华东葡萄抗逆相关基因VpSBP16原核表达及功能初步分析  

Prokaryotic Expression and Preliminary Function Analysis of Stress Tolerance Related Gene VpSBP16 from Vitis pseudoreticulata

作　　者：柴生樾 王浩[1,2] 王西平[1,2] CHAI Shengyue;WANG Hao;WANG Xiping(College of Horticulture,Northwest A&F University,Yangling,Shaanxi 712100,China;State Key Laboratory of Crop Stress Biology in Arid Areas,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100 [2]旱区作物逆境生物学国家重点实验室,西北农林科技大学,陕西杨凌712100

出　　处：《陕西农业科学》2022年第3期78-84,共7页Shaanxi Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31872071、30671446);基本科研业务费科技创新专项重点项目(2452019170)。

摘　　要：SBP-box基因在植物响应逆境胁迫中起着非常重要的作用,通过制备华东葡萄(V.pseudoreticulata)转录因子VpSBP16基因抗血清,为深入探究葡萄抗逆相关基因VpSBP16的功能试验提供基础。将已构建的pGEX-VpSBP16原核表达载体转入大肠杆菌中,利用IPTG诱导表达的蛋白作为抗原注射新西兰兔,从而获得VpSBP16基因抗血清,利用Western blot检测手段,以所获得的VpSBP16基因抗血清作为一抗检测毛葡萄‘商-24’叶、茎、花序、卷须和果实不同发育时期的VpSBP16蛋白表达情况。结果表明,原核表达载体pGEX-VpSBP16在大肠杆菌菌株DE3中高效表达,Western blot检测出与预期结果一致的分子量约为87kD的GST-VpSBP16融合蛋白。在新西兰兔的背部及腹股沟注射纯化后的目的融合蛋白获得了免疫-抗原反应良的多克隆抗血清。提取葡萄总蛋白后检测到VpSBP16蛋白表达量在花序中含量最低,而在果实发育的绿果期表达量较高。大肠杆菌表达系统中,pGEX-VpSBP16融合蛋白可以在27℃、0.6 mmol/L IPTG诱导条件下获得高表达。因此,笔者研究制备的多克隆抗血清具备效价高、特异性强的特点。VpSBP16基因不仅增强植物非生物胁迫的抗性,也可能会影响葡萄花发育和葡萄果实成熟,参与调节葡萄从营养生长到生殖生长的转变。The SBP-box gene plays a very important role in plant response to stress. The objective of this study is to prepare antiserum of the transcription factor gene VpSBP16 from Vitis pseudoreticulata for providing a basis on the function of stress resistance related gene VpSBP16. The constructed pGEX-VpSBP16 prokaryotic expression vector was transferred into E. coli, and the protein induced by IPTG was used as an antigen to inject New Zealand rabbits to obtain VpSBP16 gene antiserum. Western blot was used to detect the expression of VpSBP16 protein in leaves, stems, inflorescence, tendrils and berries at different development stages of Vitis quinquangularis ‘Shang-24’. Prokaryotic expression vector pGEX-VpSBP16 was efficiently expressed in DE3, and Western blot detected GST-VPSBP16 fusion protein with molecular weight of about 87 kD was consistent with the expected results. The purified target fusion protein was injected into the back and groin of New Zealand rabbits to obtain polyclonal antiserum with good immune-antigen response. After extracting total grape protein, the expression of VpSBP16 protein was the lowest in the inflorescence, but the expression was higher at the green berry stage of berry development. In the expression system of E. coli, pGEX-VPSBP16 fusion protein could be highly expressed under the conditions of 27℃ and 0.6 mmol/L IPTG induction, and the prepared polyclonal antiserum had high titer and specificity. The VpSBP16 gene not only enhances the resistance of plants to abiotic stress, it is likely to affect the development of grape flower or grape berry ripening, and participate in the regulation of the transition from vegetative growth to reproductive growth of grapes.

关 键 词：葡萄 VpSBP16 抗血清 蛋白表达 

分 类 号：S436[农业科学—农业昆虫与害虫防治]