Introduction Of Salt-tolerant Gene NHX Into Brassica Napus Of Xinjiang Mediated By Agrobacterium Tumefaciens

Xinjiang has the biggest poor soil area and salted region in China. 11million hectares of salted area occupy 1/3 of whole salted soil in the china and 6.6% of land area of Xinjiang. Currently there has 1/3 secondary salted soil of whole cultivation land in Xinjiang,the slight and middle degree of salted land would reduce output of 10%～50% and the heavy salted oil almost harvest nothing. This is the major cause of low yield and severe block of the economic development of Xinjiang. Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the salt tolerance of crops,keeping steady-state growth and increasing the output in the saline environment. NHX1,a tonoplast Na+/H+ antiporter energized by the ?pH across the tonoplast,facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance,an active antiporter would function to sequester Na+ into the vacuole, which results in avoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel,vacuolar In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis. Overexpressing AtNHX1 can accumulate the large numbers of antiporter in the transgenic Arabadopsis, oilseed rape and tomato, at the same time improve their salt tolerance. Rape widely distributed in China,America and Canada as the major oil crop. It is an important economic oil and vegetable crop,so developing transgenic varieties of rape has the potential value. So far,31% of high quality rape is transgenic variety in the world. Because many varieties are sensitive in the tissue and cell culture, and other advantages such as simple operation,low cost,high transgenic efficiency, moreover, foreign gene can be integrated into the genomic DNA of plant stably and inherit to the offsprings by Mendel's law, made it much suitable as the plant material for genetic transformation. Suaeda is a kind of important halophyte. Mediated by Agrobacterium tumefacines method, salt-tolerant gene NHX from Suaeda was transformed into Brassica napus,The goal is to develop salt-resistant variety which retains original good characters and increases output in salted soil, and hope to do something for promotion the economic development of Xinjiang. Tissue culture of plant is the basis of genetic transformation mediated by Agrobacterium tumefacines, high efficiency of regeneration would benefit for transformation. We established the regeneration system of 912,9806 and 1khp11 spring rape variety by adventitious shoot way with cotyledon petiole and hypocotyl. The regeneration rate of cotyledon petiole and hypocotyl can be obviously increased when precultured in medium supply with 1.0 mg/L 2 , 4 –D,which showed that 2 , 4 –D had strong inducing function. During shoot inducing of cotyledon petiole in different varieties,the concentrations of 6-BA and NAA needed vary in a small range. The experiment indicated that appropriate NAA can improve the shoot induction rate, high concentration of NAA (0.1mg/L) induced abundant roots,although NAA was seldom supplied in media in previous reports, and it showed obviously different between genotypes. When induction of shoot with hypocotyl, the concentrations of 6-BA and NAA varied in a wide range between different varieties. Increasing agar concentration was able to reduce the vitrification degree by comparing of various factors. Because the transformation efficiency of cotyledon petiole was higher than hypocotyl in the transformation according to previous report, and in our study, 1khp11 had stable seed germination and high regeneration rate of cotyledon petiole by comparing with different varieties,this variety was chosen as the most suitable material for transformation. Many factors which included bacterium concentration,AS supplement or not,preculture time of explant,infection time,co-cultured time, AgNO3's concentration and the suitable kan concentration in the different growth phase all affected transformation efficiency significantly. OD600 ranging from 0.24 to 1.96 was considered as the logarithm growth phase,the bacterium grew very well within this range and most suitable for transformation. In our experiments,bacterium culture during logarithm growth phase was diluted to the range of needed concentrations, then transformed and got the different efficiency of green kan–resistant shoot, which showed that the concentration of diluted bacterium culture,and physical growth condition were very important to the transformation rate. Cotyledon petioles of oilseed variety-1khp11 were transformed with strain ofAgrobacterium tumefaciens EHA105,which harboring Na+/H+ antiporter gene of Suaeda heteroptera. So far we had got some kanamycin resistant plantlets. In the research, major factors of transformation were discussed. Cotyledon petioles which were precultured for 3 days with 1mg/L2,4-D were considered as the best physical and transformation condition. Concentration of bacterium culture was another important factor. When explants immerged in OD600 0.3～0.4 bacterium culture for 7min, 14.67% kanamycin resistant plantlets could be obtained. By PCR, Dot blot and PCR-Southern identification, the preliminary results showed that NHX gene had integrated into genomic DNA of oilseed rape.