梨Ty1-copia反转录转座子的克隆及IRAP分子标记体系的建立  

Cloning of Ty1-copiaretrotransposons in pear(Pyrus bretschneideri Rehd.)and establishment of the inter-retrotransposon amplified polymorphism(IRAP)marker system

作　　者：周鹏[1] 张士伟[1] 翟锐[1] 王志刚[1] 徐凌飞[1] 

机构地区：[1]西北农林科技大学园艺学院,农业部西北地区园艺作物生物学与种质创制重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2016年第2期97-104,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31171925);高校基本科研业务费项目(ZD2013005)

摘　　要：【目的】从早酥梨及其红皮芽变基因组中分离Ty1-copia反转录转座子全长序列,分析序列特点,并基于其中长末端重复序列(LTR)建立IRAP分子标记体系。【方法】以早酥梨及其红皮芽变和极早熟芽变植株叶片为试材,根据已知的Ty1-copia反转录转座子保守区域设计引物,利用同源克隆技术得到目的基因。根据基因两端LTR序列设计引物,建立IRAP分子标记体系,并对早酥梨及其芽变进行遗传分析。【结果】从早酥梨基因组中获得2条Ty1-copia反转录转座子全长序列,分别命名为PbTYZS1和PbTYZS2,长度分别是9 363和9 632bp。从红色芽变基因组中获得1条序列,命名为PbTYRM1,长度为9 652bp。经结构分析,获得的3条序列都是典型的Ty1-copia反转录转座子,但其开放阅读框(ORF)的完整性发生了变化。利用反转录转座子LTR保守区域设计引物成功建立了IRAP分子标记体系,早酥梨与红皮芽变的平均遗传距离为0.104,与极早熟芽变的遗传距离为0.115,并且早酥梨与极早熟芽变的多态性为20.41%,较之与红皮芽变的多态性(18.89%)更为丰富。【结论】早酥梨及其红皮芽变基因组中都含有完整结构的Ty1-copia反转录转座子,建立的IRAP分子标记体系可以得到清晰稳定的多态性指纹图谱。【Objective】The aim of this study was to clone Ty1-copia retrotransposons,analyze the characteristics in genome of‘Zaosu＇pear（Pyrus bretschneideri Rehd.）and its red mutants,and establish inter-retrotransposon amplified polymorphism（IRAP）molecular marker system using long terminal repeat（LTR）sequences of Ty1-copiaretrotransposon.【Method】DNA were extracted from the leaves of‘Zaosu＇pear,its red mutants and extremely early mutants.The primers were designed according to the known conserved regions of Ty1-copiaretrotransposons and the desired genes were obtained by homology-based cloning technology.According to both ends of retrotransposons,primers were designed from the LTR sequences to establish IRAP molecular marker system for genetic analysis of‘Zaosu＇pear and its mutants.【Result】Two Ty1-copiaretrotransposons full-length sequences obtained from ‘Zaosu＇pear genome were named PbTYZS1（9 363bp）and PbTYZS2（9 632bp）,respectively.One sequence obtained from the red mutant pear genome was named PbTYRM1（9 652bp）.Structural analysis showed that all three sequences were typical Ty1-copia retrotransposons with changes in ORF integrity.Based on PbTYZS1 sequence,IRAP molecular marker was established and 5pairs of IRAP marker primers were developed.The average genetic distance between‘Zaosu＇pear and the red mutants was 0.104 and that between‘Zaosu＇pear and the extremely precocious mutants was 0.115.The polymorphism of‘Zaosu＇pear and the red mutants was18.89%,and that of‘Zaosu＇pear and the extremely precocious mutants was 20.41%.【Conclusion】The genomes of‘Zaosu＇pear and its red mutants contained complete structure of Ty1-copiaretrotransposons.The established IRAP molecular marker system showed clear and stable fingerprint.

关 键 词：梨 芽变 反转录转座子 进化分析 IRAP 

分 类 号：S661.2[农业科学—果树学] Q78[农业科学—园艺学]