关键词: 小麦, 基因拷贝数, 数字PCR, 内参基因

Abstract:

【Objective】In order to explore the analysis technology of wheat gene copy number based on digital PCR, and improve the analysis efficiency of target gene copy number, and promote the research of wheat genome and gene engineering.【Method】Using the transgenic wheat with high resistance to wheat yellow mosaic disease transformed by nib8 gene created by the wheat stress resistance molecular breeding group in the Crop Science Institute of Chinese Academy of Agricultural Sciences as the experimental material, and used the single copy homozygous endogenous gene PIN-D1b (grain hardness gene) in wheat D genome as the internal reference gene, and four pairs of specific primers and corresponding probes were designed according to the sequence of the transformed disease resistance gene nib8, and the optimal concentration of probe and primer, the optimal annealing temperature of the system, to find the most appropriate template concentration were determined through experiments. Then, the copy number of nib8 genes in the nib8 transgenic wheat was detected by digital PCR; the accuracy of the above-mentioned copy number assay results were verified through using methods including the real time PCR and Southern blot; the copy number of the Wx012 (waxy gene) and SSII (starch synthesis gene) were detected by using the PINb-D1b as reference gene, and copy number of nib8 were detected by using the Wx012 and SSII as reference genes in the nib8 transgenic wheat, to verify the accuracy of the results using the PINb-D1b as reference gene; the specific primers were designed in different regions of the nib8 gene to compare the difference of the copy number analysis results of the nib8 transgenic wheat. Finally, a high-throughput assay method based on digital PCR was established to detect the copy number of target gene in wheat genome. 【Result】Finally, the detection system of target gene copy number in wheat genome based on digital PCR was determined. The optimal final concentration of primer and probe was 500 and 250 nmol·L ^-1, respectively. The optimal annealing temperature was 59℃ and the optimal amount of DNA template was 40 ng. PINb-D1b gene was used as the internal reference gene to detect the copy number of nib8 gene in the nib8 transgenic wheat. The results showed that the copy number of nib8 gene in the 12th, 16th, 17th, 23rd, 29th and 30th lines was 7, 1, 1, 1, 1, 1, 7, respectively. At the same time, the results were consistent with the real-time PCR and Southern blot results. In the nib8 transgenic lines, the PINb-D1b was used as the internal reference gene to detect copy number of the Wx012 and SSII, and we found that the results of copy number of nib8 gene in nib8 transgenic wheat by using these three kinds of internal reference gene analysis were consistent, which indicated that these three internal reference genes were all suitable for digital PCR method; the primers were designed in the upstream, middle and downstream regions of nib8 gene, and the results of copy number using different primer were consistent. 【Conclusion】The method and reaction system to detect target gene copy number in wheat genome based on digital PCR method were optimized, and the detection system to detect target gene copy number in wheat genome based on digital PCR method was established. The results of digital PCR analysis are stable and reliable, and the detection efficiency is significantly improved, which has a certain application prospect.

Key words: wheat, copy number of genes, the digital PCR, the reference genes