| Increasingly severe soil salinization or secondary salinization can affect the normal growth and development of crops. According to the latest incomplete statistics of the UNESCO and FAO, there were about 954.3 million hectares of saline-alkali soil in the world and China accounted for about 99.13 million hectares .Most of the crops cannot grow in heavy salinized farming areas, and ultimately seriously affect the crop production yield, quality and efficiency.Rice is China's largest food crops since ancient times, and rice-based food accounted for about 65% of the population. Rice is the bio-improved pioneer crops for saline-alkali soil, so the cultivation of salt-tolerant rice is of great significance.There are about 500 kinds of halophytes in China. It is one of the very effective ways to improve the salt-tolerance of crops by transferring the salt-tolerant genes of these halophytes into crops by biotechnology. A wild salt-tolerant Phragmites australis Trin.,which grows on the northwest salt-cap, belonging to the gramineous plants, has a strong salt-tolerance. According to study and utilize its functional genes related to salt-tolerance,which can provide important exogenous gene for improving the salt-tolerance of gramineous crops such as rice.The ordinary binary vector which has been widely used in plant genetic transformation, can only transfer exogenous DNA fragments within 20 Kb. so this type of carrier can carry a few functional genes each time as well. While the binary bacterial artificial chromosome (BIBAC) vector can achieve multi-gene or transfer of large DNA fragment as large as 150Kb. BIBAC vectors can be used to clone large DNA fragment to construct gene library, and to direct transformation of plants by Agrobacterium-mediated as well. The dual-function of the BIBAC vector is important for introducing those unknown genomic DNA into the crop and cloning salt-tolerant genes in the future.Inserting high molecular weight DNA into BIBAC vector is the key to construct the gene library, meanwhile, obtaining high-quality high molecular weight nuclear DNA(HMW-DNA) is the most essential prerequisite for large DNA inserting into BIBAC vector.Conventional SDS method and CTAB method, even nucleus lysis method of extracting the genomic DNA fragment, cannot meet the requirements of BIBAC vector construction because of the small size. Zhang's method has become a basic method for preparing HMW-DNA since Zhang et al. (1995) proposed the preparation of high molecular weight nuclear DNA by embedding nuclei with low melting agarose. This method requires the use of etiolated seedling as the material for the preparation of HMW-DNA. However the wild salt-tolerant Phragmites australis Trin. materials we use,are collected from the field,the mature Phragmites australis Trin. leaves are rich in polyphenols, polysaccharides, tannins,lipids and other secondary metabolites, especially polyphenols, which are tightly bonded with DNA to form brown colloidal complexes, as chloroplast, mitochondrial and other organelles DNA contamination will affect the quality of HMW-DNA as well.Based on the method of Zhang et al. (1995), the preparation method of HMW-DNA suitable for wild Phragmites australis Trin. was proposed by using mature leaf of wild salt-tolerant Phragmites australis Trin. as material, and separated by pulsed field electrophoresis The results showed that the improved nucleus embedding plug method in extracting HMW-DNA could overcome the adverse effect of non-etiolated seedling material that is rich in polyphenol polysaccharide and reduce the cost of preparation of nuclear HMW-DNA and improve effectiveness of the connection of succeeding experiment.The BIBAC vector was successfully constructed by using the large fragment of Phragmites australis Trin., which laid the foundation of the construction of the BIBAC library of salt-tolerant Phragmites australis Trin. and the genetic transformation of DNA fragment as well. |
PDF Full Text Request