梨自交不亲和基因cDNA芯片杂交条件优化及应用

发布时间：2018-04-24 15:54

  本文选题：梨 + cDNA芯片　； 参考：《生物技术》2017年01期

【摘要】：[目的]优化梨自交不亲和基因(S-RNase或S基因)c DNA芯片杂交条件,利用芯片检测梨品种S基因型。[方法]提取梨品种雌蕊RNA,Cy3标记引物RT-PCR获得S基因荧光标记特异c DNA序列。设置不同杂交条件,用已知S基因型品种荧光标记的PCR产物在不同条件下分别与芯片杂交,杂交信号分析芯片杂交效果。用芯片优化杂交体系鉴定梨品种未知S基因型,DNA测序验证芯片鉴定结果。[结果]芯片杂交最佳条件:杂交温度42℃,杂交时间8~9 h,PCR纯化产物终浓度为200 ng·μl-1。优化杂交条件下芯片鉴定晚咸丰、秀水、丽江马占梨1、湘菊、木通梨、甘甜、弥渡小红梨、丽江大中古、金晶和弥渡火把等梨品种S基因型分别为:Pp S15Pp S52、Pp S4Pp S5、Pb S22Pp S37、Pp S1Pp S2、Pp S1Pp S3、Pp S13Pp S15、Pp S12Pb S42、Pb S21Pb S22、Pp S3Pp S60和Pp S5Pp S5。DNA测序验证各品种所含S基因与芯片鉴定结果一致。[结论]梨自交不亲和基因c DNA芯片优化杂交条件后可准确鉴定梨品种所含已鉴定的S基因资源。
[Abstract]:[objective] to optimize the conditions of S-RNase or S-gene DNA microarray hybridization of pear self-incompatibility gene, and to detect the S-genotypes of pear varieties by microarray. [methods] the C DNA sequence of S gene fluorescent marker was obtained from the primer RT-PCR labeled with Cy3 in pistil of pear cultivars. Under different hybridization conditions, the PCR products labeled with known S genotypes were hybridized with microarray under different conditions, and the hybridization signal was analyzed by chip hybridization. DNA sequencing of unknown S genotypes of pear cultivars identified by microarray optimized hybridization system was used to verify the results of microarray identification. [results] the optimal conditions for microarray hybridization were as follows: hybridization temperature 42 鈩，

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