Cultivation Of Safe Wheat Variety For Celiac Patients

Celiac disease is a primary malabsorption syndrome in human characterized by the lesions in mucous membrane of small intestine due to patients’intolerance to gluten proteins. The autoimmume reactions of patients are caused against the epitopes of relevant fractions from wheat grain storage proteins, as well as the storage proteins from barley, rye and oak. The symptom only gets severe when gluten proteins are consumed with the major symptom being diarrhea, sometimes abdominal distension and borborygmy, and steatorrhea as a typicality. For celiac patients, malabsorption of nutrients such as iron, folate, calcium, fat-soluble vitamins A, D, E, K, and protein can occur, leading to nutritional deficiencies such as anemia and osteoporosis. Additional complications of untreated celiac disease include infertility in women and men, miscarriage, lymphoma, and the possible development of other autoimmune diseases. The only effective therapy for patients is the strict dietary abstinence from these grains.As one of the major food source, Low molecular weight (LMW) glutenins and gliadins from wheat are the major antigen sources to trigger autoimmune response for celiac patients. To make wheat grain a reliable food source for celiac patients, all LMW glutenin and gliadin fractions need to be eliminated. The gene expression control between different protein groups in barley and wheat has the similar methylation patterns, which can distinguish HMW glutenins from LMW glutenins and gliadins. This intrigues us to engineer the methylation patterns within promoters of wheat LMW glutenin and gliadin genes in wheat’s developing endosperm, so as to improve the protein composition in wheat seed for celiac patients.Bi-functional DNA glycosylase DEMETER (DME) is in charge of demethylation process extensively in plant genomes, and is a key factor to redesign the methylation patterns of gene promoter of wheat prolamins which cause celiac disease. A315bp fragment from wheat DME was amplified and radioactively labeled to screen hexaploid variety Chinese Spring BAC DNA library.3unique BAC clones (1946D08,2106P11,2159B03) were identified from the positive BAC clones by genome-non-specific primers and sequenced by454sequencing.3full-length DME sequence (GenBank accession numbers:JF683316, JF683317, JF683318) were obtained from3clones, respectively. The3DME were mapped to the long arm of wheat group5chromosomes. The further assignment of DME homoeologues to genome locations of bread wheat was performed using homoeologues-specific primers to amplify the genomic DNA of diploid wheat progenies T. urartu (AA), Ae.Speltoides (BB), Ae Tauschii (DD), with the unambiguous assignment of2159B03to chromosome5A,1946D08to chromosome5B and2106P11to chromosome5D. Sub-genome assignment of DME homoeologues TaDME-5B was further validated by the use of wheat chromosome5B specific terminal and interstitial deletion lines to a sub-centromeric-bin, encompassing98.78Mb of genomic DNA on5BL.Three wheat DME sequences differ in length from each other ranging from12.27kb to12.63kb. Necleotide diversity analysis shows the length difference of the3homoeologues is mostly due to insertions and deletions (InDels) in the introns. A large number of point mutations and small InDels (HSVs) within DME homoeologues also exist in exons. Intrestingly, the frequency of HSVs causing amino acide changes was kept low in the vicinity of all5domians of5-methylcytosine DNA glycosylases. This indicates a natural selection against the functional point mutations and InDels in these domains and reflects their functional importance.Three wheat DME sequences were analyzed by Matchpoint and a small fragment with highest target score was used to construct a transformation vector for specifically silencing DME activity in wheat’s developing endosperm. The silencing trigger is an inverted repeat big hairpin construct, with an185bp arm including73bp showed perfect homology with3DME genes. The intron is a568bp fragment from wheat TAK14. The hairpin was thereafter constructed in between wheat HMW glutenins promoter and nos terminator. Particle bombardment transformation was performed to wheat embryo callus with stringent following selections. Based on the assays to genomic DNA and DME transcript level in developing endosperm,21transformants from To were selected and regenerated into282T1plants. In both To and T1generations, DME transcription in developing endosperm showed varying levels of suppression levels up to93.72%by real-time PCR assay. SDS-PAGE gel and RT-HPLC analysis indicated a significant reduction in all LMW and gliadin fractions in certain T1lines, from which extra amino acids accumulated also contributed to increased expression of HMW glutenins. This points out a new way for creating celiac safe wheat variety, and has the potential to be a practical way to finally provide celiac patients a quality life with a natural dietary recipe from wheat.