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新麦草YUCCA基因克隆及其表达特性分析
任晓敏1, 云岚2, 艾芊,等1
1.内蒙古农业大学 草原与资源环境学院;2.内蒙古农业大学 草原与资源环境学院,草地资源教育部重点实验室
摘要:
【目的】克隆新麦草分蘖关键基因YUCCA并明确其表达特性,为该基因功能验证和育种利用奠定基础。【方法】以不同分蘖类型的新麦草分蘖节为材料,采用PCR法克隆新麦草YUCCA基因并进行同源序列比对,构建1300-cYFP-YUCCA过表达载体,利用烟草瞬时转染及蛋白质印迹法分析新麦草YUCCA蛋白的表达位置及表达情况,通过RNA-Seq数据及qRT-PCR分析验证YUCCA基因的相对表达量。【结果】成功克隆了新麦草YUCCA基因全长,大小为1 336 bp,最大阅读框为1 236 bp,并成功构建了1300-cYFP-YUCCA过表达载体。多序列比对结果显示,新麦草YUCCA主要与麦类作物的亲缘关系较近。表达特性分析发现,YUCCA基因在多分蘖型新麦草中的相对表达量远低于少分蘖型新麦草;与叶、根等组织相比,YUCCA基因在分蘖节中的相对表达量最高。烟草瞬时转化和蛋白质印迹法分析均显示,YUCCA蛋白可以正常表达,其亚细胞定位于细胞质中。【结论】YUCCA基因在调控新麦草分蘖中起下调作用,可用于后续YUCCA基因功能的研究。
关键词:  新麦草  YUCCA  过表达载体  亚细胞定位  基因克隆
DOI:
分类号:
基金项目:内蒙古自治区自然科学基金重点项目(2023ZD07);内蒙古自治区种业创新重大示范工程“揭榜挂帅”项目(2022-JBGS0040)
Cloning and expression analysis of YUCCA gene in Psathyrostachys juncea
REN Xiaomin,YUN Lan,AI Qian,et al
Abstract:
【Objective】The key gene YUCCA of Psathyrostachys juncea was cloned and its expression characteristics were clarified to provide basis for its functional verification and breeding utilization.【Method】The tillering nodes of P. juncea with different tillering types were used as materials.The YUCCA gene of P. juncea was cloned by PCR and the 1300-cYFP-YUCCA overexpression vector was constructed.The expression position and expression of YUCCA protein in P. juncea were analyzed by tobacco transient transfection and Western blot.The relative expression ofYUCCA gene was verified by RNA-Seq data and qRT-PCR analysis.【Result】The full-length YUCCA gene of P. juncea was successfully cloned,with a size of 1 336 bp and a maximum reading frame of 1 236 bp.The 1300-cYFP-YUCCA overexpression vector was constructed.Multiple sequence alignment showed that YUCCA of P. juncea was mainly related to wheat crops.Relative expression level of YUCCA gene in multi-tillering P. juncea was much lower than that in less-tillering P. juncea.The relative expression of YUCCA gene in tillering node was the highest among different tissues,such as leaf and root.Both tobacco transient transformation and Western blot showed that YUCCA protein could be expressed normally and its subcellular localization was in the cytoplasm.【Conclusion】YUCCA gene plays a down-regulated role in regulating the tillering of P. juncea,and this study provide basis for its subsequent functional verification.
Key words:  Psathyrostachys juncea  YUCCA  overexpression vector  subcellular localization  gene cloning