Cloning Of Pepc1 From Jatropha Curcas L. And Its Role In Regulation Of Protein And Oil Synthesis In Tabacco

Jatropha curcas L. is widely distributed in tropic and subtropic. In China, it is existed or cultivated or semi-wide in tropic and dry-hot valley region. Jatropha curcas L. has an huge development potential as an oil tree substituting fossil fuels in future, because of its high oil content in seeds. But sutibale lands for planting is so limited, and improving oil content in seeds is one of breeding goals. PEPCase is reported involved in oil synthesis payway. Therefore, down-regulated expression of pepc may enhance oil content in seeds antisense approach.In this study, pepcl was cloned an a member of pepc gene family using RT-PCR and RACE techniques from development seeds of Jatropha curcas L.. Its amino acid sequence was analyzed. Expression vectors with sense and antisense of gene were contructed. These vectors were introduced into Agrobacteria and used to transform to tobacco. Transgenic taboccos were confirmed by Southern blot, real-time PCR, and further characterized by determining the activity of PEPC, content of protein and fatty acid. The expression patterns were analyzed in different tissues and different stages in developing seeds. Those results reflected expression of pepcl in level of transtription and translation. The main results are as followed:1. The 3'-fragment of pepc from Jatropha curcas L. development seeds was isolated via RT-PCR, and 5'-fragment was amplified by RACE. The cDNA length of pepc is 3124 bp. The open read frame (ORF) contains 2898 bp,965 amino acids.2. The molecular weight of PEPC is 110.6 kD with the the acidic amino acid account for 14.6%, alkali amino acid 13.5% of the total, and pⅠat 6.18. The single peptide chain was not stabily because of its low stability coefficient. The amino acid secondary structure is coil mainly, a-helical andβ-strand in occasion. The hydrophilic protein has not transmembrane region and signal peptide. The protein belongs to PEPC1 in pepc gene family, and the gene named pepcl. The homology of PEPC 1 of Jatropha curcas L. and Ricinus communis is the highest, and higher with tobacco.A typical large Ppc functional domain span 11-965aa was also found using the CDD program an NCBI. In addition,46 activity sites were found belonged to 7 types by searching in website at http://cn.expasy.org/prosite/.3. The housekeeping gene of Jatropha curcas L., actin was cloned. The temporal and spatial expression patterns of pepcl Jatropha curcas L. were studied using real-time PCR. During different stages from flower to mature fruit, the pepcl expression shows increasing in first four stages, and decreasing in late five stages. The expression level of pepcl is low in leaves.4. The sense expression vector pCAMBIA-KSpepc and antisense expression pBI-BXpepc were constructed based on the character of pepcl sequence. The sense pepcl and antisense pepcl gene were transformed into tobacco by Agrobacterium-mediated method. Three plants with sense pepcl and ten plants with antisense pepcl were obtained after kanamycin selection, PCR and PCR-Southern analysis. The transgenic tobacco plants were analyzed by real-time PCR. The expression level of pepc of tobacco endogenous changed little, but up-regulated of Jatropha curcas L. in sense pepcl plant. Of the ten antisense pepcl plants, seven plants have the expression level of both tobacco pepc and Jatropha curcas L. pepcl down-regulated.5. The activity of PEPCase, contents of total protein and fat were determined from leaves of transgene tobacco. Three sense pepcl tobacco plants were found higher than wild type. Two of the three transgenic plants were lower than wild type in fat content. In ten antisense pepcl gene plants, nine plants were lower than wild type in activity of PEPCase, but five plants decreased in total protein content and four plants increased in fat content. The fatty acid composition of transgene plants were found changed little. Therefore, two sense pepcl gene tobacco and four antisense pepcl tobacco lines were obtained with expected result.