纽荷尔脐橙叶片和幼果内生细菌群落多样性及其功能预测

Diversity and Functional Prediction of Endophytic Bacterial Community in Leaves and Young Fruits of Newhall Navel Orange

【目的】柑橘溃疡病是脐橙上的一种重要病害,影响其发生的因素较多。通过了解纽荷尔脐橙内生细菌群落组成和其潜在的生物学功能,探索内生细菌在脐橙感染溃疡病中的作用。【方法】从纽荷尔脐橙树上随机采集未表现任何病害症状的幼果和嫩叶,嫩叶分别在春、夏、秋3个季节进行采样,每个样品设3个重复。对4个样品进行表面消毒后从中提取DNA,PCR在25μL反应体系中进行,扩增产物经20 g/L琼脂糖凝胶电泳检查,选取主条带清晰的400~500 bp片段进行测序。采用基于16S rRNA基因V3-V4可变区的高通量测序技术及Tax4Fun功能预测对纽荷尔脐橙内生细菌菌群及其功能进行了测定和分析。各样本数据在FLASH V1.2.7上进行拼接,利用QIIMEV1.7.0对产生的原始数据进行质控,用Uparse V7.0.10012对有效数据进行聚类,在97%的相似度水平上生成OTU。为每个OTU挑选一个代表序列,并使用SSUrRNA数据库进行物种注释分析使用,获得分类学信息并分别在各个分类水平统计各样本的群落组成,并用MUSCLE(V3.8.31)进行α多样性(样本内)和β多样性(样本间)分析。通过UProC和PAUDA 2种方法将功能信息对应到SILVA数据库中,实现功能注释。【结果】季节和采摘部位对脐橙内生细菌群落组成的多样性和物种丰富度存在一定的差异,脐橙幼果中的内生细菌数量多于叶片,夏季相较于其他季节内生细菌群落丰富度更高;脐橙内生细菌群体在门水平上的组成相似,但丰度占比不同,且厚壁菌门、变形菌门和蓝菌门为主要优势门,各优势细菌纲数目、组成及其丰度随不同脐橙样本而异,芽孢杆菌属为主要优势属;Tax4Fun功能预测显示纽荷尔脐橙内生细菌包含大量关于膜运输、碳水化合物代谢、氨基酸代谢、翻译、复制和修复、能量代谢、辅因子和维生素的代谢等基因信息,其中功能基因丰度较大的为碳水化合物代谢和氨基酸代谢,且脐橙夏天叶片的细胞过程和环境信息处理功能信息高于其他3种样品,幼果的遗传信息处理、新陈代谢、有机系统和人类疾病功能信息高于其他3种样品。【结论】纽荷尔脐橙内生细菌群落多样性丰富,具有大量代谢相关的有益菌,研究结果可为今后探索纽荷尔脐橙内生细菌菌群的利用和柑橘溃疡病的生物防治提供思路。

[Objective]Citrus canker is a disease on navel orange,and there are many factors affecting its occurrence.This study aims to understand the community composition and potential biological functions of endophytic bacteria in Newhall navel orange,thus exploring the role of endophytic bacteria in navel orange infected with canker disease.[Method]The young fruit and young leaves without any disease symptoms were randomly collected from the Newhall navel orange tree.The young leaves were sampled in spring,summer and autumn respectively,each sample had three replicates.After disinfecting the surface of the four samples,DNA was extracted from the four samples.PCR was performed in a 25μL reaction system.The amplification product was examined by 20 g/L agarose gel electrophoresis,and the 400-500 bp fragment with clear main band was selected for sequencing.The endophytic bacterial flora and its function were determined and analyzed by highthroughput sequencing technology based on 16S rRNA gene V3-V4 variable region and Tax4Fun function prediction.The sample data were spliced on FLASH V1.2.7,the original data were quality controlled by QIIMEV1.7.0,and the effective data were clustered by Uparse V7.0.10012 to generate OTU at the similarity level of 97%.A representative sequence was selected for each OTU,and the SSUrRNA database was used for species annotation analysis,the taxonomic information was obtained and the community composition of each sample was counted at each classification level.MUSCLE(V3.8.31)was used forαdiversity(within samples)andβdiversity(between samples)analysis.Functional information was mapped to the SILVA database by UProC and PAUDA methods to achieve functional annotation.[Result]The results showed that there were differences in the diversity and species richness of endophytic bacterial communities in navel oranges between seasons and picking sites,the number of endophytic bacteria in navel orange fruits was more than that in leaves,and the richness of endophytic bacteria in summer was higher than that in other seasons.The composition of endophytic bacteria in navel orange was similar at the phylum level,Firmicutes,Proteobacteria and Cyanobacteria were the main dominant phyla.The number,composition and abundance of each dominant bacterial class varied in different navel orange samples.Bacillus was the main dominant genus.Tax4Fun function prediction revealed that endophytic bacteria in Newhall navel orange contained a large amount of genetic information about membrane transport,carbohydrate metabolism,amino acid metabolism,translation,replication and repair,energy metabolism,metabolism of cofactors and vitamins.Among them,carbohydrate metabolism and amino acid metabolism were the most abundant functional genes,and the cell process and environmental information processing function information of navel orange leaves collected in summer was higher than that of the other three samples,the genetic information processing,metabolism,organic system and human disease function information of young fruit was higher than that of the other three samples[.Conclusion]The endophytic bacterial community of Newhall navel orange is rich in diversity and has a large number of beneficial bacteria related to metabolism.The results can provide ideas for the utilization of endophytic bacterial flora in Newhall navel orange and the biological control of citrus canker disease in the future.