向日葵HaACO1基因的表达分析及功能验证  

Expression Analysis and Functional Verification of the HaACO1 Gene in Sunflower

作　　者：孙瑞芬[1] 张艳芳[2] 牛素清[1] 郭树春[1] 李素萍[1] 于海峰[1] 聂惠[1] 牟英男 SUN Rui-fen;ZHANG Yan-fang;NIU Su-qing;GUO Shu-chun;LI Su-ping;YU Hai-feng;NIE Hui;MOU Ying-nan(Inner Mongolia Academy of Agricultural&Animal Husbandry Sciences,Huhhtot 010031;College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Huhhtot 010011)

机构地区：[1]内蒙古农牧业科学院,呼和浩特010031 [2]内蒙古农业大学园艺与植物保护学院,呼和浩特010011

出　　处：《生物技术通报》2021年第9期114-124,共11页Biotechnology Bulletin

基　　金：内蒙古自治区自然科学基金项目(2017MS(LH)0312);内蒙古自治区科技计划项目(2020GG0052)。

摘　　要：为加强对向日葵ACC氧化酶基因的利用,以前期从盐诱导的向日葵中克隆的ACC氧化酶基因HaACO1(GenBank accession number.KP966508)为对象,进行了该基因在不同胁迫条件下的表达分析及在烟草中的超表达研究。结果表明,该基因受病原菌、机械损伤、低温、NaCl和水杨酸胁迫诱导表达,且在不同的胁迫下表现出不同的表达模式;HaACO1在向日葵根、下胚轴和叶中均有表达,但在叶中的表达量最高,在根中的表达量最低。利用瞬时表达载体进行亚细胞定位分析,发现HaACO1-GFP在洋葱表皮细胞的细胞质中有表达。构建HaACO1植物表达载体进行过表达分析,表明在含有NaCl的分化培养基上,转基因烟草叶色失绿程度较野生型的轻,分化能力较野生型的高;低温、干旱和NaCl胁迫下,转基因烟草的HaACO1相对表达量高于野生型;NaCl胁迫下,转基因烟草的可溶性蛋白(soluble protein)、脯氨酸(proline,PRO)和叶绿素(chlorophyll)含量及过氧化物酶(peroxidase,POD)和超氧化物歧化酶(superoxide dismutase,SOD)活性提高;脯氨酸合成关键酶基因P5CS及抗氧化相关基因POD、MnSOD和GuZnSOD表达上调。HaACO1过表达提高了烟草的耐盐性,这将为进一步理解向日葵耐盐分子机制以及利用该基因进行作物抗逆性状改良奠定基础。To exploit genes encoding ACC oxidase in sunflower more profitably,we studied one ACC oxidase gene HaACO1(GenBank accession number:KP966508)cloned from previous salt-induced sunflower plants.The expression patterns of HaACO1 under different stress types were investigated.Additionally,the overexpression of this gene in tobacco was carried out.The results showed that the expression of HaACO1 in sunflower was induced by pathogens,mechanical damage,low temperature,NaCl and salicylic acid stress,and expression patterns were distinct under these types of stress.HaACO1 expressed in the roots,hypocotyls and leaves of sunflower plants,with the highest expression in the leaves and the lowest expression in the roots.By taking advantage of a transient expressive vector,the subcellular localization of HaACO1-GFP was found to be largely in the cytoplasm of onion epidermal cells.A plant vector of expressing HaACO1 was constructed to analyze the overexpressing of HaACO1.The results indicated the decrease in green color of leaves of transgenic tobacco was smaller than that of the wild plants placed on differentiation medium with NaCl,while the leaf differentiation was higher than that of the wild plants.Under low temperature,drought and NaCl stresses,the relative expression of HaACO1 in the transgenic tobacco plants was higher than that of the wild plants.During NaCl stress,the contents of total soluble protein,proline and chlorophyll and the activities of peroxidase and superoxide dismutase in the transgenic tobacco plants increased;and the expression of a key gene P5CS for proline biosynthesis and three antioxidant related genes POD,MnSOD and CuZnSOD were all up-regulated.These results indicate that overexpression of HaACO1 in tobacco plants enhances the plants’tolerance to high concentrations of NaCl,providing foundations for further understanding the molecular mechanism of salt tolerance in sunflower,and laying a foundation for using this gene in crop stress resistance improvement.

关 键 词：向日葵 HaACO1 胁迫应答 亚细胞定位 过表达 

分 类 号：S565.5[农业科学—作物学]