姚全胜 杨倩 柳凤 詹儒林

摘  要：由柑橘黃单胞菌芒果致病变种（Xanthomonas citri pv.mangiferaeindicae， Xcm）引起的芒果细菌性角斑病是芒果上的一种重要的细菌性病害。利用同源基因克隆技术克隆了Xcm上内切葡聚糖酶（endoglucanase）基因CEL的全长，经测序后运用多种生物信息学软件对其序列进行分析。结果表明：CEL基因的完整阅读框包含1134 bp，编码377个氨基酸;预测分子量为40.72 kDa;等电点为9.01;不稳定指数39.93，为稳定蛋白;亲水性为–0.081，有信号肽;总磷酸化位点有51个，无跨膜螺旋;在二级结构预测中，α-螺旋、β-转角、无规则卷曲和延伸结构分别为35.28%、6.10%、16.45%和42.18%。经保守结构域预测，具有Cellulase保守结构域。系统进化树结果显示，该基因的氨基酸序列与X. citri pv. punicae str. LMG 859（CCF67690.1）的亲缘关系最近。以gyrB、GAPHD和rpoD作为内参基因，经qRT-PCR分析CEL在Xcm侵染芒果叶片12 h后表达量持续上升，明显高于对照，在72 h处为最大表达量，由此推断该基因在病菌侵染时发挥重要作用。

关键词：芒果;细菌性角斑病;内切葡聚糖酶;克隆;序列分析

中图分类号：S435      文献标识码：A

Cloning and Expression Analysis of Endo-1，4-β-D-glucanase Gene of Xanthomonas citri pv. mangiferaeindicae

YAO Quansheng， YANG Qian， LIU Feng*， ZHAN Rulin

South Subropical Crops Research Institute， Chinese Academy of Tropical Agricultural Sciences / Key Laboratory for Post-harvest Physiology and Technology of Tropical Horticultural Products of Hainan Province， Zhanjiang， Guangdong 524091， China

Abstract： Mango bacterial black spot which is caused by Xanthomonas citri pv. mangiferaeindicae is one of the most important bacterial diseases in mango. In this study， the CEL gene from Xcm was cloned using the homologous cloning method， the biochemical feature， tertiary structure and phosphorylation sites of the CEL protein were predicted by a variety of bioinformatics softwares. The results showed that the total length of cDNA was 1134 bp， encoding 377 amino acid polypeptides. The molecular weight of the protein was 40.72 kDa， the theoretical isoelectric point was 9.01， the instability index was 39.93， which was a stable protein， the hydrophilicity was –0.081， and there was a signal peptide in the endoglucanase. The total phosphorylation sites were 51， with no transmembrane helix. In the secondary structure prediction， the α - helix， β - corner， irregular curl and extended structure was 35.28%， 6.10%， 16.45% and 42.18%， respectively. According to the prediction of conservative domain， it had cellular conservative domains. Phylogenetic analysis showed that CEL gene had the closest genetic relationship with X. citri pv. punicae str. LMG 859 （CCF67690.1）. Using gyrB， GAPHD and rpoD as internal reference genes， quantitative real-time PCR analysis revealed that CEL expression was increasingly higher with the Xcm infecting mango leaves after 12 hours. The maximum expression was at 72 hours， which suggested that the gene played an important role in the infection of Xcm.

Keywords： mango; Xanthomonas citri pv. mangiferaeindicae; endoglucanase; cloning; sequence analysis

DOI： 10.3969/j.issn.1000-2561.2021.04.022

植物細胞壁是植物体抵御各种逆境（生物胁迫和非生物胁迫）和正常生存的第一道防线，同时也是生物因子侵染植物体与植物相互斗争的重要场所。细胞壁降解酶作为病原菌重要的致病因子，其主要功能是降解植物细胞壁，降解的物质既可为病原菌提供营养物质，使其顺利在植物体内定殖、繁殖和扩散，又能破坏植物的细胞壁导致寄主植物细胞分离和组织溃烂死亡[1-3]。芒果细菌性角斑病是由柑橘黄单胞菌芒果致病变种引起的一种细菌性病害，危害芒果的叶片、枝条、花芽、花和果实。……