陆地棉GHWAT1-35基因的克隆及亚细胞定位

Cloning and subcellular localization of the GHWAT1-35 gene in Gossypium hirsutum

【目的】研究陆地棉GHWAT1-35基因在棉花纤维发育过程中的作用。【方法】选用陆地棉系9花后不同时期的纤维为材料,克隆GHWAT1-35基因的全长cDNA序列,并进行生物信息学、实时荧光定量(qRT-PCR)分析及亚细胞定位。【结果】该基因全长1125 bp,编码374个氨基酸,相对分子量为40.231 kDa,理论等电点为8.74。GHWAT1-35蛋白与亚洲棉亲缘关系最近。GHWAT1-35基因在开花后15 d表达量显著升高,亚细胞定位预测表明该蛋白定位于细胞质膜上。将GHWAT1-35基因与pCAMIA1300-35S-YFP载体重组,构建融合表达载体,利用冻融法转化农杆菌,注射到烟草后观察到GHWAT1-35蛋白定位在细胞质膜上。【结论】陆地棉系9 GHWAT1-35基因在开花后15和20 DAP的表达量显著高于其他时期,pC1300∷35S-WAT-YFP融合蛋白定位于细胞质膜上。

【Objective】To explore the role of GHWAT1-35 gene in the development of cotton fibers.【Methods】In this study,the fiber at different developmental stages post-anthesis of Gossypium hirsutum variety Xi9 were used as materials.The full-length cDNA sequence of the GHWAT1-35 gene was successfully cloned and subjected to bioinformatics analysis,real-time fluorescence quantitative(qRT-PCR)analysis,and subcellular localization.【Results】The length of the GHWAT1-35 gene was 1125 bp,encoding 374 amino acids,with a relative molecular weight is 40.23 kDa and a theoretical isoelectric point of 8.74.Protein multiple sequence alignment and construction of a systematic evolutionary tree analysis showed that the GHWAT1-35 protein was most closely related to the Gossypium arboreum.The expression of the GHWAT1-35 gene significantly increased at 15 days after flowering,and subcellular localization predicted that the protein was located on the cytoplasmic membrane.The GHWAT1-35 gene was recombined with the pCAMIA1300-35S-YFP vector to construct a fusion expression vector,which was then transformed into Agrobacterium using the freeze-thaw method and after being injected into tobacco.The GHWAT1-35 protein was observed to be located on the cytoplasmic membrane.【Conclusion】The GHWAT1-35 gene plays an important role in fiber development,providing a foundation for further exploration of its biological function in Gossypium hirsutum.