Bioinformatics Analysis And Functional Study Of Mulberry MAPK Gene Family

Biotic and abiotic stress factors, such as against pathogens, drought, salinity and high temperature and so on, seriously affect the growth, yield and quality of crops. The response to the different stresses in plants is a complex process invloved in genes, signal transduction pathways and processes, and gene expression products. This process can be divided into four phases:plant perception of stress signals, transferring of stress signals, identification of stress signals by membrane receptors, and expression of the corresponding gene. Among these, the protein kinases are an important class of plants regulatory enzyme, they perceived the external environmental stress signal by membrane receptors, to regulate the changes of some ions and molecular from intracellular concentrations, and then to activate protein phosphorylation pathway. Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses during the plant life cycle. Those cascades are highly conserved signaling components in all eukaryotic cell signal transduction.Mulberry (Moraceae morus) is a perennial woody plant and is ecologically and economically important. In addition, mulberry can adapt to many different environments, including cold, waterlogged, drought and saline environments. Although the function of MAPK has been identified in some plants, but there has been little research into its stress physiology, biochemistry and molecular biology in mulberry tree. There is also very little mulberry MAPK information in the public databases (such as the NCBI, EMBL, etc.). After complete analysis of the M. notabilis genome had been undertaken, it became possible to analyze the defense genes, such as MAPKs, involved in abiotic and biotic responses in mulberry at the genome wide level, which has a great significance to reveal MAPK stress signal transduction regulation mechanism of plants and biotic and abiotic stress adaptation mechanisms. These provide the foundation for the study of mulberry molecular improvement.This study identified47mulberry Morus notabilis MAPK (MnMAPK) family genes:32MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. In addition, we investigated the upstream promoter conservative elements and conserved domains among the ten MAPKs by multi alignment of protein sequences. After about two months, mulberry seedlings that were about25cm tall were subjected to the abiotic stresses of:high temperature, low temperature, salinity and dehydration. There were also signal substance treatments, consisting of:abscisic acid (ABA), salicylic acid (SA), hydrogen peroxide (H2O2) and methyl jasmonate (MeJA). Further expression profile analysis using the quantitative real time-polymerase chain reaction technique (qRT-PCR) showed that most of the MAPK genes from mulberry were induced by various stresses. The candidate gene MnMAPK6was chosen by the analysis of stress-induced expression profiling mulberry MAPK. The subcellular localization of MnMAPK6was conducted. Agrobacterium-derived plant overexpression vector was constructed to transform Arabidopsis and obtained transgenic Arabidopsis plants. WT and transgenic plants were treated by high temperature, drought, salinity and H2O2to observe the growth and development of plants and in response to abiotic stress tolerance. The main findings of this study are as follows:1. Cloning and sequence identification of MAPKs from mulberryBased on an analysis of the morus genome database,32MnMAPKKK, five MnMAPKK and ten MnMAPK genes from M. notabilis were identified. Ten MnMAPK genes were successfully cloned and sequenced. The information analysis of MnMAPK showed that the CDS sizes for MnMAPK genes ranged from1107bp to1902bp and they encoded proteins ranging from368to633amino acids (AA) in size. The deduced protein molecular weights were between42.4kD and70.9kD and the pIs ranged from5.0to9.28. The MnMAPK genes were found in groups A-E by multiple sequence alignment and amino acid sequence phylogenetic analysis.2. Expression profiles of MnMAPK responses to various stresses and signalsThe primer pairs for the qRT-PCR analysis of ten mulberry MAPK genes were designed to explore the responses of mulberry MAPK genes to abiotic stresses. M. multicaulis cv. Husang No.32mulberry seedlings, after two months of growth, were subjected to high or low temperatures, drought and NaCl treatments, as described in the Materials and Methods. Then qRT-PCR was performed using the cDNAs obtained from the treated and untreated M. multicaulis cv. Husang No.32leaves as templates. The results of the qRT-PCR analysis for the different abiotic stresses showed mulberry MnMAPKs could be induced by various abiotic stress. Different MnMAPKs have different stress response patterns to different stress treatment.3. Subcellular localization and function identification of MnMAPK6MnMAPK6had different tissue expression patterns in different abiotic stresses at different times, suggesting MnMAPK6was involved in abiotic stresses signal transduction pathway.We constructed35S-MnMAPK6::EGFP vector for transient expression, and the expression vector construct35S-EGFP as a control, to transform onion epidermal cells with Agrobacterium infection. The result showed MnMAPK6::EGFP fusion protein is located in the nucleus.We constructed MnMAPK6overexpression vector to transform Arabidopsis by floral dip method and got transgenic Arabidopsis plants. The GUS Expression levels of the various over-expressing plants and the tissues showed some difference, which OE6transgenic lines expressing the highest level of GUS amount transgenic plants, OEl lines in the minimum amount of GUS expression. The expression of MnMAPK6in transgenic plants were different. MnMAPK6transgenic lines OE6had highest expression level, OE5and OE3followed and lowest OE1of expression level. Choosing OE6, OE5and OEl three transgenic lines to study the stress tolerance of abotic stresses. The results show that compared with the wild-type Arabidopsis, overexpressing transgenic plants showed increased tolerance ability to salinity and H2O2, while sensitive to heat and drought.