Bioinformatic And Functional Analysis Of Mulberry Genes Involved In PAMP Pathway

Recognition of microbes depends on a series of pattern recognition receptors (PRRs).The pattern of characteristic molecular structures shared with large groups of microbes are called pathogen-associated molecular patterns (PAMPs). PAMPs play important roles as activators of the innate immune response in animals, similarly, as elicitors of defense response in plant.The specific recognition in the different kingdoms arose independently by convergent evolution to recognize conserved molecules, which is important for innate immune system. Plants can perceive structures (like the cell-wall polysaccharide chitin and sterol ergosterol in fungi). Plants also have been reported to recognize structures such as bacteria-lipopolysacharides (LPS), bacterial cold-shock protein (CSP), flagellin, and EF-Tu. In plants, a variety of molecular patterns (PAMPs) have been identified. However, the functions of PRRs, the downstream of transcription factor for corresponding response, defense proteins, and as the relationship of each other are not very clear.Mulberry(Morus L.) belonging to family Moraceae, is an important economic tree in our country. It has been widely planted in china and the mulberry leaves are the major nutrition resource for silkworm. Mulberry is a woody perennial and constant pruning is critical for the accumulation of leaves that can feed more silkworms. So it is susceptible to pests and pathogens and thus results to the reduction of output and its effect in medicinal and ecological protection. For understangding the genetic basis of mulberry inmmue system, we focused on the analyses of genes involved in the PAMP pathway in the mulberry genome. The target genes have been identified, combined with transgenic technology can enhance resistance in mulberry.The main results of this paperas follows:1. Bioinformatic analysis of PAMP-related genes in the mulberry genomeThe14LysM proteins,56WRKY transcription factors and20chitinase are identified from mulberry genome database. It revealed that most of these genes have conserved domains in each gene families. The upstream regulation sequences of these genes contain some plant immune regulatory elements (JA, SA, fungi, elicitors, and the wounding). Phylogenetic analysis was conducted using the protein sequences from Arabidopsis, other reported proteins and mulberry. The results of bioinformatics analysis indicated these genes may be involved in the PAMP defense immune of plants.2. Expression analyses of key genes involved in PAMP pathway by biotic and biotic-like stressesThe expression profiles of11genes were detected in response to different stress through the qPCR. Results suggested that MnCERK1, as an upstream receptor protein, was highly expressed by biotic stress and biotic-like stress and MnCERK1gene expressions are different in response to four treatments. And for four WRKY transcription factors of mulberry, the espressions of their encoding genes showed different degree of induction by chitin treatment and silkworm feeding. In the previous reports, four transcription factors as the key proteins in the PAMP pathway, are able to respond to chitin induction. But, inscet biting always triggers JA immune pathway. This result may indicate the four transcription factors could participate in the downstream of the two different immune pathways with positive regulation. Plant chitinase is generally regared to be involved in plant defens. Six chitinases of different classes of mulberry can be induced by different stresses. Only Mnchil6significantly was induced after silkworm wounding, Mnchi8also was induced significantly by fungus infection. Difference of expression of chitinase by biotic stress and biotic-like stress are obvious.3Gene cloning of Mn WRKY29and Mnchil4and the genetic transformation of tobaccoIn order to further study Mnchil4and MnWRKY29, we constructed three gene expression vectors and tranformed tobacco. We then analyzed the expression level in transgenic tobacco after chitin and chitosan treatment. The results showed that both of Mnchil4and MnWRKY29genes were respectively induced by chitin and chitosan, suggested that they are involved in the immune response of mulberry.