Cloning Of MAPK3 Gene From Aquilaria Sinensis And Preliminary Study Of Expression

MAPK signaling cascade system plays a very important role in plant growth and development, biotic and abiotic stress reaction, hormones and other signaling pathways. It is responsible for the exotic reception, transfer and amplification, so as to start or regulate the expression of genes. Rare and endangered Aquilaria sinensis is an important source of Chinese eaglewood. Agarwood can be formed only when the healthy tree was wounded. In the previous study, we identified a sesquiterpene synthase gene ASS1, which was significantly induced by wound. A series of transcription factors and protein kinses that expressed coordinately with ASS1 were also indentified preliminary of which MAPK signaling cascade system.On this basis, this paper focused on cloning and expression analysis of AsMAPK3 gene:1. Using RT-PCR and RACE method, the full-length cDNA of AsMAPK3 was cloned from Aquilaria sinensis. The open reading frame was 1110 bp, encoding 369 amino acids. On the basis of amino acid sequences of MAPK3 gene, phylogenetic analysis indicated that the gene from Aquilaria sinensis formed a small branch with one from melon. It indicated that AsMAPK3 was predicted as a hydrophilic and uncertain protein with the unstable coefficient 41.15. The isoelectric point pI is 5.42. Subcellular localization prediction showed AsMAPK3 maybe mainly express in the nucleus.2. Used root, stem and leaf of A. sinensis as experimental material, tissue expression of AsMAPK3 gene was analyzed, and the results showed that the AsMAPK3 mainly expressed in leaves, followed by the stem, the minimum amount of expression in the flowers. Suspended cells were treated with two kinds of chemical damages by added methyl jasmonic acid and salicylic acid, the expression pattern of AsMAPK3 was analyzed. The results showed that the damage signal can regulate AsMAPK3 gene expression. In terms of overall trend, the expression of genes was improved in the damage process within 24 h; after 24 h, all have significantly reduced. Gene expression level under different damage is different.3. On the basis of gene gun technique, subcellular localization of AsMAPK3 was analyzed. The results showed AsMAPK3 in onion epidermal cells located in the cell membrane and the nucleus.4. An prokaryotic expression vector, PET-28a-AsMAPK3, was constructed. To find out the suitable conditions for exogenous expression of AsMAPK3, different temperatures, speeds, and IPTG concentrations were designed, the results showed the fused protein could be successfully induced through 0.4 mM IPTG induction 4 h in 37℃.This paper will lay the foundation for further research about the function of AsMAPK3 in wound-induced agarwood sesquiterpene formation and its molecular mechanism.