Cloning And Functional Analysis Of Disease-resistant Related Genes In Camellia Sinensis

Camellia sinensis is an important economic crop in China,but the occurrence of disease seriously restricts the development of tea industry.The discovery and identification of disease-resistant genes in tea plants contribute to the breeding of resistant varieties of tea plant and the implementation of green control of tea plant diseases and insect pests.In order to excavate disease resistance related gene of tea plant,two candidate genes involved in disease resistance were cloned from the Arabidopsis thaliana mutants that showed sensitive to fungi leading to disease in tea plants,using map-based cloning.After identification of the candidate genes from A.thaliana,these genes were used to clone disease resistance related genes in tea plants through homologous gene cloning method.Finally,the function analysis of candidate genes from C.sinensis was performed through overexpression of these genes in Nicotiana benthamiana.The main results are as follows.1.The AtDEFL and AtMYB81 genes were cloned from A.thaliana using RT-PCR and recombined with the plant vector PCXSN.The plant overexpression vectors PCXSN-AtDEFL and PCXSN-AtMYB81 were constructed and transferred into Agrobacterium GV3101.Subsequently,the PCXSN-AtMYB81 was transformed into N.benthamiana and regenerated plants overexpressing AtMYB81 were obtained,which laid the foundation for the future functional identification.More over,a transient assay system based on N.benthamiana plants was developed in the present work aimed to test the function of AtDEFL using Alternaria longipes as tested phytopathogen.These results suggested that overexpression of AtDEFL enhanced the resistance of N.benthamiana to A.longipes infection.2.The coding sequences of CsDEF2 were obtained from C sinensis transcriptome database by sequence alignment and bioinformatics analysis was performed.The open reading frame of CsDEF2 consisted of 246 bp and polypeptides encoded 81 amino acids with a deduced molecular weight of 8.6 kDa and the theoretical isoelectric point of 9.12.Sequence alignment and phylogenetic analysis showed that the related sequences in tea plants were not found to be homologous,indicating that CsDEF2 is a novel gene.The amino acid sequence encoded by CsDEF2 is similar to the amino acid sequence of the Leguminosae defensin family,and the similarity with cowpea(RDY09218.1)is 78%.Proteindomain prediction revealed the presence of a thionin domain followed by eight cysteines in the amino acid sequences of CsDEF2.In addition,analysis of the modelled three-dimensional structure of CsDEF2 detected the presence of a triple stranded ?-sheet with one ?-helix in parallel.3.CsDEF2 gene was cloned from tea leaf and its biological activity examined.The expression profiling of CsDEF2 was investigated to gain insight into the mode of action of the elicitors from pathogenic fungus.After inoculation with Fusarium roseum,CsDEF2 showed enhanced expression level in tea leaves,which suggested that Cs DEF2 might play a role of positive regulation in the response to fungi infection.Furthermore,a recombinant overexpression vector PCXSN-CsDEF2 was constructed using gene recombinant technique and it was transformated into Agrobacterium GV3101.Then,the Agrobacterium-mediated transient assay systempresented here was used to test the function of CsDEF2.And the result showed that overexpression of CsDEF2 siginificantly increased the resistance of N.benthamiana to A.longipes infection.