Study On Transferring Capsicum Frutescens L High-lysine (cflr) Gene Into Wheat

Being one of important food crops, the application of genetic engineering on wheat improvement has been attracted great attention recently. In contrast to rice, maize and other crops of agronomic importance, genetic transformation of wheat is still in the exploratory stage. In the study, biolistics-mediated transformation was employed. The transformation, development and regeneration systems are as the following: immature embryos of common wheat cv. Alondra was used as the receptor with the diameter less than 0.5mm, 1mm or greater than 1.5mm, coated by 0.6 and/or 1μm gold particles; screening and selection were performed on 1-7mg/L"Basta"culture medium.As one of the essential amino acids on human nutrient, lysine content is relatively low in wheat which reduces its nutritional quality. Since the situation is the same to other cereal crops, it seems insignificant to perform traditional crossing with the distal wheat varieties. Biolistics-mediated transformation can make it possible through the introgression of genes encoding lysine-rich proteins, a key enzyme in lysine biosynthesis and the proteins and/or enzymes inhibiting or decreasing lysine biolysis process.cflr(accession number: EU367999), one gene coding high-lysine protein isolated from pepper, was constructed into the vector pAHC25 and then transformed into wheat immature embryos by gold particle bombardment. CFLR consists of 223 amino acids, of them the number of lysine 40, 21.2% (w / w), and the number of threonine 24, 10.3% (w / w), respectively.Main results: In the process of"bullet"preparation, 0.6μm gold powder can make good results with hard caking, good dispersion, high transformation efficiency, while 1μm or larger particles can precipitate easily and not be dispersed easily in the carrier membrane. Plantlets can be regenerated easily using 1mm immature embryos while smaller or larger ones make bad results. A Basta concentration of 3-4mg/L was more favorable for screening. In total, 144 T0 generation positive plants were obtained. Of them, 5 had produced offsprings. 28 of 33 lines in total were positive in T1 generation using PCR assay at RNA level. Sequencing confirmed the occurrence of cflr transcripts. The content of lysine increased by 7.4% compared to the control.