摘要
SlDREB2基因是利用rd29A基因启动子的DRE元件通过酵母单杂交技术从番茄幼苗(丽春)cDNA文库中筛选得到的属于EREBP家族的一个转录因子基因。利用构建病毒VIGS载体,分别用含pBINTRB6、pTV00::SlDREB2-1和pTV00::SlDREB2-2重组载体的农杆菌GV3101菌液灌根与叶片注射法侵染番茄植株,转化7 d后进行病毒DNA检测与验证。测定了干旱胁迫下侵染成功的番茄植株与野生型株系的生理指标,并通过Real-time qPCR分析了VIGS介导的SlDREB2转录因子基因的表达特征。番茄植株干旱3周胁迫处理后测定结果表明,野生番茄中脯氨酸和可溶性糖水平要高于被VIGS病毒侵染的番茄植株,而丙二醛水平要低于被侵染植株,并且指标显示被pTV00::SlDREB2-2重组载体的农杆菌GV3101菌液侵染的番茄植株抗旱性明显降低;复水1周后,受VIGS浸染株系丙二醛含量明显高于野生型,而可溶性糖与脯氨酸累积却低于野生型。Real-time qPCR结果表明,经VIGS病毒侵染过的番茄植株在胁迫处理时期SlDREB2基因的相对表达量都明显低于野生型株系,表明番茄SlDREB2基因保守域末端459 bp的特异片段具有较显著的沉默效果,说明VIGS介导的SlDREB2基因的表达受干旱诱导,该基因可以作为抗旱型作物品种改良的有价值基因。
SIDREB2 gene was isolated by yeast one-hybrid system using DRE element of rd29A promoter through hybridization with tomato seedlings （ Lichun ） eDNA library, and it is a transcription factor belonging to EREBP family. In this study, the VIGS virus vectors were constructed, and transformed into the GV3101 Agrobacterium strains, and the resulting reeombinants carrying pBINTRB6, pTVOO-SIDREB2-1 and pTVOO-SIDREB2-2 vectors were used to infect leaves of tomato plants respectively by injection of liquid filling respectively. All the plants infected after 7 days were identified by viral DNA testing and the physiological index of wild-type strains of tomato plants as well as infected plants successfully were determined under drought stress for three weeks, and Real-time qPCR was performed to analyze expression profile of the SlDREB2 transcription factor. Data showed that the contents of prolinc and soluble sugar in wild tomato plants were higher than that of tomato plants infected hy VIGS virus, and malondialdehyde levels in wild plants were lower than that of plants infected hy VIGS virus. When all tomato plants stressed by drought for three weeks were re-watered for one week, the contents of proline and soluble sugar in wild tomato plants were lower than that of tomato plants by VIGS virus infection, and the contents of malondialdehyde in wild plants were higher than that of plants infected by VIGS virus infection. Real-time qPCR results indicates that the expression of SlDREB2 in plants by VIGS virus infection was lower significantly than that of in wild type plants under drought stress, indicating that 500 bp of specific fragment at the end of SlDREB2 gene conservative domain has better silence effect, and the expression of SlDREB2 mediated by VIGS was inhibited by drought stress, and indirectly implying that the SIDREB2 gene can be a valuable candidate gene for improving crop varieties with drought resistance.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第12期93-100,共8页
Biotechnology Bulletin
基金
国家转基因重大专项(2011ZX002-5)
