高品质花生DNA提取:CTAB法改良及多维度品质评价OA

Because peanut leaves are rich in polyphenols,polysaccharides and other secondary metabolites,conven-tional methods or commercial kits cannot extract high-quality DNA from peanut leaves,which seriously hinders the process of peanut molecular breeding.In this study,the traditional CTAB method was modified and reconstituted according to the roles played by each component of the CTAB lysis buffer in the DNA extraction process,as well as the phase separation characteristics of DNA and substances such as polyphenols and polysaccharides in cells in space.Firstly,a pretreatment solution composed of PVP and high concentration EDTA was used to pre-treat peanut tissue to block phenolic oxidation from the source and remove excessive polyphenols,polysaccharides and other impurities,and effectively reduced the viscosity of peanut leaf tissues.Then,high concentration of CTAB(3×)lysate was used to completely remove impurities such as proteins and neutral polysaccharides.The results showed that,compared with the traditional CTAB method,the purity of peanut leaves genomic DNA extracted by the modified CTAB method was extremely higher,with an OD260/OD280 ratio of 1.8-1.9 and an OD260/OD230 ratio greater than 2.0,the extraction results have good stability at the same time.The DNA integrity is high,large DNA se-quences of about 4 kb can be effectively amplified.The stability of DNA was improved,and there was no obvious sign of degradation when stored at room temperature(25℃)for two months.In addition,the DNA extraction efficiency of the modified CTAB method was greatly improved,with 3 μg total DNA obtained from 10 mg leaves,and high-quality DNA could be efficiently extracted even using senescent leaves.These studies show that the modified CTAB method can im-prove the purity,integrity,stability and extraction efficiency of peanut genomic DNA,and is suitable for molecular breeding of peanut,especially for genetic identification of rare materials.Simultaneously,it provides an important refer-ence for the extraction of DNA from samples rich in secondary metabolites such as polyphenols and polysaccharides.