【摘要】 土壤盐碱化是小麦(Triticum aestivum)生产的主要限制因子之一。锌转运体(zinc transporter,ZTP)属于锌铁调控蛋白(ZRT,IRT-like protein,ZIP)蛋白家族,并参与调控非折叠蛋白应激响应,是一个优良的抗盐基因。本研究采用同源克隆的方法获得了ZTP29在小麦中的直系同源基因TaZTP29,利用生物信息学、qRT-PCR、绿色荧光蛋白示踪技术和转基因拟南芥(Arabidopsis thaliana)过表达的方法明确了TaZTP29的保守域信息、表达模式、亚细胞定位信息和功能。序列分析结果表明,TaZTP29基因(GenBank登录号:KY610283)含有81 bp的5’UTR、834 bp基因编码区及117 bp的3’UTR,编码277个氨基酸。TaZTP29蛋白具有典型的ZIP保守域,具有8个跨膜域,且在第V跨膜域具有完全保守的组氨酸蛋白残基,定位于质膜上,属于ZIP蛋白家族。序列同源性和进化分析表明,TaZTP29与节节麦(Aegilops tauschii)ZIP29蛋白同源性最高;与拟南芥(Arabidopsis thaliana)ZTP29蛋白同源性最远。TaZTP29强烈响应锌和盐胁迫,上调表达,在高温与干旱胁迫下,下调表达;且TaZTP29主要在地下部分的根中表达,在地上部分的组织中表达量较低。抗盐性鉴定表明,在不加NaCl的条件下,野生型与过表达株系总根长基本一致,在100和150 mmol/L NaCl处理的培养基中,3个转基因株系的总跟长显著长于野生型拟南芥,表明过表达株系对NaCl胁迫具有较好的耐性。转入TaZTP29后,在拟南芥中过表达能够提高拟南芥对盐胁迫的抗性,TaZTP29具有抗盐功能,为进一步改良小麦抗逆性等分子机理研究提供了新的依据。更多还原

【Abstract】 High salt is one of the major constraint that adversely affects wheat yield in China.The zinc transporter (ZTP) belongs to the ZRT,IRT-like protein (ZIP) family,and ZTP is involved in the response to salt stress through regulating induced the unfold protein response pathway,zinc levels to induced the unfold protein response pathway.In this study,the TaZTP29,which is the orthologous gene of At ZTP29,was isolated from wheat (Triticum aestivum) by RT-PCR and the expression pattern was analyzed through qRT-PCR.The subcellular localization of TaZTP29 was also investigated through green fluorescent protein (GFP) method.Meanwhile,the salt resistance of TaZTP29 was confirmed by over-expressing of the TaZTP29 in Arabidopsis thaliana.The sequencing analysis showed that TaZTP29 gene (GenBank accession: KY610283) consisted of834 bp CDS,81 bp 5’UTR and 117 bp 3’UTR,and encoded 277 amino acids.The conserved domain analysis demonstrated that TaZTP29 had the typical ZIP domain,which was consisted of 8 transmerabrane domains and the V transmerabrane domain had the completed conserved amino acid histine (His).TaZTP29 was localized to the endoplasmic reticulum with proyien predicted.The subcellular location were validated by means of fusing TaZTP29 with N-terminal GFP and then expressed in wheat protoplast using polyethylene glycol (PEG) transfection method.The green fluorescence was observed in endoplasmic reticulume.The information of conserved domain proved that TaZTP29 belonged to the ZIP protein family.The 16 homologues genes from 16 different species of TaZTP29 were searched and found in NCBI database,and the identity of amino acid between 16 homologous genes and TaZTP29 ranged from 64.03% to 94.5%.The highest identity of amino acid was between Aegilops tauschii (EMT27056.1) and TaZTP29,while the lowest identity of amino acid was between Arabidopsis thaliana (Q940Q3.1) and TaZTP29.Then phylogenetic tree of TaZTP29 was constructed using these 16 genes and TaZTP29.The expression pattern disclosed the expression level of TaZTP29 was increased immediately under Zn Cl2 treatment and NaCl stress.However,TaZTP29 in response to heat stress was passive.When wheat suffered heat stress and drought stress,expression of TaZTP29 dropped rapidly.Tissue expression pattern analysis showed that TaZTP29 expressed in multiple tissues.Root accumulated higher TaZTP29 transcription when comparing to the other tissues of ground.The seed germination rate and total root length of wild type was similar with that of TaZTP29 transgenic Arabidopsis lines in normal growth condition.While,the seed germination rate and total root length of TaZTP29 transgenic lines were significantly higher and longer than those of wild-type plants under salt treatments (100 and 150 mmol/L NaCl),respectively.These results indicated that TaZTP29 transgenic lines had higher salt tolerance than wild type,and TaZTP29 could increase the salt tolerance of Arabidopsis thaliana,and could be used as a candidate gene for salt tolerance.This study provides a new foundation for further understanding the molecular mechanism of wheat stress resistance.更多还原