枇杷AS-PCR反应体系的建立和优化

【目的】建立和优化枇杷AS-PCR反应体系,为开展枇杷S基因快速鉴定奠定基础。【方法】以‘大五星’、‘早钟6号’和‘龙泉5号’为试材,通过正交实验设计对影响枇杷AS-PCR反应较大的Mg2+等5个因素的浓度进行筛选,并对扩增反应程序进行优化,运用正交设计直观分析法和DPS 7.05统计软件对扩增结果进行方差分析。【结果】优化后的枇杷AS-PCR反应分析体系为:25μL反应体系中,含10×buffer2.5μL,Mg2+浓度2.0 mmol·L-1,Taq酶1.5 U,引物0.5μmol·L-1,模板DNA80ng,dNTPs0.4 mmol·L-1。反应程序为94℃预变性1 min;94℃变性30 s,退火温度30 s,72℃延伸1 min,35次循环;72℃延伸5 min,4℃保存。【结论】建立了基于S基因保守序列设计引物的枇杷AS-PCR反应体系,利用该体系成功确定了‘大五星’的S基因型为S2-S41,其中S41为新分离鉴定的枇杷S-RNase基因,它们在GenBank上的登录号分别为JQ228451和JX217035。

Establishment and optimization of AS-PCR reaction system for Eriobotrya

【Objective】 The objective of the study is to establish and optimize the AS-PCR reaction system of loquat,lay a foundation for identifying quickly of S-allele Genotypes of loquat cultivars.【Method】'Dawuxing','Zaozhong 6' and 'Longquan 5' were used as materials.The concentrations of Mg2+,Taq polymerase,dNTPs,primer and template DNA are critical to PCR analysis.Orthogonal design of above components in AS-PCR of loquat was thus optimized in this study,time and temperature of various procedures was also improved.The intuitive analysis method by orthogonal design and DPS 7.05 software were adopted to analysis of variance.【Result】Results showed that the optimized PCR cocktail of 25 μL contained 10×buffer 2.5 μL,2.0 mmol·L-1 Mg2+,1.5 U Taq polymerase,0.5 μmol·L-1 primer,80 ng template DNA,and 0.4 mmol·L-1 dNTPs.The optimized reaction procedure was:94 ℃ for 1 min,followed by 35 cycles of 94 ℃ for 30 s,annealing temperature for 30 s,72 ℃ for 1 min,and was terminated with a 5 min extension step at 72 ℃.【Conclusion】The AS-PCR system was established based on conserved sequence of S gene.The S-genotype of 'Dawuxing' was S2-S41 by utilizing the system,and they were deposited in GenBank under the accession numbers of JQ228451 and JX217035,respectively.