| Caragana korshinskii Kom, a widely grown cultivar in Inner Mongolia, is cold, drought, and heat-resistant, salt and alkali tolerance. It has significant economic value for it’s an sand fixation species, a good forage for animals and so on. The previous research of Caragana korshinskii Kom are mostly focus on physiology and ecology. However, the study of quality improvement and molecular is increasingly attractive. Therefore, plant regeneration and genetic transformation become the limiting factor for Caragana korshinskii Kom’s research.Lignin is one of the major structural component in the biosphere, being second only to cellulose in abundance. It is essential for mechanical support, water transport and pathogen defense in plant, and has important influence on humanity’s life and production. Cinnamyl alcohol dehydrogenase (CAD) catalyse the reduction of the cinnamyl aldehydes to the corresponding cinnamyl alcohols which play an important role in three lignin polymer biosynthesis. So, it’s necessary to study the characterization of lignin biosynthesis gene CAD.The major object of this study is to induce multiple shoots using cotyledonary node explants and get regenerate plants of Caragana korshinskii Kom. At present, we have established the regeneration system of Caragana korshinskii Kom initially which is a basic research for others. The degenerate primers were designed according to the conserved domain of the well-known CADs from other plant species, and the corresponding intermediate fragments were amplified from the cDNA and gDNA of Caragana korshinskii Kom firstly by RT-PCR and RACE technique. The amplified gene from cDNA was named CkCAD (accession number HQ829861in GenBank).1. The best disinfection time for Caragana korshinskii Kom seeds is75%alcohol for10min,10%sodium hypochlorite for40min. In this way, the seedling grow healthy with low pollution.2. The callus, induced from cotyledon and hypocotyls cultured on MS medium supplement with0.5mg/L BA (w/v)and2mg/L2,4-D, grow well on MS proliferation culture medium containing0.5mg/L BA and2mg/L2,4-D. Unfortunately, the callus fail to differentiation. 3. We made a test program of orthogonal design on induce multiple shoots from cotyledonary node to choose the best culture condition. The number of shoots was highest when the6-day-old explant cultured on B5medium supplemented with2.0mg/L BA which concentration is determined by an optimized test. In average, the number of multiple shoots was2,8shoots maximum, and3-5shoots in common.4. We made a test program of orthogonal design on rooting to choose the best culture condition. On the1/8MS medium containing0.5mg/L IBA and0.5mg/L NAA, the maximum rooting ratio is66.67%, and the number of roots is2.91per an explant.5. Rooted plantlets were acclimatized in water for hardening, then transferred to sterilized potted soil and grown under greenhouse conditions. The survival ratio is93.22%.6. The full-length cDNA of the CkCAD is1,287bp with an open reading frame of1,074bp and95bp5’UTR,118bp3’UTR, polyA(11). The deduced protein has357amino acids with ATG as the initiation codon and TGA as the terminator codon. The predicted physico-chemical property of the deduced CkCAD protein reveals that its molecular weight is about38.75kD with the pI at about5.44. The predicted secondary structure of CkCAD is making up with alpha helix, beta sheets and random coils. The tertiary structure also been predicted. Phylogenetic analysis revealed that the CkCAD shared the closest homology with CAD2from Medicago sativa and CAD from Leucaena leucocephala, the two legume species.7. The full-length gDNA of the CkCAD is2,016bp, contains five exons and four introns with GT as the left-boundary and AG as the right-boundary, which is obey the rule of eukaryote.8. We constructed the overexpression binary vector35S::CAD, and transform the wild type Arabidopsis thaliana. Finally, we get the homozygous CkCAD overexpression transgenic plants. |
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