The Construction Of BIBAC Vector For HMW-DNA Of Salt-tolerant Phragmites Australias Trin And Rice Genetic Transformation

The formation of saline-alkali land mainly comes from the changes of man-made economic activities and natural environment.At present,the global saline-alkali land area has already reached 1 billion hectares,which has reduced the arable land area in the world.In the highly saline crop areas,many crops can not grow normally or even die.Eventually,the yield,benefit and quality of their production will be seriously affected,which hinders the sustainable development of agriculture.There are many varieties of salt-tolerant plants in China.The introduction of Salt-tolerant Genes from salt-tolerant plants into other crops through biotechnology can effectively improve the salt tolerance of crops.The wild salt-tolerant reed(Phragmites Australis trin)is mainly distributed in the northwestern part of China.It has strong salt tolerance and belongs to gramineous plants.It is a high-quality donor that provides salt-tolerant functional genes.The salt-tolerant exogenous functional genes provided by salt-tolerant reeds play an extremely important role in improving the salt tolerance of gramineous crops such as rice.Binary bacterial artificial chromosome(BIBAC),which has been widely used in plant genetic transformation,is different from ordinary binary vectors.The foreign DNA fragments that can be loaded into the common binary vector will not exceed 20 KB,and only a few functional genes can be carried.The binary bacterial artificial chromosome(BIBAC)vector can shuttle between E.coli and Agrobacterium.It can not only directly transfer large multi-gene DNA into plants through Agrobacterium-mediated transformation,but also construct large-segment DNA libraries.In this study,we constructed a large fragment DNA-BIBAC vector of reed genome and transformed rice to obtain innovative salt-tolerant rice germplasm,proposed a large fragment transformation technology system,and laid the foundation for salt-tolerant gene cloning of reed.The results are as follows:1.By preparing genomic DNA of Mb-grade salt-tolerant reed,the large fragments of reed DNA were separated into 50-100 Kb,100-150 Kb and 100-350 Kb by pulsed electrophoresis after digestion,and linked with BIBAC-S recovered by digestion.The large fragments of DNA-BIBAC vector with different sizes of salt-tolerant reed were successfully constructed and then transduced.The positive recombinant was screened out by blue and white spot in the competent cells of E.coli.The stable inheritance of large fragments in the positive recombinant was detected by pulsed field electrophoresis.Finally,the large fragment BIBAC vector was constructed by cloning and preservation.2.Large fragments of DNA-BIBAC with different sizes inserted into salt-tolerant reeds were transformed into Agrobacterium tumefaciens EHA105.Rice genetic transformation was carried out by Agrobacterium tumefaciens-mediated transformation using mature embryo callus from Zhonghua 11 of Japonica rice as receptor.Agrobacterium tumefaciens EHA105 mediated the infection of callus,through hygromycin screening,differentiation,rooting,obtained pseudo-transgenic rice plants.The genomic DNA of pseudo-transgenic rice plants was detected by PCR,and 23 transgenic positive plants were obtained.The results showed that BIBAC vector with different large fragments mediated by Agrobacterium tumefaciens had been transformed into Zhonghua 11 rice,which laid a technical foundation for screening salt tolerant mutants,creating new salt tolerant rice varieties and improving salt tolerance of rice.