黄龙病菌侵染晚锦橙叶脉和根早期病原蛋白组学的比较分析

【目的】黄龙病(Huanglongbing,HLB)是世界柑橘生产上最具毁灭性的病害,由于黄龙病病原菌无法离体培养,其关键的致病机制仍不清楚。为此,通过蛋白组学分析探究黄龙病菌侵染晚锦橙早期时病原蛋白的表达情况。【方法】利用叶圆片嫁接技术对晚锦橙(Citrus sinensis Osbeck)进行嫁接传毒,在传毒2个月时,对感病后的根和叶脉组织进行蛋白组测序,以黄龙病亚洲种(Candidatus Liberibacter asiaticus,CLas)基因组为参考,对测序结果进行注释,并以感病叶脉为对照,筛选病原菌差异表达蛋白,最后利用qRT-PCR对显著性差异表达蛋白质进行验证。【结果】共鉴定出38个病原蛋白,其中有6个显著性表达差异蛋白;GO功能注释揭示这些病原蛋白主要与核酸结合和物质转运活性相关;qRT-PCR结果显示,6个显著差异蛋白在根部组织中的表达水平显著高于叶片组织;其中ABC转运系统重要组成成分CLasMalE(ACT56611.1)转运蛋白基因的表达倍数高达595倍。【结论】在黄龙病菌侵染柑橘根和叶的早期,与病原菌物质运输和核酸代谢相关基因的表达差异明显,暗示其在黄龙病菌侵染寄主中起着重要作用,为黄龙病菌致病机制研究提供了基因资源和理论基础。

Proteomics analysis of early pathogens after infection of Candidatus Liberibacter asiaticus with Jincheng Orange(Citrus sinensis Osbeck)

【Objective】Huanglongbing(HLB)is the most devastating disease in citrus production throughout the world.Because Candidatus Liberibacter asiaticus(CLas),the most virulent pathogen of HLB identified at present,cannot be cultured in vitro,its main pathogenic mechanism is still unrevealed.Therefore,proteomics analysis was used to explore the expression of pathogenic proteins during the early CLas infection stage in Citrus sinensis Osbeck.【Methods】In this study,the leafdisc grafting was used to infect the healthy Jincheng Orange(Citrus sinensis Osbeck)to transmit the CLas.Two months later,the CLas was detected by PCR,then the infected roots and leaf veins were collected for iTRAQ proteome Sequencing.The sequencing result was annotated while the genome of the CLas was used as a reference,and the infected leaf veins were used as control to screen the proteins of the CLas which expressed differently.Finally,qRT-PCR was used to verify those significantly expressed differently proteins.【Results】A total of 38 pathogenic proteins were identified,13 pathogenic proteins were up-regulated and 14 pathogenic proteins were down-regulated while there were 7 pathogenic proteins with unknown functions.The proteome of the infected roots was screened,the infected leaf veins were used as control,the difference fold value more than 1.5 times were used as a significant up-regulation and less than 1/1.5 as a significant down-regulation standard.As a result,there were 2 proteins(ACT56611.1.ACT66821.4)were significantly up-regulated,and 4 proteins were significantly downregulated(ACT56726.1,ACT56912.1,ACT57006.1,ACT57090.1).Gene ontology(GO)analysis was performed on the 38 identified pathogenic proteins.The results showed that pathogenic proteins were mainly involved in 10 biological processes,but were mainly related to nucleic acid binding and material transport activities.All pathogenic proteins were involved in 10 molecular functions and there were 22 related to the cellular biosynthetic process,accounting for 57.9%of the total proteins,12 pathogenic proteins related to the cellular macromolecule biosynthetic process,accounting for 31.6%of the total proteins.Among 38 pathogenic proteins,11 pathogenic proteins were related to the molecular function of ribonucleotide binding,accounting for 28.9%of the total proteins,5 pathogenic proteins were related to transporter activity,accounting for 13.2%of the total proteins.The 38 pathogenic proteins were used to construct a protein interaction network diagram which showed that more proteins interacted with ACT57362.1,ACT56735.1,ACT57090.1,ACT57110.2,and ACT56789.1.The protein annotation clarified that ACT57362.1 was inosine 5’-monophosphate dehydrogenase,which catalyzes the conversion of inosine 5’-phosphate(IMP)to xanthine 5’-phosphate(XMP).It is the first important and rate-limiting step in the de novo synthesis of guanine nucleotides,and it plays an important role in regulating cell growth;ACT56735.1 was the 30S ribosomal protein S10,which is involved in the binding of tRNA and ribosomes;ACT57090.1 was the alpha subunit of ATP synthase,which converts ADP to ATP in the presence of a proton gradient on the membrane;ACT57110.2 was the chaperone protein DnaK,which plays an important role in the process of protein folding.ACT56789.1 was theβsubunit of phenylalaninetRNA ligase.The results of these 5 pathogenic proteins annotations showed that the key proteins related to growth and development of cell and protein folding were expressed to response to the CLas at the early HLB infection stage in Jincheng Orange.The result of qRT-PCR showed that the expression of 6 significantly differentiated proteins(ACT56611.1,ACT66821.4,ACT56726.1,ACT56912.1,ACT57006.1,ACT57090.1)in the roots were significantly higher than that in the leaves;the expression of CLasMalE(ACT56611.1)transporter proteins,an important component of the ABC transport system,is as high as 595 times.The bioinformatics analysis of ClasMalE showed that its open reading frame was 1122 bp and there was 1 exon but no intron,so it encodes 373 amino acids.The protein primary structure prediction showed that the molecular weight(MW)of the protein was 4.4 ku,and the theoretical pI was 9.02,so it was a hydrophilic protein.The secondary structure of the CLasMalE mainly includedα-helix,random coil,extended strand and beta turn.The conserved domain prediction revealed that its protein sequence contained a MalE domainand its number was COG0767.Finally,this study analyzed the expression characteristics of the CLasMalE in citrus,it showed that its expression in the roots of susceptible species was significantly higher than that in the leaf veins,and the expression in the old leaves was significantly higher than that in the young leaves,but there was no significant difference in tolerant species sour pomelo.【Conclusion】The high-level expression of ABC transport system’s important component ClasMalE in the infected Jincheng roots was found,indicating that the ABC transport system would play an important role in the host infection of the CLas.