Cloning And RNAi Vector Construction Of PEPC Gene Consertative Sequence In Oil Sunflower

Oil sunflower is one of the five oilseed crop, with a cold drought, salinity tolerance barren, wide adaptability and poor soil dependent characteristics and so on. Back in the 50s of this century, geneticists have reported that the quality of rapeseed protein and fat content was highly negatively correlated, which plays an important role is phosphoenolpyruvate carboxylase enzyme (PEPC). Now in rape and soybean, PEPC has been knocked and its oil production does have a certain increase.Since the PEPC gene in oil sunflower hasn't been reported before, we have cloned its PEPC mRNA partial sequence by alignment with close relatives of this species. Compared with other homologous PEPC genes, two evolutionary conserved regions are found. RNAi expression vectors are constructed with these two regions. We expect to improve oil content in oil sunflower. The results of this study are as follows:(1) Obtain PEPC gene and partial sequence of mRNA by using homology cloning methods. Comparison to GENEBANK showed that it is highly homologous with the sequences of PEPC in other species, especially with same family (Asteraceae) plants—F.trinervia and Artemisia annua. And the highest homology with F.trinervia PEPC gene is 92% of similarity, and illustrate the sequence can be judged as sunflower PEPC gene sequences.(2) Find two conserved regions in PEPC gene of sunflower by comparing. According to the size of RNAi requires and pTCK-303 vector restriction sites, design two pairs of primers to clone the two fragments, and link them into the T vector. Then sequence the fragments and ensure it is correct. Positively and negatively, connected it to carrier pTCK-303. Construct RNAi vector pTCK303-FP1-RP1 and pTCK303-FP2-RP2.(3) By comparison, the best explant in tissue culture is cotyledonary nodes. The callus induction and shoot induction rates are higher than other explants. The best medium for shoot induction is MS + IAA (0.03 mg·L^-1) +6- BA (1.4 1.6 mg·L^-1), the induction rate reached 76.67%. In the medium 1/2MS + NAA (0.3 mg·L^-1).All adventitious buds can root, and fibrous roots are more conducive to survive the transplanted seedlings. After exploreing transformation of sunflower pollen tube pathway, determine their seeds can be positively transformed, and lay the foundation for further study.