【作者】 姜翠翠；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2012， 博士

【摘要】 （木奈）（Prunus salicina Lindli. var cordata J.Y.Zhang et al.）属蔷薇科（Rosaceae），李属（Prunus），是原产福建的名、特、优水果之一，果桃形李实，酸甜适度，风味极佳，是南方的一种重要水果。但在果实采收以及贮藏期间，发现了果实褐变现象。发生褐变的（木奈）果果实，虽然外观表现正常，但是果肉已经发生褐变，以果腔附近的果肉褐变尤为严重，这种果肉褐变在采前和采后贮藏过程中均会发生，导致果实的食用品质和耐贮藏性下降，甚至丧失其商品价值，严重影响（木奈）果的市场声誉，给生产带来极大的经济损失。因此，（木奈）果肉褐变问题成为制约（木奈）果产业发展的瓶颈。本研究以油（木奈）褐变初期的果肉为试材，应用SMART（Switching Mechanism At5′end of the RNA Transcript）建库技术和DSN（Duplex-Specific Nuclease）均一化处理相结合的方法，构建了油（木奈）褐变果实均一化全长cDNA文库，进行了大规模的5′EST测序，并进行基因功能注释，根据所有EST基因的功能预测和前人对果实褐变的研究进展及结果，选取了6个可能与（木奈）果实褐变相关的基因，编号为：NFH-102、NFH-C23、NFH-F21、NFH-17、NFH-633、NFH-99，并对这些基因进行了生物信息学分析，采用染色体步移技术分离这些基因的启动子，并对启动子序列进行分析，同时，采用Real-time PCR技术分析了这些基因在（木奈）果实采后各种生物和非生物胁迫下的表达模式，根据这些研究结果，可以初步推测，（木奈）果实褐变过程是一个复杂的基因调控网络。主要研究结果如下：1.应用SMART建库技术和DSN均一化处理相结合的方法，构建了油（木奈）果实褐变初期的均一化全长cDNA文库，经检测，该文库的初始滴度为2.0×106pfu/ml，重组率为99%，插入片段平均大小为1.8kb，文库质量良好。随机挑取720个阳性克隆进行5′EST测序，经序列拼接、去掉污染序列，突变移码序列后，共获得684条有效的ESTs（Expressed SequenceTags）序列，对该684条有效序列进行拼接后，58条被组装成21个重叠群（contigs），单拷贝（singlets）基因有626条，共得到647个单基因簇，文库冗余率为8.4%。用Blast2go在线软件对其测序结果进行注释和归类分析，已知功能基因与能量代谢、蛋白质合成与降解、次生代谢物质、细胞壁代谢和转录因子有关。2.根据候选基因的功能预测以及前人对果实褐变的研究结果，本研究选取6个可能与（木奈）果实褐变相关的基因（苯丙氨酸解氨酶基因、膨胀素1，2，3基因、NAC和WRKY类转录因子），分别从基因生物信息学分析、启动子分离以及（木奈）果实在采后生物和非生物胁迫下的表达模式进行了研究。具体结果如下：（1）本试验分离的苯丙氨酸解氨酶，全长2497bp，包含2154bp的开放阅读框，编码718个氨基酸，蛋白质分子量为78kDa，理论等电点为6.6，包含119bp的5′UTR和224bp的3′UTR。系统进化树分析表明，与蔷薇科樱桃属于同一簇，具有苯丙氨酸解氨酶-组氨酸解氨酶（PAL-HAL）和苯丙氨酸解氨酶（PAL）保守区域。在各种胁迫和乙烯处理条件下，与对照相比，PsPAL在转录水平上呈现不同程度的差异表达。在响应机械损伤和低温处理后，PsPAL呈明显的上调表达趋势；高温和无氧处理后该基因呈先上升后下降的趋势；乙烯处理后，PsPAL呈上调-下调-上调的趋势。（2）本研究从均一化全长cDNA文库中分离得到3个膨胀素基因，分别命名为PsEXP1、PsEXP2和PsEXP3，基因登录号分别为：JN675711, JN675712和JN675713。A：3个基因分别长1303bp，1392bp和1384bp，开放阅读框为759bp,780bp和759bp，编码253到260个氨基酸，其分子量为26.76kDa,27.99kDa和26.80kDa，理论等电点为7.3,9.5和9.2；B：同源性比对分析表明，共含有8个保守的半胱氨酸残基和4个色氨酸残基，并含有一个保守的HFD域，这个区域可能是45-家族内葡糖基酶的一个催化位点；系统进化树分析表明，PsEXP1，PsEXP2和PsEXP3分别落入α-膨胀素不同的亚家族，PsEXP1包含在B亚族，PsEXP2包含在A亚族，PsEXP3包含在C亚族，不同的亚族具有不同的生物功能；对3个基因进行基因组DNA序列扩增表明，都含有3个外显子和2个内含子；C：分别获得PsEXP1，PsEXP2和PsEXP3上游899bp，744bp和596bp的调控序列，均含有启动子的典型结构特征，如TATA-box，CAAT-box等，同时都包含有厌氧诱导元件、胚乳表达元件和光反应元件；D：对3个膨胀素基因在其果实发育的不同时期以及果实采后各种胁迫处理下的表达模式分析，结果表明，除了PsEXP1在花后1周即果实刚发育时期没有表达外，3个基因在（木奈）果整个生长发育期都有表达；3个基因的表达模式分为3种：（a）：PsEXP1基因的表达呈下调-上调的表达模式；（b）：PsEXP2基因的表达呈上调-下调-上调的表达模式；（c）：PsEXP3基因的表达呈上调-下调的表达模式；E:3个基因在（木奈）果实采收各种胁迫下的表达模式，3个基因受乙烯和损伤的诱导表达，不受高、低温以及无氧的诱导。（3）经均一化全长cDNA文库5′EST测序，发现编号为NFH-633的序列与苹果的MdNAC基因同源性很高，我们将其命名为PsNAC，基因登录号为：JN675710。其序列包含1077bp的开放阅读框，编码359个氨基酸，蛋白质分子量为39kDa，理论等电点为9.14。用半定量分析表明，该基因在（木奈）褐变果实中表达而在正常果实中未表达。与苹果、杨树、脐橙的NAC基因的氨基酸序列同源性均有超过60%的同源性；蛋白结构域分析表明，PsNAC基因编码的氨基酸序列N末端含有可能包含DNA结合域的NAC类转录因子的保守序列（NA-D）；同源进化树分析PsNAC属于NAC家族的NAP亚类，NAP亚类的基因大多数与植物的衰老有关；酵母试验研究表明，该基因在C末端具有转录激活活性；通过亚细胞定位分析该基因定位于细胞核中；在（木奈） PsNAC基因的5′上游获得825bp的调控序列，这些序列经在线软件PlantCARE进行分析，都含有启动子的典型的结构特征，如TATA-box，CAAT-box等，还含有厌氧诱导元件、与干旱诱导相关的元件（MYB元件，TAACTG）、低温相关的元件（LTR core element，CCGAAA），全部的这些元件可能与胁迫和逆境相关；Real-time PCR检测PsNAC基因在机械损伤、乙烯、无氧处理下呈上调表达；而在高温处理下呈下调表达；在低温处理下无明显变化，特别是在乙烯诱导下，PsNAC基因表达量明显增强。（4）WRKY类转录因子是从文库中筛选到的另一个转录因子，将其命名为PsWRKY基因，登录号为：JN675708。其序列长1836bp，包含1599bp的开放阅读框，编码533个氨基酸，蛋白质分子量为58kDa，理论等电点为8.3。用半定量分析表明，该基因在（木奈）褐变果实中的表达量比正常果实中高。该基因编码的氨基酸序列具有WRKY家族的特征，含有两个WRKY保守结构域，两个C2-HC型（C-X4-C-X22-H-X1-H）和（C-X4-C-X23-H-X1-H）锌指结构域和一个预测核定位信号PKRR。酵母试验研究表明，PsWRKY具有转录激活活性；通过亚细胞定位分析该基因定位于细胞核中；在PsWRKY基因的5′上游获得916bp的调控序列，这些序列经在线软件PlantCARE进行分析，都含有启动子的典型的结构特征，如TATA-box，CAAT-box等，激素响应元件（茉莉酸甲酯）以及抗病相关的元件（W-Box）；Real-time PCR检测PsWRKY基因在机械损伤和高温处理下呈下调表达，在乙烯、无氧、低温处理下呈上调表达。3.从（木奈）成熟叶片均一化全长cDNA文库中新分离得到了一个PPO基因，命名为PsPPO2，基因登录号为：JF681036.1。经NCBI-BLAST分析，其核苷酸序列与蔷薇科果树中的河北鸭梨和富士苹果果实的PPO基因具有很高的同源性，分别为81%和80%；与作者先前分离克隆的PsPPO1和PsPPO3基因同源性分别为55%和56%，说明此克隆是PPO基因家族的新成员。通过RT-PCR分析,PsPPO1,PsPPO2和PsPPO这3个基因在（木奈）不同生长发育时期和果实受损伤后的表达模式。在叶片发育过程中，PsPPO2表达量都很高；PsPPO1在嫩芽和幼叶中表达；PsPPO3只在嫩芽中表达；在果实发育的不同时期，PsPPO2和PsPPO3在早期表达，随着果实成熟表达量下降，PsPPO1在褐变果中表达量较高。受机械损伤后，PsPPO1受诱导，而PsPPO2和PsPPO3不受诱导。更多还原

【Abstract】 “Nai”(Prunus salicina) belongs to the prunus family, Rosaceae, and believed tobe originated from FuJian province, southern of China. Nai is one of the fomous,special and excellent fruits which has the shape of peach and the pulp of plum, themediate sweet and sour taste. Fruit browning always occur at the stage of fruit harvestand storage. The browning fruit has normal appearance, but brown pulp, especiallythe pulp near the fruit cavity. Fruit browning not only deteriorate fruit edible qualityand storage property, but also deprive of the value of commodities, seriouslyinfluenced the market reputation of Nai fruit and economic benifit. So, the issue of thefruit browning has become the bottleneck of industry development of Nai fruit.This research took Oil Nai fruit at early stage of fruit browning as material,combined the SMART library-construction technology and DSN homogenizationmethod, constructed a full-length normalized cDNA library of Oil Nai fruit withbrowning. After massive5’EST sequencing and function annotation analysis, theselection of six genes (numbered NFH-102, NFH-C23, NFH-F21, NFH-17,NFH-633and NFH-99, respectively) possibly related to Oil Nai fruit browning havebeen undergone bioinformatics analysis, promoter cloning analysis, Real-time PCRanalysis and the various gene expression of these genes under different biotic stressand abiotic stress, according to the result of EST gene function anticipation andprevious research related to fruit browning. The objectives were to understand themolecular mechanisms of Oil Nai fruit browning.1Total RNA was extracted from the fruit of Oil Nai with beginning of browning,then reverse transcribed into cDNA by two-step amended SMARTTMcDNAsynthesized method which has higher proportion of full-length cDNA. The full-lengthnormalized cDNA library was constructed by the combination of long distance PCR,DSN (Duplex-Specific Nuclease) treatment and directed cloning. The titer of primarylibrary was about2.0×106pfu/ml and99%clones were recombinant, and the lengthof insert cDNAs were about1.5kb. The cDNA library was well normalized with8.4%redundancy. A total of720random clones in this cDNA library were thensequenced by5’EST. A total of valid684ESTs were attained by fragment assembly,removing the contaminated sequences and frameshift mutation. Then647unigenes were attained, which included21contigs and626singlets. Annotation by the onlineBlast2go search against GenBank database on NCBI web server revealed that mostof genes with predicted function were related to energy metabolism, protein synthesis,degradation of secondary metabolites, cell wall metabolism and transcription factors.These results will lead to further research of mechanism of browning fruit inmolecular level and develop good foundation of cloning related important genes ofbrowning from Oil Nai.2According to the function prediction of the six selected genes and the result ofprevious research related to fruit browning, the analysis of the six selected genes hasconducted from bioinformatics, promoter cloning and the various gene expression ofthese genes under different biotic stress and abiotic stress, the main result asfollowed:(1) Phenylpropanoid metabolism pathway is one of the important pathways ofsecondary metabolism. Phenylalanine ammonialyase (PAL) which catalyze the firststep of phenylpropanoid metabolism pathway, is the key enzyme and rate–limitingenzyme of phenylalanine metabolism pathway. This research has separated PAL genewhich has a full sequence of2497bps containing2154bps open read frame, a119bps5′UTR and a224bps3′UTR, respectively, encoding718amino acids withmolecular weight of78kDa and the isoelectric point of6.6. Systematic cladogramanalysis revealed that PAL gene cloned and the counterpart from Prunuspseudocerasus belong to the same cluster, which had conserved regions ofPhenylalanine ammonialyase–Histidine ammonialyase (PAL-HAL). Compared withcontrol, PsPAL showed a differential expression mode in varying degrees attranscriptional level under various abiotic stress and ethylene treatment. After woundand low temperature treatment, PsPAL showed a obvious up-regulated expressionmode. After high temperature and oxygen free treatment, PsPAL gene wasup-regulated first and then down-regulated. The gene expression showed up-down-up mode under ethylene treatment.(2) Three expansin genes have been separated from the full-length normalizedcDNA library, named PsEXP1, PsEXP2and PsEXP3with Genbank accession numberJN675711, JN675712and JN675713, respectively. The full length of these genes were 1303bp,1392bp and1384bp with open read frame of759bp,780bp and759bp,respectively. Systematic cladogram analysis revealed that PsEXP1, PsEXP2andPsEXP3belonged to different sub-family with various biological function ofα-expansin. Genome DNA sequence amplification revealed that all of these threegenes contained three exons and two introns. At the upstream of PsEXP1, PsEXP2and PsEXP3, three regulatory sequence with length of899bp,744bps and596bpswere acquired respectively. All of these regulatory sequences had the specialstructural character, such as TATA-box, CAAT-box, anaerobic induction element,endosperm expression element and photoreaction element et al. The result ofexperiment showed that all of the three genes have transcribed through the entiregrowth and developmental period except PsEXP1was absent during the early stage offruit development. The expression of these three genes were induced by ethylene andwound, but not by high and low temperature and oxgen free.(3) It revealed that the sequence of NFH-633shared high homogeneity with thesequence of MdNAC from Malus domestica by5′EST sequencing of the full-lengthnormalized cDNA library. This clone named PsNAC and submitted to Genbank withaccession number of JN67510has a1077bp length of ORF with359aa and39kDamolecular weight. The homogeneity between PsNAC and the counterpart from Malusdomestica, popar and Navel orange was more than60%. Bioinformatics analysisshowed that the expression of PsNAC might be relevant to decrepit and environmentalstress etc. Real-time PCR revealed that PsNAC transcript was up-regulated by wound,ethylene and oxygen free treatment, especially by ethylene, whereas it wasdown-regulated by high temperature and unchanged under low temperature treatment.(4) WRKY transcription factor was another transcription factor separated from thefull-length normalized cDNA library. So far as, this WRKY transcription factorseparated here, named PsWRKY, was reported the first one from Nai fruit. It has beensubmitted to Genbank with accession number of JN675708, with full length of1836bp and encoding533amino acids. Semi-quantitative analysis demonstrated that it hadhigh expression level in browning fruit than normal fruit. Bioinformatics analysis ofthe sequence of amino acids deduced by this gene showed that it has the character ofWRKY family with two WRKY conservative domain, two zinc domain of C2-HC style and a putative nucleus-located signal PKRR. PsWRKY protein could bind to Wbox to activate the GUS gene expression. Transient expression analysis in onionepidermal cells suggested35S:: PsWRKY-GFP fusion protein was localized in thenucleus,Real-time PCR revealed that PsNAC transcript was down-regulated by woundand high-temperature treatment, whereas it was up-regulated by ethylene, oxygen freeand low temperature treatment.3Polyphenol oxidase (PPO) has been regarded to be a critical enzyme in foodtechnology. A new clone, named PsPPO2, was separated from cDNA library preparedfrom mature leaf of the Prunus salicina. The deduced amino acid sequences shared81%and80%identity with the PPO from Hebei Yali pear and Fuji apple and shared55%and56%identity with the PPO gene from P.salicina leaf and fruit whichprevious isolated, respectively. RT-PCR showed that three PPO gene display uniquepatterns of expression in developmental specific manner in leaf and fruit tissue. In theprocess of leaf development, the PsPPO2mRNA was detectable at all stages. Incontrast, PsPPO1and PsPPO3were expressed in young tissue. PsPPO2and PsPPO3expression are higher at the early stages of fruit development, then dramaticallyreduced as the fruit ripened. The PsPPO1mRNA was detectable only at the browningfruit. Upon wounding, PsPPO1was significantly induced in mature fruit, whereas thelevel of PsPPO2and PsPPO3were not affected by mechanical damage.更多还原