Improvement Of Wheat (Triticum Aestivum L.)Nutritional Quality By Introduction Of Bacterial Carotenoid Biosynthetic Genes CrtB And CrtI

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid and trace amount of provitamin A content. Few studies on carotenoid metabolic engineering in wheat have been reported due to our poor knowledge of wheat carotenoid biosynthesis. Study on wheat breeding has traditionally focused on improving milling and bread making qualities. The identification of key carotenoid biosynthetic genes progressed slowly due to the large size and complexity of the wheat genome. Meanwhile, the bottleneck in the genetic transformation has hindered carotenoid metabolic engineering of wheat.The nutritional values and healthy benefits conferred by carotenoids have been realized by human beings. So far, the scientists have made a lot of successful attempts to enhance the carotenoid content in crops by metabolic engineering, such as in rice, canola, maize and potato. As one of the most important staple food crops, enhancement of the carotenoids in wheat grains will not only improve the nutritional value of wheat, but also provide nutritional benefits for human health. In order to enrich the carotenoid content in wheat grains and understand the mechanism regulating wheat carotenoid biosynthesis, the bacterial phytoene synthase gene CrtB and carotene desaturase gene CrtI were transformed into wheat cultivar Bobwhite. After selection by phosphinotricin and genomic PCR, we obtained the transgenic wheat lines of three transformation combinations(CrtB, CrtI, CrtB and CrtI). Expression of CrtB or CrtI alone could slightly increase the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grains phenotype, accompanied by the total carotenoid content increase of approximately8-fold, achieved to4.76μg g-1seed dry weight, β-carotene increased65-fold, to3.21μg g-1seed dry weight and the provitamin A content (sum of a-carotene, β-carotene and β-cryptoxanthin) increased76-fold, to3.82μg g-1seed dry weight. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat is also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in wheat carotenoid biosynthetic pathway. In addition, our results revealed the important roles of LCYs and HYDs in biosynthesis of provitamin A. The present results also demonstrated that LCYE and LCYB are the key enzymes in the downstream of wheat carotenoid biosynthetic pathway.Furthermore, we have generated the transgenic wheat lines with the highest provitamin A content ever reported through co-expressing of the crtB and crtI genes. In conclusion, it is promising to achieve higher carotenoid accumulation in the wheat endosperms by regulating several steps of the carotenoid biosynthetic pathway simultaneously. Our attempt to enhance the provitamin A content in hexaploid wheat through the genetic engineering will not only strengthen the knowledge of carotenoid biosynthetic regulation in wheat endosperm, but also supply predictable and sustainable metabolic engineering strategies for breeding the transgenic wheat varieties with high carotenoid content in future.