向日葵E3泛素连接酶基因的分析定位和表达鉴定

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Cam-35S-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。

Subcellular Localization Analysis and Expression Verification of E3 Ubiquitin Ligase Gene HERC2 of Sunflower (Helianthus annuus L.)

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 up regulated between the salt tolerant hybrid P50 and salt sensitive hybrid P29 under NaCl 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.