【作者】 刘佳；

【导师】 徐秉良；

【作者基本信息】 甘肃农业大学 ， 作物保护， 2013， 博士

【摘要】 本文以有壳美洲南瓜和裸仁美洲南瓜为试材，进行了种皮PAL基因全长序列的克隆，生物信息学及表达分析，研究了植株不同发育时期不同组织部位PAL酶活和PAL基因的表达特性变化，明确了接种灰霉菌后裸仁美洲南瓜植株各个部位防御酶活性变化及其与PAL基因表达水平之间的关系。现取得以下结果：1.利用RT-PCR技术，克隆获得了有壳美洲南瓜（P3，P5）和裸仁美洲南瓜（P13，P15）种皮PAL基因的cDNA片段。分析结果表明P3、P13、P5、P15的PAL基因核苷酸和氨基酸序列与其他植物中已分离的PAL基因有很高的同源性，其氨基酸序列具有PAL-HAL功能域和酶活性中心特征序列（GTITASGDLVPLSYIA），同属于Lyase_I_Like超家族。有壳和裸仁美洲南瓜种皮PAL基因核苷酸序列及其推导的氨基酸序列比对结果显示P3和P13有25个核苷酸位点发生变异，15个位点氨基酸发生变化。P5和P15发生变异的核苷酸位点有20个，P15编码的氨基酸序列的中间部分与P5编码的氨基酸序列完全一致。2.利用RT-PCR，结合RACE技术克隆得到了有壳美洲南瓜（P3）种皮PAL基因的全长序列。将该基因命名为CP-PAL，序列长为1720bp，含有一个1359bp的ORF，可编码452个氨基酸，分子量为48.86KDa，等电点为6.55。通过BlastX比对表明P3种皮PAL基因序列及其氨基酸序列与黄瓜PAL基因及其氨基酸序列的相似性分别为85%和94%。CP-PAL包含PAL-HAL、PLN02457及phe_am_lyase三个结构域及酶活性中心序列（GTITASGDLVPLSYIA），属于Lyase_I_Like超家族。CP-PAL不具有导肽及信号肽，为非跨膜蛋白，可能定位于细胞质及内质网上，属可溶性蛋白。系统进化树分析表明P3种皮PAL基因和黄瓜PAL基因的亲缘关系最近。CP-PAL蛋白三级结构主要以α-螺旋为主要结构元件，β-转角和无规卷曲较少。3.克隆获得裸仁美洲南瓜（P13）种皮PAL基因的全长序列。将该基因命名为CPL-PAL，序列长为2340bp，含有一个2115bp的ORF，可编码704个氨基酸，分子量为77.38KDa，等电点为6.34。P13种皮PAL基因核苷酸序列及其推导的氨基酸序列同黄瓜PAL基因及其氨基酸序列相似性分别为80%和85%。CPL-PAL包含PAL-HAL、PLN02457及phe_am_lyase三个结构域，属于Lyase_I_Like超家族。CPL-PAL蛋白不具有导肽及信号肽，为非跨膜蛋白，可能定位于细胞质、内质网及叶绿体上，为亲水性蛋白。系统进化树表明P13种皮PAL基因和黄瓜PAL基因的亲缘关系最近。三级结构分析可知CPL-PAL蛋白以α-螺旋为主要结构元件，β-转角和无规卷曲较少。4.在整个种皮发育过程中，PAL基因在裸仁美洲南瓜（P13，P15）中的相对表达量均低于其在有壳美洲南瓜（P3，P5）中的相对表达量。PAL基因在P3和P13种皮发育中的呈现相反对应的变化趋势，P3中PAL基因在自交授粉后20d后表达量增加，P13中20d后表达量则下降。有壳和裸仁美洲南瓜PAL基因在叶、茎、根、花瓣中均有表达，其中在叶和花瓣中的相对表达量均较高。有壳美洲南瓜植株不同组织部位PAL酶活及PAL基因的相对表达量在整个生育期均高于裸仁美洲南瓜，叶、茎、根中PAL的酶活性与其对应组织部位的PAL基因的表达量存在协同增加的趋势，均在开花期达到峰值。5.裸仁美洲南瓜抗病和感病品种接种灰霉病菌后，随着发病时间的延长，病情指数增加，植株叶片、叶柄、茎中的叶绿素含量下降，POD、PPO、PAL酶活性上升。抗病品种叶片、叶柄、茎中叶绿素含量、PAL、POD、PPO酶活性均高于感病品种。裸仁美洲南瓜抗病品种叶片、叶柄、茎中PAL基因的相对表达量均显著高于感病品种，叶片和茎中PAL基因达到表达高峰的时间也早于感病品种。灰霉病菌诱导后裸仁美洲南瓜不同组织部位PAL基因的表达水平与PAL酶活性的变化趋势存在差异。更多还原

【Abstract】 Taken the hulled and hull-less Cucurbita pepo as materials，cloned the full-length sequence ofPAL gene respectively from seed coat and analysed the bioinformatics and expression characteristics,studied the variations in PAL activities and PAL gene expression in different tissues of the plant duringthe different development, maked clear the changes of several defense enzyme activities in differentparts of the plant and the relationship with PAL gene expression level after inoculation of Botrytiscinerea. The experimental results are as follows:1. Using RT-PCR technique, the cDNA segment of PAL gene were cloned respectively from seedcoat in hulled (P3, P5) and hull-less (P13, P15) C. pepo. Homology analysis showed that the PALnucleotide sequences and deduced amino acids of P3, P5, P13, P15were highly homologous to otherPAL nucleotide sequences and amino acids from different plant species, the sequences of amino acidshad a PAL-HAL functional domain and PAL enzyme activity centre region (GTITASGDLVPLSYIA),and they were one member of Lyase_I_Like superfamily. Comparing the PAL sequences of hulled andhull-less C. pepo, the result indicated that25nucleotide sites and15amino acid sites of P3and P13were changed;25nucleotide sites of P5and P15were varied, the intermediate amino acid sequences inP15were in full accord with deduced amino acid sequences in P5.2. The PAL gene sequence from seed coat in hulled C. pepo (P3) were obtained by RT-PCR andRACE technique. The full-length sequence of PAL (designated as CP-PAL) was1720bp, andcontained a complete open reading frame (ORF) of1359bp, which encoded452amino acid residues,the molecular weight of this protein was48.86KDa, pI was6.55. Homology analysis showed that thePAL nucleotide sequences and deduced amino acids of P3were highly homologous to Cucumis sativusPAL nucleotide sequences and amino acids, the number were85%and94%respectively. The aminoacid sequences of CP-PAL contained three functional domains of PAL-HAL, PLN02457,phe_am_lyase and the region of PAL enzyme activity centre (GTITASGDLVPLSYIA), and it was onemember of Lyase_I_Like superfamily. The CP-PAL was most likely to be located in the cytoplasm andendoplasmic reticulum, without signal peptide and leader peptide. And it was non-transmembrane andsoluble protein. The neighbor-joining phylogenetic tree indicated that PAL gene in seed coat of P3hada very close relationship with PAL gene in C. sativus. The main structural element in CP-PAL proteintertiary structure was α-Helix, less in β-Turn and random coil.3. The PAL gene sequence from seed coat in hull-less C. pepo (P13) were obtained. The resultsshowed that CPL-PAL cDNA completed sequence contained2340bp and the ORF contained2115bpand coded704amino acids, the molecular weight and pI were predicted as77.38KDa,6.34separately. The PAL nucleotide sequences and deduced amino acids of P13showed the highest homology of80%and85%with C. sativus, respectively. The amino acid sequences of CPL-PAL contained threefunctional domains of PAL-HAL, PLN02457, phe_am_lyase, and was one member of Lyase_I_Likesuperfamily. The CPL-PAL was most likely to be located in the cytoplasm, endoplasmic reticulum andchloroplast, without signal peptide, leader peptide and transmembrane region. And the protein washydrophilic. The neighbor-joining phylogenetic tree indicated that PAL gene in seed coat of P13hadthe most of familiar genetic relationship with PAL gene in C. sativus. The main structural element inCPL-PAL protein tertiary structure was α-Helix, less in β-Turn and random coil.4. In the whole process of seed coat development, the relative expression of PAL gene in hull-lessC. pepo (P13, P15) was lower than the relative expression in hulled C. pepo (P3, P5). Real-time PCRanalysis revealed that PAL gene in seed coat of P13showed the opposite tendency to P3, the relativeexpression of PAL gene in seed coat of P3was increased, but the relative expression in P13wasdecreased after20d of self-pollination. The PAL gene in hulled and hull-less C. pepo were expressed inleaves, stems, roots, petals, the relative expression in the leaves and petals were higher. During thewhole growth period, the activity of PAL and PAL gene expression in different tissues of hulled C. pepowere higher than that of hull-less C. pepo. The PAL activity and PAL gene expression in the leaves,stems and roots had synergistic increase trend, and reached its peak during the flowering period.5. After inoculation of Botrytis cinerea, the disease index of resistant and susceptible varieties ofhull-less C. pepo were increased, the chlorophyll content of leaves, petioles and stems were decreasedand the activities of POD, PPO and PAL were increased with the extension of onset time. Thechlorophyll content, PAL, POD and PPO activity in leaves, petioles and stems of resistant variety werehigher than that of susceptible variety. Real-time PCR results displayed PAL gene expression in leaves,petioles and stems of resistant cultivar were significantly higher than that susceptible cultivar, and thepeak times of PAL gene expression in leaves and stems were earlier than that of susceptible cultivar.There were differences between PAL gene expression level and trend of PAL activity in different tissuesof hull-less C. pepo.更多还原