Studies On Molecular Manipulation Of Condensed Tannin Synthesis And Genetic Transformation Of Alfalfa (Medicargo Sativa L.)

Alfalfa (Medicago sativa L.) has been taken as the "king of forage" for its' excellent forge quality, rich nutrition and strong palatability. It is the most widely used and most important legumes in Asia and North America. However, the ruminant livestock feeding with fresh alfalfa would easily cause bloat disease, this has affected its use in grazing area. It is generally accepted that low content of condensed tannins (CT) in alfalfa leaf is the major reason that caused ruminant livestock's bloat. Through genetic manipulation, promoting synthesis and accumulation of CT in alfalfa leaves is the most important research topic of alfalfa breeding, and has great significance in theory and practice.The condensed tannin biosynthesis is through the shikimate pathway and produced phenylalanine, after the latter form the CT after series of reactions and there has many enzymes involved in it. Dihydroflavonol reductase (DFR) is the key enzyme of the biosynthesis of CT, regulating the expression level of DFR gene can change the accumulation of CT, but the expression level of dfr in genetically modified alfalfa would be further improved. PNZIP (pharbitis nilleu zipper) promoter is a tissue-specific promoter that will drive the target gene expressed in photosynthetic tissues efficiently, while in non-photosynthetic tissues at very low levels of. In order to improve the content of CT in alfalfa leaves, stems and other organs of livestock feed, achieve new germplasm materials for breeding anti-bloat alfalfa, the experiment carried out DFR gene and PNZIP tissue-specific promoter cloning, PNZIP promoter drived DFR gene plant expression vector construction and genetic transformation on alfalfa, achieved the following result:1. RT-PCR was employed to clone cDNA fragment of DFR gene from Medicargo truncatula young fruit. This fragment is 1018 bp, encode 337 amino-acid residues. Sequence analysis showed that 99.8% of the DFR were homologous to those in GenBank. The two base mutation did not affect the translation of amino-acid, based on inferred online there is only one amino acid change. DFR gene Prokaryotic expression vector named pETDFR was constructed; and DFR gene has expressed at high level in the prokaryotic expression vector, achieved the expected size 39.8 KD enzyme protein. All these indicated that the coned DFR gene has complete coding sequence.2. By PCR method, the 1487 bp PNZIP promoter was cloned from pharbitis nil choisy Morning Glory genomic DNA. Sequence analysis showed that:In addition to a typical eukaryotic core promoter region (-60--10 bp) and a number of TATA-box, CAAT-box and other promoter elements, there are 6 light effect elements such as I-box, ACE, G-box, CAANNNNATC components, Box-II, CCAAT-box in it, indicating the sequence has a regulatory role in photosynthetic tissues microbial metabolism. Taking green fluorescent protein (GFP) as reporter gene, the plant expression vector named pPNGFP which GFP gene was drived by PNZIP promoter was constructed, in transgenic tobacco green tissue especially in mesophyll and stem GFP gene have been expressed efficiently, indicating that the cloned PNZIP promoter has the specificity expressed in photosynthetic tissue3. Based on pBI121 vector, the constitutive promoter CaMV 35S and photosynthetic tissue-specific promoter PNZIP drived DFR gene plant expression vector pBIDFR and pPNDFR were constructed. With agrobacterium (EHA105) engineered bacteria containing pBIDFR, pPNDFR transformed tobacco (Nictiana tabacum L.) varieties "Honghuadajingzi'(2n=4 X=48),27 and 23 Kan resistant plants were received respectively. Hydrochloric acid method was used to determine the CT content in wild tobacco (control), different promoter drived DFR gene transformed tobacco leaves, stems and roots organs, the results show that the constitutive promoter drived DFR gene (pBIDFR) transformed tobacco every organs' CT content was significantly higher than the control, in leaves up to 4.27 mg/g,75% higher than the control leaves'. The photosynthetic tissue specific promoter drived DFR gene (pPNDFR) transgenic tobacco stems and leaves'condensed tannins were significantly higher than wild type tobacco, which leaves'condensed tannin contents was 4.01 mg/g,64.34% higher than the control leaves. This indicated that the PNZIP promoter activity is near to the constitutive promoter CaMV35S, from the point of energy PNZIP promoter drived gene expression pattern is more economical.Taking alfalfa varieties "Zhongmuyihao" as material, the Agrobacterium engineering bacteria pPNDFR/EAH105 transformation system was studied. Obtained with 5-7d old cotyledons as explants, pre-cultured 3 d, OD 600 0.3 engineering bacteria concentration, infected for 30 min, co-cultured 3 d transformation system, which can get 70% resistant callus induction rate. After induction the resistant callus eventually acquired 6 resistant transgenic plants and a number of resistant buds.