不同赤眼蜂品系ISSR-PCR条件优化及分子鉴定

Optimization of ISSR-PCR analyses and molecular identification of different strains of Trichogramma spp

赤眼蜂Trichogramma不同品系在寄生能力等生物学特性上存在差异,而这些差异可能被用于优良品系的筛选。通过优化影响赤眼蜂不同品系ISSR-PCR的主要参数,建立适于鉴别赤眼蜂不同品系的ISSR-PCR反应体系和扩增程序。在20μL反应体系中,各反应物的最适含量为10×PCR Buffer 2.0μL,MgCl22.0μL(25mmol/L),dNTPs1.6μL(2.5mmol/Leach),正反向引物各1μL(25μmol/L),DNA模板1.0μL,Taq酶0.4μL(2.5U/μL),和ddH2O。ISSR-PCR的扩增程序为:95℃预变性5min,94℃变性50s,52℃退火1min,72℃延伸1min20s,35个循环,72℃延伸10min。结果表明,在上述优化条件下,采用ISSR-PCR可实现对7个赤眼蜂优良品系的分子鉴定。该研究对于赤眼蜂优良品系的筛选具有潜在的应用价值。

Different Trichogramma strains are differentiated from each other in biological characteristics, such as parasitic capability, which might be used for screening of superior Trichogramma strains. Here Inter-Simple Sequence Repeats （ISSR）-PCR was used to investigate the genetic variation of 7 candidate strains, and the molecular markers specific to different strains were subsequently identified. By optimizing the main parameters, the optimal ISSR-PCR reaction conditions for Trichogramma were firstly established. The results showed that the optimum concentrations of different reagents in a total of 20 μL volume were： 2.0 μL MgCl2 （20 mmol/L）, 1.6 μL dNTPs （2.5 mmol/L each）, 1 μL ISSR primer （10 μmmol/L）, 1.5 μL genomic DNA and 0.4 μL Taq DNA polymerase （2.5 U/μL）. The suitable PCR procedures were determined to be： pre-denaturing at 95℃ for 5 min, followed by 35 cycles of denaturing at 94℃ for 50 s, annealing at 52℃ for 1min and extension at 72℃ for 1 min 20 s, with a final extension at 72℃ for 10 min. Under the optimal conditions, the 7 Trichogramma strains tested eould be discriminated by using ISSR-PCR. It is therefore suggested that this technique is helpful in identification and screening of Trichogramma strains.