梨自交不亲和基因cDNA芯片杂交条件优化及应用

Optimization and application of pear self-incompatibility gene cDNA microarray hybridization assay

[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。

[Objective]To optimize the hybridization condition of self-incompatibility gene（S-RNase or S gene） c DNA microarray and by the chip to identify S-genetypes of pear cultivars. [Methods]Total RNA was extracted from pear pistils,with which fluorescently-labeled specific c DNA sequences of S-RNase were amplified by RT-PCR amplification using Cy3-labeled specific primers. The hybridization effects of that the purified PCR products of pear cultivars with known S-genotype were hybridized with the c DNA microarray in different condition were analysed. Then pear cultivars with unknown S-genetypes were identified by the microarray at the optimal conditions and the hybrided results were verified by TA cloning and DNA sequencing. [Results]The optimized conditions for hybridization include a hybridized temperature of 42 ℃,a hybridized time of 8-9 hours,and final concentration of 200 ng·μl-1purified PCR products of pear cultivars. The S-genotypes of ten pear cultivars including Wanxianfeng,Xiushui,Lijiang Mazhanli 1,Xiangju,Mutongli,Gantian,Midu Xiaohongli,Lijiang Dazhonggu,Jinjing and Midu Huoba were identified by the microarray at the optimal conditions and their S-genotypes respectively are Pp S15 Pp S52,Pp S4 Pp S5,Pb S22 Pp S37,Pp S1 Pp S2,Pp S1 Pp S3,Pp S13 Pp S15,Pp S12 Pb S42,Pb S21 Pb S22,Pp S3 Pp S60and Pp S5 Pp S5. DNA sequence verification information were consistent with the microarray analyses. [Conclusion]The S-RNase c DNA microarray could be used to accurately detect the S-genotypes of pear cultivars after the optimization.