【作者】 张春蕊；

【导师】 杨传平； 王超；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2017， 硕士

【摘要】 水通道蛋白（aquaporins,AQPs）是一类高效特异转运水分子的整合膜蛋白。盐胁迫环境中,植物调节水分进出细胞的机制主要是由AQPs介导的,它能够通过多种活性调控方式影响细胞膜的通透性,对维持细胞渗透平衡有着重要作用。为了加深对AQPs功能的认识,完善木本植物的耐盐调控机制,本研究拟在盐胁迫下ThTIP时空表达特性、ThTIP上游调控因子以及ThTIP对植物的耐盐生理调节三个方面系统研究柽柳ThTIP基因在盐胁迫应答过程中的功能。本研究从柽柳转录组数据中鉴定出一条盐胁迫响应的水通道蛋白基因,命名为ThTIP,该基因全长759bp,编码252个氨基酸,编码蛋白相对分子质量26099.25Da,理论等电点（PI）为4.6。ThTIP包含6个跨膜区,2个水通道蛋白特有的NPA（Asn-Pro-Ala）单元。通过构建ThTIP植物过表达载体pROKⅡ-ThTIP,获得T₃代转基因拟南芥株系,对转基因及野生型拟南芥株系进行NaCl和Mannitol胁迫,胁迫条件下两个转基因株系的种子萌发率、根长、鲜重及生长势都要优于野生型植株,表明ThTIP能够提高转基因拟南芥的抗旱、耐盐能力。通过构建抑制表达载体pFGC5941-ThTIP,利用瞬时侵染方法将pROKⅡ-ThTIP、pFGC5941-ThTIP及空p ROKⅡ转入柽柳中,获得过表达（OE）、抑制表达（IE）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O₂^-和H₂O₂含量明显低于Control和IE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。叶绿素含量、脯氨酸Pro含量、H₂O₂含量、丙二醛MDA含量、过氧化物酶POD和超氧化物歧化酶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;IE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,ThTIP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。采用启动子5’端系列缺失方法,获得一系列ThTIP启动子5’端缺失片段,分别为N1（-1^-1731 bp）,N2（-1^-1299 bp）,N3（-1^-867bp）,N4（-1^-432bp）,N5（-1^-181 bp）共五个片段,将这五个片段分别替换表达载体pCAMBIA1301上的CaMV 35S启动子,与GUS基因融合构建植物表达载体,并转化拟南芥。GUS染色结果表明,N1-N4片段转基因拟南芥能够被染色,N5区段不能被染色,说明N4-N5之间长度为252 bp的序列（即-181^-432bp）为ThTIP启动子核心区段。PlantCARE在线分析发现该核心区段中含有三个顺式作用元件,这三个元件可能结合上游调控因子进而调控TIP启动子,因此将元件逐一突变,将逐一突变的启动子核心片段构建到pCAMBIA1301-35S::GUS载体上,转化拟南芥,盐胁迫下GUS酶活测定结果表明,ABRE1和ABRE2元件可能是ThTIP启动子上重要的盐胁迫响应顺式作用元件。通过酵母单杂交技术,鉴定ABRE元件与ThABFs结合情况。首先,构建报告载体pHIS2-ABRE和效应载体pGADT7-Rec2-ThABFs的效应载体,利用酵母单杂交技术筛选ABRE元件可能结合的ABF家族基因,结果表明部分ABRE/ABF家族基因ThABF1,ThABF2,ThABF7,ThABF8转录因子能识别并结合于ThTIP启动子上ABRE元件来调控ThTIP基因表达。为了验证酵母单杂交的结果,将ABRE元件和进行3次重复构建到46bp小启动子置换35S后的pCAMBIA1301-46bp报告载体上,pROKⅡ-ThABF1,pROKⅡ-ThABF2,pROKⅡ-ThABF7,pROKⅡ-ThABF8为效应载体,利用农杆菌介导法将报告载体与效应载体瞬时侵染烟草,通过GUS染色来检测能否启动下游GUS基因的表达。结果显示,ThABF1,ThABF2,ThABF7,ThABF8与ABRE元件均有不同程度的结合,从而证实ThABF1,ThABF2,ThABF7,ThABF8转录因子能与ABRE元件互作。更多还原

【Abstract】 Aquaporins（AQPs）are integrated membrane proteins of a class of highly efficient and specific transporting water molecules.In the environment of salt stress,the mechanism of plant regulation of water in and out of cells is mainly mediated by AQPs.It can affect the permeability of cell membrane through various activity regulation methods,which plays an important role in maintaining cell osmotic balance.TIPs（tonoplast intrinsic proteins）are a subfamily of the plant AQP family,located in the vacuole membrane,and thus become the vacuolar membrane intrinsic protein.In order to deepen the understanding of AQPs function,improve the resistance to salt regulation mechanism of woody plants,this study intends to ThTIP under salt stress expression of time and space characteristics,ThTIP upstream regulatory factors and ThTIP of plant salt tolerance physiological regulatory system in three aspects:the research of tamarisk ThTIP gene in the process of salt stress response function.In this study,a aquaporin gene,named ThTIP,was identified from the data of Tamarix chinensis.The gene was 759 bp in length and encoded 252 amino acids.It encodes the protein relative molecular mass 26099.25Da,the theoretical isoelectric point（PI）is 4.6.Amino acid sequence analysis showed that ThTIP contained six transmembrane regions and two water channel protein-specific NPA（Asn-Pro-Ala）units.After constructing the plant overexpression vector of ThTIP,Arabidopsis thaliana was transformed and the transgenic Arabidopsis plants of T₃ generation were stained,The wild type and transgenic lines of Arabidopsis were treated under NaCl and Mannitol.The results showed that compared with WT plants,transgenic lines had higher germination rate,root length,fresh weight and growth.The two lines of the T₃ generation of ThTIP transformed plants displayed significantly improved tolerance to salt and drought.A suppression vector of pFGC5941-ThTIP was constructed.By using transient transformation method,three kinds of transgenic T.hispida plants were generated,including plants overexpressing ThTIP（transformed with pROKⅡ-ThTIP）,RNAi-silencing ThTIP plants（transformed with pFGC5941-ThTIP）,and control plants（transformed with empty pROKⅡ）.DAB,NBT,and Evans Blue staining showed that compared with control plants and RNAi-silenced ThTIP plants,plants overexpressing ThTIP has lowest ROS content,and suffers lowest cell membrane damage.These results suggested that overexpression of ThTIP significantly improved abiotic stress tolerance of plants.Physiological indexes sush as water loss rate,chlorophyll content,proline content,H₂O₂ content,MDA content,POD and SOD activity were measured to analysis the stress tolerance in the three kinds of plants.The results showed that,the water loss rate,ROS and MDA content in transgenic plants overexpressing ThTIP were lowest,and the overexpression plants showed highly increased chlorophyll content,proline content,SOD and POD activity.These Indicated that overexpression of ThTIP in transgenic T.hispida plants increased the reactive oxygen species（ROS）scavenging capability,decreased cell membrane damage,and then improved the plants’tolerance to stress.The deletion fragments of the 5’end of the ThTIP promoter were obtained by a set of 5’deletion method,named N1（-1^-1731 bp）,N2（-1^-1299 bp）,N3（-1⁸67bp）,N4（-1^-432bp）and N5（-1¹81 bp）.The five fragments were replaced with the CaMV 35S promoter on the expression vector pCAMBIA1301,and the plant expression was constructed with GUS gene Vector,and transformed Arabidopsis thaliana.The results of GUS staining showed that N1-N4 fragment could be stained blue,but the fragment of N5 could not be stained,that means the 251 bp（-181^-432bp）sequence of N4-N5 was the core region of ThTIP promoter segment.There are three cis-acting elements in the core segment by Plant CARE online analysis,which may be combined with the upstream regulatory factors and then control the TIP promoter,so we mutatied of the core fragment promoter one by one and then built to pCAMBIA1301-35S::GUS vector.The transformation of Arabidopsis thaliana under salt stress showed that the ABRE1 and ABRE2 element might be an important response on the ThTIP promoter under salt stress.The binding of ABRE and ThABFs was identified by yeast single hybridization.First,the effect vector of pHIS2-ABRE and the effect vector pGADT7-Rec2-ThABFs was constructed.The ABF family gene of ABRE was screened by yeast single hybridization.The results showed that some ABRE/ABF family genes ThABF1,ThABF2,ThABF7,ThABF8 recognizes and binds to the ABRE element of the ThTIP promoter to regulate ThTIP gene expression.In order to verify the results of yeast single hybridization,the ABRE element was reconstructed on a pCAMBIA1301-46bp reporter vector with 35bp small promoter substitutions for 35S,and pROKⅡ-ThABF1,pROKⅡ-ThABF2,pROKⅡ-ThABF7,pROKⅡ-ThABF8 was constructed as the expression vector of GUS gene,and then transiently infect tobacco with reporter vector and effect vector by Agrobacterium tumefaciens-mediated method.The expression of GUS was detected,the results showed that ThABF1,ThABF2,ThABF7,ThABF8 and ABRE were differentially bound with ABRE elements,which confirm that ThABF1,ThABF2,ThABF7and ThABF8 transcription factors could interact with ABRE elements.更多还原