Studies On Construction Of Antisense Expression Vector Containing PEPCase Gene And Screening Of The Suitable Genotype For Genetic Transformation In Peanut

Peanut (Arachis hypogaea L.) is the fourth largest oil crop in the world. It is also the highest single yield, highest total yield and highest export income in our country. So, increasing the lipid content of peanut, is a important goal for peanut breeding. Now technology of genetic transformation of plant can combine the favorite characters from different species into one cultivar, and provides an effective mean to reach the goal. In theory, it can transfer single or a few genes with some halts, and avoid the interfere from large numbers of disadvantage genes, need not large population and the cycle of breeding is short, so it is very effective. The development of biotechnology established on a stability foundation of increasing the lipid content of peanut breeding by gene technology. Antisense RNA technology can prevent the expression of target gene in cells, through a single-stranded RNA, which is connected reversely to a promoter and be introduced into a cell, that is complementary to a messenger RNA strand transcribed within a cell. In the synthesis pathway of plant oil and protein, the product of glycolysis - phosphoenolpyruvate (PEP) can be used as the common substrate for fat and protein biosynthesis. PEPCase (phosphoenolpyruvate carboxylase) is a potential key point, which control the fat synthesis pathway. The expression of gene for encoding PEPCase in peanut seeds was inhibited by use of antisense RNA technology in order to reduce the amount of PEPCase, that can reduce the amount of PEP in the protein synthesis and increase the amount of PEP flow to fatty acid synthesis, provide more substrate for the synthesis of oils, which can effectively improve the oil content of peanut seeds.The objective of this study was to got the cDNA fragment of PEPCase gene in peanut through homology cloning technology, the fragment connected reversely with the CaMV 35S promoter, and build antisense expression vector; The Agrobacterium GV3101 containing plasmid pGBVE (portableγ-vitamin E methyltransferase gene (γ-tmt) and selection marker gene bar) was infected the leaflet of peanut which have pre-cultured for 4 day for genetic transformation. The leaflet of 17 peanut cultivars (eleven large kernel peanut cultivars and six small kernel peanut cultivars) were cultured to compared the efficiency of differentiation,regeneration and genetic transformation, and screened the suitable peanut genotype for genetic transformation. The integration of target gene in anti-PPT transgenic plants was detected by Polymerase chain reaction technique . The major results are abstracted as follows:1. Cloning of peanut gene fragment encoding PEPCase and construction of its antisense expression vectorThe primers were designed according to the published sequence of PEPCase gene on Genbank (EU391629).Extracting total RNA from peanut Lipu big peanut, the fragment of PEPCase was obtained by RT-PCR.The fragment from PEPCase was cloned into pGM-T easy vector for sequencing and named pGMPEP. Sequencing analysis indicated that the inserted fragment of nucleotide and amino acid sequence was showing 99.77% and 100% homology to the reported sequence PEPCase. The recombinant plasmid pGMPEP and pBI121 were digested by BamHⅠand SacⅠ, recover the aimed 886bp fragment and the longer of pBI121 resected GUS gene. The result proved that the PEPCase gene was reversely inserted into the normal vector of pBI121. And the antisense expression vector of PEPCase gene was constructed.2. Screening of the Suitable genotype for genetic transformation of peanutThe results indicated that the 17 genotypes have significantly different in differentiation, regeneration and transformation rates, and genotype is a major factor, which can affect peanut genetic transformation. The results showed that five genotypes have higher differentiation rate, they are Fenghua2, 05A110, Huaxuan9, Linhua5 and Junan2, and the rate of differentiation are 28.43%, 18.40%, 14.48%, 12.07% and 10.52%, respectively. And they are also have higher regeneration rate, and the rate of regeneration are 67.57%, 32.93%, 21.41%, 24.77% and 21.21%, respectively. Four genotypes with higher transformation efficiency are Junan2, Linhua5, HY-1 and Fenghua2, and the rate of transformation are 3.36%, 1.99%, 1.57% and 0.47%, respectively. Considering the development of transformed explants, Junan2, Linhua5, Fenghua2 and HY-1 were the feasible materials for the research of peanut genetic transformation. PPT-resistant plants mediated by Agrobacterium tumefacien are obtained. Polymerase chain reaction of anti-PPT plants total genome results prove that gene bar and geneγ-tmt were integrated into peanut genome.