The Creation Of High Cotton Oil Germplasm By Regulating GhPEPC And GhDGAT

Cotton is an important economic crop in China and also is the main material of textile industry. Because the oil content of cottonseed is about 30%, cotton is also called an important oil crop. Presently, more than 60% of edible oil depends on import in China. So improving the oil content of edible oil crops can ease the problem of China’s oil shortage.According to the metabolic pathway of vegetable oil, phosphoenolpyruvate carboxylase and acetyl coenzyme A carboxylase are considered as the two major enzymes regulating the flow of carbon source. These two enzymes control the synthesis of protein and lipid via competing the same substrate- pyruvate. Therefore, through silence PEPCase by modern biotechnology, more carbon source may flow into oil synthesis pathway. The current study is based on this theory. So we hope to downregulate the expression of PEPCase by RNAi technology for improving oil content in transgenic cotton seeds. In addition, we also foucs on DGAT(diacylglycerol acyltransferase), which is the key gene in lipid synthesis pathway. Over-expression vector with two promoters(Ca MV 35 S promoter and the α-globulin promoter) were constructed in order to improve the gene expresstion in seeds and then the oil content. Results are as follows.1.We searched Gh PEPC(Gen Bank: AF008939.1) gene sequence in NCBI and blasted the conserved region of PEPC. Respectively, two pair of primers were designed: the conserved domain of Gh PEPC gene(PPC) primers and Gh PEPC1 3 ’end(PEPC1) primers. These two regions of Gh PEPC were cloned from Gossypium hirsutum cultivar YZ1, and both gene sequences were completely consistent with Gh PEPC1 in NCBI. Also, α- globulin seed specific promoter(Gen Bank: AX795651.1) of Gossypium hirsutum and Gh DGAT gene were cloned, then primers were designed for vector constrction.2. PPC and PEPC1 were connected to the RNAi vector p Hellsgate 4 while αGP and Gh DGAT were connected to over-expression vector p K2GW7.0 for cotton genetic transformation. Then, PCR and enzyme digestion were applied to verify these vectors. Finally, we obtained newly recombinant vectors which were expected.3. Two Gossypium hirsutum varieties YZ1 and Y668 were used for genetic transformation and seveal putative transgenic plants were obtained with different foreign genes. Currently, 22 transgenic PPC lines and 14 transgenic PEPC1 lines were abtained using YZ1 as the genetic background; Also, using Y668 as the genetic background,we obtained 11 transgenic PPC lines and 21 transgenic PEPC1 lines. Moreover, 10 transgenic 35s-Gh DGAT T0 plants and 8 transgenic αGP-Gh DGAT T0 plants were abtained with YZ1 as explants.4. PCR positive detection and Southern hybridization of seveal T0 transgenic plants, which were used YZ1 as the genetic background, confirmed the intergration of foreign gene into cotton genome. Then, RT and q RT-PCR analysis revealed the expression of PPC and PEPC1 were downregulated in different transgenic plants. T2 seeds from PPC-1, PPC-1-8 and PPC-8 transgenic lines have been harvested and they were YZ1 genetic background. Metabolite analysis indicated the seed protein content in transgenic lines was decreased by 16.5%-18.5% when compared to wildtype control. However, seed oil content was 0.7%-11.0% higher than wide-type.