核桃早实性相关SCAR标记(1-1_(500))基因末端序列的克隆

【目的】克隆SCAR标记的核桃早实性相关基因片段1](AFLP早实分子标记转化成的SCAR标记)的末端序列,为验证其功能和早实核桃分子育种奠定基础。【方法】采用RACE技术对SCARE标记的核桃早实性相关基因片段设计特异性PCR引物,并扩增其末端序列。【结果】分别获得了长度为453 bp和463 bp的片段,通过与NCBI核酸数据库中已经发表的序列进行比对分析,发现该基因3′末端含有358个核苷酸非编码序列。其核酸序列与葡萄假定蛋白相应部位同源性为55.26%,与葡萄重叠群相应部位同源性为38.38%,与线虫粘粒Y38F2AR基因全序列相应部位相似性为40.86%,和野猪免疫球蛋白超家族成员相应部位的相似性为39.75%,具有poly(A)尾。5′末端含有248个核苷酸非编码序列,其核酸序列与葡萄重叠群基因组鸟枪全序列相应部位同源性为39.07%,与拟南芥基因组DNA 3号染色体相应部位的同源性为42.22%,与嗜热四膜虫假定蛋白基因序列相应部位相似性为44.53%,和斑马鱼DNA序列DKEY-120E17克隆2号连锁群全序列相应部位相似性为42.30%。【结论】试验采用的3′和5′末端快速扩增技术(3′RACE和5′RACE技术)能很好地扩增核桃早实性相关基因的末端序列。

Cloning of the Terminal Sequence of the SCAR Marker(1-1500) that Linked to Early-bearing Genes in Walnut

【Objective and Method】Cloning of the terminal regions of SCAR marker related to early-bearing genes in walnut(AFLP early-bearing molecular markers which was transformed to SCAR markers),this study laid a sound foundation for verifying its function and molecular breeding.Using RACE technology to design special PCR primer that related to early-bearing genes in walnut and amplify it's terminal regions.【Result】The 453 bp and 463 bp segments of walnut were obtained.The 3′terminal region that related to early-bearing genes in walnut contained 358 nucleotide non-coding sequence and a poly(A) tail.The homology between nucleotide sequence and hypothetical protein in Vitis vinifera is 55.26%;with Vitis vinifera contig the homology is 38.38%;with the complete sequence of Caenorhabditis elegans cosmid Y38F2AR,the homology is 40.86% and with Sus scrofa immunoglobulin superfamily the homology is 39.75% respectively.The 5′terminal region that related to early-bearing genes in walnut contained 248 nucleotide non-coding sequence.The homology between nucleotide sequence and whole genome shotgun sequence of Vitis vinifera contig VV78X238793.8,chromosome 3 of Arabidopsis thaliana genomic DNA,and complete sequence of Zebrafish DNA sequence from clone DKEY-120E17 in linkage group 2 are 39.07%,42.22%,44.53%,and 42.30% respectively.【Conclusion】The results showed that the techniques of 3′and 5′RACE could amplify the terminal regions that related to early-bearing genes in walnut effectively.