果树学报2013，Vol.30Issue(1)：62-68,7.
枇杷AS-PCR反应体系的建立和优化
Establishment and optimization of AS-PCR reaction system for Eriobotrya
摘要
Abstract
[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 2.5μL, 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.关键词
枇杷/AS-PCR/正交设计/反应体系Key words
Eriobotrya Lindl./ AS-PCR/ Orthogonal design/ Reaction System分类
农业科技引用本文复制引用
杨芩,付燕,王永清,陶炼,邓群仙,范建新,邓仁菊..枇杷AS-PCR反应体系的建立和优化[J].果树学报,2013,30(1):62-68,7.基金项目
贵州省教育厅自然科学项目(项目编号:黔教科20090085) （项目编号:黔教科20090085）
凯里学院院级规划课题(项目编号:Z1006) （项目编号:Z1006）
四川省"十二五"农作物育种攻关项目(2011NZ0098-8) （2011NZ0098-8）
凯里学院植物学省级重点扶持学科 （）
