| Kiwifruit is known as the "king of fruits" for its rich nutrients.In recent years,with the continuous growth of kiwifruit cultivation area,its disease situation is increasingly serious,especially the occurrence of bacterial canker disease.The disease has the characteristics of strong pathogenicity,fast transmission speed,which can cause a large area of tree death in a short period of time,seriously affecting the development of the world kiwifruit industry.In this study,29 kiwifruit cultivars(lines)from the National Crop Germplasm Resources Platform Kiwifruit Platform were selected to evaluate their disease resistance,In addition,the high resistant variety of ‘Huate' and the high susceptible variety of ‘Hongyang' were selected as materials to analyze the gene expression changes at different time points after infected with Psa using RNA-Seq sequencing technology,and screened four kinds of genes related to disease resistance.On this basis,the structure and function of pathogenesis-related protein 1(PR-1)were studied in detail.The main results were as follows:1.Evaluation of resistance of kiwifruit varieties(lines)against bacterial canker disease and correlation analysis among evaluation indexes.(1)29 kiwifruit varieties(lines)were inoculated with Psa,and the phenotype was observed day by day: six days after Psa inoculation,some cultivars(lines)began to show symptoms.The inoculation spots became reddish-brown and exhibited a small amount of milky-white mucus,with the prolongation of inoculation time,the diameter of lesions became larger and larger,21 days after Psa inoculation,disease index was calculated and disease resistance was evaluated,the results showed that C2-72 and CHongyang showed high susceptibility;D6-65,C1-74,CHort16-A,D14-57 and D6-20 showed susceptibility;C2-43,CJintao,CJinyan and DYouxi-52 showed tolerance;DXuxiang,DHward,DBulunuo,AHongbaoshixing,ASE1-5,AP1-2,AP4-2,EN4-32,AP6-1,AP3-3,A11-17,EN4-25,EN14-9,DJinkui and DTML showed resistance;EN12-16,EN12-31 and EHuate showed high resistance.The above results showed that there were significant differences in disease resistance among different kiwifruit varieties,among which A.Chinese had the worst disease resistance,followed by A.deliciosa,and A.eriantha and A.arguta had the highest disease resistance.(2)The correlation analysis of lesion length and disease index of isolated shoot showed that there was a significant positive correlation between them,the correlation coefficient R was 0.957,the higher the disease index was,the longer the lesion length was,lesion length could also be used as an important index for identification of kiwifruit canker diseases.(3)Flow cytometry was used to detect the ploidy of 29 kiwifruit Cultivars(lines),the results showed that among A.chinensis,the disease resistance of tetraploid cultivars were stronger than that of diploid cultivars,but the effect of interspecific ploidy on disease resistance was not significant.2.The high resistant variety of ‘Huate' and the high susceptible variety of ‘Hongyang' were selected as materials,RNA-Seq sequencing was carried out at 0,12,24,48 and 96 h after Psa inoculation,the main results were as follows:(1)Transcriptome analysis showed that ‘Huate' was able to respond rapidly after Psa infection,the defense response of kiwifruit to canker disease might be concentrated on the early response before 12 hours of infection,early differentially expressed genes might play a more important defensive role in the disease resistance response.(2)Weighted gene co-expression network analysis results showed that there were significant differences in gene expression of five modules between ‘Huate' and ‘Hongyang'.We performed KEGG enrichment analysis of the genes in these five modules and found that the differentially expressed genes of MEgreenyellow and MEyellow module were mainly concentrated in plant-pathogen interaction and endocytosis,the differentially expressed genes of MEblack module were mainly concentrated in protein processing in endoplasmic reticulum and plant-pathogen interaction,which might be closely related to the response of kiwifruit to disease resistance.3.The high-resistant variety ‘Huate' and high-susceptible variety ‘Hongyang' were used as materials.We cloned the cDNA sequences of PR-1 genes of ‘Huate' and ‘Hongyang'(named htPR-1 and hyPR-1),respectively,the main results were as follows:(1)The full length of htPR-1 and hyPR-1 genes were 522 bp,without introns.The difference of these sequences was that twelve sites of htPR-1 and hyPR-1 cDNA sequences had mutations,resulting in seven different amino acids,but they all encoded 173 amino acids,which showed that mutations in base sites did not lead to early termination of translation.(2)Bioinformatics analysis showed that similar to Arabidopsis thaliana and Nicotiana tabacum PR-1 gene,both htPR-1 and hyPR-1 genes had SCP-PR-1-like conservative domains,a signal peptide with 26 amino acid sequences at the N-terminal,Subcellular localization predicted that PR-1 was a secretory protein.The difference was that htPR-1 had five alpha-helix structures and three beta-folding structures,while hyPR-1 had five alpha-helix structures and four beta-folding structures.These results indicated that htPR-1 and hyPR-1 genes were homologous genes of PR-1 in Actinidia.(3)Through blast multiple sequence alignment and phylogenetic tree analysis,it was found that htPR-1 and hyPR-1 genes had high homology with PR-1 genes of many plants,in which,they had high homology with Solanum lycopersicum and Solanum tuberosum,and had close genetic relationship which were clustered into one group,but were distant from Zea mays.(4)The results of quantitative real-time PCR analysis showed that kiwifruits were inoculated with Psa,PR-1 gene was significantly induced in both ‘Huate' and ‘Hongyang',but its expression level in ‘Huate' was significantly higher than that in ‘Hongyang'(5 times),indicating that PR-1 gene may be involved in the immune response of kiwifruit to Psa infection.(5)Subcellular localization experiments showed that the green fluorescence of pCAM35s-PR-1-GFP fusion protein was mainly distributed in the cytoplasm and cell membrane.(6)In order to study the functions of htPR-1 and hyPR-1 genes,overexpression vectors were constructed,respectively.The htPR-1 and hyPR-1 genes were transiently expressed in tobacco.The symptoms were observed on the 14 th day after inoculation with Psa.The results showed that both htPR-1 and hyPR-1 genes could enhance the resistance of tobacco to Psa. |
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