摘要
以香榧Torreya grandis‘Merrillii’基因组DNA为材料,对影响简单序列重复区间扩增-聚合酶链式反应(ISSR-PCR)的Taq酶用量、Mg2+浓度、模板DNA用量、磷酸碱基脱氧核苷酸(dNTPs)浓度和引物浓度等进行了优化试验。优化后的香榧ISSR-PCR扩增体系为:总容积20μL,包括30 ng模板DNA,16.67 nkat Taq酶,0.350μmol.L-1引物,1.625 mmol.L-1 Mg2+,0.250 mmol.L-1 dNTPs,10×PCR buffer 2μL。PCR扩增程序:94℃变性5.0 min;35个循环:94℃变性30 s,退火45 s,退火温度视引物而定,72℃延伸1.5 min;最后72℃延伸7.0 min,4℃保温。在此基础上,从77个ISSR引物中筛选出34个适合香榧ISSR分析的引物,且通过梯度PCR确定了各自最适的退火温度。研究结果为香榧遗传图谱构建打下了基础。
With genomic DNA of Torreya grandis ‘Merrillii＇, an optimization experiment was performed in terms of Taq DNA polymerase, Mg^2＋, DNA template, deoxynucleotide triphosphates （dNTP）s, and primer concentration. The optimized system was as follows： a total polymerase chain reaction （PCR） volume of 20 μL included 30 ng DNA, 16.67 nkat Taq polymerase, 0.350 μmol·L^-1 primer, 1.625 mmol·L^-1 Mg^2＋, 0.250 mmol ·L^-1 dNTPs, and 2 μL 10 x PCR buffer. Then, a PCR amplification program was established with denaturing at 94 ℃ for 5.0 min followed by 35 cycles of denaturing at 94 ℃ for 30 s, annealing for 45 s （annealing temperatures varied with primers） with extension at 72 ℃ for 1.5 min, extension at 72 ℃ for 7 min, and finally maintained at 4 ℃. Based on this optimized system, 34 inter simple sequence repeat （ISSR） primers suitable for the species were screened from 77 primers with gradient PCR being used to propose the optimal annealing temperature. This experimental result has laid a foundation for genetic map construction in T. grandis ‘Merrillii＇ [Ch, 4 fig. 1 tab. 12 ref.]
出处
《浙江林学院学报》
CAS
CSCD
北大核心
2010年第1期87-92,共6页
Journal of Zhejiang Forestry College
基金
"十一五"浙江省科技计划项目(2008C2200)
浙江省重中之重学科开放基金资助项目(200505
200508
200602)
关键词
经济林学
香榧
ISSR—PCR
引物筛选
cash forestry
Torreya grandis ‘Merrillii＇
chain reaction（PCR）
primer screening inter simple sequence repeats （ISSR）-polymerase
