The Effect Of Citrus Yellow Vein Clearing Virus Infection On Gene And Small RNA Expression In Citrus Limon

Citrus yellow vein clearing virus(CYVCV)is an important virus that has recently been found in our country that is harmful to lemon.At present,there are few studies on the pathogenesis of CYVCV.In this study,the one-year-old lemon seedling were used as the host plant.The lemon leaves which inoculated and mock-inoculated with CYVCV were analysised by transcriptome sequencing and small RNA sequencing.Utilizing bioinformatics techniques to mine CYVCV infection related genes and miRNAs,to study the function of these genes and miRNA in the CYVCV stress of lemonade response.The main results are following:1.The CYVCV-infected and control lemon leaf samples collected at 30 d,60 d and 90 d were mixed and tested for transcriptomes sequencing.The results of transcriptome sequencing show that 679 genes were up-regulated and 637 genes were down-regulated in the lemon sample infected by CYVCV.Among these gene,1114 and 453 were annotated to GO function and KEGG pathway,respectively.The GO function of these gene were mainly concentrated in biological processes and molecular functions.The most significant enrichment genes were intercellular information transfer,redox process,defensed against to fungi,enzyme activation and enzyme inhibition.KEGG pathway analysis showed that the genes were mainly cpncentrated in metabolic pathways.Total of 68 genes were significantly enriched to the photosynthesis,chlorophyll biosynthesis and metabolism,carbon metabolism,carbon fixation and photosynthesis pathway.It is presumed that the changes in gene expression of these metabolic pathways may be related to the lemonade and chlorosis and chlorosis of leaves in the leaves after CYVCV infection.2.Totally,16 differentially expressed genes which related to photosynthesis,chlorophyll biosynthesis and metabolism,carbon metabolism and amino acid biosynthesis pathways were analysised by qRT-PCR.The results showed that these genes were dynamic change with time.Six of these genes were first down regulated and then up-regulated,and four genes were first up regulated and then down regulated.At the same time,five AGO homologous genes and three DCL homologous genes were analisised by qRT-PCR.The results showed that there were seven genes has differential expression,and the gene expression on the 60 d were significantly different from that on the 30 d and 90 d.Based on the qRT-PCR results of differentially expressed genes,six significant differerntially expressed gene were selected and analysised by qRT-PCR in sweet orange,lemon and lime plants which were infected by CYVCV,the results showed that the same genes were expressed in similar patterns.In addintion,qRT-PCR method were used to detect the leaf samples of lemon plants inoculated by CYVCV,we find the virus can be detected by 30 d after inoculated CYVCV with lemon,on the 60 d the virus content was the highest and decreased slightly to 90 d.3.There are 17 170 461 and 18 472 346 Clean reads were obtained from CYVCV-infevted(CY)and contral lemon(MC),respectively,and 5,360,339 and 6,043,365 reads were comparable to the orange genome in CY and MC respectively.Total of 159 miRNAs were predicted from two groups of samples,include 56 known miRNAs in sweet oranges and 103 newly predicted miRNAs from lemons.Among these miRNA,13 conserved miRNAs and 20 newly predicted miRNAs were differentially expressed.4.The 20 new predicted miRNAs were amplication by miRNA stem-loop premer RT-PCR method.The sequences of the 12 amplified products were identical to the predicted sequences.Sequencing results showed that all of the 12 newly identified miRNAs,only two were down-regulated and the others were up-regulated.The expression levels of four known miRNAs and four newly identified miRNAs were analyzed by qRT-PCR.The results showed that the expression level of miRNAs were changed dynamically 30-90 days after inoculation by CYVCV.The conservative miRNA were down-regulated and the novel miRNA were first down-regulated and then up-regulated.Using transcriptome sequencing data as reference,25 differentially expressed miRNAs were used for target gene prediction.Two newly identified miRNAs and six known miRNAs predicted 52 target genes.The function analysis of these target gene showed that the target gene involved in biological processes such as stress response and metabolism.In addition,differentially expressed miRNAs were targeted in the sweet orange genome for prediction of target genes.Functional analysis of the target genes showed that these miRNAs are related to plant responses to abiotic and biological stresses.KEGG analysis of the conserved miRNA targed genes show that these genes were obvious enrichment in plant stress response and photosynthesis and energy metabolism related pathways.