The Extraction Of Genomic DNA And The Detection Of Antifreeze Protein Gene Mpafp149 In Transgenic Plant Cotton

The cold damadge of low temperature is one of the abiotic stresses limiting the geographical distribution of crops and periodically account for significant losses in plant productivity. So, it is important to improve the cold resistance of crops for agriculture. With the advances in the research of molecular mechanism in plant cold resistance, the studies of genetic engineering for improving cold resistance in plants have also been made quite progress. To ensure that the plant can be protected from frost damage, ice inside and outside of cells should be avoided. Insect antifreeze protein (AFP) can reduce the freezing point of a solution in a noncolligative manner without affecting the melting point.As a result, AFP functions to protect organsims avoiding the damage of freezeing.The insect antifreeze gene Mpafp149 from Microdera puntipermis dzungarica,was cloned into plant expression vector pCAMBIA1302 and pBI121 for cotton genetic transformation by Pollen-tube Pathway method. The seeds were bred up in laboratory. The positive transgenic cotton were determined first by Hygromycin and Kanamycin resistant, then screened by PCR and RT-PCR. The transgenic plant cotton and wild-type cotton were treated at low-temperature 2℃for 3 days for testing the cold resistant ability. Compared with wild-type, the transgenic cotton showed better phenotype and kept the lower malondialdehyde (MDA) content and electricity leakage (EI) in the leaves. The test results indicated that the antifreeze protein gene Mpafp149 can confer the freezing tolerance to the transgenic cotton.The high quality and sufficient yield of cotton genomic DNA is the foundation for cotton molecular biological research. Michaels methods, improved SDS(Sodium Dodecyl Sulfate), and improved CTAB(Cetyltrimethyl Ammonium Bromide)methods were used to extract the cotton genomic DNA from the cotton tenderleaves and old leaves.The DNA extraction effect was compared by agarose gel electrophoresis for the completeness of the genomic DNA , by ultraviolet spectrophotometry for the ratio of OD260/OD280, and OD260/OD230, and by PCR for the amplification of especial gene.The results showed that the extraction effect of Michaels's method is much better than the improved SDS method and the improved CTAB method to genomic DNA. The genomic DNA from the tender leaves tend to keep higher quality. Sample leaves preserved at different temperatures appeared to have different DNA quality, suggesting that temperature influence the DNA stability of the preserved samples in it. The genome DNA from leaves conserved in unsealed plastic bag seems to be much better than that from the samples preserved with traditional ways both in quality and yeild.