Effect Of CoMAPKs Gene On Self-incompatibility In Camellia Oleifera

Camellia oleifera is the most important woody edible oil tree species in South China,which belongs to the late self-incompatibility(LSI)plant,which planted the main varieties should be allocated planted in corresponding pollination varieties.In order to analyze the molecular mechanism of self-incompatibility in the late stage of Camellia oleifera,we constructed the omics database of self-pollination and cross-pollination pistils of Camellia oleifera.It was found that mitogen activated protein kinases(MAPKs)are closely related to self-incompatibility.In order to further explore the effect of CoMAPKs gene on self-incompatibility of Camellia oleifera,the main cultivars of Camellia oleifera ’Huashuo’ and’Huajin’ were used as experimental materials,carried out the cloning,bioinformatics analysis,expression pattern,prokaryotic expression,subcellular localization,overexpression and effect of MAPKs inhibitor apigenin on Camellia oleifera pollen in vitro culture.The main results are as follows:1.Inhibitory effect of MAPKs inhibitor on Camellia oleifera pollen culture in vitro.The pollen of Camellia oleifera ’Huashuo’ was treated was different concentrations of MAPKs inhibitor and observed after in vitro culture.The results show that with the increase of MAPKs inhibitor concentration,the inhibition effect of pollen germination rate and pollen tube length was more obvious.2.The cloning and bioinformatics analysis of CoMAPKs gene.The Camellia oleifera cultivar ’Huashuo’ was used as experimental material to clone the CoMAPKs genes of Camellia oleifera by RT-RCR.Including MAPKs(5),MAPKKs(2)and MAPKKKs(1),which was named CoMPK1,CoMPK2,CoMPK3,CoMPK6,CoMPK9,CoMKK3,CoMKK5 and CoMEKK1.The full-length CDS sequence of CoMAPKs genes is 1119 bp,1119 bp,1098 bp,1197 bp,1764 bp,1554 bp,1093 bp and 1737 bp,the number of amino acids encoded by the corresponding protein is 372 aa,371 aa,365 aa,398 aa,587 aa,518 aa,363 aa and 578 aa.The amino acid sequences of CoMAPKs and CsMAPKs were 98.07%-1 00%similar.Phylogenetic tree showed that CoMPKl,CoMPK2,CoMPK3,CoMPK6,CoMPK9,CoMKK3,CoMKK5 and CoMEKKl were closely related to AtMPK1 and AtMPK2,AtMPK3,AtMPK6,AtMPK15 and AtMPK18,AtMKK3,AtMKK4 and AtMKK5 and AtMEKK1.The analysis of conserved domains shows that CoMAPKs proteins have eleven conserved domains(Ⅰ-Ⅺ),and there is a TxY motif between Ⅶ and Ⅷ domains.Both CoMAPKKs and CoMAPKKKs have corresponding conserved domains,which is consistent with the structural characteristics of MAPKs signal transduction pathway.3.The expression pattern of CoMAPKs gene.The expression pattern of CoMAPKs in different tissues,styles and ovaries at different treatments and different time periods by qPCR.In different tissues,CoMPK1,CoMPK6,CoMPK9 and CoMKK5 were highest expression levels in pollens,CoMPK2 was highest relative expression levels in ovary and pollen,CoMPK3,CoMKK3 and CoMEKK1 were highest relative expression levels in ovaries.In styles and ovaries,the expression levels of CoMPK6,CoMPK9 and CoMEKK1 in self-24 h styles were significantly higher than those in cross-and non-pollination.The expression ovaries of CoMPK9 in cross-48 h ovaries were significantly higher than those in self-and non-pollination.The expression ovaries of CoMKK5 in cross-2 h and 24 h ovaries were significantly higher than those in self-and non-pollination.4.Prokaryotic expression of CoMAPKs gene.The prokaryotic expression vectors pCold-CoMAPKs of eight genes were constructed by homologous recombination and transformed into E.coli(DE3)competent cells.SDS-PAGE electrophoresis analysis that the recombinant vector pCold-CoMAPKs could induce a corresponding target protein band under different concentrations of IPTG,which was consistent with the molecular weight of the theoretical fusion protein.5.Subcellular localization of CoMAPKs gene.The subcellular localization vectors pCAMIB1300-CoMAPKs of eight genes were constructed and transformed into tobacco by transient expression method.The results showed that CoMPK1,CoMPK3,CoMKK5 and CoMEKK1 proteins were expressed in chloroplast,CoMPK2,CoMPK6,CoMPK9 and CoMKK3 proteins were expressed in endoplasmic reticulum.6.Genetic transformation of CoMAPKs to Arabidopsis.The overexpression vectors pCAMIB1304-CoMAPKs of eight genes was constructed.Transformed into wild-type Arabidopsis by the pollen tube pathway method,obtained these eight genetically modified T3 generation homozygotes.The phenotypes of eight transgenic Arabidopsis have no significantly different.The flower buds of wild-type Arabidopsis and T3 transgenic CoMPK6,CoMPK9,CoMKK5 and CoMEKK1 Arabidopsis plants were collected as experimental materials,measure plant endogenous hormones(acyl-coa synthetase(ACCS),salicylic acid(SA),jasmonic acid(JA)and abscisic acid(ABA)under a multifunctional microplate reader with a wavelength of 450 nm.The analysis found that the content of SA in CoMPK6 transgenic Arabidopsis plants is lower than that in wild-type Arabidopsis,the content of SA in CoMPK6 transgenic Arabidopsis plants is longer than that in wild-type Arabidopsis,the content of ABA in CoMPK6 transgenic Arabidopsis plants is significantly longer than that in wild-type Arabidopsis,the content of ACCS in CoMPK6,CoMPK9,CoMKK5 and CoMEKK1 transgenic Arabidopsis plants is significantly longer than that in wild-type Arabidopsis.