Cloning Of Phosphoenolpyruvate Carboxylase Gene(PEPC)from Isoetes Shangrilaensis X. Liu And Construction Of Its Expression Vector

Isoetes L.is the only surviving genus of Isoeteceae.It originates from ancient Lycopodiophyta plants and is one of the earliest vascular plant groups.It has a special evolutionary status and can adapt to series of Habitat type.Besides,it is also the oldest plant group with Crassulacean Acid Metabolism（CAM）,which is a typical representative of aquatic CAM plants.However,the origin and evolutionary mechanism of the CAM metabolic pathway of the Isoetes plants are not clear.The PEPC gene（phosphoenolpyruvate carboxylase gene）encodes the key enzyme of the CAM metabolic pathway——phosphoenolpyruvate carboxylase（PEPCase）.The PEPC gene family consists of multiple members,including several plant-type PEPC and at least one Bacterial-type PEPC which is of low origin.PEPC gene plays an important role in plant photosynthesis carbon fixation,fatty acid synthesis,and plant stress resistance.At present,the research on PEPC genes is mainly concentrated on terrestrial C4 and CAM plants.The characteristics,functions and evolution of PEPC genes in aquatic CAM plants are still unclear,and further research is needed.Early transcriptomics studies have shown that the Isoetes sinensis Palmer is highly expressed in the submerged state at night,which at the molecular level verifies that the PEPC gene is involved in the expression regulation of the CAM pathway of the Isoetes plant.In this study,the Chinese peculiar species of Isoetes shangrilaensis X.Liu was taken as the research object.The PEPC gene（Is PEPC）of Isoetes shangrilaensis X.Liu was cloned,the sequence characteristics,phylogenetic type,protein structure and function were analyzed.Transgenic Arabidopsis thaliana with Is PEPC gene was constructed,and the main results are as follows:1.Isolation and identification of Is PEPC geneUsing transcriptome data analysis and specific primer amplification,two PEPC family genes were isolated and cloned from the c DNA of Isoetes shangrilaensis X.Liu.These two genes have the typical structural characteristics of the PEPC family genes and are named Is PEPC2 and Is PEPC4,respectively.Using NCBI online blast function,the nucleotide and amino acid sequences of Is PEPC2 and Is PEPC4 were searched for homology,and the results showed that Is PEPC2 had the highest homology with Pd PEPC2 of angiosperm Dicotyledonae Prunus dulcis Pall.at nucleotide levels,It had the highest homology with Sm PEPC2 of Selaginella moellendorffii Hieron.at the amino acid level;Is PEPC4 has the highest homology with Sm PEPC4 of Selaginella moellendorffii Hieron.at nucleotide and amino acid levels.2.Phylogenetic type and adaptive evolution significance of Is PEPC geneThe phylogenetic analysis based on nucleotide sequence showed that the Is PEPC gene of Isoetes shangrilaensis X.Liu were separated into two branchs.Among them,the Is PEPC2 gene was located in the plant-type PEPC gene branch,and the Is PEPC4gene was located in the bacterial-type PEPC gene branch.It had the closest genetic relationship with Sm PEPC gene of Selaginella moellendorffii Hieron.,which gathered at the base of each branch and had an older history of origin.The phylogenetic analysis based on amino acid sequences got the same results.Besides,the plant-type Is PEPC2 gene might be related to CO₂ fixation and participate in the CAM photosynthesis pathway,indicating that the early PEPC gene might differentiate into functions related to CO₂fixation and led to the origin of the CAM pathway of the Isoetes plant.The bacterial type Is PEPC4 may be related to Sporulation and plant stress resistance.3.Correlation between structural characteristics and functions of protein encoded by Is PEPC geneThe CDS coding region of the Is PEPC2 gene was 2928 bp and encoded 975 amino acids,the molecular weight of the encoded protein was about 110.69 k D,and there were relatively conserved phosphorylation regions（Ser residues）at the N-terminus;the CDS coding region of the Is PEPC4 gene was 3060 bp and encoded 1019 Amino acids,the molecular weight of the encoded protein was about 115.26 k D,there was no conserved phosphorylation region（Ser residue）at the N-terminus.The theoretical isoelectric point（PI）of Is PEPC2 and Is PEPC4 proteins were both 5.65,they were instable and hydrophilic proteins with negatively charged.The prediction analysis of Is PEPC secondary structure showed that both Is PEPC proteins are mainlyα-helix,followed by random coil,and the proportion of extended chain structure andβ-turn is small.The protein signal peptide prediction showed that there was no signal peptide cleavage site for these two proteins,and no signal peptide.The analysis results of transmembrane area showed that there was no obvious transmembrane area of these two proteins.Conservative domain analysis revealed that there were PEPcase conserved domains in both Is PEPC2 and Is PEPC4 proteins.Both PEPC proteins were conserved at the C-terminus.Is PEPC2 had one PEPcase conserved domain and Is PEPC4 had two PEPcase conserved domains.Conservative motif analysis found that there were 10 conserved motifs in both Is PEPC2 and Is PEPC4protein,but the positions of the conserved motifs were different.It was further shown that Is PEPC2 was a plant-type PEPC and Is PEPC4 was a bacterial-type PEPC.The difference in domains and conserved motifs indicated that the two proteins had different functions.The tertiary structure analysis of Is PEPC protein showed that the Is PEPC2 protein of Isoetes shangrilaensis X.Liu had 193α-helices,50 extended chain structures and288β-turns,and the Is PEPC4 protein had 194α-helices,59 extended chain structures and 392β-turn angle.Is PEPC2 protein contained 240 cys（cysteine）,Is PEPC4 protein contained 264 cys（cysteine）,but no disulfide bond was found,neither of the two Is PEPC proteins contained calcium ion binding sites,zinc ion binding Location.Moreover,Isoetes shangrilaensis X.Liu was a representative plant of the aquatic CAM pathway,and Ananas comosus（Linn.）Merr.was a representative plant of the xerocole CAM pathway of the Bromeliaceae.Compared with the Ac PEPC protein of Ananas comosus（Linn.）Merr.,the position and number of the conserved domains of Isoetes shangrilaensis X.Liu was basically the same,but there were certain differences in the secondary and tertiary structures of the protein,and the Ac PEPC4 protein of pineapple had lost the motif 10.On the one hand,this difference might be related to the evolutionary history of the phylogenetic development of the PEPC gene,on the other hand,it might be related to the different environmental selection pressures that they had suffered.The origin of PEPC gene in Isoetes shangrilaensis X.Liu was earlier,and the structure of the encoded protein was more conservative.Moreover,the taxonomic groups with CAM metabolism had their own independent evolutionary history.In aquatic CAM plants and terrestrial CAM plants,the evolutionary relevance to the structure and function of the protein encoded by the PEPC gene needs further study.4.Overexpression of Is PEPC2 gene in transgenetic Arabidopsis thalianaWe inserted the Is PEPC2 gene into p CAMBIA-2300-N-e GFP and p MD plasmid vectors by enzyme digestion and ligation.Agrobacterium-mediated inflorescence dip method was used to transfer the two recombinant vectors into wild-type Arabidopsis thaliana,and through resistance screening,g DNA level identification and RNA level identification,we initially identified T1 generation 26 p C2300-N-e GFP-Is PEPC2transgenic lines and 32 p MD-Is PEPC2 transgenic lines,we futher identified 3 T3generation p C2300-N-e GFP-Is PEPC2 homozygous overexpression lines and 4 p MD-Is PEPC2 homozygous overexpression lines,and subcellular displayed Is PEPC2 gene positioning on the cell membrane,it provides a foundation for further research on the function and expression mechanism of the PEPC gene of the isoetes plant.