Isolation And Transformation Of DFR And ANR Genes Of Tannins Synthetic Way In Medkago Sativa L

Medicago sativa L. is an excellent leguminous fodder crop with high output, good qualityand palatability. It's praised as the "king of grass". However, feeding the ruminant livestock withfresh Medicago sativa would easily cause bloat, which has affected its use in grazing pastures. It isgenerally accepted that low content of condensed tannins (CT) in Medicago sativa was the majorreason that caused ruminant livestock's bloat. Thus, improving tannins content of alfalfa couldreduce the bloat incidence. It has the important practical significance.We studied the two genes involved Medicago sativa tannins synthetic way: dihydroflavonol4-reductase (DFR) and anthocyanidin reductase (ANR). The main results of this study are asfollowing:1. The complete coding cDNA sequence of DFR was amplified from Medicago sativa podsmRNA by reverse transcription polymerase chain reaction (RT-PCR). Sequencing analysis revealsfull length of open reading frame of DFR was1020bp and encode a protein of339amino acidsresidues. The sequence alignment analysis by using blast online software revealed that thesequence share high homology with DFR protein from other plants. It exhibited homologous of97%with Medicago truncatula DFR. The gene encode a dihydroflavonol4-reductase (MsDFR) inMedicago sativa, and the Genbank accession number is JF931173.2. Real-time PCR was performed to reveal that transcript level of MsDFR in different tissuesand under different stresses. The results indicated that MsDFR transcription was abundant in pods;And the expression of MsDFR was induced by NaCl and PEG treatments; Furthermore, MsDFRwas induced by exogenous hormone GA and wouding stress.3. We constructed the fusion protein expression vector pCAMBIA1302-MsDFR-GFP, andtransformed it into onion epidermal cells using a gene gun. Subcellular localization of transientlyexpressed MsDFR-GFP fusion protein was detected by a confocal laser scanning microscope. Theresult revealed that MsDFR is localized in cytoplasm.4. The coding region of MsDFR was inserted into the expression vector pBI121toconstructed the recombined overexpression vector pBI121-MsDFR-GUS. Then the vector wastransformed into Arabidopsis by floral dip method. After been selected by antibiotics, theregenerated plants were obtained. PCR analysis showed they were all positive lines. The tanninscontent of transgenic plants were slightly higher than that of non-trasgenic plants, but there was nosignificant difference between them.5. The open reading frame of the MsANR gene was1017bp in length, encoding338aminoacid residues. The sequence alignment analysis by using blast online software revealed that thesequence share high homology with ANR protein from other plants and the similarity to Medicagotruncatula ANR was the highest with95%. The gene encode an anthocyanidin reductase (MsANR)in Medicago sativa, and the Genbank accession number is HM754630.6. DNA gel blot analysis indicated MsANR gene is an one-copy gene, and the gene's DNA sequence contains six exons and five introns.7. Using DNA walking method,MsANR gene promoter sequence was obtained. Except thebasal elements TATA-box and CAAT-box, the promoter be predicted include some other specialelements. Such as light sensing element(ACE, Box-4, BoxI, G-Box, GA-motif, GAG-motif,GT1-motif, Chs-Unit1ml, Sp1and TCT-motif,abscisic acid response element(ABRE),Gibberellic acid response element(GARE-motif and TATC-Box),MYB transcription factorbinding region,ERF transcription factor binding region and resist stress response elemen(tTC-richrepeats and anti-oxidation element ARE).8. Real-time PCR was performed to reveal transcript level of MsANR in different tissues andunder different stresses. The results indicated MsANR transcription was abundant in pods, and theexpression of MsDFR was induced by NaCl and PEG treatments. Furthermore, MsANR wasinduced by Exogenous hormone GA, ABA and wouding, dark stresses.9. We constructed the fusion protein expression vector pCAMBIA1302-MsANR-GFP andtransformed it into Arabidopsis protoplast using PEG–calcium fusion. Subcellular localization oftransiently expressed MsANR-GFP fusion protein was detected by a confocal laser scanningmicroscope. The result revealed that MsDFR is localized in nucleus and cytoplasm.10. The open reading framgment of MsANR was inserted into pET-30a(+) to construct theexpression vectors, and the recombinant plasmids were transformed into E.coli BL21(DE3). Thefusion protein expressed under the induction of IPTG at37℃. SDS-PAGE analysis revealed thatthe full-length gene could be expressed and the fusion protein was highly expressed.11. The expression vector pCAMBIA1302-MsANR-GFP was transformed into Arabidopsis,expecting to gain MsANR overexpression plant. After been selected by antibiotics, the regeneratedplants were obtained. PCR analysis showed they were all positive lines. The tannins content oftransgenic plants were significantly higher than that of non-trasgenic plants. These resultssuggested that MsANR gene play an important role in tannins synthetic way.