Study On Transformation Of HAL1 Gene Related To Salt-Tolerance In Tobacoo And Wheat

Salinization of soil is one of the major abiotic-stresses that influence agricultural production and environment.With the increasing of world's population and the decreasing of field,how to develop and utilize salinized-soil becomes an urgent problem in agriculture production and ecological environmental protection. It provides a new way to increase food production for the need of population growing and maintain sustainable development of agrieulture;we can use the correlation salt tolerance gene about the transformation technique to improve the salt tolerance of the cultivar and to discover and utilize salinized-soil. HAL1 gene is a key factor of salt tolerance,it's expression can regulate Na+/K+ balance.Thought HAL1 gene is not a kind of transfererd protein,it can keep lower Na+/K+ with other gene and transfererd system.So HAL1 gene have great potentiality in the gene engineering of salt tolance.HAL1 gene was transformed into tobacco by transfection of Agrobacterium tumefaciens and into wheat by particle Bombardment ,the factors effecting on frequency of gene transfer were examined was analysised.The main results are as follows:1.The agrobacterium tumefaciens transformation system was be superior : the study did some research on the level of Kan-resistent with the Qin 96 and Qin 97,and provided some efficiency gists for the transgene plants choices .The experiment of Kan-concentration ladder screening show that scanning the explants with 50 mg / L kanamycin (Kan)and degerming with 800 mg / L carboxymethyl benzylpenicillin (Cb), then it got the ideal result. The optimization conditions of Agrobacterium-mediated genetic transformation were: dipping the explants which were not been pre-training (pre-culture 0 d)in the diluted 150 times Agrobacterium bacilli (OD600 0.5)for 1 min, then transferred the explants to the medium MS1. After training 48 h, washing the explants with sterilized distilled water and then dipping them in volume scores 2% NaClO for 15 min ,rinsing with sterilized distilled water four to five times, and dipping the explants in 800 mg / L Cb + quality scores of 0.1 % mannitol about 12 h.And then put the tobacco explants on the MS1 medium to induce the buds,use 50 mg /L kanamycin to screening the resistent bud of the Qin 96 and Qin 97 after agrobacterium tumefaciens transformation;Then the resistent buds breeded their roots on the MS3 medium .2.Got the HAL1 transgene plant of tobacco: by the agrobacterium tumefaciens-mediated and the Kan-resistent scanning, we totally got 69 resistent plants. The molecule test show that from the PCR amplification we can see six transformants obtained the special bands at 879bp. HAL1 gene had bene integrated into the genome of part of tobacco plants.3.The particle Bombardment transformation system was be superior : In the system of the transformation of the callus of wheat immature embryos with the particle Bombardment,the best plasmid DNA concentration is 1.0μg per gun;the acceptor was bombed 1 time ; an efficient acceptor system for gene transformation was established in callus of wheat immature embryos by improving the condition of osmotic treatment before bombardment.In this experiment,the acceptors were from 2 wheat cultivars including Xiaoyan 22 and 213.Particle bombardment was carried out using pROKⅡcontaining the NPTⅡgene and HAL1 gene.The factors affecting the Biolistic transformation efficiency like physiological state of acceptor cells and time of osmotic treatment with different osmosis concentration were studies.The result showed that osmotic treatment can enhance the differentiation frequency of bombarded callus.The callus before embryo switching on was optional.Sucrose as osmosis has a less inactive effect than mannitol before bombardment. The callus treated with 0.4mol/L sucrose for 6 hours before bombardment but 18 hours after bombardment could significantly achieved optional transformation efficiency. The antibiotic,which was normally used in plant transformation for the selection gene NPTⅡwas tested for the optional concentrations in wheat transformation. Kanamycin of 100mg/L used for the selection of NPTⅡwas available.The medium of MS with 1.0mg/L 2,4-D,0.5 mg/L KT and 0.01 mg/L NAA has tested to be an optional cultural condition for regeneration of the callus after bombardment and in selection.4.Got the Kan-resistent rebirth wheat : by the particle Bombardment and the Kan-resistent scanning ,we totally got 26 resistent transplants.