Cloning And Genetic Transformation Of Genes Encoding Key Enzymes In Carotenoids Biosynthsis Pathway In Wheat (Triticum Aestivum L.)

Wheat is the word's important food crop, and improving the nutritional quality of wheat is an important objective in wheat molecular breeding programs. Carotenoids are essential components of human diets, primarily as precursors of vitamin A. Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthamia and keratomalacia, leading to total blindness. Furthermore, vitamin A deficiency exacerbates affilictiona, such as diarrhea, respiratory diseases, and childhood diseases such as measles. However, there is low carotenoid content in common wheat (less than 3μg/g).In this thesis, firstly, the EST (Expressed Sequence Tag) sequences of several wheat carotenoids biosynthesis key enzymes, such as phytoene synthase (PSY), phytoene desaturase (PDS) andζ-carotene desaturase (ZDS), were analyzed. Then the full-length cDNA sequences and the expression characteristics of these genes were identified by molecular biology methods, on the basis of which, the function of these genes were identified in genetic transformation and the new pathway of increasing the wheat carotenoids content are discussed. The main results were as follows.1. The full-length cDNA sequences of phytoene desaturase gene (TaPDS) andζ-carotene desaturase gene (TaZDS) were isolated from common wheat (Triticum aestivum. L.) by using RACE (Rapid Amplification of cDNA Ends) method. Otherwise, the complete coding sequence of phytoene synthase gene (PSY) was isolated from the same wheat variety by using homologous sequence separation. Through semi-quantitative RT-PCR method, the expressions of PSY, PDS and ZDS genes were detected. These genes showed higher expression in photosynthetic tissues (leaves and flowers), and lower expression in non-photosynthetic tissues (seeds). Since the first full-length cDNA cloning of these enzymes in common wheat are reported here, the implications for the genetic modification of the carotenoids (pro-vitamin A) content in wheat are also discussed.2. In order to increase the carotenoid content of common wheat endosperm, transgenic wheat has been produced by over-expressing a maize psy1 gene coding phytoene synthase driven by endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with a bacterial carotene dusaturase gene CrtI from Erinia uredovora under the constitutive CaMV 35S promoter control. This combination covers the requirements forβ-carotene synthesis and, as hoped, a clear increasing of carotenoids content was detected in the T₀ generation of transgenic wheat seeds, which showed a light yellow colour. We observed an increase in total carotenoids content from about 0.5μg/g up to 3.8～4.9μg/g.3. The "minimal expression cassettes" transformation procedure has been developed and optimized. Stable transgenic plants were recovered from the immature embryos with transgenes on the linear gene cassettes lacking vector backbone sequences, corresponding to promoters, open reading frames and terminators. The efficiency of transformation using gene cassettes reached 0.7%, and it's similar to the conventional transgenic technology.The results of present research provided with valuable reference for improving wheat nutritional quality through genetic engineering.