

Fig. 2 Protein purification resultsA： MshA protein purification， in which M is marker， 1 is running through liquid， 2 is buffer A elution， 3~9 are the eluents of 20， 40， 60， 80， 100， 150， 200 mmol L-1 imidazole concentration， respectively； B： EpsH protein purification， in which 1 is the flow through solution， 2~6 are 60， 80， 100， 150， 200， 300 mmol L-1 imidazole concentration eluates， respectively， M is marker； C： WbnH2 protein purification， in which M is marker， 1 is running through liquid， 2~6 are 20 mmol L-1 GSH elution； D： TuaG protein purification， in which M is marker， 1 is running through liquid， 2~4 is 10 mmol L-1 GSH elution， 5-6 is 10 mmol·L-1 GSH+5 mmol L-1 DTT elution

图3 4种糖基转移酶对不同UDP-糖的水解能力

Fig. 3 Hydrolysis ability of four glycosyltransferases to different UDP sugars

图4 GST pull down结果A：WbnH2与MshA蛋白的结合，M—Marker，1—GST与MshA蛋白结合，2—MshA蛋白，3—WbnH2与MshA蛋白结合；B：TuaG与MshA蛋白的结合，M—Marker，1—GST与MshA蛋白，2— MshA蛋白，3—TuaG与MshA蛋白结合；C：WbnH2、TuaG蛋白分别与EpsH蛋白结合，M—Marker，1—WbnH2与EpsH蛋白结合，2—GST与EpsH蛋白结合，3—EpsH蛋白，4—GST与EpsH蛋白结合，5—TuaG与EpsH蛋白结合

Fig. 4 GST pull down resultA： Binding between WbnH2 and MshA proteins， M—Marker， 1—GST binds to MshA protein， 2—MshA protein， 3—WbnH2 binds to MshA protein； B： Binding between TuaG and MshA proteins， M—Marker， 1—GST binds to MshA proteins， 2—MshA protein， 3—TuaG binds to MshA protein； C： Binding between WbnH2 and TuaG proteins to EpsH protein， respectively， M—Marker， 1—WbnH2 binds to EpsH protein， 2—GST binds to EpsH protein， 3—EpsH protein， 4—GST binds to EpsH protein， 5—TuaG binds to EpsH protein

图5 Western-blot 检测结果A：WbnH2与MshA蛋白结合，1—GST与MshA蛋白结合，2—MshA蛋白，3—WbnH2与MshA蛋白结合；B：TuaG与MshA蛋白结合，1—GST与MshA蛋白结合，2—MshA蛋白，3—TuaG与MshA蛋白；C：WbnH2与EpsH蛋白结合，1—GST与EpsH蛋白结合；2—EpsH蛋白；3—WbnH2与EpsH蛋白结合；D：TuaG与EpsH蛋白结合，1—GST与EpsH蛋白结合；2—EpsH蛋白；3—TuaG与MshA蛋白结合

Fig. 5 Western blot test resultA： Binding between WbnH2 and MshA proteins. 1—GST binds to MshA protein； 2—MshA protein； 3—WbnH2 binds to MshA protein； B： Binding between TuaG and MshA proteins. 1—GST binds to MshA protein； 2—MshA protein； 3—TuaG binds to MshA protein； C： Binding between WbnH2 and EpsH proteins， 1—GST binds to EpsH protein； 2—EpsH protein； 3—WbnH2 binds to EpsH protQein； D： Binding between TuaG and EpsH proteins， 1—GST binds to EpsH protein， 2—EpsH protein； 3—TuaG binds to MshA protein

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