【作者】 郭晓红；

【导师】 姜静；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2009， 博士

【摘要】 植物遭受非生物胁迫后,活性氧将会大量产生,对植物产生毒害作用。为了清除活性氧植物形成了一些保护机制,主要包括2种:酶促脱毒机制和非酶促清除机制。过氧化物酶是植物酶促脱毒机制中的一种酶,该酶对H₂O₂的亲和力是过氧化氢酶的1000倍,在活性氧的清除中起着重要的作用。本研究通过柽柳过氧化物酶基因特定引物将该基因从柽柳根cDNA文库中分离出来,将其命名为ThPOD。本论文对ThPOD基因的表达调控及基因功能进行了初步研究,主要结论如下:1生物信息学分析表明,ThPOD属于植物第Ⅲ类过氧化物酶超家族成员之一,为典型的分泌型过氧化物酶。2柽柳ThPOD能够对盐、冷、重金属、干旱引起的氧化损伤而产生应答反应,并且其在根中的表达量明显高于叶中的表达量。此外,ABA能够诱导ThPOD基因在根和叶组织中大量表达,推测该基因为ABA依赖型。3ThPOD在大肠杆菌系统中实现了高效表达,并纯化出ThPOD融合蛋白,进而以其为抗原免疫大白兔,获得抗ThPOD的多克隆抗体。该抗体对柽柳过氧化物酶具有特异性反应,应用该抗体分析了在20%PEG和100μmol/L ABA条件下柽柳过氧化物酶基因表达情况。4根据ThPOD的蛋白质结构功能域,构建了两种短肽缺失克隆,即RG/pET32a-ThPOD（aa41-337）（编码植物过氧化物酶功能区）和RG/pET32a-ThPOD（aa73-337）（编码抗坏血酸过氧化物酶功能区）,以及ThPOD全长cDNA的Rosetta gami重组菌。SDS-PAGE、Western blot分析表明,这些重组子表达的特异条带均为目的多肽。重组菌株在NaCl和PEG胁迫条件下的生长曲线和存活率分析表明,在NaCl和PEG胁迫条件下,这些重组蛋白及多肽均能提高大肠杆菌抵御非生物胁迫的能力,其中pET32a-ThPOD（aa73-337）重组蛋白赋予大肠杆菌抵御非生物胁迫的能力最强,RG/pET32a-ThPOD（aa73-337）菌株的存活率分别是对照RG/pET32a的2倍和10倍。5为了进一步验证ThPOD的功能,我们将该基因克隆至含CaMV 35S启动子控制的pROKⅡ植物表达载体。利用农杆菌介导法转化山新杨。共获得卡那霉素抗性株系17个,其中有14个株系经PcR验证为阳性。从14个株系中选择5个株系（TL1,TL3,TL5,TL8,TL12）做进一步的Southern及Northern检测,结果证明该基因已成功地整合到山新杨基因组中,并能转录出相应的产物。将转基因山新杨和非转基因对照植株分别接种到含有150 mmol/L和200 mmol/L NaCl的培养基中培养15 d后,过量表达ThPOD的转基因山新杨植株生长状态好于对照植株。转基因植株叶片抗氧化胁迫分析表明,0.8mol/L H₂O₂胁迫8 h后各转基因株系（除了TL3）叶绿素含量显著地高于非转基因对照植株叶绿素含量;各转基因株系（除了TL3）相对电导率显著低于非转基因对照植株的电导率。由此可见,氧化胁迫条件下,转基因山新杨受害程度明显小于对照,转基因山新杨中过量表达ThPOD在逆境条件下对光系统有保护作用,减轻膜脂过氧化程度,具有更高的抗氧化能力。更多还原

【Abstract】 After suffering from abiotic stresses,the active oxygen would arise in the cells of plants, posing damaging poison to plants.In order to scavenge the active oxygen,plants have engendered some protective mechanics,which mainly included enzymatic detoxication mechanics and non-enzymatic scavenging mechanics.Peroxidases play a key role in the detoxification of reactive oxygen species because they have an almost 1000-fold higher affinity for H₂O₂ when compared to catalases.A gene encoding peroxidase（named ThPOD） was isolated from a T.hispida NaCl-stress root cDNA library.The preliminary research on transcriptional regulation and genetic function of ThPOD gene were studied.The primary conclusions are as follows:1 Bioinformaties analysis of ThPOD showed that this gene belongs to the classⅢperoxidase multigenic family and is typically secretory peroxidase.2 The expression of ThPOD could be induced by abiotic stresses such as cold,salt, drought and exogenous abscisic acid.These findings suggested that ThPOD might be involved in the plant response to environmental stresses and ABA treatment.Moreover,mRNA transcript level of this gene was higher in roots than leaves.3 The ThPOD performed efficient expression in E coli and the fusion protein was purified used as antigen to immune rabbit for subsequent preparing for polyclonal antibody to ThPOD. This antibody specifically responsed to ThPOD.This antibody was applied to analyze the expression of peroxidase in Tamarix hispida under different stress conditions such as 20% PEG and 100/amol/L ABA.4 To elucidate the function of this gene,recombinant plasmids expressing full-length ThPOD as well as ThPOD_{（aa 41-337）}（encoding of the plant peroxidase functional region）,and ThPOD_{（aa 73-337）}（encoding of the ascorbate peroxidase functional region） truncated polypeptides were constructed.SDS-PAGE and Western blot analyses of the fusion proteins revealed that the molecular weights of ThPOD,YhPOD_{（aa41-337）} and ThPOD_{（aa 73-337）} were～57,～50 and～47 kDa,respectively.The survival rate and growth curve assay showed that expression of these polypeptides in host cells could confer protective function against abiotic stresses.Especially,ThPOD_{（aa 73-337）} could confer higher salt and drought tolerance in Escherichia coli than others.The survival ratio of E.coli harboring ThPOD_{（aa 73-337）} Was over 2-and 10-fold higher than that of control E.coli grown on LB plates supplemented with 0.6% NaCl and 20%PEG,respectively.This finding could potentially be used to improve plant tolerance to drought stress via gene transfer.5 To further study the function of the ThPOD gene in high plant,the nucleotide sequence was cloned into pROKⅡvector which could express aimed protein by the control of CaMV 35S promoter.The gene was introduced into popular by Agrobacterium tumefaciens.It was totally obtained 17 independent lines with tolerance to kanamycin.Among the seventeen,the expected 1086 bp amplification product was detected in 14 lines.These five transgenic lines （TL1,TL3,TL5,TL8,TL12） were selected from the fourteen putative transformants for further analysis.The results of Southern and northern blot analysis indicated that ThPOD was expressed in the five transgenic lines,and had integrated into the plant genome and could successfully transcript.The transgenic and thenon-transgenic plants were cultured on the medium with 150 mmol/L NaCl or 200 mmol/L NaCl for 15 days.Growth of transgenic plants was better than non-transgenic plants under Salt-stress conditions.Assay of oxidation stress of transgenic and non-transgenic plant showed that the chlorophyll contents of non-transgenic plants were significantly lower than those of transgenic plants（except for TL3）.The relative electrical conductivity of each transgenic line except for TL3 was significantly lower than that of non-transgenic plants.The experiments reflected that,under oxidative stress,the transgenic plans suffered far less damage than control.The results also reflected that the over-expression of the gene in transgenic plants was beneficial for protecting photo system as well as alleviating oxidative membrane lipid injury which boosted the anti-oxidative ability.更多还原