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柽柳THNAC7基因响应高盐胁迫的调控机理研究
霍琳
学位类型硕士
导师王玉成
2016
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词柽柳 Nac转录因子 抗逆 转录激活 瞬时表达
摘要NAC转录因子是植物所特有的并广泛存在于植物当中的一个超家族。其典型的特征是N端含有一个高度保守的NAC结构域,并且在其C端存在一个多变的转录调节区域。NAC转录因子参与包括花的发育、次生壁的生成以及细胞分裂、茎顶端分生组织的形成以及叶片衰老在内的多个过程,同时也在生物和非生物胁迫响应的过程当中发挥重要的作用。 本实验室从柽柳当中鉴定出一条响应盐胁迫的NAC基因,命名为ThNAC7(GenBank number:JQ974961)。此基础上,本研究首先通过转录激活实验证明,ThNAC7转录因子具有转录激活活性,其转录激活结构域由位于C端的连续三个独立的区域组成;然后通过构建了35S:ThNAC7过表达载体,并将其转化到拟南芥中,获得过表达ThNAC7的T3代转基因拟南芥幼苗。对其中的2个转基因株系(OE2和OE5)和野生型(WT)拟南芥进行NaCl和Mannitol胁迫,并对拟南芥的根长和鲜重等进行了统计与分析。结果表明,过表达株系的抗旱和耐盐能力明显强于野生型植株,表明过表达ThNAC7基因提高了转基因拟南芥的抗旱和耐盐能力;对OE2、OE5和WT进行100 mM NaCl胁迫,通过对SOD活性、POD活性、MDA以及叶绿素含量等生理指标的测定与分析,结果显示过表达株系OE2和OE5的SOD和POD酶的活性显著比WT高,MDA也比WT低,说明在盐胁迫下,过表达ThNAC7基因的细胞膜完整性好于野生型对照,进一步说明ThNAC7转基因拟南芥的耐盐能力较野生型显著提高。 为了快速鉴定基因的抗逆功能,本研究在已建立瞬时转化系统的基础上,以整株烟草为试材,通过分析不同菌液浓度、乙酰丁香酮浓度、Tween20浓度、蔗糖浓度、不同浸染时间、及有无激素对GUS基因表达的影响,研究其瞬时表达的最佳条件。结果显示:用OD600值为0.2的农杆菌侵染3 h,在1/2 MS培养基中添加120 μM的乙酰丁香酮,1.5 mg/L KT,0.5 mg/L NAA,3%的蔗糖,共培养3 d,能够得到高效的GUS基因瞬时表达。该体系的建立为利用瞬时转化体系快速分析柽柳基因的抗逆功能奠定了基础。
其他摘要NAC (NAM, ATAF1/2 and CUC2) transcription factors, which distribute widely in many land plant species, constitute one of the largest families of plant-specific transcription factors. The NAC family members have a highly conserved NAC domain in N-terminal ends with about 150 amino acids and a variable transcriptional regulatory region in the C-terminal region. NAC TFs have been found to participate in various processes, including flower development, formation of secondary walls and cell division, shoot apical meristem formation, leaf senescence, as well as biotic and abiotic stress responses. In our previous study, a NAC gene from Tamarix hispida that is respond to salt stress was identified, and named ThNAC7 (GenBank number: JQ974961). Yeast two-hybrid analysis revealed that ThNAC7 has transcription activity, and the transcription activity domain locates in its C-terminal constituted by three independent transcription activity domains. The 35S:ThNAC7 expression vector was constructed and transformed into Arabidopsis, and T3 transgenic lines with overexpressing ThNAC7 was used in study. Measurement of the length of roots and fresh weight showed that the ThNAC7 transformed plants displayed significantly improved tolerance to NaCl and Mannitol compared with WT under NaCl and Mannitol. Additionally, SOD and POD activity, and chlorophyll content in OE2, OE5 were significantly higher than WT, but MDA content in OE2 and 5 lines were significantly lower than WT when exposed to 100 mM NaCl, furter suggested that ThNAC7 could confer stress tolerance to transgenic plants. In order to build a method that could identify the stress tolerance of genes quikly, a transient genetic transformation was established with the whole tobacco (Nicotiana tabacum L.) seedlings as explants. Effects of A. tumefaciens concentration, acetosyringone concentration, Tween20 concentration, sucrose concentration, infection time, and hormone on the transient expression of GUS gene were analysed to determine the optimal transformation condition. A optimal transient genetic transformation condtions were: OD600 value of A. tumefaciens for infiltration 0.2, infiltrating for 3 h, adding 120 μM acetosyringone, 1.5 mg/L KT, 0.5 mg/L NAA, and 3% sucrose to co-culture medium, and co- culture for 3 days
学科领域生物工程
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14694
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
推荐引用方式
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霍琳. 柽柳THNAC7基因响应高盐胁迫的调控机理研究[D]. 北京. 中国科学院大学,2016.
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