Cloning And Functional Analysis Of Jasmonic Acid Signal Transduction Related Genes Derived From Self-incompatibility Of Camellia Oleifera

Camellia oleifera is a woody edible oil tree species that is late-acting self-incompatibility(LSI).In our previous research,we constructed the transcriotome database of self-pollination and cross-pollination of Camellia oleifera,and conducted in-depth bioinformatics analysis.The analysis found that many differential genes of Camellia oleifera self-pollination and cross-pollination are enriched in several important plant hormone metabolism pathways,and the jasmonic acid signal transduction pathway is one of them.In order to further explore the molecular mechanism of Camellia oleifera self-incompatibility,especially the relationship between the jasmonic acid signal transduction pathway and the self-incompatibility of Camellia oleifera,the related genes and functions of the jasmonic acid signal transduction pathway in Camellia oleifera were studied.The effects of jasmonic acid on the pollen tube germination and flower opening of Camellia oleifera were studied.The main results are as follows:1.Screened and cloned 9 genes related to jasmonic acid signal transduction in Camellia oleifera.Nine genes related to jasmonic acid signal transduction were screened from the transcriptome database of Camellia oleifera self-pollination and cross-pollination,and these genes were cloned.Related genes are:C.oleifera jasmonic acid-isoleucine synthase gene CoJAR1,C.oleifera jasmonin receptor protein gene CoCOI1,C.oeifera jasmonic acid response inhibitor CoJAZl,C.oleifera transcription activator CoMYC2,C.oleifera transcription activator CoMYC4,C oleifera transcription factor CoTIFY6B,C.oleifera Transcription Factor CoTIFY9,C.oleifera Transcription Factor CoTIFY9-like,C.oleifera Transcription Factor CoTIFY10A;the full-length CDS sequence lengths of each gene are:1770 bp,1776 bp,702 bp,1455 bp,1947 bp,1197 bp,663 bp,639 bp,885 bp,encoding 589,591,233,484,648,398,220,212,294 amino acids,respectively.The analysis found that the 9 proteins all have typical conserved domains and are closely related to Camellia sinensis related proteins.2.Revealed the expression pattern of 9 genes related to jasmonic acid signal transduction in C.oleifera.CoJAR1,CoMYC4,CoTIFY6B,CoTIFY9 genes are significantly expressed in anthers;CoCOI1,CoTIFY10A genes are significantly expressed in pollen;CoJAZ1,CoTIFY9-like genes are notably expressed in ovary,CoMYC2 is notably expressed in filaments,but not in pollen.The 9 jasmonic acid signal transduction genes accumulate in different positions in the natural state.In C.oleifera styles,the expression trends of CoJAR1 after self-pollination and outcross pollination were the same,but the expression level of self-pollination was significantly lower than that of cross-pollination;CoJAZ1 was extremely significantly expressed at 24 h after selfling,and then declined;CoMYC2 and CoMYC4 was extremely significantly expressed at 24 h after self-pollination.In the ovary of C.oleifera,the above-mentioned 9 genes showed similar expression patterns after pollination,showing a "double-peak curve" expression trend:the expression level increased at 2-12 h,then decreased at 24 h,the expression level increased again at 36 h,and at 48 h After gradually reducing.It shows that the jasmonic acid signal transduction of C.oleifera participates in the process of self-pollination,and thus participates in the process of plant defense.When the pollen tube grows slowly in the style,the ovary may receive the JA signal,which initiates the molecular mechanism that inhibits the growth of the self-pollen tube.3.Prokaryotic expression and subcellular localization of genes related to jasmonic acid signal transduction.The gene was recombined with the pCold-TF vector with His tag,and a small amount of recombinant protein expression was induced by IPTG.The gene was fused with a carrier protein with GFP fluorescence,and the fluorescent expression sites were observed by laser confocal.CoJARl,CoCOIl,CoJAZ1 recombinant proteins are expressed in the nucleus,and CoMYC2,CoMYC4,CoTIFY6B,CoTIFY9,CoTIFY9-like,and CoTIFY10A proteins are expressed in the endoplasmic reticulum.4.Eukaryotic expression of genes related to jasmonic acid signal transduction.The related genes were transformed into Arabidopsis thaliana to obtain A.thaliana as no obvious phenotypic difference compared with the wild-type plants.Endogenous hormone assays found that the JA content of overexpression plants did not change significantly,and the content of methyl jasmonate,ethylene synthase and gibberellin were different.Among them,the MeJA content of CoCOI1,CoMYC2,and CMYC4 overexpression plants was significantly higher than that of wild-type plants The ABA content of CoCOI1,CoJAZ1,CoMYC2,and CoMYC4 overexpressing plants is slightly higher than that of the wild type,and the ACCS and GA3 content of the CoCOI1,CoJAZ1,CoMYC4 overexpressing plants are significantly different than that of the wild type plants,indicating that these genes are involved in the interaction of jasmonic acid and jasmonic acid.The interaction of other hormones jointly regulates the gro wth and development of plants.5.Jasmonic acid and methyl jasmonate have inhibitory effects on the germination of C.oleifera pollen.The in vitro germination test of C.oleifera pollen was conducted with JA and MeJA.The results showed that 1.0 mM JA and MeJA can significantly inhibit pollen tube germination and pollen tube elongation,and the inhibitory effect of jasmonic acid is more obvious.9 genes were extremely significantly expressed under jasmonic acid treatment,but under MeJA treatment,only CoJAR1 gene expression increased.6.Methyl jasmonate has the effect of affecting the blooming process of C.oleifera flowers.MeJA at a concentration of 0.05 mM can promote the process of C.oleifera from the bud stage to semi-opening,shorten the time required for the flower to open at this stage;it can inhibit the process of C.oleifera flowers from semi-opening to fully open,and prolong the blooming time of C.oleifera.Under natural conditions(CK),with the extension of C.oleifera flower opening,the expression of 9 genes gradually increased,and under the treatment of 0.05 mM MeJA concentration,bud may respond to the signal transduction of jasmonic acid in advance,and gene expression The amount is significantly higher than that of CK,but with the opening of flowers,the amount of gene expression gradually decreases.This is consistent with the results of exogenous hormone spraying.That is,0.05 mM MeJA can speed up the development of C.oleifera from the bud stage to half-opening,and delay the development process from half-opening to full-opening.In summary,the genes related to jasmonic acid signal transduction in Camellia oleifera are involved in the jasmonic acid signal transduction mechanism and the molecular mechanism of Camellia oleifera self-incompatibility.It may play an important role in the self-incompatibility of Camellia oleifera by participating in the signal recognition and interaction mechanism of style-ovary.And jasmonic acid can inhibit the germination of Camellia oleifera pollen and regulate the opening process of Camellia oleifera flower.