柽柳锌指蛋白基因ThZFP3的功能研究

发布时间：2018-06-04 16:32

  本文选题：柽柳 + ThZFP3　； 参考：《东北林业大学》2016年硕士论文

【摘要】：ZFP (Zinc Finger Proteins)转录因子是植物中的一大类重要转录调控子,主要在植物生长发育、抗逆境胁迫等方面具有重要的作用。本研究从柽柳转录组数据中鉴定出一条盐胁迫响应的锌指蛋白基因,命名为ThZFP3,该基因全长1335 bp,编码444氨基酸,属于CCCH型锌指蛋白基因家族,编码蛋白相对分子质量48 491.6 Da,理论等电点(PI)为8.08。构建植物表达载体pBI121-GFP-ThZFP3,利用基因枪技术瞬时转化洋葱表皮研究亚细胞定位,结果表明ThZFP3蛋白定位在细胞核中。利用酵母双杂交技术鉴定ThZFP3转录激活结构域,确定其转录激活区位于C端的225～280氨基酸区段。利用实时荧光定量RT-PCR技术分析显示,该基因受高盐、干旱诱导上调表达,暗示ThZFP3可能在柽柳抗旱耐盐过程中发挥重要作用。构建ThZFP3基因的过表达载体,转化拟南芥,获得T3代转基因株系。对野生型和转基因株系拟南芥进行NaCl和Mannitol胁迫处理,胁迫条件下转基因株系的萌发率、根长、鲜重及长势都优于野生型株系,表明ThZFP3基因能提高转基因拟南芥的耐盐性和耐旱性。构建RNAi载体pC5941-ThZFP3,利用瞬时转化技术将农杆菌pROKⅡ-ThZFP3、 pC5941-ThZFP3和pROKⅡ转入柽柳,获得过表达(OE)、抑制表达(RNAi)和对照(Control)转基因植株,分析三种转基因植株在NaCl和Mannitol胁迫下的抗逆能力。DAB、NBT和Evans blue组织化学染色结果显示,过表达植株(OE)细胞内的02-和1-1202含量明显低于比Control和RNAi植株,说明过表达株系受胁迫后细胞膜结构较为完整,受损伤程度低,而抑制表达株系细胞受损伤程度最严重。测定三种转基因植株的失水率、叶绿素含量、PRO含量、H202含量、MDA含量、POD和SOD活性等生理指标,结果显示过表达植株(OE)的失水率、ROS含量和MDA含量相对最低,叶绿素含量、PRO含量、POD活性和SOD活性相对最高;RNAi植株的失水率、ROS含量和MDA含量最高,叶绿素含量、PRO含量、POD和SOD活性相对最低。以上研究表明,ThZFP3能够提高转基因植株活性氧(ROS)清除能力,降低细胞受损伤程度,从而提高植株的抗逆能力。
[Abstract]:ZFP Zinc Finger proteins is a kind of important transcription regulators in plants, which plays an important role in plant growth and development, stress resistance and so on. In this study, a zinc finger protein gene, named ThZFP3, was identified from Tamarix chinensis transcriptional data. The gene is 1335 BP in length and encodes 444-amino acid. It belongs to the CCCH type zinc finger protein gene family. The relative molecular weight of the encoded protein is 48 491.6 Da, and the theoretical isoelectric point (Pi) is 8.08. The plant expression vector pBI121-GFP-ThZFP3 was constructed, and the subcellular localization of onion epidermis was studied by gene gun technique. The results showed that the ThZFP3 protein was located in the nucleus. Yeast two-hybrid technique was used to identify the transcriptional activation domain of ThZFP3. Real-time fluorescence quantitative RT-PCR analysis showed that the gene was overexpressed by high salt and up-regulated by drought, suggesting that ThZFP3 might play an important role in the process of drought and salt tolerance in Tamarix chinensis. Overexpression vector of ThZFP3 gene was constructed and transformed into Arabidopsis thaliana. Under the stress of NaCl and Mannitol, the germination rate, root length, fresh weight and growth of transgenic Arabidopsis were better than those of wild type. The results showed that ThZFP3 gene could improve salt tolerance and drought tolerance of transgenic Arabidopsis thaliana. RNAi vector pC5941-ThZFP3 was constructed. Agrobacterium tumefaciens pROK 鈪，

本文编号：1978056