向日葵E3泛素连接酶基因的分析定位和表达鉴定

摘要

In this study,utilizing the salt tolerant gene E3 ubiquitin ligase HERC2 sequence in sunflower which acquired previously,we constructed the transient expression vector Cam-35S-HERC2-GFP.Subcellular localization was carried out by transforming onion epidermal cells with particle bombardment.The expression analysis,that of root,hypocotyl and leaf between the salt-tolerant hybrid P50 and the salt-sensitive hybrid P29 under 120 mmol · L-1 NaCl stress,was done by RT-qPCR.Plant expression vector pPZP221-HERC2 was constructed.HERC2 was transferred into tobacco to verify salt-tolerant functionality by Agrobacterium-mediated.The results showed that:(1) subcellular localization showed that HERC2 protein expressed in cell membrane,cytoplasm and nucleus;(2) the expression of HERC2 gene was upregulated between the salt-tolerant hybrid P50 and salt-sensitive hybrid P29 under NaC1 stress,but the expression quantity of the former is higher than that of the latter;(3) the salt tolerance of transgenic HERC2 tobacco was stronger than that of the wild type tobacco.The study laid a foundation for analyzing of salt tolerance mechanism in sunflower and breeding salt-tolerant cultivar.%该研究利用前期获得的向日葵耐盐相关基因E3泛素连接酶基因序列(HERC2),构建瞬时表达载体Cam35S-HERC2 GFP,采用基因枪法转化洋葱表皮细胞进行亚细胞定位;采用RT PCR技术,分析盐胁迫下HERC2在耐盐品种P50和盐敏感品种P29根、下胚轴和叶中的表达差异;构建HERC2植物表达载体pPZP221-HERC2,采用农杆菌介导法将HERC2导入烟草,进行耐盐功能验证.结果表明:(1)HERC2蛋白定位在细胞膜、细胞质和细胞核中.(2)受到NaCl胁迫后,HERC2基因在耐盐品种P50和盐敏感品种P29中均上调表达,但耐盐品种中的表达量较高.(3) HERC2基因的表达,能够提高转基因烟草的耐盐性.该研究结果为进一步解析向日葵对盐胁迫的响应机制,以及耐盐新品种的选育奠定了基础.