Transformation Of Rice With High-lysine Gene Lys, CFLR And HLFgene

Lysine is the first limiting amino acid of rice, its content was up to less than theWHO recommended standards, lacking of lysine will affect the absorption of otheressential amino acids, resulting in protein and amino acids in the rice cann't be fullyabsorbed by the human body. Besides, the iron content in rice was0.02~0.28mg/kg,only1/10can be used in the milled rice Especially in the underdeveloped areas, somegroups of people lived on rice suffering from iron deficiency anemia. The high-lysineprotein gene (Lys), was cloned from winged bean (Psophocarpus tetragonolobus L.),the lysine content in the protein by the gene encoded was up to11.4%; so far, thelysine content from pepper (Capsicum frutescens L.) is the highest of the naturalprotein gene, which up to21.2%. As a natural immune protein of the human body,human lactoferrin has many biological functions such as antioxidant effects, involvedin iron metabolism, anticarcinogenic activity, anti-infective and immune regulationand so on. Therefore, if the high-lysine gene and the human lactoferrin gene wereintroduced into rice, not only can increase the lysine and iron content in rice but alsoenhance the resistance of the plants.In this present study, using genetic engineering techniques, making high-lysinegene and human lactoferrin gene introduced into rice by twin T-DNAco-transformation, in order to obtain functional rice with maker–free.The main resultsare as follows:1constructing two double T-DNA plant expression vector pCDMAR-Lys-hLF-hptand pCDMAR-Cflr-hLF-hpt. Both have in common is two different sources of highlysine gene are drived by rice endosperm specific promoter Gt1, Marker gene (hyg)and purpose gene which located in different T-DNA area. The difference is that thehLF gene of vector pCDMAR-Cflr-hLF-hpt. was under the control of theConstitutive promoter ubiquitin-1, another is drived by glutelin promoter GluB-1.2The two double T-DNA plant expression vectors were transformed into ahigh-quality rice varieties"TG-9" by agrobacterium-mediated. The plant expressionvector pCDMAR-Lys-hLF-hpt have obtained76strains of the transgenic plants inT0, PCR analysis of these T1generation lines obtained by self cross, of which20do notcontain the marker gene marker-free. HPLC analysis, of which8rice lines' the ratioof Lys amino acid/total amino acid markedly increased by13.38%compared to thenon-transgene seeds. But only resistant callus gained from the plant expressionvector pCDMAR-Cflr-hLF-hpt, and these resistant callus are in differentiation.