Huang Bangbang, Wei Meiling, Wei Rongchang, Hou Wenhuan, Tang Xingfu, Zhao Yanhong, Liao Xiaofang, Zhou Ruiyang
College of Agriculture, Guangxi University, Nanning 530004, China.
Soil and Fertilizer Work Station, Lingshan 535499, China.
Genes (Basel). 2025 Apr 23;16(5):479. doi: 10.3390/genes16050479.
The aim of this study was to develop a genetic transformation system to construct an overexpression vector for the mitochondrial gene in tobacco, thereby providing a foundation to investigate the functional roles of mitochondrial genes in this species.
A full-length coding sequence (CDS) of the gene from a sterile line was cloned, along with the mitochondrial leader peptide sequence of from tobacco, using cDNA from kenaf UG93A anthers as a template. An overexpression vector for plants was constructed by employing In-Fusion technology, and wild-type tobacco plants were transformed via -mediated transformation. Transgenic tobacco plants were then subjected to resistance screening and PCR validation.
The overexpression vector PBI121---EGFP, which includeds the mitochondrial leader peptide sequence, was successfully constructed. PCR validation using two pairs of primers targeting different sites on the overexpression vector confirmed the stable expression of the target gene in six transgenic tobacco plants (H1, H3, H4, H5, H7, and H8) via both primer pairs. A phenotypic analysis and iodine-potassium iodide (I-KI) staining of pollen grains from transgenic tobacco plants revealed the presence of shriveled and malformed pollen grains with reduced viability. These findings suggest that the gene, including the mitochondrial signal peptide, induces pollen abortion in tobacco.
The genetic transformation system developed for the vector overexpressing the mitochondrial gene from kenaf provides a valuable framework to investigate the molecular regulatory mechanisms underlying the role of the gene in kenaf cytoplasmic male sterility (CMS).
本研究的目的是开发一种遗传转化系统，用于构建烟草中线粒体基因的过表达载体，从而为研究该物种中线粒体基因的功能作用提供基础。
以红麻UG93A花药的cDNA为模板，克隆了不育系中该基因的全长编码序列（CDS）以及烟草中该基因的线粒体前导肽序列。采用In-Fusion技术构建植物过表达载体，并通过农杆菌介导的转化方法转化野生型烟草植株。然后对转基因烟草植株进行抗性筛选和PCR验证。
成功构建了包含线粒体前导肽序列的过表达载体PBI121---EGFP。使用针对过表达载体上不同位点的两对引物进行PCR验证，证实了目标基因在六株转基因烟草植株（H1、H3、H4、H5、H7和H8）中通过两对引物均稳定表达。对转基因烟草植株的花粉粒进行表型分析和碘-碘化钾（I-KI）染色，发现存在皱缩和畸形的花粉粒，活力降低。这些结果表明，包含线粒体信号肽的该基因可诱导烟草花粉败育。
为过表达红麻线粒体基因的载体开发的遗传转化系统，为研究该基因在红麻细胞质雄性不育（CMS）中作用的分子调控机制提供了有价值的框架。
