丁香假单胞菌番茄致病变种DC3000甘油激酶基因的克隆与功能研究

河南农业科学 ›› 2018, Vol. 47 ›› Issue (6): 70-75.

丁香假单胞菌番茄致病变种DC3000甘油激酶基因的克隆与功能研究

刘晓柱,李银凤^*,于志海,刘晓辉

(贵州理工学院食品药品制造工程学院，贵州贵阳 550003)

收稿日期:2018-01-15 出版日期:2018-06-15 发布日期:2018-06-15
通讯作者: 李银凤（1984-），女，山西朔州人，硕士，主要从事农业资源开发与保护研究。E-mail：liyinfeng614@126.com
作者简介:刘晓柱（1984-），男，河南商丘人，博士，主要从事微生物分子育种与发酵研究。E-mail：liuxiaozhu_840914@163.com
基金资助:
贵州省教育厅创新群体重大研究项目(黔教合KY字［2017］046)

Received:2018-01-15 Published:2018-06-15 Online:2018-06-15

摘要/Abstract

摘要： 为研究丁香假单胞菌番茄致病变种（Pseudomonas syringaepathovar tomato,Pst）DC3000甘油激酶（Glycerol kinase，GK）的功能，根据GenBank中登录的Pst DC3000 GK氨基酸序列设计引物，克隆了Pst DC3000 GK基因，对其生物信息学特征进行分析，然后构建了GK基因敲除突变体，探讨其对菌体生理特性的影响。序列分析表明，GK基因全长1 506 bp，可编码一条501个氨基酸的多肽。生物信息学分析结果显示，PstDC3000 GK蛋白分子质量55.8 ku，等电点5.54，富含无规卷曲结构。敲除Pst DC3000 GK后，可降低菌体在基本培养基与丰富培养基中的生长速率，增加细胞中甘油含量，同时也使菌体在拟南芥叶片上的增殖能力降低。因此，Pst DC3000 GK基因参与调控菌体的生长过程，影响菌体甘油代谢。

关键词: 丁香假单胞菌番茄致病变种DC3000, 甘油激酶, 基因克隆, 功能分析

刘晓柱,李银凤,于志海,刘晓辉. 丁香假单胞菌番茄致病变种DC3000甘油激酶基因的克隆与功能研究[J]. 河南农业科学, 2018, 47(6): 70-75.

参考文献

参考文献:
［1］Xin X F,He S Y.Pseudomonas syringae pv.tomato DC3000:A model pathogen for probing disease susceptibility and hormone signaling in plants［J］.Annual Review of Phytopathology,2013,51(1):473-498.
［2］Wang X,Hou S,Wu Q,et al.IDL6HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv.tomato DC3000 in Arabidopsis leaves［J］.Plant Journal,2017,89(2):250-263.
［3］Spoel S H,Johnson J S,Dong X,et al.Regulation of tradeoffs between plant defenses against pathogens with different lifestyles［J］.Proceedings of the National Academy of Sciences of the United States of America,2007,104(47):18842-18847.
［4］Yang Y,Wang M,Yin Y,et al.RNAseq analysis reveals the role of red light in resistance against Pseudomonas syringae pv.tomato DC3000 in tomato plants［J］.BMC Genomics,2015,16:120.
［5］Nomura K,Mecey C,Lee Y N,et al.Effectortriggered immunity blocks pathogen degradation of an immunityassociated vesicle traffic regulator in Arabidopsis［J］.Proceedings of the National Academy of Sciences of the United States of America,2011,108(26):10774-10779.
［6］Hou Z,Zhang H,Li M,et al.Structure of 2haloacid dehalogenase from Pseudomonas syringae pv.tomato DC3000［J］.Acta Crystallographica Section D(Biological Crystallography),2013,69(6):1108-1114.
［7］夏峰峰，金平，王领民，等.Klebsiella pneumoniae甘油发酵代谢调控研究进展［J］.化学与生物工程，2009，26（10）：1-4.
［8］Deocaris C C,Takano S,Priyandoko D,et al.Glycerol stimulates innate chaperoning,proteasomal and stressresistance functions:Implications for gerontomanipulation［J］.Biogerontology,2008,9(4):269-282.
［9］Bahieldin A,Sabir J S,Ramadan A M,et al.Control of glycerol biosynthesis under high salt stress in Arabidopsis［J］.Functional Plant Biology,2014,41(1):87-95.
［10］ Wei Y,Shen W,Dauk M,et al.Targeted gene disruption of glycerol3phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen［J］.Journal of Biological Chemistry,2004,279(1):429435.
［11］Holtman C K,Pawlyk A C,Meadow N D,et al.Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo［J］.Journal of Bacteriology,2001,183(11):3336-3344.
［12］Usuki T,Takato T,Lu Q,et al.Behavioral and pharmacological phenotypes of brainspecific diacylglycerol kinase δknockout mice［J］.Brain Research,2016,1648:193201.
［13Kang L,Li J,Zhao T,et al.Interplay of the Arabidopsis nonhost resistance gene 〖STBX〗NHO1〖STBZ〗 with bacterial virulence［J］.Proceedings of the National Academy of Sciences of the United States of America,2003,100(6):35193524.
［14］Agosto J A,Mccabe E R.Conserved family of glycerol kinase loci in Drosophila melanogaster［J］.Molecular Genetics and Metabolism,2006,88(4):334-345.
［15］Fodor E,Hellerud C,Hulting J,et al.Glycerol kinase deficiency in adult hypoglycemic acidemia［J］.The New England Journal of Medicine,2011,364(18):1781-1782.
［16］格林M R，萨姆布鲁克J.分子克隆实验指南［M］.4版.北京：科学出版社，2017.
［17］Miyamoto M,Furuichi Y,Komiyama T,et al.The highosmolarity glycerol and cell wall integrityMAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM1［J］.Yeast,2012,29(11):475-485.
［18］Fukuda Y,Abe A,Tamura T,et al.Epistasis effects of multiple ancestralconsensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp.NT3060［J］.Journal of Bioscience and Bioengineering,2016,121(5):497-502.
［19］Shemesh M,Chai Y.A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling［J］.Journal of Bacteriology,2013,195(12):2747-2754.
［20］Buell C R,Joardar V,Lindeberg M,et al.The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv.tomato DC3000［J］.Proceedings of the National Academy of Sciences of the United States of America,2003,100(18):10181-10186.
［21］Lu M,Tang X,Zhou J,et al.Arabidopsis NHO1 is required for general resistance against Pseudomonas bacteria［J］.The Plant Cell,2001,13(2):437-447.

[1]	高文, 闫庚洋, 左俊凯, 许乐, 侯蕊利, 景其昌, 任昊. 蜀葵ArbHLH148 基因克隆与表达分析[J]. 河南农业科学, 2023, 52(9): 133-140.
[2]	姚帅涛, 吴文静, 王少瓷, 詹为民, 刘通, 姜良良, 杨建平. 玉米ZmHY5和ZmHY5L基因的克隆及其转录丰度对不同光质处理的响应[J]. 河南农业科学, 2023, 52(3): 26-35.
[3]	杨翠凤, 滕峥, 韦春, 刘正鲁. 百香果苯丙氨酸解氨酶基因PePAL克隆及表达分析[J]. 河南农业科学, 2023, 52(10): 100-108.
[4]	白天亮, 李杰, 冉杰, 杨辉, 乔承彬, 李培富, 田蕾. 水稻产量构成因素QTL 精细定位的研究进展[J]. 河南农业科学, 2022, 51(8): 1-8.
[5]	杨立均, 鲁雅雯, 丁超, 金维环, 郭红祥. 烟草HPL基因的生物信息学分析与抗逆功能分析[J]. 河南农业科学, 2021, 50(2): 50-57.
[6]	刘艳阳, 武轲, 梅鸿献, 杜振伟, 崔承齐, 江晓林, 郑永战. 芝麻抗裂蒴基因SiIND1的克隆与原核表达[J]. 河南农业科学, 2020, 49(5): 63-68.
[7]	张元臣，蔡新，张国强，董丽丽，王景顺. 黏虫溶菌酶基因Mswly的克隆与真核表达[J]. 河南农业科学, 2019, 48(5): 70-77.
[8]	李亚，王珠娜. 长春花法尼基转移酶β-亚基基因的克隆及其在黄龙病菌感染过程中的表达分析[J]. 河南农业科学, 2019, 48(5): 78-84.
[9]	牛杨莉, 朱小佩, 杨亚萍, 葛晓敏, 杜晓华, 刘会超. 不同耐热三色堇材料HSP70基因克隆及热胁迫下的表达分析[J]. 河南农业科学, 2019, 48(10): 127-132.
[10]	郑玲,张欢欢,张延召. 棉花SBP家族基因的鉴定与分析[J]. 河南农业科学, 2019, 48(03): 39-48.
[11]	刘晓柱，李银凤，赵燕，张学文. 生长素对荠菜心皮发育的影响[J]. 河南农业科学, 2018, 47(8): 88-94.
[12]	曹路遥，周岩，魏琦超，陈燕绘，陈亚东，蔺一帆，田瑞婷. 玉米磷酸烯醇式丙酮酸羧化酶基因的克隆及其表达载体构建[J]. 河南农业科学, 2018, 47(5): 16-23.
[13]	樊爽爽，张雪梅，刘晓贺，刘姣扬，明胜利，褚贝贝，杨国宇，王江. 猪Gasp1和Gasp2基因对C2C12细胞分化的影响[J]. 河南农业科学, 2018, 47(3): 121-127.
[14]	杨雪，雒军，杨彩霞，王振恒，夏琦，王引权. 当归MYB4转录因子基因的克隆及表达分析[J]. 河南农业科学, 2018, 47(12): 48-56.
[15]	刘振，张侠，尹海波，陈世华，郭善利. 互花米草BADH基因的克隆与定量表达分析[J]. 河南农业科学, 2018, 47(11): 45-49.