1a.Objectives (from AD-416):
The general objective of our proposed research is to elucidate the regulatory network of Or and to reveal novel strategies for breeding agricultural important crops with enhanced Beta-carotene level. Specifically, our objectives include:
1). To identify and functionally characterize genes and proteins in the regulatory network of Or in mediating high Beta-carotene accumulation. This includes to isolate the protein(s) that physically interacts with OR, and the genes and processes that are associated with Or. 2). To define genetic and functional allelic variation of them using cucurbit crops as models.
1b.Approach (from AD-416):
Two complementary approaches will be used for the identification of the genes and proteins associated with Or in regulating carotenoid accumulation. A sensitive yeast two-hybrid will be employed to isolate the OR-interacting protein(s). As a systems approach provides a unique opportunity to identify components of metabolic pathways and map points of regulatory control, the systems approach that integrates transcriptome and bioinformatics tools will also be utilized to define the key genes and metabolic processes that impact carotenoid accumulation. Their involvement will be accessed genetically in melon and functionally confirmed in Arabidopsis callus system. The allelic variation in association with Beta-carotene content in melon will be examined in the germplasm collection to uncover the favorable alleles for breeding high Beta-carotene crops.
Carotenoids play an indispensable role to human nutrition and health in providing the primary dietary sources of provitamin A and in reducing the risk of certain chronic diseases. We have demonstrated that regulation of chromoplast biogenesis to enhance sink strength for greater biosynthesis and higher storage capacity represents a critical mechanism by which carotenoid levels are regulated in plants. The Or gene governs this process. This project investigates how Or regulates this process and how it controls other genes and metabolic pathways in ultimately conferring high levels of B-carotene accumulation in melon fruit. The following is a short description of the progress we made during our first year. We did preliminary RNA-Seq analysis of orange and green-flesh melon fruits during three stages of ripening. RNA was extracted and RNA-Seq libraries were constructed. A single lane of an Illumina GAII run was performed for 12 melon libraries (2 varieties x 3 stages x 2 repeats) and an average of over 4 million 86-bp reads from each library were produced. Our preliminary examination of these data indicates that a number of carotenoid cleavage dioxygenases show differential expression between orange and white flesh melon during fruit development, and they are being further characterized. We also performed site-directed mutagenesis of the Or gene to examine the effect of specific amino acid in the OR protein on carotenoid accumulation. We transformed the mutagenized constructs into Arabidopsis and will test their effect when the transgenic plants are ready.