Research On Salt-tolerant Genes ScHAL1 And ZmHKT Genetic Transformation In Soybean

Soybeans,one of the most important crops food and oil crops in China,Soybean products are a great source of nutrition in our national diet.Due to the threat of soil salinization in the area of agricultural crops in China,the crops can't grow properly and the yield loss is serious.At present,how to resist the threat of salinization of crops and improve the salt-telerance of plants is the primary task for plant breeding.In conventional breeding Due to shortage of salt-tolerant germplasm it isdifficult toimprove the salt-tolerant of soybean.With rapidly development transgentic technology,combing with the molecular biology,it is possible to improve the salt-tolerant of planted therefore enrich the salt-tolerant genetic resources.Combing conventional breeding with genetic engineering technology with is an effective technological system for salt-tolerantce improvement in soybean breeding.The main results are as follows:In order to improve the salt-tolerance of soybean,the salt-tolerance gene of ScHAL1 and ZmHKT was coloned and the plant expression vector pCAMBIA3301-HAL1 and pCAMBIA3300-HKT were constructed.The gene were tansformed into the soybean variety Bert mediated with agrobacterium Transgenic plants were identified by Bar Quick Stick,PCR,RT-PCR,Southern blot.In this study,total 5200 cotyledonary nodes wee infected.45 and 40 transgenic plants for ScHAL1 and ZmHKT respectively were determined by PCR and Southern blot.9 and 7 transgenic plants with salt-tolerance,identified with 1.5%NaCl for gene ScHAL1 and ZmHKT respectively were obtained.1.Construction of plant expression vector.Designed primers with Best II and Noc I enzyme restriction sites at both ends of the HAL1 gene.The gene was amplified by PCR.pCAMBIA3301 plasmids were restricted by Best IIand Noc I enzyme.The HAL1 fragment was ligated to pCAMBIA3301 with T4 DNA ligase and plant expression vector pCAMBIA3301-HAL1 was contructed.Designed primers with BamHI and SacI enzyme restriction sites at both ends of the HKT gene.The gene was amplified by PCR.pCAMBIA3300 plasmids were restricted by Bam Iand Sac I enzyme.The HKTfragment was ligated to pCAMBIA3300 with T4 DNA ligase and plant expression vector pCAMBIA3300-HKTwas contructed.pCAMBIA3301-HAL1 and pCAMBIA3300-HKTvector was transferred into Agrobacterium EHA105 by freeze-thawing method2.Genetic transformation of soybean.In this study,ScHAL1 and ZmHKT were imported into soybean by agrobacterium mediated method.In this experiment,This experiment inoculation soybean number is 5200,the induction rate of buds was 91%,The elongation rate was 27%,the rooting rate was 80%,and the conversion rate was 2.5%.The Positive transgenic soybeans gains genes of HAL1 45 strains,genes of HKT 40 strains.3.Molecular detection of transgenic plants.Design primer of ScHAL1 gene and ZmHKT gene and then transformation obtained soybean plants were identified by PCR,RT-PCR,Southern blot biochemical molecules and Bar protein test,To verify that the ScHAL1 and ZmHKT genes were successfully expressed in soybeans and integrated into the soybean genome by the genetic transformation method.4.Salt tolerance identification of bud and seedling stage.Bud and seedling stage in the positive transgenic soybean plants salt resistance identification,Comparison of tolerance between positive and negative plants for saline irrigation(1.5%NaCl).The plant has a good tolerance for salt-tolerant genes.