Introduction Of Salt-tolerant Gene Into Brassica Napus And Tobacco

Salt stress is the major factor that limits and decreases the output of crops in many parts of the world, particularly for the irrigated land. Improve the salt tolerance of plants by transform salt-related genes into will become an Effective way to solve this problem. The laboratory tested experiments of introduction of Salt-tolerant Gene into BrassicaNapus of and tobacco Mediated by Cre/lox system during 2007-2009.The main results were as follows:1.In this series of experiments,ThNHX1,a high salinity related gene coding from Thellungiella halophila , was isolated from Thellungiella halophila genome by PCR method and replace the GFP gene harboring pX6-GFP vector,forming the recombinant vector pX6-ThNHX1 with the function of Cre/loxP-mediated site-specific DNA recombination.2.In vitro high frequency regeneration system established in the use of Agrobacterium-mediated carrier of the new vector pX6-ThNHX into the process of oilseed rape varieties.The most suitable optimum medium for the differentiation of 5-7d of vaccine cotyledon explants of yangyou 6th and shilifeng was MS+6-BA 5mg/L +NAA 0.2 mg/L +AgNO3 5mg/L, In this mediu, the rate of differentiation was highest of yangyou 6th rate of highest differentiation was 57.43% and shilifeng rate of highest differentiation was 61.54%。.The most suitable optimum medium for the differentiation of 5-7d of hypocotyl vaccine was MS+6-BA 4mg/L +ZT 2mg/L+AgNO3 5mg/L, In this mediu, the rate of differentiation was highest of yangyou 6th rate of highest differentiation was 34.72% and shilifeng rate of highest differentiation was 40.54%。3. Appropriate Kana will improve the rate of Genetic transformation and get more health positive plants.In the experiments optimum concentration was elected by comparison of different concentrations of the shoot growth. when the concentration of was 5mg/L,66.67%Kanamycin resistant plantlets could be obtained ,when 10mg/L obtained 13.33%,When the concentration was higher than 20mg/L, obtained none. More plantlets abtained after cultrue in Kana 5mg/L,but most of them dead ,PCR identification was false positive,if cultrue in 10mg/L Kana plantlets was few.So we culture plantlets first in 5mg/L Kana and then 10mg/L,by this way will get more plantlets4. Kanamycin resistant plantlets after using Agrobacterium-mediated carrier of the new vector pX6-ThNHX into cotyledon of rape .Cotyledon petioles which were precultured for 2 days with 1mg/L2,4-D and then culture in MS+6-BA 4mg/L +ZT 2mg/L+AgNO3 5mg/L were considered as the best physicaland transformation condition..When explants immerged in OD600 0.3～0.4 bacterium culture for 5min,first culture in 5mg/L Kana then10mg/L,5 yangyou 6th and 2 shilifeng Kanamycin resistant plantlets could be obtained.By PCR identification , the preliminary results showed that NHX gene had integrated into genomic DNA of oilseed rape.5 Using Agrobacterium-mediated method to import the new vector constructed pX6-ThNHX1 into tobacco plants.3 Kanamycin resistant plantlets were obtained after PCR identification.Fragments were eliminated from the transformants under the inducement of 5μM/Lβ-estradiol to produce marker-free transgenic plants.PCR identification unveiled that ThNHX1 gene already integrated into the cole and tabacoo genome.6.It hoped these results can promote the genetic transformation of other important crops in the future.