The Identification Of TPS And HMGR Gene Family In Pear And Functional Analysis Of PcHMGR1 Gene

Terpenoids,a class of secondary metabolites that is widely present in plants.Plant terpenoids play an important role in plant growth and plant defense responses and they are the major allelopathic substances.In addition,some terpenoids such as paclitaxel,artemisinin have important commercial and medical value.Therefore,the terpenoid metabolic project has become an important part of the secondary metabolic engineering research and it is more important to further discover the key enzymes and the genes encoding the key enzymes in the synthesis of terpenoids.Pear is a representative economic crop in the rosaceae plants,but till now there are fewer reports to explore functions of the key enzyme gene family of the terpene synthesis pathway in the pear genome.Since pear genome has been decoded,it has been possible to analyze the key enzyme gene family from the genomic perspective of the terpene synthesis pathway.In this study,we used the bioinformatics method to identify the two key enzymes(TPS and HMGR)gene families in the terpene synthesis pathway,and analyzed the physical and chemical properties of proteins,gene structure,protein domain.In addition,in order to further understand the function of PcHMGR1,PcHMGR1 expression vector was constructed and successfully transformed into Arabidopsis thaliana.The main results are as follow:(1)Based on the data of pear genome,50 TPS protein sequences in GeneBank were used as the query sequence by blast and domain screening.Finally,33 TPS gene were identified in the pear genome,named PbrTPS1-PbrTPS33 by reference to the TPS in Arabidopsis thaliana.The phylogenetic tree of pear TPS protein sequence were clustered and analyzed and it was divided into five subfamilies.(2)Pear TPS gene chromosome mapping results showed that 33 TPS family members were located in 8 pear chromosomes.Most TPS proteins are localized on chromosome 12.There are fewer gene sequences located on chromosomes 8,10 and 17.Subcellular localization results showed that most of the 33 TPS proteins were located in the cytoplasm,the few were distributed in mitochondria,chloroplast and endoplasmic reticulum.?-helix and random coil are the main constituent elements of the secondary structure.(3)Four HMGR genes in the pear genome,named PcHMGR1,PcHMGR2,PcHMGR3 and PcHMGR4 were identified.Analysis showed that all proteins were stable protein except for PcHMGR3.Subcellular localization analysis showed that PcHMGR1 and PcHMGR3 were localized in the cytoplasm,PcHMGR2 was located in the endoplasmic reticulum and PcHMGR4 was located in the chloroplast.The results of phylogenetic analysis showed that the PcHMGR1 and PcHMGR4 were more closely related to the evolutionary relationship.The genetic structure of PcHMGR was analyzed using the GSDS online site.The results show that PcHMGR1,PcHMGR2 and PcHMGR3 have a substantially identical exon intron structure.(4)HMGR protein conserved domain analysis from MEME showed that pear HMGR protein contained four conserved domains,including two HMG-COA binding sites(EMPVGYVQIP and TTEGCLVA)and two NADP(H)binding sites(DAMGMNM and GTVGGGT).GO functional annotation results analysis showed that the four genes were also annotated to the following eight functional groups,involved in the redox process,the metabolic process of coenzyme A and the synthesis of isoprenoids,but also has a variety of binding and catalytic activity and cell membrane system correlation.(5)The results of tissue expression pattern analysis showed that PcHMGR1 was highly expressed in young leaves and stems,PcHMGR2 and PcHMGR3 were expressed in young leaves and shoots,PcHMGR4 was highly expressed in roots.Analysis of expression patterns under biotic and abiotic stress conditions showed that PcHMGR1 and PcHMGR4 have similar expression patterns,PcHMGR2 and PcHMGR3 expression patterns are highly similar.(6)PBI122+PcHMGR1 expression vector were constructed and transformed into Arabidopsis thaliana,transgenic plants were identified for genomic and transcriptional levels.The results showed that overexpressing PcHMGR1 transgenic Arabidopsis plants were successfully obtained.At the rosette stage,AtAACT1 and AtHMGR2 were upregulated.The expression of AtAACT1,At AACT2,AtFPS1,AtFPS2 and AtDXS gene was significantly increased in the flowering stage.These results indicate that overexpression of PcHMGR1 in Arabidopsis thaliana affects the expression of upstream and downstream genes in its pathway.(7)Overexpression of PcHMGR1 increased the production of terpenoids metabolites such as chlorophyll and carotenoid and the species of downstream volatiles.After oxidative stress,the wild-type plants showed more severe wilting and de-greening than the transgenic Arabidopsis thaliana,the transgenic lines had higher activities of antioxidant enzymes SOD,APX and CAT and lower MDA content indicating that overexpression of PcHMGR1 increased Arabidopsis antioxidant capacity.