[1]陈飞飞,黄金思,王翕韫,等.吡咯伯克霍尔德氏菌JK-SH007多聚半乳糖醛酸酶基因的克隆及表达分析[J].南京林业大学学报(自然科学版),2018,42(04):127-133.[doi:10.3969/j.issn.1000-2006.201708013]
 CHEN Feifei,HUANG Jinsi,WANG Xiyun,et al.Cloning and expression analysis of polygalacturonase gene(BpPG) from Burkholderia pyrrocinia JK-SH007[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(04):127-133.[doi:10.3969/j.issn.1000-2006.201708013]
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吡咯伯克霍尔德氏菌JK-SH007多聚半乳糖醛酸酶基因的克隆及表达分析
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
42
期数:
2018年04期
页码:
127-133
栏目:
研究论文
出版日期:
2018-07-12

文章信息/Info

Title:
Cloning and expression analysis of polygalacturonase gene(BpPG) from Burkholderia pyrrocinia JK-SH007
文章编号:
1000-2006(2018)04-0127-07
作者:
陈飞飞黄金思王翕韫刘婉慧叶建仁
南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏 南京 210037
Author(s):
CHEN FeifeiHUANG JinsiWANG XiyunLIU WanhuiYE Jianren*
Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
关键词:
吡咯伯克霍尔德氏菌 多聚半乳糖醛酸酶基因 分子克隆 表达分析
Keywords:
Keywords:Burkholderia pyrrocinia BpPG gene molecular cloning expression analysis
分类号:
Q939.96; S763.1
DOI:
10.3969/j.issn.1000-2006.201708013
文献标志码:
A
摘要:
【目的】克隆吡咯伯克霍尔德氏菌JK-SH007(Burkholderia pyrrocinia JK-SH007)多聚半乳糖醛酸酶基因(BpPG),并阐明BpPG基因在B. pyrrocinia JK-SH007定殖中的生物学功能。【方法】通过设计特异性引物(PG-F、PG-F)克隆BpPG基因,并对其全长基因进行生物信息学分析; 采用MEGA 5.0软件进行系统发育树分析; 将B. pyrrocinia JK-SH007接种杨树,利用实时荧光定量PCR方法,分析BpPG基因在B. pyrrocinia JK-SH007定殖过程中的差异表达。【结果】BpPG基因全长2 001 bp,编码666个氨基酸,预测蛋白质分子量69.23 ku,理论等电点为8.37; BpPG蛋白有6个蛋白质结合位点,属于Glycosyl hydrolases family 28家族,为亲水性蛋白,不具有信号肽。二级结构主要包括α-螺旋、β折叠、延伸链和无规则卷曲,三级结构预测结果与二级结构相符。系统进化分析表明,BpPG与PG(GenBank 序列号WP 034181566.1)具有同源性。实时荧光定量PCR结果显示B. pyrrocinia JK-SH007定殖过程中,不同时期的BpPG基因表达量存在差异。【结论】克隆得到BpPG基因,BpPG基因在进化上是保守的,其极有可能在B. pyrrocinia JK-SH007定殖过程中发挥作用。
Abstract:
Abstract: 【Objective】In this study, we aimed to isolate the polygalacturonase gene(BpPG)from Burkholderia pyrrocinia JK-SH007 and clarify the biological characteristics of the BpPG in colonizing B. pyrrocinia JK-SH007.【Method】The BpPG was isolated from B. pyrrocinia JK-SH007 by PCR using primers PG-F and PG-F. Bioinformatics and homology analysis were conducted using various online software products and the phylogenetic tree was constructed with MEGA 5.0. The RT-qPCR method was used to analyze differences in the specific expression of the BpPG gene during B. pyrrocinia JK-SH007 colonization of poplar. 【Result】The sequence of the BpPG was 2 001 bp in length, and encoded a protein of 666 amino acids with a molecular weight of 69.23 ku and a theoretical isoelectric point of 8.37. The BpPG was homologous to polygalacturonase(WP 034181566.1), with 99% similarity, and contained six protein binding sites. BpPG is a hydrophilic protein belonging to the glycosyl hydrolase family 28, and lacks a signal peptide. The secondary structures of the protein are comprised mainly of alpha helices, beta turns, extended strands, and random coils, which contain six PbH1 motifs. The predicted tertiary structure was consistent with the secondary structure. RT-qPCR analysis revealed that the relative expression of the BpPG differed during various periods of B. pyrrocinia JK-SH007 colonization of poplar. 【Conclusion】The BpPG isolated from B. pyrrocinia JK-SH007 had a conserved sequence. In addition, the results suggest that the BpPG plays an important role during B. pyrrocinia JK-SH007 colonization of poplar.

参考文献/References:

[1] OBRIEN P A. Biological control of plant diseases[J]. Australasian Plant Pathology, 2017,46(4): 1-12. DOI: 10.1007/s13313-017-0481-4.
[2] HONG C E, PARK J M. Endophytic bacteria as biocontrol agents against plantpathogens: current state-of-the art[J]. Plant Biotechnology Reports, 2016, 10(6): 353-357. DOI: 10.1007/s11816-016-0423-6.
[3] SIQUEIRA K A D, BRISSOW E R, SANTOS J L D, et al. Endophytism and bioactivity of endophytic fungi isolated from Combretum lanceolatum Pohl ex Eichler[J]. Symbiosis, 2016, 71(3): 1-12. DOI: 10.1007/s13199-016- 0427-6.
[4] 张华姣. 具杀虫活性雷公藤内生菌的分离与筛选[D].杨凌:西北农林科技大学, 2012. ZHANG H J. Isolation and screening of endophytes from Tripterygium wilfordii with insecticidal activity[D]. Yangling: Northwest A&F University, 2012.
[5] TAN Y, CUI Y, LI H, et al. Diversity and composition of rhizospheric soil and root endogenous bacteria in Panax notoginseng during continuous cropping practices[J]. Journal of basic microbiology, 2017, 57(4): 337-344. DOI: 10.1002/jobm.201600464.
[6] KUREPIN L V, PARK J M, LAZAROVITS G, et al. Involvement of plant stress hormones in Burkholderia phytofirmans induced shoot and root growth promotion[J]. Plant Growth Regulation, 2015, 77(2): 179-187. DOI: 10.1007/s10725-015-0049-7.
[7] COMPANT S, REITER B, SESSITSCH A, et al. Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN[J]. Applied and Environmental Microbiology, 2005, 71(4): 1685-1693. DOI: 10.1128/AEM.71.4.1685-1693.2005.
[8] CNOSSEN-FASSONI A, BAZZOLLI D M S, BROMMONSCHENKEL S H, et al. The pectate lyase encoded by the pecCl1 gene is an important determinant for the aggressiveness of Colletotrichum lindemuthianum[J]. Journal of Microbiology, 2013, 51(4): 461-470. DOI: 10.1007/s12275-013-3078-9.
[9] 许春景, 吴玉星, 戴青青, 等. 苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能[J]. 中国农业科学, 2016, 49(8): 1489-1498. DOI: 10.3864/j.issn.0578-1752.2016.08.006. XU C J, WU Y X, DAI Q Q, et al. Function of polygalacturonase genes Vmpg7 and Vmpg8 of Valsa mali[J]. Scientia Agricultura Sinica, 2016, 49(8): 1489-1498.
[10] ROPER M C, GREVE L C, WARREN J G, et al. Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines[J]. Molecular Plant-Microbe Interactions, 2007, 20(4): 411-419. DOI: 10.1094/MPMI-20-4-0411.
[11] HUANG Q, ALLEN C. Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants[J]. Physiological and Molecular Plant Pathology, 2000, 57(2): 77-83. DOI: 10.1006/pmpp.2000.0283.
[12] MOSTAJERAN A, AMOOAGHAIE R, EMTIAZI G. The participation of the cell wall hydrolytic enzymes in the initial colonization of Azospirillum brasilense on wheat roots[J]. Plant and Soil, 2007, 291(1/2): 239-248. DOI: 10.1007/s11104-006-9189-x.
[13] ABDALLAH R A B, MOKNI-TLILI S, NEFZI A, et al. Biocontrol of Fusarium wilt and growth promotion of tomato plants using endophytic bacteria isolated from Nicotiana glauca organs[J]. Biological Control, 2016, 97: 80-88. DOI: 10.1016/j.biocontrol.2016.03.005.
[14] 范晓静, 杨瑞先, 邱思鑫, 等. 果胶酶基因在内生芽孢杆菌中的过表达及其对定殖的影响[J]. 应用与环境生物学报, 2013, 19(5): 805-810. DOI: 10.3724/SP.J.1145.2013.00805. FAN X J, YANG R X, QIU S X, et al. Overexpression of pectinase gene in endophytic Bacillus strains and its effect on colonization[J]. Chin J Appl Environ Biol, 2013, 19(5): 805-810.
[15] REN J H, YE J R, LIU H, et al. Isolation and characterization of a new Burkholderia pyrrocinia strain JK-SH007 as a potential biocontrol agent[J]. World Journal of Microbiology and Biotechnology, 2011, 27(9): 2203-2215. DOI: 10.1007/s11274-011-0686-6.
[16] 任嘉红. 杨树溃疡病拮抗细菌的筛选及抗菌物质的研究[D].南京:南京林业大学, 2009. REN J H. Screening of antagonistic bacteria against poplar canker and study on antimicrobial substances of antagonistic bacteria [D]. Nanjing: Nanjing Forestry University, 2009.
[17] WU B Y, YE J R, HUANG L, et al. Validation of reference genes for RT-qPCR analysis in Burkholderia pyrrocinia JK-SH007[J]. Journal of Microbiological Methods, 2017, 132:95-98. DOI: 10.1016/j.mimet.2016.10.004
[18] 李曼, 郑佩晶, 怀宝玉, 等. 小麦条锈菌果胶酶基因PsPL1的克隆与功能分析[J]. 西北农林科技大学学报(自然科学版), 2016, 44(11): 155-160. DOI: 10.13207/j.cnki.jnwafu.2016.11.022. LI M, ZHENG P J, HUAI B Y, et al. Cloning and functional analysis of PsPL1 from Puccinia striiformis f.sp.tritici[J]. Journal of Northwest A&F University(Natural Sciences Edition), 2016, 44(11): 155-160.
[19] 李孟娇, 吕蕊花, 余建, 等. 果桑肥大性菌核病菌多聚半乳糖醛酸酶基因(CsPG1)的克隆及功能分析[J]. 作物学报, 2016, 42(2): 190-200. DOI: 10.3724/SP.J.1006.2016.00190. LI M J, LÜ R H, YU J, et al. Cloning and functional analysis of polygalacturonase genes fromCiboria shiraiana[J]. Acta Agronomica Sinica, 2016, 42(2): 190-200.
[20] ZHANG W, LIU B, LU Y, et al. Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference[J]. Archives of Insect Biochemistry and Physiology, 2017, 94(4):e21382. DOI: 10.1002/arch.21382.
[21] CHENG Z, CHEN D, WANG Q, et al. Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production[J]. Journal of Bioscience and Bioengineering, 2017, 123(6): 665-672. DOI: 10.1016/j.jbiosc.2017.01.013.
[22] MARCHLER-BAUER A, BO Y, HAN L, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures[J]. Nucleic Acids Research, 2017, 45(D1): 200-203. DOI: 10.1093/nar/gkw 1129.
[23] YANG H, SHI P, LIU Y, et al. Loop 3 of fungal endoglucanases of glycoside hydrolase family 12 modulates catalytic efficiency[J]. Applied and Environmental Microbiology, 2017, 83(6): 3116-3123. DOI: 10.1128/AEM.03123-16.
[24] BARONCELLI R, MATARESE F, MONCINI L, et al. Two endopolygalacturonase genes in Trichoderma virens: in silico characterization and expression during interaction with plants[J]. Journal of Phytopathology, 2016, 164(1): 18-28. DOI: 10.1111/jph.12414.
[25] BARAD S, SELA N, KUMAR D, et al. Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum[J]. BMC Genomics, 2016, 17(1): 330. DOI: 10.1186/s 12864-016-2665-7.
[26] 范晓静. 植物内生细菌GFP标记及两个基因与定殖的关系[D].福州:福建农林大学, 2009. FAN X J. Labeling endophytic bacteria with green fluorescent protein and relations of two genes in colonization[D]. Fuzhou: Fujian Agriculture and Forestry University, 2009.
[27] CHEN X, LI L, ZHANG Y, et al. Functional analysis of polygalacturonase gene RsPG2 from Rhizoctonia solani the pathogen of rice sheath blight[J]. European Journal of Plant Pathology, 2017: 149(2):491-502. DOI: 10.1007/s10658-017-1198-5.

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备注/Memo

备注/Memo:
基金项目:国家林业公益性行业专项项目(201304404); 国家自然科学基金项目(31470645); 江苏高校优势学科建设工程资助项目(PAPD) 第一作者:陈飞飞(ffchen1951@163.com)。*通信作者:叶建仁(jrye@njfu.com.cn),教授。
更新日期/Last Update: 2018-07-27