Transformation Of Salt-tolerance Gene OsMYB56 Into Rice

Rice is one of the most important food crops and widely distributed in the world. Rice originated in China, and has been cultivated in China for a long history. Due to soil salinization increased and tillage area decreased year by year, the crop growth and yield were influenced seriously. Thus, increasing the salt tolerance of rice is one of the most important tasks in plant breeding. With rapid development of molecular biology and modern gene engineering technology, genetic traits of plants can be improved by transgenic technology, the barriers of reproductive isolation between species can be breaken, the genetic resources have been enriched, the shortage of traditional breeding resources can be supplemented, the low breeding efficiency and accuracy can be improved and the further crop breeding work will be developed. Therefore, new varieties breeding of salt resistant transgenic rice has become important research work of rice breeding by genic engineering.The main results of this study are as follows:1.Cloning and bioinformatics analysis of MYB transcription factor gene Os MYB56 of Rice. Primers were designed according to Os MYB56(Gen Bank accession number: NM001052096.1) gene sequence and Os MYB56 gene was cloned by RT-PCR method. The biological information softwares were applicated to analysis Os MYB56 gene nucleic acid sequence and amino acid sequence, physical and chemical properties, hydrophobicity / hydrophilicity, cross membrane domain, signal peptide, subcellular localization, protein domain, secondary structure and tertiary structure, and a phylogenetic tree was constructed. The results show that the length of the c DNA is 666 BP, the encoded protein cotains 221 amino acids, isoelectric point is 6.13. It is a hydrophilic protein with no transmembrane helices and signal peptide. The protein with two SANT domains was localized in the nucleus, and is belonged to the R2R3-MYB type, containing 31.67% alpha helix, 8.6% beta sheet, 50.23% random coil and 9.5% extended strand. It is a loose globular protein, containing 6 alpha helix, without disulfide bonds. The similarity of Os MYB56 transcription factor of rice and At MYBC1 transcription factor of Arabidopsis is the highest in evolution homology.2. Construction of Plant expression vector. Designed primers with Bam HI and Sac I enzyme restriction sites at both ends of the Os MYB56 gene. The gene was amplified by PCR and ligated to p EASY-Blunt cloning vector. After sequencing both p EASY-Blunt-Os MYB56 plasmids extracted and p CAMBIA3300 plasmids were restricted by Bam H I and Sac I enzyme. The Os MYB56 fragment was ligated to p CAMBIA3300 with T4 DNA ligase and plant expression vector p CAMBIA3300-Os MYB56 was contructed. p CAMBIA3300-Os MYB56 vector was transferred into Agrobacterium EHA105 by freeze-thawing method.3. The genetic transformation of rice. Os MYB56 gene was transferred into rice mediated with Agrobacterium. In this experiment, 573 seeds were inoculated and 492 seeds produced callus, the callus induction rate was about 86%. One thousand culluses were infected, 63 PPT resistant plants were obtained and plant regeneration rate was 6.3%. Fifty five positive plants were obtained and transformation efficiency was 5.2%.4. Molecular idification of transgenic plants. Transgenic plants were confirmed by PCR, RT-PCR, Southern blot and Liberty Link quick stick. The results show that Os MYB56 gene has been sucessfully integrated into the genome of rice.