Sequence Analysis And Transformation System Of NBS-LRR Type Resistance Genes In Sugar Beet (Beta Vulgaris L.)

Disease resistance of crops is an important topic in scientific field. In this paper, we (1) analyzed NBS-LRR-type resistance gene analogues in the Sugarbeet (Beta vulgarisL.) Genome; (2) established a tissue culture or regeneration system and (3) an effective transformation system by Agrobacterium for sugar beet. The main results follow as:1. The analysis of TIR-type resistance gene analogues in the sugar beet genomeThe majority of known plant resistance genes encode proteins with conserved nucleotide binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic and sequence alignment analysis revealed that NBS-LRR type resistance genes of sugar beet belong to non-TIR-type NBS-LRR and all other clones are this subfamily. PCR amplifications based on the primers specific for TIR were researched in EST database of sugar beet that contains 16,000 sequences. The results showed that we could not find the representative TIR NBS-LRR-containing resistance genes. This indicated that TIR NBS-LRR-containing resistance genes might miss in sugar beet genome. This instance was the fist reported in dicotyledon and only covered in monocotyledon (such as Oryza sative L.).2. Establishment of regeneration system of sugar beetIn order to establish an effective regeneration system of sugarbeet and research key factors, we investigated the concentration of NAA, BAP,TIBA and sucrose, the time of pre-culture and the type of explant forregeneration of sugar beet. The results showed that the regenerationfrequency reached 76.7% by medium with MS+MS+NAA0. 4mg/L+BAPl. Omg/L+TIBA0. 4mg/L+3%Scurose+0. 8%agar, the bestexplant is petioles, and the most condign time is three weeks. Therhizogenesis mediums are MS+KT0.1mg/L+NAAO. 8mg/L and MS+NAA1. Omg/L, andthe rhizogenesis frequency reached 100% in those mediums. The livabilityof regeneration plantlet is 100%. This formula is fit for other cultivarsin Beta vulgar is L.3 Establishment of an effective transformation system by Agrobacteriumof sugarbeet varietyTo effective eliminate Agrobactiu/nand screen of transformated cells after co-cultivation, sensitivity of cultivar Shuang-eight of sugar beet cultured in vitro to four antibiotics was tested. Results showed that petiole was sensitive to knamycin and the suitable concentration is 150mg/L. Among carbenicillin, ampicillin and cefotaxime used for elimination of hgrobacterium, carbenicillin should be preferable based on their effects on shoot regeneration.In order to establish an effective transformation system of sugarbeet, several key factors (the concentration of Agrobacterium, pre-culture time, infective time, co-culture time and polarization temperature) on in the process of Agrobacten'urn-mediated transformation were optimized based on the frequency of transient expression of gus gene. The highest frequency was observed when explants that had been pre-cultivated two days were immersed in bacterium suspension with OD600 value about 1. 0 for 15 minutes followed by co-cultivated for about three days, and then they were cultured in 25-30T.