沃柑叶片响应柑橘溃疡病菌侵染的转录组分析

Transcriptome analysis of Orah leaves in response to citrus canker infection

【目的】了解沃柑叶片响应柑橘溃疡病侵染的分子机制及易感品种与柑橘溃疡病菌的互作应答机制,筛选出柑橘溃疡病菌危害沃柑叶片时的相关应答基因,为抗性育种提供基因基础。【方法】以接种溃疡病菌后0、2、4、6和8 d的沃柑叶片为试材,利用Novaseq 6000平台进行转录组双向测序,并对数据进行生物信息学相关性分析。【结果】接种无菌水(CK)后0、2、4、6和8 d获得的Clean reads分别是48 482 127、47 270 288、50 998 549、53 201 972和47 924 731条,接种柑橘溃疡病菌(JZY)后0、2、4、6和8 d获得的Clean reads分别是51 042 967、49 552 248、46 734 029、47 940 345和45 371 891条。接种后0、2、4、6和8 d的上调差异表达基因数量分别为1、947、1081、656和2108个,下调差异表达基因数量分别为1、343、753、303和1908个。在接种后2、4、6和8 d有374个基因均表达差异,其中上调基因为61个,下调基因为313个。GO功能富集分析结果显示,沃柑叶片响应溃疡病不同时期的差异表达基因,主要集中在生物进程、分子功能和细胞组分中。KEGG注释分析结果显示,差异表达基因主要参与次生代谢物的生物合成、植物与病原体相互作用、植物激素信号转导、过氧化物酶体和蛋白质内质网合成等途径。【结论】植物与病原体相互作用、植物激素信号转导、过氧化物酶体和蛋白质内质网合成4条通路为柑橘感抗病相关的重要代谢通路。研究结果可作为深入研究柑橘种质资源感抗溃疡病基因和探究柑橘与病原菌互作分子机制的理论提供参考依据。

【Objective】Orah is a hybrid with high yield, good flavor and superior quality. It is sensitive to citrus canker, caused by Xanthomonas citri subsp. citri. The mechanisms response to the susceptibility remains unknown. In this study, the transcriptome changes of Orah leaf upon the infection by X. citri subsp. citri were explored. Therefore, the molecular mechanism of citrus canker occurrence and the interaction between susceptible cultivars and X. citri subsp. citri were studied on Orah leaves. Transcriptome sequencing technology was used to screen the related response genes of citrus canker leaves, providing a genetic basis for disease resistance breeding.【Methods】Orah leaves with citrus canker were extracted from the Research Base of Agricultural College of Guangxi University to prepare citrus canker pathogen. Then the leaves with X. citri subsp. citri were inoculated by in vivo injection. The number of inoculum holes was determined by the size of the leaf until the bacterial suspension covered one side of the leaf. Centered on the veins of the leaves, one side was inoculated with sterile water as control(CK), and the other side was inoculated with citrus ulcer pathogen as experimental treatment(JZY).Samples were taken at 0, 2, 4, 6 and 8 d after inoculation. Three replicates were set for each treatment.The control sample(CK) and the treatment sample(JZY) were wrapped in tin foil, treated with liquid nitrogen and put into the refrigerator at-80 ℃ for later use. DNA was extracted by Biospin bacterial genomic DNA extraction kit, RNA was extracted by Trizol method, and transcriptome sequencing was completed by Beijing Novo Biotechnology Co., LTD. Raw data were obtained using the sequencing platform Novaseq 6000 and clean reads were obtained through quality control. The reference genome used in this experiment was the sweet orange genome. Sequencing data were analyzed and functional annotation was performed. HTSeq software was used to analyze gene expression levels. The threshold of gene expression level was FPKM>1. DESeq was used to standardize clean reads, and then DESeq software was used for differential expression analysis. FDR was calculated by BH, p value was calculated by negative binomial distribution, and the screening criterion for differential genes was p value <0.05. On this basis, GOseq software was used to carry out GO enrichment analysis and functional annotation of differential genes based on Geneontology. Usage threshold FDR≤0.05. KOBAS(2.0) was used for Pathway enrichment analysis, and KEGG database was used to analyze the metabolic pathways of the differential genes. Use a threshold: p < 0.05 for correction. Differential genes were classified according to GO and KEGG data, and differential genes were screened for functional annotation and metabolic pathway analysis.【Results】The quality of original data was evaluated from two aspects:basic content and error rate. The original data obtained ranged from 46 538 959 to 54 504 629, of which about 97% were filtered sequences. Q value was greater than 93%, and the quality of transcriptome sequencing met the standard. The obtained data can be used for subsequent analysis. Clean reads were compared with the sweet orange genome and the comparison rate was about 92%. Clean reads respectively obtained at 0, 2, 4, 6 and 8 d after inoculation with sterile water(CK) were 48 482 127, 47 270 288,50 998 549, 53 201 972 and 47 924 731. At 0, 2, 4, 6 and 8 d after vaccination by X. citri subsp. citri(JZY) the clean reads were 51 042 967, 495 248, 46 734 029, 47 940 345, 45 371 891, respectively. At0, 2, 4, 6 and 8 d after inoculation, the number of differentially expressed up-regulated genes was 1,947, 1081, 656 and 2108, and the number of differentially expressed down-regulated genes was 1, 343,753, 303 and 1908, respectively. At 2, 4, 6 and 8 d after inoculation, 374 differential expression genes were found, including 61 up-regulated genes and 313 down-regulated genes. The results of GO functional enrichment analysis showed that citrus canker disease mainly concentrated on biological processes,molecular functions and cellular components in different periods. There were 374 Differential expression genes in different periods, among which 169 genes were annotated in biological processes, 48genes in cell components, and 234 genes in molecular functions. KEGG annotation analysis of JZY_0 h vs CK_0 h showed that the differential genes were annotated into glutamate metabolism pathway.KEGG annotation analysis of JZY_2 d vs CK_2 d showed that all differential genes were mainly annotated in the biosynthesis and metabolic pathway of secondary metabolites, and were significantly enriched in the biosynthesis of secondary metabolites. KEGG annotation analysis of JZY_4 d vs CK_4 d showed that all genes were significantly enriched in secondary metabolites, amino sugars and nucleotide sugars metabolism, phenylpropanoid biosynthesis, photosynthesis, and photosynthesis-antenna protein biosynthesis and metabolism pathways. KEGG annotation analysis of JZY_6 d vs CK_6 d showed that all differentially expressed genes were enriched in secondary metabolites, phenylpropanoid biosynthesis and photosynthetic biosynthesis and metabolism pathways, while down-regulated genes were enriched in photosynthesis and other pathways. KEGG annotation analysis of JZY_8 d vs CK_8 d showed that all genes were enriched in amino acid biosynthesis, secondary metabolite biosynthesis and metabolic pathway, carbon fixation, carbon metabolism, glycolysis/gluconeogenesis, phenylpropanoid biosynthesis and other photosynthetic biological pathways. After of X. citri subsp. citri pathogen inoculation,Orah differentially expressed genes that were involved in the metabolic pathway were more. Through the further analysis of enrichment KEGG pathway, four main ways relating to disease defense response were analyzed: plant-pathogen interaction, plant hormone signaling transduction, peroxisome and protein processing in endoplasmic reticulum.【Conclusion】Pathways of plant-pathogen interaction, plant hormone signaling transduction, peroxisome and protein processing in endoplasmic reticulum, all four ways are important metabolic pathways related to citrus disease resistance, which can be used as the theoretical reference basis for deeply studying on the disease resistance gene of citrus germplasm resources and exploring the molecular mechanism between plants and pathogens.