猕猴桃6个LOX基因家族成员实时定量PCR引物特异性的检测与应用

从基因家族成员的视角开展基因表达研究是阐明基因功能的重要组成内容，实时定量PCR（QPCR）技术是分析基因表达的有效手段.以猕猴桃脂氧合酶（LOX）基因家族6个成员为对象，分析了引物特异性的检测方法.该方法整合了熔点曲线分析、琼脂糖电泳、交叉PCR扩增和PCR产物测序等分析手段，有效消除其他成员的交叉扩增干扰，为利用QPCR检测基因家族成员表达提供了特异、准确与可行的途径.

Specificity Determination and Its Application of Real-time Quantitative PCR Primers for Six Members of Kiwifruit LOX Gene Family

The redundancy of plant genomes leads to large gene families, and the expression analysis of multi-members of a gene family sometimes is critical to understand the exact roles of a gene in plants. Real-time quantitative PCR (RT-QPCR) is an effective method to evaluate gene expressions with satisfactory specificity, sensitivity and simplicity. The specificity of primers used in these assays are cuicial for profiling the expression of an entire gene family, and yet the discrepancy of different gene family members is a great challenge, especially in fluorochrome, like SYBR Green, based detection systems. Lipoxygenases (LOX) are non-heme iron-containing dioxygenases with multiple isoforms identified in plants. The present study illustrated the strategies to design specific primers for six LOX gene family members from kiwifruit, integrated melting curve analysis, agarose gel analysis, cross-amplification PCR and amplicon sequencing were subsequently performed. Our primer pairs were able to prevent cross-amplification from other LOX family members, and our RT-QPCR method was a sensitive and specific way to analyze the expressions of different gene family members.