Analysis Of The PEPC Family Genes And Transformation Of Antisense PEPC1 Gene In Peanut(Arachis Hypogaea L.)

Vegetable oil is one of the main nutrients needed for human daily life and also important industrial raw materials.With the increase of population,economic development,agricultural products consumption structure changes and other reasons,the domestic and foreign vegetable oil market demand increased rapidly.At present the contradiction between supply and demand of vegetable oil is outstanding,and the domestic supply rate is less than 35%.To improve the oil content of oilseed crops is an effective way to increase oil production.Peanut is one of the most important oil crops in China,compared to the main oil crops such as rapeseed and soybean,peanut oil production per unit area is the highest.The oil content in peanut seeds generally ranges from 45% to 56%,which has the potential to further be improved.In recent years,along with the peanut genome sequencing work and peanut transgenic technology is gradually perfected,it is possible to cultivate high oil peanut varieties by the transgenic and molecular breeding methods.There is some competition in the oil synthesis and protein synthesis.The two metabolic processes require the use of phosphoenolpyruvate(PEP)as substrate.Phosphoenolpyruvate carboxylase(PEPC)catalyzes PEP to yield oxaloacetate,which is required for protein biosynthesis;Acetyl-CoA carboxylase(ACCase)catalyzes PEP to yield acetyl-CoA,which is required for fatty acid synthesis.PEPC has a variety of physiological functions in plants,such as PEPC is involved in carbon / nitrogen uptake and fatty acid regulation and is related to salt and drought stress responses,but the understanding of its function is still very limited.This study analyzed the expression characteristics of peanut PEPC gene family.On this basis,we constructed the reverse expression vector of AhPEPC1 gene,then transformed the peanut,and analyzed the traits of transgenic plants.The findings are helpful to increase oil content through genetic engineering.The main results are as follows:1.We analyzed the characteristics of the PEPC family gene sequence,through the bioinformatics analysis methods such as the amino acid sequence alignment,conservative domain analysis,gene structure prediction and phylogenetic analysis.AhPEPC1,AhPEPC2,AhPEPC3 and AhPEPC4 belong to the plant-type PEPC,AhPEPC5 belongs to the bacterial-type of PEPC.The structure of AhPEPC1,AhPEPC2,AhPEPC3 or AhPEPC4 gene consists of 10 exons and 9 introns;the structure of AhPEPC5 gene consists of 20 exons and 19 introns.2.Quantitative real-time PCR was employed to detect the expression patterns of AhPEPCs in roots,stems,leaves,seeds and at the different stages of seed development.The results reveal that all five PEPC genes are expressed in the four peanut tissues investigated,but they have different expression levels and spatial patterns.The highest expression of AhPEPC1 gene is in seeds;The expression of AhPEPC3 gene in roots was higher;The expression of AhPEPC4 gene was higher in leaves.3.The antisense expression vector pCAMBIA1301-PEPC was transfected into peanut.The agronomic traits of transgenic peanut did not change.The oil content of transgenic peanut seeds increased by 5.7%-10.3%,while the relative content of fatty acid components did not change,but the seed protein content decreased by 7.5%-17.0%.The tolerance to salt stress was enhanced in transgenic peanut.4.The transcriptome of the transgenic plants was sequenced,and 110 differentially expressed genes were selected.Among them,the expression of the 25 genes was up-regulated and the expression of the 85 genes was down regulated.Differentially expressed genes were analyzed by KEGG enrichment,and 34 genes were successfully obtained by KEGG annotation.There are two genes(Aradu.M0JX8,Aradu.FE0Z7)down regulated in the biosynthesis of amino acids.These data are consistent with the reduction of protein content in transgenic peanut seeds.