检测葡萄无核基因DNA探针的合成与应用

Application and synthesis on the DNA probe for detecting seedless genes in grapevine

利用细菌质粒 M13克隆葡萄无核基因的 RAPD标记 UBC- 2 6 94 50 后 ,采用自动荧光 DNA序列分析仪对其测序。结果表明 ,UBC- 2 6 94 50 由 4 84对核苷酸及其特定序列构成。按照该序列 ,人工合成两个寡聚核苷酸5′ CCAGT TCACT CTCAA TAGGT CC3′和 5′ CCAGT TCGCC CGTAA ATG3′分别作引物 ,当用获得该标记及其序列的亲本和无核杂种后代作模板进行 PCR分析 ,凡是可以扩增出约 5 90 bp片段即为无核基因携带者和表达者。进一步用其原始无核祖先无核白和商业化无核品种及 C78- 47× B52 - 1 2 2 杂交组合后代做模板进行 PCR扩增 ,凡出现约 5 90 bp的特殊标记即为无核基因携带者和无核性状表达者。研究结果证明 ,18bp寡聚核苷酸 5′ CCAGTTCGCC CGTAA ATG3′具有检测葡萄无核基因的探针作用 ,定名为检测葡萄无核基因的探针 1号 (GSL P1)。

Bacteriophage M13 was used for cloning the RAPD marker linked to the Seedless genes in grapevine,fluorescence based specific DNA sequences 484 bp were obtained by an automated fluorescent DNA sequencer systems (Model 373A Version 2.0).Based on the sequences obtained,two oligomers were artificially synthesized and additionally used as specific primers for amplifying the parents and seedless hybrids with the sequence.Thus,the DNA band about 590 bp was obtained,this was the carrier and expression of seedless genes of grapevine.The initial Thompson seedless,and commercial seedless varieties,and the hybrids from another seedless combination were further used as template DNA for amplification with specific oligomers.The about 590 bp specific fragment suggested that those were carrier and expression of seedless genes of grapevine.Therefore,our research showed that the oligomer 5′CCAGT TCGCC CGTAA ATG3′ as the DNA probe would be used to identify seedless genes of the materials and hybrids from seedless grape breeding program with PCR assay,so that it is named probe No.1 for detecting the seedless genes of grapevine.