蓖麻标雌/单雌/两性系花序MSAP反应体系的建立

Establish Castor Marked Female Lm Per Female Amphoteric Lineinflores Cence MSAP Reaction System

为了建立Lm型雌性系蓖麻不同发育时期的标雌/单雌/两性系花序MSAP反应体系,提取不同类型不同发育时期的花序基因组DNA,混合成基因池;用EcoRⅠ和HpaⅡ/MspⅠ的组合,12 h可将DNA酶切完全;16℃条件下,将酶切产物与EcoRⅠ、HpaⅡ/MspⅠ接头过夜连接;连接产物用于预扩增。优化后的预扩增反应体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.5μL、Mg2+(25 mmol/L)2.0μL、模板2.0μL、E0扩增引物(10pmol/μL)1.5μL、H0扩增引物(10 pmol/μL)1.5μL、LA Taq(5 U/μL)0.25μL、dd H2O 12.75μL。预扩增产物稀释10倍后用于选择性扩增。优化后的选择性扩增体系为10×PCR Buffer 2.5μL、d NTPs(10 mmol/L)2.0μL、Mg2+(25mmol/L)4.0μL、模板3.0μL、EX扩增引物(10 pmol/μL)1.0μL、H/MX扩增引物(10 pmol/μL)1.0μL、LA Taq(5U/μL)0.30μL、dd H2O 11.2μL。

The purpose of this experiment was to establish Lm-type female system castor different developmental stages marked female,per female,amphoteric system inforescence establish MSAP reaction system. MSAP reaction system established to determine differences in inflorescence type genes at different developmental stages of research to lay the foundation. The results were as follows: extraction of different types at different developmental stages of inflorescence genomic DNA mixed into the gene pool. EcoRⅠ and HpaⅡ / MspⅠ digested gene pool 12 h.The digestion products and EcoR Ⅰ,Hpa Ⅱ / Msp Ⅰ adapter overnight connection for pre-amplification. The opti-mized system for the pre-amplification reaction 10 × PCR Buffer 2. 5 μL,d NTPs( 10 mmol / L) 2. 5 μL,Mg^(2+)( 25 mmol/L) 2. 0 μL,template 2. 0 μL,E_0 amplification primer( 10 pmol / μL) 1. 5 μL,H_0 amplification primer( 10 pmol/μL) 1. 5 μL,LA Taq( 5 U/μL) 0. 25 μL,dd H_2O 12. 75 μL. Pre-amplification products were diluted 10-fold for the selective amplification. Selective amplification system optimized for 10 × PCR Buffer 2. 5 μL,dNTPs( 10 mmol/L) 2. 0 μL,Mg2 +( 25 mmol/L) 4. 0 μL,template 3. 0 μL,EXamplification primer( 10 pmol / μL)1. 0 μL,H / Mxamplification primer( 10 pmol / μL) 1. 0 μL,LA Taq( 5 U / μL) 0. 30 μL,dd H_2O 11. 2 μL.