山新杨蔗糖合酶基因PCR介导的重组现象研究

The phenomenon of PCR-mediated recombination by using SUS genes of Populus davidiana×P.bolleana

【目的】山新杨(Populus davidiana×P.bolleana)是林木基因工程育种基础和应用研究的良好材料;以山新杨蔗糖合酶基因(PdbSUS)为例研究聚合酶链式反应(polymerase chain reaction,PCR)过程介导的重组现象,在此基础上区分每一个PdbSUS基因的两种单倍型,为后续研究提供准确的序列信息,并为判断木本植物基因真实的单倍型遗传信息提供参考。【方法】分别采集番茄(Lycopersicon esculentum)和山新杨新鲜嫩叶,以番茄基因组为内参,利用流式细胞仪测定山新杨基因组大小及其倍性水平。参考毛果杨(P.trichocarpa)蔗糖合酶序列设计引物,分别以山新杨基因组DNA和cDNA为模板进行同源克隆,将PCR扩增产物回收纯化,连接测序载体并转化大肠杆菌感受态细胞,挑取阳性克隆进行菌液PCR验证,将整合有外源片段的转化子进行一代测序。利用SnapGene及DNAMAN软件对测序结果进行分析比对。【结果】山新杨为二倍体植物,获得了7个PdbSUS基因的全长序列,并分别在基因组和转录水平鉴定出每个基因的两种单倍型序列。同时,检测到PCR介导的重组现象并通过限制性酶切多态性(RFLP)进行了验证。在测序的209个克隆中,发现有18个含有嵌合产物,占比8.6%,不同基因产生嵌合产物的概率为0~33.3%。检测到的嵌合产物均来自同一个位点的不同等位基因之间发生重组,未发现不同蔗糖合酶基因之间发生重组的现象。【结论】PCR介导的重组是PCR反应过程中能够衍生重组分子的一种高频事件,该现象可能是由于引物延伸不完全或聚合酶模板转换导致。在利用PCR技术克隆木本植物或其他基因组杂合度较高物种的基因时,需识别产物中重组分子才能够准确区分单倍型的遗传信息。

【Objective】Populus davidiana×P.bolleana(Shanxin poplar)is an ideal material for basic and applied research in tree genetic engineering.This study aimed to determine the ploidy level of Shanxin poplar,reveal the phenomenon of PCR-mediated recombination via a case study of PdbSUS genes,and define two haplotypes of each gene to provide precise sequence information for subsequent genetic engineering studies and serve as a reference for cloning and determining the haplotypes of genes in woody plants.【Method】Fresh young leaves of tomato(Lycopersicon esculentum)and Shanxin poplar were separately collected.The genome size and ploidy level of Shanxin poplar were analyzed by influx flow cytometry,using the tomato genome as an internal reference.Primers designed based on the sequences of sucrose synthase(SUS)genes in P.trichocarpa,were used to amplify the full-length genomic DNA and the cDNA sequences of SUS genes in Shanxin poplar.The PCR amplicons were visualized on 1%agarose gels by GelRed staining,then appropriate bands were excised from the gels and purified using the AxyPrepTM DNA Gel Extraction Kits.All purified products were separately ligated to the pEASY-Blunt vector and transformed into Escherichia coli strain DH5αcompetent cells.Colonies of E.coli were randomly selected from individual LB agar plates containing kanamycin and screened for target fragments by using colony PCR.Independent bacterial clones harboring the recombinant plasmids were sequenced using the Sanger method,then the sequences for each gene were aligned and analyzed using DNAMAN(version 8)and SnapGene(version 1.1.3)software with default parameters.【Result】Shanxin poplar is a diploid plant.Seven PdbSUS genes were obtained,and the haplotypes of each gene were identified at the genomic and transcriptional levels.Recombination mediated by PCR was verified by analyzing restriction fragment length polymorphisms(RFLP).Among the 209 sequenced clones,18 harbored chimeric products,accounting for 8.6%,and the frequency of chimera generation in individual genes ranged from 0 to 33.3%.All chimeric products were recombinants derived from heterozygous alleles located at the same locus of homologous chromosomes.Chimeras of different PdbSUS genes were undetectable.【Conclusion】The present findings indicated that PCR-mediated recombination is not rare,but is rather a high-frequency event leading to PCR-derived recombinants,which might arise via incomplete primer extension or polymerase template switching.Thus,chimeric products should be differentiated from parental haplotypes when cloning genes from woody plants or other species with highly heterozygous genomes by using PCR.