Rice OspPLA Gene Family Identification And Basic Analysis Of OspPLAⅢα Function

Patatin-related phospholipase A (pPLA) is a class similar to animal calcuim independent phospholipase A2. pPLAs hydrolyze the ester bonds in the sn-1 or sn-2 positions of phospholipids to generate lysophospholipids and a free fatty acid. Polyunsaturated free fatty acids are the precursors for the production of oxylipins such as jasmonic acid, involved in various stress responses. Arabidopsis contains 10 pPLA genes and some of them have been characterized. However, pPLA (OspPLA) in Oryza sativa is totally unknown. To investigate the rice pPLA family, the rice genome was searched by BLAST to identify genes with sequence similarities to AtpPLAs and the resulting sequences were analyzed phylogenetically. The expression pattern of OspPLAs in various development stages and the specific tissues of rice were determined using semi-quantitative RT-PCR. To investigate the functions of individual OspPLAs, T-DNA inserted mutant for OspPLAⅢαwas isolated and characterized for its effect on rice response to jasmonate treatment and to X. oryzae pv. Oryzae. In addition, the full length genomic DNAs of OspPLAⅡηand OspPLAⅢβwere cloned and overexpresed in rice plants by Agrobacterium mediated transformation. The main results are as follows:The rice gemone has twenty one OspPLA genes. They were found in rice genome by BLAST and key word research (using patatin's Pfam accession no. PF01734), and are grouped into four subfamilies,Ⅰ,Ⅱ,Ⅲ,Ⅳ. The sequences in the catalytic domains of pPLAs are highly conserved between AtpPLAs and OsPLAs, but, the primary gene and protein structures of the OspPLA family are more complex that those of AtpPLAs.OsPLAs exhibit different and overlapping expression patterns in leaves and roots at two developmental stages, four-leaf and booting. The expression level of OspPLAⅡγ,δ,η,ι,λand OspPLAⅢβ,γ,ζwas, higher in both tissues of booting stage than that of four-leaf stage, whereas the level of OspPLAⅡβ,Ⅲα,Ⅳγwas higher in the four-leaf than bolting stage.A homozygous T-DNA inserted mutant OspPLAⅢα-KO was isolated. The loss of OspPLAⅢαresulted in the alteration in the expression pattern of genes involve in JA/SA dependent pathway in response to the 50μM JA treatment. Moreover OspPLAⅢα-KO mutant displayed less resistance to X. oryzae pv. Oryzae than WT. The deficiency of OspPLAⅢαmay have a lower basal level of JA and less resistance to the attack of blight bacteria.The full-length genomic DNAs of OspPLAⅡη(Os09g28770) and OspPLAⅢβ(Os03g43880) have been cloned and constructed into an expression vector. The two OspPLAs were transformed into rice via Agrobacterium mediated transformation. Several transgenic rice plants containing OspPLAⅡηand OspPLAⅢβwere confirmed by PCR.