【作者】 杨霞；

【导师】 张红生；

【作者基本信息】 南京农业大学 ， 应用植物基因组学， 2007， 博士

【摘要】 以拟南芥金属离子转运体基因AtZIP1（GenBank登录号:AAC24197）的氨基酸序列为信息探针,从水稻（Oryza sativa L.）中分离得到OsZIP7a和OsZIP8两个锌铁转运体基因,OsZIP7a和OsZIP8分别编码384和390个氨基酸残基的产物。OsZIP7a和OsZIP8与ZIP家族成员有着高度的同源性,均包含8个跨膜区,有着高度保守的ZIP结构,在第3和第4跨膜区之间存在一个可变区,可变区富含组氨酸。半定量RT-PCR分析结果表明,OsZIP7a和OsZIP8在水稻根、茎、叶和幼穗等组织中均呈低水平表达;在缺铁处理时,OsZIP7a基因在水稻幼苗根部的表达量增多,而在地上部的表达未明显增多;在缺锌处理的水稻幼苗中,OsZIP8基因的表达量显著增多。构建OsZIP7a::GFP瞬时表达载体,采用农杆菌介导法转化洋葱表皮细胞,结果表明,OsZIP7a::GFP定位于洋葱表皮细胞的质膜上。构建酵母表达载体pFL61-OsZIP7a和pFL61-OsZIP8,利用醋酸锂方法分别转入酵母双突变株fet3fet4DEY1453和zrt1zrt2ZHY3中,设pFL61空载体为阴性对照,分别在低铁和低锌的酵母YPD培养基中进行酵母功能互补实验。结果表明,OsZIP7a和OsZIP8分别互补酵母铁吸收突变株fet3fet4DEY1453和锌吸收突变株zrt1zrt2ZHY3在低铁条件和低锌条件下生长,推测它们可能分别具有吸收和转运胞外铁和锌到胞内的功能。以往研究表明水稻TFⅢA型锌指蛋白基因ZFP182的表达受低温、干旱、高盐和ABA的诱导,ZFP245的表达受低温和干旱的诱导。构建ZFP245::GFP瞬时表达载体,采用农杆菌介导法转化洋葱表皮细胞,发现ZFP245::GFP定位于洋葱表皮细胞的细胞核。将ZFP182和ZFP245基因的启动子区1980bp分别替换35S启动子来驱动pCAMBIA1304载体中的GFP::GUS融合基因,采用农杆菌介导法分别转化水稻中花11和日本晴。组织化学检测表明正常生长条件下,ZFP182基因启动子的转基因水稻中花11中,几乎检测不到GUS报告基因的表达;而ZFP245基因启动子的转基因水稻日本晴的各个组织中,GUS的表达量很高;在这两个基因启动子的转基因水稻叶和根中,GFP的活性均显著受150mM NaCl和低温（4℃）胁迫诱导。通过农杆菌介导法将ZFP182基因及其反义结构分别转化水稻品种中花11,将ZFP245基因及其反义结构分别转化水稻品种日本晴。转基因水稻T₀代植株的农艺性状未发现有明显变化。对转基因水稻的耐逆性分析表明,ZFP182和ZFP245的过量表达提高了转基因水稻T₂代植株的耐盐性、耐冷性和耐旱性;而ZFP182和ZFP245的反义表达减弱了转基因水稻T₂代植株的耐逆性。以水稻中花11（WT）作为对照,对ZFP182过量表达的转基因水稻S25株系的T₂代进行cDNA微阵列分析,发现有163个基因的表达受上调,243个基因的表达受下调。通过NCBI的BLAST检索,推测了受ZFP182转录因子调控的部分基因的功能。更多还原

【Abstract】 In order to isolate ZIP genes from rice,the amino acid sequence of Arabidopsis ZIP transporter gene AtZIP1 was used as a query probe to search Rice Genome Database of China through tBLASTn algorithm program.Two cDNA fragments containing complete ORFs of 1,287bp and 1,207bp were respectively cloned from total RNAs prepared from rice seedlings by the RT-PCR approach,and designated as OsZIP7a（Accession No: AY275180） and OsZIP8（Accession No:AY324148）.The predicted proteins OsZIP7a and OsZIP8 display high similarity to other plant ZIP proteins,with 384 and 390 amino acid residues in length respectively.Both of them contain 8 TM domains and a highly conserved ZIP signature motif,with a histidine-rich region in the variable sequence between TM domainsⅢandⅣin each protein.The semi-quantitative RT-PCR analysis showed that the expression profiles of OsZIP7a and OsZIP8 were constitutively and weakly expressed in roots,culms,leaves and flowering spikes at the adult stage.OsZIP7a was significantly induced by iron-deficiency only in rice roots.OsZIP8 was significantly induced by zinc-deficiency in both roots and shoots of rice seedlings.The fusion protein OsZIP7a::GFP was introduced to onion epidermal cells.The results by histochemical detection showed that the fusion protein was localized in the cytosolic membrane of onion epidermal cells,while the control of pBI121-GFP was distributed throughout the cells.The complementation analysis in yeast transformed with OsZIP7a and OsZIP8 were performed to determine whether OsZIP7a or OsZIP8 had Fe or Zn transporting capacity.When expressed in yeast（Saccharomyces cerevisiae） cells,OsZIP7a reversed the growth defects of the yeast iron-uptake mutant,and OsZIP8 reversed the growth defect of the yeast zinc-uptake mutant.It is suggested that these two proteins might function as iron or zinc transporters in rice respectively.The previous research showed that ZFP182 was up-regulated by cold,drought,high salinity and ABA treatment,ZFP245 up-regulated by cold and drought stress.In this study The fusion protein ZFP245::GFP was introduced to onion epidermal cells.The results showed that the fusion protein ZFP245::GFP was localized in the nucleus of onion epidermal cells,while the control of pBI121-GFP was distributed throughout the cells. About 1,980 bp sequences in the promoter region of the ZFP182 and ZFP245 were respectively fused to GUS::GFP reporter genes in the vector of pCAMBIA1304,and then transformed into rice by Agrobacterium-mediated transformation.Histochemical analysis revealed that the GUS activity of ZFP245 is more than of ZFP182 in the root,culm,leaf and seed of rice transgenic plants under the normal condition.GFP activities in the leaf and root of transgenic plants were increasely induced under 150 mM NaC1 or the cold（4℃） treatment.Transgenic rice plants of cultivars Zhonghua 11 and Nipponbare with overexpression or suppressexpression of both ZFP182 and ZFP245 were respectively generated by Agrobacterium-mediated transformation.There was no significantly difference in agronomic traits between 35S:ZFP182 and 35S:ZFP245 transgenic plants and vector transgenic or non-transgenic plants.Transgenic plants in the T₂ generation were analyzed for abiotic stresses.The results showed that overexpression of ZFP182 and ZFP245 in rice conferred tolerance to salt,cold and drought stress.Microarray analysis was used to characterize the expression profiles of transgenic rice of over-expressing ZFP182 at transcriptional levels.There were 163 up-regulated genes and 243 down-regnlated genes. The possible functions of some genes regulated by ZFP182 transcriptional factor were analyzed through the BLAST（basic local alignment search tool） of NCBI（national center of biological information）.更多还原