Wang Chunyu, Zhuge Junhao, Tang Siqi, Zhou Xiang, Zhou Lifeng, Guo Kai
School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou, China.
National Joint Local Engineering Laboratory for High-Efficient Preparation of Biopesticide, Changsha, China.
Front Microbiol. 2023 Nov 30;14:1234895. doi: 10.3389/fmicb.2023.1234895. eCollection 2023.
The gene localization technique of (pinewood nematode, PWN) is used for study gene expression in PWNs. Two hybridization methods, namely, whole-mount hybridization and the cut-off method are used widely. To compare the effects of these two hybridization methods, the present study investigated the patterns of two functional genes expression in PWNs. The gene (GenBank accession number: OR228482), related to pathogenicity, and the gene (GenBank accession number: OR228481), related to sex determination, were selected to map related genes in the whole-mount and amputated PWNs at different ages using these hybridization methods. Based on the overall statistical comparison, we found that compared to the cut-off method, the whole-mount method exhibited higher staining rates and correct staining rates for the gene and gene. However, considering the correct staining aspect, the cut-off method yielded better staining effects on pinewood nematode sections than the whole-mount method, with clearer hybridization signal locations and less non-specific staining. In other words, the cut-off method demonstrated more precise gene localization. Both methods are applicable for gene localization, but considering the overall staining pattern, analysis of experimental results, and comprehensive experimental operations, we believe that the whole-mount method is more suitable for gene localization and expression analysis of development-related genes in pinewood nematodes. This is because intact pinewood nematodes are better suited for showcasing the continuous developmental process of development-related genes. On the other hand, considering the experimental time, accuracy of staining site, and the amount of non-specific staining, the cut-off method is more suitable for disease-related genes. Additionally, to achieve better performance, the cut-off method can be selectively applied to samples during the experimental process.
松材线虫基因定位技术用于研究松材线虫中的基因表达。两种原位杂交方法，即整装原位杂交和截断法被广泛应用。为比较这两种原位杂交方法的效果，本研究调查了松材线虫中两个功能基因的表达模式。选择与致病性相关的基因（GenBank登录号：OR228482）和与性别决定相关的基因（GenBank登录号：OR228481），使用这些原位杂交方法在不同年龄的整装和截断的松材线虫中定位相关基因。基于总体统计比较，我们发现与截断法相比，整装法对该基因和该基因的染色率和正确染色率更高。然而，从正确染色方面考虑，截断法对松材线虫切片的染色效果比整装法更好，杂交信号位置更清晰，非特异性染色更少。换句话说，截断法显示出更精确的基因定位。两种方法都适用于基因定位，但考虑到总体染色模式、实验结果分析和综合实验操作，我们认为整装法更适合松材线虫中与发育相关基因的基因定位和表达分析。这是因为完整的松材线虫更适合展示与发育相关基因的连续发育过程。另一方面，考虑到实验时间、染色位点的准确性和非特异性染色量，截断法更适合与疾病相关的基因。此外，为了获得更好的效果，截断法可以在实验过程中选择性地应用于样本。
