南海芋螺ITS-PCR体系的建立与优化

Establishment and Optimization of ITS-PCR System for Conus spectrumin South China Sea

为了建立适合南海芋螺的ITS-PCR体系以研究不同种类芋螺的遗传多样性,以多种芋螺的3种不同组织为材料,利用海洋生物基因组试剂盒法提取芋螺基因组DNA为模板,采用正交试验对ITS-PCR过程中的关键影响因素进行优化,以最优ITS-PCR体系扩增获得不同种类芋螺的ITS序列并进行测序鉴定。结果表明:最佳ITS-PCR反应体系(25μL)为Taq酶0.3U,dNTPs 3mmol/L,Mg^(2+)1.0mmol/L,引物2.0μmol/L,模板40ng,10×PCR Buffer(不含Mg^(2+))2.5μL,ddH_2O补足25μL。采用该最佳体系对芋螺基因组DNA进行PCR扩增,产物经单向测序获得芋螺ITS部分序列,经NCBI数据库比对其匹配度为83%。

The genomic DNA of different Conus spectrum is extracted from three different tissues by Marine Genomes Extraction Kit method.The key factors influencing ITS-PCR process are optimized by an orthogonal test.ITS sequence of different C.spectrum acquired by the optimum ITS-PCR system is sequenced and identified to establish the suitable ITS-PCR system of C.spectrum in South China Sea for studying genetic diversity of C.spectrums.Result:The optimum ITS-PCR system(25μL)includes 0.3 U Taq DNA polymerase,3 mmol/L dNTPs,1.0 mmol/L Mg^2+,2.0μmol/L primer,40 ng template DNA,2.5μL 10×PCR Buffer and 25μL ddH2O.The genomic DNA of C.spectrum is amplified by the optimal ITS-PCR system.The identity between part ITS sequence of C.spectrum obtained by sequencing the amplified product and NCBI database reaches 83%.