【作者】 赵凌；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 HPL(脂氢过氧化物裂解酶)是细胞色素P450家族成员，是植物脂氧化LOX(脂氧合酶)途径的参与酶之一，催化裂解植物LOX的产物-脂氢过氧化物，生成短链醛和含氧酸。HPL的催化产物不仅与植物抗病、抗虫有关，也是植物特异气味的主要成分之一。目前对HPL的研究主要集中于双子叶植物，对于单子叶植物HPL的基因克隆及相关的研究，还鲜有报道。本试验建立了水稻的HPL酶活性测定体系，测定了水稻萌发过程中的HPL酶活性，并进行了水稻HPL基因的克隆及表达研究，结果如下： 1、水稻HPL的酶活性测定 在参考其它植物HPL酶活性测定方法的基础上，建立了水稻的HPL底物发生体系和酶活性测定体系，并测定了不同贮藏性水稻品种萌发过程中的种胚9-HPL、13-HPL活性和不同休眠性水稻品种种子的9-HPL、13-HPL酶活性。结果发现： 萌发过程中，不同水稻品种9-HPL、13-HPL活性都在浸种后2-3天活性最大，然后开始下降： 打破休眠前不同品种种子9-HPL酶活性差异不大，而13-HPL酶活性与休眠性之间存在正相关；打破休眠后品种间13-HPL酶活性差异不大，而9-HPL活性与休眠性呈相关关系。 2、水稻HPL基因的克隆 利用大麦HPL基因作探针，在水稻基因组数据库中搜索到一个高度同源的contig。通过对该contig的核苷酸序列分析、ORF寻找和翻译蛋白的功能分析，推测其中的一个与大麦HPL基因高度同源的ORF是水稻HPL候选基因。利用PCR的方法，克隆到了水稻HPL基因(OsHPL1，Genbank登录号：AY340020)。OsHPL1是第一个被克隆的水稻HPL基因，编码区全长1464bp，没有内元，编码一个488个氨基酸的蛋白质。Southern Blot分析结果表明OsHPL1以多拷贝的方式存在于水稻基因组中。3、水稻月甲乙基因的表达 进一步将‘污为毕忆I基因的完全编码区构建入大肠杆菌体外表达载体pET-30a，IPTG诱导表达后，得到约60KD的融合蛋白，并进一步检测了融合蛋白的生物活性.结果表明:乙枯月尹五I具有较高的13一HPL催化活性.运用RT-PCR的方法，发现051刀〕L了在水稻幼穗、未成熟胚和叶片中表达量较高，茎中表达量较低.通过软件预测，推断Os月甲L了基因起始密码子至一800bp区域是‘污月毕忆I基因的启动子区域.对‘污月，乙1基因的启动子进行了克隆及功能元件分析，结果表明口，月尹乙I基因具有组织特异表达元件，可能受sA、ABA诱导及光调节，并与冷害、千旱等胁迫有关。 关键词:水稻(o桃。:ativ。L化物;Rl’- PCR;启动子HPL酶活性;‘坛石子凭j;基因克隆;脂氢过氧更多还原

【Abstract】 Fatty acid hydroperoxide lyase (HPL) is a novel CytP-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids, and further to produce volatile flavor molecules and potential signal molecules. The aldehydes which formed by HPL are involved in plant resistant to disease and insect herbivores. They are also important constituents of the characteristic flavors of fruits, vegetables, and green leaves. Now the research on HPL focus on dicotyledon, and the research on HPL of monocotyledon still haven’t been reported. In this paper, the research on cloning of rice HPL and enzyme activity assay were carried out and several result were obtained.1. Enzyme activity assay of rice HPLBased on the method of measure HPL Enzyme activity in other plants, the means on preparation of hydroperoxides and measurement rice HPL activity were set up. The 9-HPL arid 13-HPL activity of several rice varieties with different storability and different dormancy were tested. The results showed that:9-HPL has maximum activity in seeds in 2-3 days after germination, and then began to decrease. The tendency is the same as LOX activity in germination stage.9-HPL activity has no remarkable change in seeds between varieties during dormancy, but 13-HPL activity is positive correlativity with dormancy. After dormancy was broken, 13-HPL activity become no remarkable difference among varieties, but 9-HPL activity is positive correlativity with dormancy.2. Cloning of rice HPL geneUsing a polymerase chain reaction-based cloning strategy, we isolated a HPL gene from rice (OsHPLl) that is high homologous to a recently cloned HPL from barley. OsHPLl was the first HPL gene isolated from rice, it’s CDS is 1461bp, encodes a putative55KD protein. The deduced protein sequence indicates that this gene encodes a cytochrome P-450.Furthermore, OsHPLl was cloned into an expression vector pET-30a and then expressed in Escherichia coli induced by IPTG. The recombinant enzyme showed to act on both 13-and 9-hydroperoxides, with a preference for the former. The result of southern blot indicated that OsHPLl has several copies in rice genomic. Expression profile analysis using RT-PCR indicated that OsHPLl was expressed higher in floral and embryo, lower in stem than other tissues. The 500bp upstream of first start codon was isolated, and be predicted as promoter by software. Sequence analysis of promoter showed that the tissue specific expression motif was found. Because there are answer motif in promoter sequence, OsHPLl maybe be regulated by SA, ABA and has relation with cold and drought stress.更多还原