Construction Of Vector PBI121-RC7 And Expression Of Transformed Poplar

In this study,took the chitinase RC7 gene constructed into pBI121-RC7 vector and took the vector into Agrobacterium by freeze-thaw method,while establishing vitro regeneration system of P.deltoides’XL-90’.Combined with Agrobacterium-mediated method.Then,took XL-90 as receptor material and transform the gene RC7, make the transgenic plants obtain the ability to against the pathogen.By inducing the differentiation of axillary buds, took the obtained blade as material for callus culture, adventitious bud growth and rooting cultures.By screening,optimal medium obtained as follows:A)Using MS+6-BA1.0mg·L-1+TDZ0.005 mg·L-1+sucrose30g·L-1+8g·L-1 agar, induced XL-90 stem segments with axillary buds;B)Using 1/2MS+6-BA1.0mg·L-1+NAA 0.2mg·L-1+TDZ 0.01mg·L-1+ sucrose30g·L-1+8g·L-1 agar,for XL-90 callus;C)Using MS+6-BA 0.5mg·L-1+ NAA0.2mg·L-1+sucrose30g·L-1+agar 8g·L-1, forXL-90 adventitious buds growth;D)Using 1/2MS+NAA0.3mg·L-1+sucrose 30g·L-1agar 8g·L-1, induced roots.On the base of establishment of regeneration system of XL-90,studied the optimal conditions for its genetic transformation:The Kan concentration of 20mg·L-1 was the tolerant concentration for adventitious buds;Shaking agrobacterium tumefaciens LBA4404 at 28℃,170rpm, in dark,19h can reach the logarithmic growth phase;Infected in OD600=0.6 for 15min, and co-cultured for 3 days, the genetic transformation efficiency was the highest.By using agrobacterium-mediated transformation,obtained 101 Kan-resitant adventious buds.After selecting at different levels,26 survived.After PCR molecular detection,got 3 positive plants of transgenic RC7.