【作者】 孙姝璟；

【导师】 张红生；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 水稻是重要的粮食作物,也是单子叶植物分子生物学研究的模式植物。干旱、高盐和低温等非生物逆境胁迫严重影响植物的生长发育和作物高产潜力的发挥。迄今为止,人们已经从不同植物中分离和鉴定了多种胁迫应答相关的基因,这些基因的编码产物可能直接保护植物细胞免受非生物逆境胁迫的伤害,或者通过调控其他基因的表达提高植物对非生物逆境胁迫的耐受性。本实验室前期已经从水稻中克隆了两个与非生物逆境胁迫相关的TFⅢA型锌指蛋白基因ZFP179和ZFP182。本研究在已有研究基础上,进一步研究了ZFP179和ZFP182在提高水稻耐非生物逆境胁迫过程中的作用机理。主要研究结果如下：1、用real-time PCR方法检测了水稻锌指蛋白基因ZFP179在水稻中的表达情况,结果表明ZFP179在成株期水稻根、茎、叶、幼穗和开花期穗等不同组织中均有表达,在幼穗中的表达量最高；NaCl、PEG6000、低温及ABA等处理能提高了水稻幼苗中ZFP179的表达量；利用酵母单杂交体系分析了ZFP179的转录激活活性；用农杆菌介导的转化技术获得了过量表达ZFP179的水稻转基因植株。对T3代转基因水稻幼苗进行了耐逆性鉴定,发现过量表达ZFPl79的转基因植株增强了对盐胁迫的耐受性,对外源ABA的处理表现更加敏感；在盐胁迫处理下,过量表达ZFP179的转基因植株能比野生型植株积累更多的脯氨酸和可溶性糖,能够促进一些胁：迫相关基因如OsDREB2A、OsLea3、OsPSCS和OsProT的上调表达,同时转基因植株也增强了清除活性氧的能力。基于以上研究结果,我们推测在盐胁迫条件下,锌指蛋白ZFP179可能通过了依赖于ABA的信号转导途径促进了渗透性保护物质如脯氨酸、可溶性糖和LEA蛋白等的积累,同时ZFP179也可以通过调控转录因子基因OsDREB2A的表达,进而通过不依赖于ABA的信号转导途径对胁迫相关基因的表达进行调节。2、将非生物逆境胁迫相关的TFIIIA型锌指蛋白基因ZFP182的完整编码序列和两个不同的缺失序列片段分别与酵母GAL4的BD序列融合,采用酵母单杂交体系分析了ZFP182的转录激活活性和转录激活域,结果表明,ZFP182在酵母体内具有转录激活功能,其C端的酸性氨基酸对于其转录激活活性是必需的。进一步通过酵母双杂交实验,以ZFP182N(C端缺失)为诱饵,筛选水稻冷处理的cDNA文库,获得了23个与ZFP182互作的蛋白,包括盐酸脱卤化酶类蛋白、防御反应相关的Dirigent-类蛋白、叶绿素荧光增加蛋白、棉籽糖生物合成蛋白或种子吸胀蛋白(Sipl)、泛素延伸蛋白、RING结构域的C3HC4型锌指蛋白以及核糖体蛋白等。用双分子荧光互补、体外Pull-down技术,对ZFP182与泛素延伸蛋白Z182IP7的互作进行了验证,结果表明ZFP182与Z182IP7蛋白在体外、体内都具有强烈的相互作用。更多还原

【Abstract】 Rice (Oryza sativa L.) is not only one of the most important food crops in the world, but also a model plant for molecular research in monocots. Plant growth and crop productivity are largely affected by environmental stress such as drought, high salinity and low temperature. To date, many stress-related genes have been isolated and characterized from various plant species. These genes may encode products either directly protecting plants cells from abiotic stresses or regulating expression of other genes to enhance plant tolerance to abiotic stresses. Our research focused on two stress responsive TFIIIA type zinc finger proteins ZFP179and ZFP182from rice. The main results were described as follows:The real-time PCR analysis showed that ZFP179was constitutively expressed in leaves, culms, roots and immature and flowering spikes of rice at the adult stage with the highest expression amount in immature spikes, and markedly induced in the seedlings by NaCl, PEG6000, cold and ABA treatments. Yeast one hybrid analysis showed that ZFP179exhibited trans-activation activity in yeast. Transgenic rice plants overexpressing ZFP179showed higher tolerance to high-salt stress, and more sensitivity to exogenous ABA treatment. It was found that transgenic plants increased contents of free proline and soluble sugars after NaCl treatment, and elevated expression of OsDREB2A and some other stress-related genes. It was also found that overexpression of ZFP179enhanced the reactive oxygen species scavenging ability under salt stress and increased the tolerance of rice seedlings to oxidative stress. These results suggested that ZFP179plays a crucial role in plant response to salt stress, and is useful in developing transgenic crops with enhanced tolerance to salt stress.In order to analyze whether ZFP182, a TFIIIA-type zinc finger protein involved in abiotic stress, has the trans-activation activity, the coding region of ZFP182and its two deletion mutants were fused in frame to yeast GAL4binding domain vector and transformed into yeast strain AH109respectively. The results indicated that ZFP182exhibits trans-activation activity, and the C-terminal region enriched in acidic acids was responsible for the transactivation activity. Through a yeast two-hybrid system with ZFP182N as a bait, a cold-treatment rice cDNA library were screened. It was identified that23different proteins Z182IP1～Z182IP23might be interacted with ZFP182. Through BLAST algorithm search in NCBI it was found that the Z182IP1might encode a haloacid dehalogenase-like protein, Z182IP2a AMP-dependent synthetase or ligase protein, Z182IP3a dirigent-like protein, Z182IP4and Z182IP5chloroplast post-illumination chlorophyll fluorescence increase protein, Z182IP6a raffinose synthase or seed imbibition protein, Z182IP7a ubiquitin extension protein, Z182IP8, Z182IP9and Z182IP10ribosomal proteins, Z182IP11a thylakoid formation, ZI82IPI2a DnaJ chaperone C-terminal domain-containing protein, Z182IP13a C3HC4-type zinc finger protein, Z182IP14a metallothionein-like protein, Z182IP15a ATP-dependent helicase, but other eight accessions including Z182IPs, Z182IP16～Z182IP23without the known functional groups. The Z182IP7was selected for further analyzing the interaction with ZFP182through the BiFC and in vitro pull-down assay. It was found that ZFP182can interact with Z182IP7either in vitro or in vivo.更多还原