通过微切一扩增建立植物(蚕豆Vicia faba)染色体区段特异性基因文库研究初探(英文)

A PRELIMINARY STUDY ON A SIMPLE PROCEDURE TO CONSTRUCT MICRODISSECTION LIBRARIES OF DEFINED REGION FROM AMPLIFIED CHROMOSOME SEGMENT IN PLANT CELL (VICIA FABA)

摘要:

以蚕豆（Viciafaba，2n=12）根尖为材料，采用改良方法制备染色体标本，在光镜下切割分离一段大M染色体核仁组织区（NOR）特定区段（约合0．9pgDNA），通过单一引物一聚合酶链式反应法（SingleUniquePrimer-PCR）随机扩增微切DNA后，获得近60μgDNA。经琼脂糖电泳分析测定扩增产物分子片段大小介于200-900bp。以地高新（Digoxigenin）标记蚕豆总体DNA，作为探针与扩增产物进行Southern杂交，证实扩增得到的DNA与蚕豆DNA同源，来自做切染色体。部分扩增产物经ECORI酶切后，连人经同酶切割后的pUC18载体质粒，转化大肠杆菌（EcoliJM109）。琼脂糖电泳分析得到的部分克隆，得知插人子长度介于0．25-0．9kb。本文将用于动物材料的单一引物一聚合酶链式反应法应用于植物染色体的微切微扩增，并作了一定程度简化，初步建立起一套包括微切、扩增、检测和克隆的便捷、经济的实验室制备植物染色体区域特异性基因文库的方法。

Abstract:

A simple method to create chromosome region specific library in plant materials,including microdissection, amplification, characterization and cloning, is described. A singleNOR region of chromosome I from Vicia faba was microdissected and directly amplified bySUP-PCR in a magnified system. The sizes of the PCR products ranged from 200 to 900bp. Southern hybridization with total genomic DNA demonstrated the reliability of themethod. Part of the products were cloned into plasmid PUC 18. The inserts ranged between0.25 and 0.9 kb.