Studies On The Construction Of Plant Expression Vector Of FAD2Gene And Its Transfer To Pumpkin

Pumpkin seeds with rich nutrition are the ideal dietotherapy health care commodities which are rich in oil and protein. Amongst them, Oleic acid, linoleic acid and other unsaturated fatty acids are easily absorbed by human body. Fatty acid content and composition are key indicators of pumpkin seed oil quality..Fatty acid accumulations of different pumpkin varieties were discrepant in genetics which were related with fatty acid metabolism related genes. So, the function of the fatty acid metabolism related genes was studied in seed pumpkin seed which would be significant in improving pumpkin seed quality and carrying out.the breeding of pumpkin quality.Oleoyl phosphatidylcholine desaturase (FAD2) is a catalytic polyunsaturated fatty acids enzyme which is important in the plant for mainly catalyzing oleic acid (came) to linoleic acid and is the key enzyme which decides the content of plant seeds oleic acid and linoleic acid. FAD2gene is the encoding gene of FAD2enzyme which had been cloned in pumpkin. FAD2enzyme showed obviously regulation and control function for the fatty acids content in plant. The sense expression of FAD2gene can improve linoleic acid and other polyunsaturated fatty acids content, and antisense expression can improve oleic acid content. Therefore, the function of FAD2gene was verified through genetic engineering in pumpkin seeds.Ovary injection is a plant transgenic method which the breeding works often use..This method effectively makes use of the plant natural reproduction, can obtain directly seeds, and shortens the breeding cycle effectively without tissue culture of cell or protoplast. In addition, the instrument and equipment is simple and cheap, and can be carried out in the field or the greenhouse. In short, it is a relatively simple and effective method. So, this studies injected FAD2gene into the pumpkin by ovary injection, expected to obtain the pumpkin transgenic plants, and provided a new, more efficient way for molecular breeding. It also laid a foundation for the validation of the function of pumpkin seeds FAD2gene and pumpkin quality breeding in the meanwhile. The results of the studies were listed as follows:1. Constructing the plant expression vector pCMBIA2300-NAPIN-FAD2plasmidThis study cloned FAD2gene according to the known sequence of FAD2gene and constructed the plant expression vector pCMBIA2300-NAPIN-FAD2on the base of plant expression vector pCMBIA2300+using the method of cutting by enzyme, linking, screening, and identifying.2. Exploring the ovary injection system suitable for the pumpkinThrough the guidance of pollen tube and ovary of aniline blue staining for different treatments, the results showed that (1) the transformation time of pumpkin ovary injection:pollination24hours;(2) injection process:ovary was injected after the upper part of the ovary and stigma were removed.3. Obtaining the transgenic pumpkinsThis study transferred FAD2gene into pumpkin’Yinhuil’via ovary injection. PCR and PCR-Southern indicated that7FAD2transgenic plants were acquired and conversion rate was0.66%.