Cloning Of Genes For Partial Member Enzymes Of β-carotene Biosynthetic Pathway In Grandis[Citrus Grandis (L.)Osbeck]

Carotenoid is one of the most important classes of plant pigments,have a lot of diverse functions. They are importantly nutritional in human diets, especiallyβ-carotenoid has pro-vitamin A activity, it serves as precursors for vitamin A in our body.β-carotenoid plays an important role in human health. For example,β-carotenoid functions as antioxidant to improve the immune system positively, and also have the function of reducing the risk of suffering cancers or infectious diseases.However, human body can not synthesizeβ-carotenoid by itself,most ofβ-carotenoid are come from foods.But in many plants,they don't have highβ-carotenoid contain, and it's hard to synthesizeβ-carotenoid with chemical method.Therefore, people pay more attention to search or exploit the fruits with complex source of carotenoids using modern sciences and technologies.The red-fleshed grandis is a new breed of pummelo,it's rich in nutrition, containing more vitamins andβ-carotenoid than general grandis. The red-fleshed grandis was used as testing materials, and the genes forβ-carotene biosynthetic were cloned to conduct relevant studies. Major results are descirbed as follow:(1) An improved method for RNA extraction from grandis samples was developed. In order to extract high quality RNA from grandis materials, four methods were compared, which included Trizol,CTAB,modified CTABand the reagent box, and the modified CTAB was the best one. Based on the method of modified Trizol, we obtained the pure and clean total RNA that could be used for further studies in the field of molecular biology such as PCR and RACE.(2) The consensus sequences of three genes ofβ-carotene biosynthetic pathway (psy, pds, zds) were cloned by PCR from the cDNA Reverse-transcripted from the RNA. And they were 422bp, 599bp and 569bp in length, respectively. (3) The RACE method was emplyed to obtain the 3'cDNA sequence of. The partial sequence of psy, which had a length of 1170bp and encodes a protein of 238 amino acids. Sequence analysis showed that its deduced amino acid sequence shared 98%,98%,97%,85% and 85% identity with that of psy of Citrus sinensis,Citrus unshiu,Citrus x paradise,Carica papaya,Daucus carota.(4) The RACE method was also used to acquire the 3'cDNA sequence of pds. The partial sequence of pds, which was linked by the 3'sequence and its consensus sequences, had a length of 1612bp and encodes a protein of 468 amino acids. Sequence analysis showed that its deduced amino acid sequence shared 99%,98%,99%,90%,and 87% identity with that of pds of Citrus sinensis,Citrus unshiu,Citrus x paradise,Carica papaya,Daucus carota.