短枝型苹果SSH文库构建及相关基因表达分析

doi:10.3864/j.issn.0578-1752.2013.20.013

摘要/Abstract

摘要： 【目的】寻找苹果短枝型枝条形成相关基因，为进一步探索苹果短枝型芽变形成机理及选育新的短枝型苹果品种奠定基础。【方法】利用抑制性差减杂交技术构建正反向苹果短枝型芽变枝条和正常型枝条差异表达cDNA文库，利用荧光定量RT-PCR验证文库中克隆的插入片段。【结果】从两个文库中共获得291个有效阳性克隆，插入片段大小主要分布在300—600 bp之间。利用GenBank的BLAST比对分析，其中126个表达序列标签（EST）找到了与之匹配的同源序列。获得的cDNA片段功能涉及代谢、转录、胁迫响应、转运、光合和信号转导；有165个EST片段未找到同源序列。对文库中EST序列进行实时荧光定量RT-PCR验证，结果显示这些EST序列在短枝型芽变枝条与正常型枝条之间差异性表达。【结论】通过构建短枝型苹果正反向文库，得到一些与短枝型苹果枝条节间长度相关联的基因，这些基因可能对短枝型苹果枝条的伸长具有一定作用。

关键词: 苹果 , 短枝型 , SSH , 表达序列标签 , 实时荧光定量PCR

Abstract: 【Objective】The aim of this experiment is to explore genes related to spur type shoots of apple and to try to understand the mechanism of sport for breeding new spur type variety.【Method】A forward and reverse suppression subtractive hybridization library were constructed using the spur type shoots from sport and the standard shoots. Identification of the inserted cDNA fragments in subtracted library was done using real-time PCR.【Result】A total of 291 positive clones were obtained and the size of the inserts was 300 to 600 bp. By means of BLAST comparison of GenBank, 126 ESTs were found to share considerable homology with known genes while the rest 165 ESTs had no homology with known genes. The putative function of these genes was involved in metabolism, transcription, stress response, transport, photosynthesis, and signal transduction. The ESTs were significantly differently expressed between the spur type sport and the standard shoots. 【Conclusion】 Some genes related to shoot internode length of spur type apple were obtained through construction of the forward and reverse suppression subtractive hybridization library. The research results suggested that the genes play an important role during the shoots elongation of spur type apple.

Key words: apple , spur type , suppression subtractive hybridization(SSH) , expressed sequence tags , real-time PCR

宋杨12, 吴树敬1, 张艳敏1, 陈学森1. 短枝型苹果SSH文库构建及相关基因表达分析[J]. 中国农业科学, 2013, 46(20): 4301-4309.

SONG Yang-12, WU Shu-Jing-1, ZHANG Yan-Min-1, CHEN Xue-Sen-1. Construction of the Suppression Subtractive Hybridization Library and Analysis of Related Genes of Spur-Type Apple (Malus domestica Borkh.)[J]. Scientia Agricultura Sinica, 2013, 46(20): 4301-4309.

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