The Studies On Gene Cloning Of Ammopiptanthus Mongolicus AmDHN,AmERF Gene And Genetic Transformation To Sugar Beet

Sugar beet is one of important sugar crops in the world and the second largest sugar crop in China. It plays an important role in the development of sugar beet industry. A cDNA library of strong xerophyte Ammopiptanthus has been known. We analyzed its EST sequence and compared GENBANK DNA database, and then spliced ESTs of 34 DHN genes and 15 ERF genes. A full-length cDNA sequences was got. AmDHN gene and AmERF transcription factor of Ammopiptanthus were cloned using a specific primer. And then, AmDHN gene plant expression vector, AmDHN gene, AmERF transcription factor and PPT screening for resistance gene divalent expression vector of the plant of Ammopiptanthus were built in turn. Sugar beet regeneration tissue culture system was established. Ammopiptanthus AmDHN gene was transferred into sugar beet using Agrobacterium method. Functions of transgenic sugar beet were verified. The results are as follows:1. AmDHN full length with 1106 bp was successfully cloned by PCR amplification technique. DNAman analysis indicated open read frame was 552 bp, coded 183 aa, theory MW was 18407.4 and pI was 6.22.2. Transcriptional factor AmERF of Ammopiptanthus was cloned successfully. Open read frame was 633 bp, coded 211 aa, theory MW was 23216.0 and pIwas 8.20 by DNAm- an analysis. Nucleotide sequence of transcriptional factor AmERF of Ammopiptanthus was compared by Blastx procedure, and 56 homologous sequences were founded. Consistency with Arabidopsis thaliana, Oryza sativa (japonica cultivar-group), Capsicum annuum, Medicago truncatula was 83%,72%,74% and 61% respectively.3. Mutation of base sequence CCATGG of DHN gene to CTATGG was completed successfully using PCR site-directed mutagenesis technique.4. AmDHN gene plant expression vector (pCAMBIA3301-DHN), DHN gene and divalent expression vector of AmERF transcription factor (pCAMBIA3301-DHN-AmERF) of Ammopiptanthus were built.5. A sugar beet genetic transformation and regeneration system was built, in which explants were petiole of sugar beet. 4 genotypes sugar beets (N98122,N9849,HBB-1and NeiTianDan1) which have higher ability to induce multiple shoots were screened, compan- ied with 2 differentiation inducing medium (MS+NAA0.5mg/L+6-BA1.5mg/L;MS+NAA 0.5mg/L+KT3.0mg/L) and 1 rooting inducing medium (MS+NAA2.0mg/L).6. Ammopiptanthus AmDHN gene was transferred into sugar beet integrated Agrobacterium tumefaciens with vacuum, and then got 107 transgenic plants which have stronger PPT resistance. Under drought stress, transgenic sugar beet plant has stronger resistance than non-transgenic ones.