Analysis Of Apple Chlorotic Leaf Spot Virus Small RNA In Hawthorn And Its Function Of Target Gene

Apple chlorotic leaf spot virus(ACLSV)is one kind of RNA virus that infects Rosaceae fruit trees extensively and has serious effects on economic traits.In this project,de novo assembly of the whole genome of ACLSV in hawthorn trees was obtained by high-throughput sequencing.The effects of ACLSV on gene expression levels in hawthorn trees were elucidated by transcriptome analysis.The structure and character of vsi RNAs originated from ACLSV was carried out by high-throughput sequencing of small RNAs form the hawthorn plant infected with ACLSV.And then the target genes of vsi RNAs were predicted by analysis the expression of genes of hawthorn with the complementary sequence of vsi RNA.The effect of vsi RNA on target genes in hawthorn and functions of target genes were validated by transferring the target gene and vsi RNA into plants.The main results were as follows:1.The full-length genome sequences of two ACLSV hawthorn isolates SY02 and SY03 were determined using Illumina RNA-seq.The SY02 sequence was verified by RT-PCR.And the results showed that the sequence obtained by the segmented clone was 99.70% identical to the transcriptome sequence,indicating the reliability of the transcriptome data.The genome sequences of the SY02 and SY03 isolates were uploaded to NCBI with Gen Bank accession numbers KU870524 and KU870525.The nucleic acid and amino acid sequences of the ACLSV hawthorn isolates and the other 15 ACLSV isolates in Gen Bank were analyzed and the phylogenetic tree was constructed.It was found that the genetic relationship of ACLSV largely depends upon their host species.2.The leaf transcriptome sequencing results of hawthorn plants infected with ACLSV and healthy plants showed that there were 9910 differentially expressed genes(DEGs)between the two materials(log2 fold change ?1,padj<0.01).KEGG enrichment analysis of the DEGs revealed that “plant pathogen interaction” was the most significant up-regulated way.“Flavone biosynthesis” was the most significant down-regulated way.3.Small RNA sequencing showed that there were small interfering RNAs(vsi RNAs)derived from ACLSV in ACLSV-infected samples,among which the 21 nt vsi RNA was the largest,accounting for 59.83% of total vsi RNA;the sense strand vsi RNA accounts for 51.80~54.28% of the total vsi RNA;the 5? base preference C?A?U>G;the hot spot region appears in the methyltransferase,protease and helicase of the replication-associated protein.4.The target genes of vsi RNA were predicted and functionally annotated,and it was found that vsi R1360(-)derived from ACLSV targeted hawthorn gene Cp LRR-RLK1.In addition,q RT-PCR,RLM-5?RACE and tobacco transient transformation experiments confirmed that vsi R1360(-)could silence Cp LRR-RLK1.5.The full length sequence of Cp LRR-RLK1 gene was cloned from hawthorn.The CDS length of this gene was 1794 bp,encoding 597 amino acids.Phylogenetic tree analysis showed that Cp LRR-RLK1 belonged to LRRII-RLK subfamily.Subcellular localization assay demonstrated that Cp LRR-RLK1 was localized to the cell membrane.The spatiotemporal expression characteristics of Cp LRR-RLK1 gene were analyzed by q RT-PCR,indicating that the expression of Cp LRR-RLK1 gene was the highest in mature hawthorn leaves and the lowest in tender leaves.The promoter of Cp LRR-RLK1 gene was cloned and sequenced,and many cis-acting elements related to defensive and stress responses were found in the promoter region of Cp LRR-RLK1 gene.6.The overexpression vector of Cp LRR-RLK1 gene was constructed,and the overexpression of hawthorn Cp LRR-RLK1 gene in apple and Arabidopsis thaliana was achieved by Agrobacterium-mediated genetic transformation.Four Arabidopsis positive overexpression lines and eight apple overexpression lines were obtained.The similarity of hawthorn and apple LRR-RLK1 gene was 96.77%.The 395 bp sequence with strong specificity at the 5? end of apple LRR-RLK1 gene was used as RNAi interference fragment to construct the RNAi vector of Md LRR-RLK1 gene and transform apple,and 17 RNAi lines were obtained.7.The WT and transgenic Arabidopsis were inoculated with Pst DC3000,and the results of phenotype,callose and ROS determination proved that Cp LRR-RLK1 could mediate the defense of Arabidopsis thaliana against bacteria;the transgenic apples were inoculated with Alternaria alternate and Glomerella cingulate,and the disease incidence and index of overexpressing lines were lower and the RNAi lines?s was higher than that of the control group,indicating the disease resistance of Cp LRR-RLK1 against fungi;the transgenic apples and control ?GL-3? were grafted to the apple material with ACLSV and ASGV,both RT-PCR and q RT-PCR results of the virus indicated that the Cp LRR-RLK1 gene had certain resistance to ACLSV and ASGV.