Study On Cloning, Genetic Transformation Of Phytoene Synthase Gene And The Preparation Of Antibacterial Peptide

Carotenoids are essential components in all plants.The caortenoids are the most dviesre and widespread group of pigments in nature and serve an as extraordinary variety of biologica functions in cyanobacteria,algae,animal and plant.They are essential components of photosynthesis and can capture light energy for photosynthesis in plants.They also function in pigmentation of flowers and fruits. Carotenoids conclude many important pigments from yellow to red,which can alter flower colour.Carotenoids play an important role in human health,especiallyβcarotene is the former of vitamin A,and can also prevent cardiorascular diseases,enhance human immunological competence,postpone ageing,and antitumous effete.As a medicinal and health protection,the functions of which have been paid more and more attention..It was attracted to regulate metabolism and accumulation of carotenoids,alter flowers colour,and improve quality of vegetable and fruit via genetic engineering.Carotenoids accumulation in wheat seeds determines endosperm colour,which is an important quality trait in wheat.Phytoene synthase(PSY) is a critical enzyme in the carotenoid biosynthetic pathway and is associated with the accumulation of carotenoids in endosperm.In the present research,we cloned phytoene synthase(PSY) by means of RACE from wheat and constructed plant expression vector,then the tp-crtB gene was transformed into Arabidopsis Thaliana via Agrobacterium tumefaciens.The main results were as follows.1) The fragment of PSY gene in the wheat was cloned by RACE,The sequence analysis showed that PSY cDNA was 1,150 bp in length.It encoded a polypeptide of 304 animo acids with a calculated molecular mass of 33.9 kDa. 2) A BLAST search in GenBank showed that wheat PSY showed high similarity to that in other plants(60%-90%).Especially,the identity of the wheat with rice and maize is over 90%,Phylogenic analysis showed that PSY was highly conservative in evolution of plants.Motif analysis showed that TaPSY belongs to PSY2 group, which was designated as TaPSY2.3) Both genes of crtB and tp were amplified by PCR and cloned into pMD18 - T vector and were confirmed by sequence analysis,Both genes of crtB and tp were ligated by the middle vector pBlue-script KS,named pSK-tp-crtB,tp-crtB in pSK-tp-crtB vector was cloned in plant expression vector pBI121-napA,named pBI121-tp-crtB,pBI121-tp-crtB was transformed into Agrobacterium tumefaciens strain EHA 105.4) Plant expression vector pBI121-tp-crtB was transformed into Arabidopsis Thaliana via Agrobacterium tumefaciens,fourteen transplants were obtained, germination experiment was done,the result showed that tp-crtB gene had no obvious effect on the development of Arabidopsis Thaliana.We do other two work:1) A Method for Fast Preparation of DNA Marker by PCR Technology,The bands of DNA Marker are even and clear.The price of made DNA Marker is much lower than the sales.It meets the need of research and laboratories.2) Fusion expression and efficient purification of a glycine-rich antibacterial peptide of Drosophila in Escherichia coli.