| Cucumber(Cucumis sativus L.)is a widely grown vegetable crop around the world,due to the high nutritional value,people all over the world are interested in it.However,because of its narrow genetic basis and lack of resistant resources,cucumber is very susceptible to many diseases,such as bacteria,fungi,viruses and nematodes,which seriously restricts the economic effect of cucumber production.Meloidogyne incognita,as a dominant race,poses a great challenge to cucumber production in China.Up to now,no cucumber cultivars with M.incognita resistance have been reported,and various control measures in production have obvious limitations.Utilization of wild resistance resources has become a key way of cucumber breeding for disease resistance.Successful breeding of Cucumis-hystrix introgression line IL10-1 with M.incognita resistance provides an important material basis for disease resistance breeding.Therefore,the first task of disease resistance breeding is to clarify the molecular regulation mechanism and key genes.In this study,comparative analysis of the physiological characteristics and changes in transcription and protein levels between resistant and susceptible materials(IL10-1,CC3)after inoculated with M.incognita were conducted to reveal the physiological characteristics,molecular regulatory network and core regulatory genes of resistance material IL10-1.The main results are as follows:1.Histological characteristics of resistance material IL10-1 against M.incognitaTo clarify the histological resistance characteristics of resistance line(R-line)IL10-1,comparative analysis of phenotypic differences between IL10-1 and CC3 after inoculation were conducted.The results showed that there were significant differences(P<0.05)in galls number between IL10-1 and CC3 at 3 days post inoculation(dpi).The number of galls on IL10-1 was significantly less than that of CC3.Moreover,with the time of inoculation,the difference of galls number became very significant(P<0.01).The analysis of developmental status of M.incognita showed that neither IL10-1 nor CC3 hindered the invasion of M.incognita.However,the development of nematode in IL10-1 was significantly slower than that in susceptible control CC3.At 15dpi,the differentiation of adult females did not occur in the root of IL10-1,by contrast a certain number of adult females appeared in the root of susceptible control CC3.The results showed that IL10-1 inhibited the development of M.incognita during inoculation.Physiological sections of feeding sites showed that giant cells(GCs)in IL10-1 roots presented thin cytoplasm and with no obvious multinucleus compared with it of CC3.In conclusion,IL10-1 inhibits the development of M.incognita by inhibiting the development of giant cells,thus showing resistance to M.incognita.2.Indentification of genes involved in giant cells development in IL10-1The abnormal development of giant cells led to the resistance of IL10-1 against M.incognita.To identify the key genes for the abnormal development of giant cells,homologous cloning method was used to analyze the homologous genes in IL10-1.AtMAP65-3 plays an active role in the development of giant cells in Arabidopsis thaliana.Genome-wide identification revealed that there were 6 MAP65 family genes in cucumber.Evolution and collinearity analysis showed that CsMAP65-3a and AtMAP65-3 were highly homologous,suggesting that they might have the same or similar functions.qRT-PCR analysis showed that CsMAP65-3a exhabited different expression between IL10-1 and CC3,which lower expressed in IL10-1,while highly expressed in CC3.And in situ hybridization showed that CsMAP65-3a was highly expressed in the nucleus and membrane of normal developing giant cells in CC3,which further confirmed that CsMAP65-3a was involved in the normal development of giant cells.Cis-acting element analysis showed that the expression of CsMAP65-3a might be regulated by hormone pathway(Auxin,SA,ETH).In summary,IL10-1 inhibits the expression of CsMAP65-3a during the development of giant cells through the regulation of hormone levels,thus causing abnormal development of giant cells.3.Combination of transcriptome and proteomics to analyze the molecular regulatory network and hub regulatory genes of IL10-1 against M.incognitaThe response of IL10-1 against M.incognita is a complex process involving the joint regulation of polygenes and polyproteins.In this study,the differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)in IL10-1 and CC3 after inculaation were identified by high throughput transcriptome and proteomic sequencing techniques.Functional enrichment analysis revealed that DEGs related to cation transport,transport and transporter activity were inhibited in IL10-1.The enrichment analysis of DEPs also specifically enriched GO entries such as cation binding in IL10-1.Identification of EDGs related to hormones showed that most auxin synthesis-related genes were inhibited.Quantitative analysis of IAA confirmed that the IAA content in the roots of IL 10-1 was relatively lower after inoculation with M.incognita compared with CC3.KEGG pathway enrichment analysis of DEPs revealed that flavonoid metabolic pathways were specifically enriched in IL10-1.Studies have shown that flavonoids are involved in the regulation of auxin levels.The construction of co-expression network revealed that lipid transfer protein(LTP)was the hub gene in the regulation network of IL 10-1 against nematode,and DEPs interaction network analysis showed that ribosomal protein(RP)was at the core of regulation.In conclusion,we speculate that regulates the synthesis of lipid transfer proteins and other related proteins through ribosomal proteins,thereby affecting the expression level of auxin,and then regulates the expression of resistance-related genes,thus inhibiting the development of giant cells. |
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