两种表达载体对西瓜食酸菌生物学特性的影响

doi:10.6048/j.issn.1001-4330.2018.03.008

摘要/Abstract

摘要： 【目的】分析两种常用基因功能表达载体对宿主西瓜食酸菌生物学特性的影响,为基因功能研究选择载体提供理论参考。【方法】以西瓜食酸菌野生型FC440菌株为宿主菌,分别转入表达载体pBBR1MCS-5和pHC60,从胞外纤维素酶活性、胞外蛋白酶活性、胞外多糖含量、游动性、致病性和过敏性反应、生长速率以及抗铜性等,对转化菌株进行生物学特性影响的分析。【结果】载体pBBR1MCS-5对菌株的生物学特性各方面未表现出显著影响,载体pHC60会显著抑制菌株的生长速率和游动性。【结论】表达载体本身会影响西瓜食酸菌的某些生物学特性,在基因功能研究中,应依据所研究基因的功能选择适宜的表达载体。

关键词: 西瓜食酸菌; 表达载体; 生物学特性

Abstract: 【Objective】 This project aims to analyze the effects of two types of expression vectors on the biological characteristics ofAcidovorax citrulli in the hope of providing a theoretical reference for the study of gene function.【Method】The wild type FC440 strains of watermelon acid bacteria were transferred into the expression vectors pBBR1MCS-5 and pHC60 respectively. Then the extracellular cellulase activity, extracellular protease activity, extracellular polysaccharide content, mobility, pathogenicity and hypersensitive reaction were obtained and the effects of growth rate and copper resistance on the biological characteristics of the transformed strain were analyzed.【Result】The results showed that vector pBBR1MCS-5 had no significant effect on the biological characteristics ofAcidovorax citrulli, but vector pHC60 significantly inhibited the growth rate and motility of the strain. 【Conclusion】During the study of gene function, the expression vector itself may affect some biological characteristics ofAcidovorax citrulli. It is necessary to choose an appropriate expression vector while exploring the function of a gene.

Key words: Acidovorax citrulli; expression vector; biological characteristics

中图分类号:

S188
S651

黄成文,王希东,刘君,杨东晟,古妮萨罕·麦托合提,
热孜宛·奥布力. 两种表达载体对西瓜食酸菌生物学特性的影响[J]. 新疆农业科学, 2018, 55(3): 457-467.

HUANG Cheng-wen, WANG Xi-dong, LIU Jun, YANG Dong-sheng, Gunisaha Matoheti, Reziwan Aobuli. Effects of Two Types of Expression Vectors on Biological Characteristics of Acidovorax citrulli[J]. Xinjiang Agricultural Sciences, 2018, 55(3): 457-467.

参考文献

[1] Schaad, N. W., Postnikova, E., Sechler, A., Claflin, L. E., Vidaver, A. K., & Jones, J. B., et al. (2008). Reclassification of subspecies of acidovorax avenae as a. avenae (manns 1905) emend. a. cattleyae (pavarino, 1911) comb. nov. a. citrulli schaad et al. 1978) comb. nov. and proposal of a. oryzae sp. nov. Systematic & Applied Microbiology, 31(6-8): 434-446.
[2] Schaad, N. W., Sowell, G. J., Goth, R. W., Colwell, R. R., & Webb, R. E. (1978). Pseudomonas pseudoalcaligenes subsp. citrulli subsp. nov. International Journal of Systematic Bacteriology, 28(4): 621
[3] Willems, A., Goor, M., Thielemans, S., Gillis, M., Kersters, K., & De, L. J. (1992). Transfer of several phytopathogenic pseudomonas species to acidovorax as acidovorax avenae subsp. avenae subsp. nov. comb. nov. acidovorax avenae subsp. citrulli, acidovorax avenae subsp. cattleyae, and acidovorax konjaci. International Journal of Systematic Bacteriology,42(1): 107-119.
[4] 阎莎莎,王铁霖,赵廷昌.瓜类细菌性果斑病研究进展[J].植物检疫,2011,25(3):71-76.
YAN Sha-sha, WANG Tie-lin, ZHAO Ting-chang. (2011). Research progress on bacterial fruit blotch [J]. Plant Quarantine, 25(3):71-76. (in Chinese)
[5] Bahar, O., Burdman, S., Edelstein, M., & Cohen, R. (2010). Bacterial fruit blotch: a threat to the cucurbit industry. Israel Journal of Plant Sciences, 58(1): 19-31.
[6] Rane, K. K., & Latin, R. X. (1992). Bacterial fruit blotch of watermelon: association of the pathogen with seed. Plant Disease, 76(5): 509-512.
[7] Schaad, N. W., Postnikova, E., & Randhawa, P. (2003). Emergence of Acidovorax avenae subsp. citrulli as a Crop Threatening Disease of Watermelon and Melon. Pseudomonas syringae, and related pathogens. Springer Netherlands.
[8] Sowell, G. J., & Schaad, N. W. (1979). Pseudomonas pseudoalcaligenes subsp. citrulli on watermelon: seed transmission and resistance of plant introductions. Plant Disease Reporter, 63(6): 437-441.
[9] 高杜娟,韩振华,王敏杰,等.瓜类细菌性果斑病菌hpaP基因的功能分析[J]. 农业生物技术学报,2011, 19(4):746-752.
GAO Du-juan, HAN Zhen-hua, WANG Min-jie, et al. (2011). Functional Analysis of the hpaPGene in Acidovorax citrulli [J]. Journal of Agricultural Biotechnology, 19(4): 746-752.(in Chinese)
[10] 刘君.西瓜食酸菌(Acidovorax citrulli)Tn5转座子插入突变体的制备和致病性缺失突变体的鉴定与分析[D].杨凌: 西北农林科技大学博士论文,2012.
LIU Jun. (2012 ). Creation of Tn5 insertional mutants of bacterial fruit blotch pathogen acidovorax Citrulli and identification of pathogenicity altered mutants [D]. PhD Dissertation. Northwest Agriculture and Forestry University, Yangling.(in Chinese)
[11] 刘星,王希东,刘君.西瓜食酸菌RND蛋白家族外排转运体cusB基因抗铜功能研究[J]. 微生物学通报,2016, 43(1):97-106.
LIU Xing, WANG Xi-dong, LIU Jun. (2016). Functional analysis of a RND family efflux transporter component-cus B gene associated with copper resistance in Acidovorax citrulli [J]. Microbiology China, 43(1): 97-106.(in Chinese)
[12] 魏娜,钱国良,李晓霞,等. 瓜类细菌性果斑病菌tvrR基因的克隆及其对致病性的影响[J]. 南京农业大学学报,2010,33(6): 68-74.
WEI Na, QIAN Guo-liang, LI Xiao-xia, et al. (2010). Cloning of tvrRgene from Acidovorax avenaesubsp. citrulliand its effect on pathogenicity [J]. Journal of Nanjing Agricultural University, 33(6):68-74.(in Chinese)
[13] 杨丙烨,付丹,胡方平,等. 西瓜细菌性果斑病菌鞭毛基因fliS的功能分析[J].中国农业科学,2017,50(15):2 946-2 956.
YANG Bing-ye, FU Dan, HU Fang-ping, et al. (2017). Function Analysis of Flagellar Gene fli Sin Acidovorax citrulli[J]. Scientia Agricultura Sinica, 50(15): 2,946-2,956.(in Chinese)
[14] 严婉荣.瓜类细菌性果斑病菌hrcN基因的克隆及功能分析[D].长沙: 湖南农业大学硕士论文,2012.
YAN Wan-rong. (2012). Coloning and Funtional Analysis of hrcN gene in Acidovorax citrulli[D]. Master Dissertation. Hunan Agricultural University, Changsha.(in Chinese)
[15] 陈燏,朱善良. PET质粒表达对宿主菌的影响研究[J].江苏教育学院学报(自然科学版),2011,(1):30-34, 92.
CHEN Wei, ZHU Shan-liang. (2011). Impact of PET plasmid expression on the E.coli [J]. Journal of Jiangsu Institute of Education (Natural Science Ed.) , (1): 30-34, 92.(in Chinese)
[16] King, E. O., Ward, M. K., & Raney, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med, 44(2):301-307.
[17] Cheng, H. P., & Walker, G. C. (1998). Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by rhizobium meliloti. Journal of Bacteriology, 180(19): 5,183-5,191.
[18] J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.第三版[M].北京:科学出版社,2002:93-96.
Sambrook, J. David W. Russell. (2002). Molecular Cloning: A Laboratory Manual,3rd ed. [M].Beijing: Science Press: 93-96. (in Chinese)
[19] Walcott, R. R., Gitaitis, R. D., & Castro, A. C. (2003). Role of blossoms in watermelon seed infestation by acidovorax avenae subsp. citrulli. Phytopathology, 93(5): 528-534.
[20] 陈磊.水稻黄单胞菌双精氨酸运输(Tat)系统关键基因的克隆与功能分析[D]. 南京: 南京农业大学硕士学位论文, 2008.
CHEN Lei. (2008). Cloning and fuctional study of key genes of twin-arginine translocation system of Xanthomonas oryzae[D]. Master Dissertation. Nanjing Agricultural University, Nanjing. (in Chinese)
[21] 徐荣旗.野油菜黄单胞菌野油菜致病变种新的依赖于Ⅲ型分泌系统的效应物的鉴定[D]. 南宁:广西大学博士学位论文, 2006.
XU Rong-qi. (2006). Identification of novel effectors depended on type Ⅲ secretion system in Xanthomonas campestris pv. campestris[D]. PhD Dissertation. Guangxi University, Nanning. (in Chinese)
[22] He, L. Y., Zhang, Y. F., Ma, H. Y., Su, L. N., Chen, Z. J., & Wang, Q. Y., et al. (2010). Characterization of copper-resistant bacteria and assessment of bacterial communities in rhizosphere soils of copper-tolerant plants. Applied Soil Ecology, 44(1):49-55.
[23] Chesnokova, O., Coutinho, J. B., Khan, I. H., Mikhail, M. S., & Kado, C. I. (1997). Characterization of flagella genes of agrobacterium tumefaciens, and the effect of a bald strain on virulence. Molecular Microbiology, 23(3): 579-590.
[24] 任争光,侯磊,宋治国,等.甜瓜细菌性果斑病菌致病性突变体筛选与hrc R基因的克隆[J].植物病理学报, 2009, 39(5): 501-506.
REN Zheng-guang, HOU Lei, SONG Zhi-guo, et al.(2009). screening of the pathogenicity mutants of Acidovorax avenaesubsp.citrulli and cloning of the hrcRgene [J]. Phytopathologica Sinica, 39(5): 501-506. (in Chinese)
[25] 管文静.水稻白叶枯病菌转录调控因子的分子鉴定及功能分析[D].北京:中国农科院植保所硕士学位论文,2009.
GUAN Wen-jing. (2009). Identification and function characterization of the transcriptional regulator clpxoo in Xanthomonas oryzae pv.oryzae[D]. Master Dissertation. Institute of plant protection, Chinese Academy of Agricultural Sciences, Beijing. (in Chinese)
[26] Untergasser,A., Geurts,R. and Franssen,H. pHC60-a very stable plasmid for fluorescence mircroscopy.
[27] Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pbbr1mcs, carrying different antibiotic-resistance cassettes. Gene, 166(1): 175-176.
[28] 张小飞, 李晓, 崔丽娜,等. 轮枝镰孢菌的绿色荧光蛋白基因标记[J]. 西南农业学报, 2014, 27(6):2 374-2 376.
ZHANG Xiao-fei, LI Xiao, CUI Li-na,et al. (2014).Green Fluorescent Protein Gene Transformation on Fusarium verticillioides[J]. Southwest China Journal of Agricultural Sciences, 27(6):2,374-2,376.
[29] 车建美,蓝江林,刘波.转绿色荧光蛋白基因的青枯雷尔氏菌生物学特性[J].中国农业科学,2008, 41(1): 3 626-3 635.
CHE Jian-mei, LAN Jiang-Lin, LIU Bo. (2008). GFP Tagging Ralstonia solanacearumwith gfp/lux ABmini-Tn5[J]. Scientia Agricultura Sinica, 41(1):3,626-3,635.(in Chinese)
[30] Hoffmann, F., & Rinas, U. (2004). Stress induced by recombinant protein production in escherichia coli. Advances in Biochemical Engineering/biotechnology, (89):73.
[31] Schweder, T., Lin, H., Jürgen, B., Breitenstein, A., Riemschneider, S., & Khalameyzer, V., et al. (2002). Role of the general stress response during strong overexpression of a heterologous gene in escherichia coli. Applied Microbiology & Biotechnology, 58(3): 330-337.
[32] 刘亮伟,郭晓丹,王宝.载体大小对木聚糖酶过量表达的影响[J].河南农业学,2010,44(4):448-452.
LIU Liang-wei, GUO Xiao-dan, WAN Bao. (2010). Influence of plasmid size on over-expression of xylanase[J]. Journal of Henan Agricultural University, 44(4): 448-452.(in Chinese)
[33] Fan, J., Qian, G., Chen, T., Zhao, Y., Liu, F., & Walcott, R. R., et al. (2011). The acyl-homoserine lactone (ahl)-type quorum sensing system affects growth rate, swimming motility and virulence in acidovorax avenae subsp. citrulli. World Journal of Microbiology & Biotechnology, 27(5): 1,155-1,166.
[34] 许景升,吴茂森,何晨阳.水稻白叶枯病菌TonB-Dep-Rec蛋白家族成员Tdrxoo的功能鉴定[J].微生物学报, 2010, 50(2): 155-161.
XU Jing-sheng, WU Mao-sen, HE Chen-yang. (2010). Functional identification of TonB-Dep-Rec protein family member Tdrxooof Xanthomonas oryzaepv.oryzae[J]. Acta Microbiologica Sinica, 50(2):155-161.(in Chinese)
[35] 丘献娟.十字花科黑腐病菌中一类与趋化性相关的感受蛋白基因的研究[D].南宁:广西大学硕士学位论文,2015.
QIU Xian-juan. (2015). Study on a rind of sensory protein genes involved in chemotaxis in XCC800[D]. Master Dissertation. Guangxi University, Nanning. (in Chinese)
[36] 王奕文.对苜蓿中华根瘤菌c-di-GMP合成及分解酶的功能和LuxR家族转录调控蛋白ExpR的研究[D].上海:华东师范大学博士学位论文,2010.
WANG Yi-wen. (2010). Dissecting roles of a LuxR family transcriptional regulator,ExpR in Sinorhizobium meliloti and functions of enzymes(GGDEF and EAL domain containing proteins) responsible for synthesis and degradation of c-di-GMP[D]. Master Dissertation. East China Normal University, Shanghai. (in Chinese)