【作者】 高燕；

【导师】 张新忠； 韩振海； 王忆；

【作者基本信息】 中国农业大学 ， 果树学， 2013， 博士

【摘要】 多年生木本植物的个体发育过程中要经历从童期到生殖生长期的转变。研究果树实生树阶段转变过程中的基因表达调控以及个体发育的分子机制,对缩短果树童期,提高果树育种效率有重大意义。本试验以9年生苹果杂种实生树(‘红玉’ב金冠’)为试材,利用SSH技术建立苹果实生树个体发育不同阶段的消减文库；之后通过斑点杂交和实时荧光定量PCR,半定量PCR等方法对苹果实生树童期、成年期的消减文库进行筛选验证,对不同发育阶段差异表达基因功能分析并从中挑选出可能与阶段转变密切相关的基因,对其克隆。构建了植物表达载体,转化烟草和拟南芥。研究结果如下：1、构建苹果实生树不同个体发育阶段基因差异表达文库。童期和成年期文库各768个阳性克隆经过斑点杂交验证后,童期和成年期分别得到244个和284个阳性克隆。2、对苹果实生树不同个体发育阶段差异表达基因进行分析。阳性克隆测序后分别获得童期和成年期特异表达EST85个和103个,其中基础代谢相关基因54个,细胞骨架和细胞周期相关基因10个,蛋白质代谢相关基因32个,氧化还原相关基因8个,次生代谢相关基因7个,激素响应及信号传递相关基因21个,逆境响应相关基因13个,转录因子13个,金属转运及膜转运相关基因30个。6个脂类代谢和7个次生代谢相关的EST只在成年期出现。32个蛋白质代谢相关EST中有62.5%在成年期特异出现。5个与Auxin,GAs,ABA和ETH响应相关的EST均只出现在童期。参与氧化还原反应的基因在童期和成年期不同。在阶段转变过程中不仅存在转录水平的差异,还检测到转录后的差异修饰。4个编码核酮糖-1,5-二磷酸加氧羧化酶小亚基rbcS的EST,均与苹果基因组的MDP0000731480的3’UTR序列匹配。其中3个EST在童期上调表达,1个EST在成年期上调表达。这四个EST仅在3’UTR序列上存在差异。4个位于MDP0000243585上的编码金属硫蛋白like的EST和2个编码NADP-甘油醛磷酸脱氢酶的EST的3’UTR序列也表现出显著差异。3、对差异表达基因在杂交组合内以及不同杂交组合间进行验证。用实时荧光定量PCR对‘红玉’ב金冠’(02-18-081,02-17-115和02-17-042)和‘紫塞明珠’ב富士’(07-07-115,07-07-119和07-07-133)2个组合各3株实生树样品对随机选取的差异表达基因验证。全部6个差异表达基因在6株实生树上的表达情况与SSH结果一致。说明SSH建库结果在杂交组合内以及杂交组合间的结果是稳定的。通过半定量PCR对存在3’UTR的差异修饰的4个rbcS EST验证。成年期上调表达的88.9%与SSH结果相符,童期上调表达的rbcS EST得到85.2%验证4、克隆了阶段转变相关的基因,并构建遗传转化载体,通过农杆菌介导的叶盘法转化烟草和花序侵染法转化拟南芥。T3代拟南芥及T1代烟草中检测到报告基因GFP及转入的外源基因。本研究表明,苹果实生树阶段转变过程中不仅存在转录水平的调控,也存在转录后水平上的调控；激素响应、氧化还原调控、次生代谢和蛋白质代谢均与苹果阶段转变密切相关。更多还原

【Abstract】 A woody perennial plant has to undergo a gradual and continuous process of transition from juvenility to reproductive maturity. To better understand the underlying mechanism of ontogenesis, our aim in this study was to find out differentially expressed genes between juvenile phase and adult phase not only among different seedlings but also different hybrid crosses. Two reciprocal subtracted cDNA libraries of juvenile versus adult phases were constructed using suppression subtractive hybridization （SSH） in an apple （Malus domestica Borkh.） hybrid seedling （Jonathan x Golden Delicious）. The expression uniformity of genes between the two ontogenetic phases was reconfirmed by real-time PCR （RT-qPCR） in three seedlings from the same population and also in three seedlings from different hybrid populations. The results were shown as follows:1. Subtracted cDNA library of juvenile and adult phase of apple seedlings were constructed. Suppression subtractive hybridization （SSH） was used to isolate mRNAs that differentially expressed in samples of juvenile and adult phase of nine-year-old hybrid seedling02-18-034（Jonathan x Golden Delicious）.384clones from both juvenile library and adult library were picked and amplified. Through dot bloting,244positive clones from juvenile library and284positive clones were selected and sequenced.2. All sequenced ESTs were analyzed. The SSH libraries generated179unambiguous juvenile phase up-regulated transcripts and230adult phase up-regulated transcripts by sequencing. These transcripts were analyzed using BLASTN and BLASTX at both NCBI and the M. domestica genome database. Eighty-five ESTs from the juvenile phase and103ESTs from the adult phase were annotated and classified as nine function group, including54genes related to primary metabolism, ten genes renlated to cytoskeleton and cell cycle,32genes related to protein metabolism, eight genes related to redox, genes related to secondary metabolism,13genes related to hormone response and signal transduction,13genes related to stress response,13transcription factors and30genes related to transportOf the188differentially expressed genes,54（28.7%） were associated with primary metabolism, and32（17%） were involved in protein biosynthesis and catabolism. The gene transcription of enzymes related to lipid metabolic processes apparently increased in the adult phase. All of the seven genes involved in secondary metabolism were adult-induced. The expression of genes encoding enzymes and factors involved in protein metabolism varied differentially between juvenile and adult phases. Nine of16（56.25%） transcripts related to protein biosynthesis were abundantly expressed in the adult phase. Six genes related to lipid metabolism and seven genes related to secondary were adult-induced only.62.5%genes of32involved in protein metabolism genes were adult-induced.. Eleven of16（68.75%） transcripts of protease and other enzymes participated in protein degradation, including subunit and adaptor proteins of clp protease, and enzymes related to ubiquitin were up-regulated in the adult phase. two genes concerning chloroplast ribosomal proteins,60₅48_A and44531_A, and three genes encoding subunit and adaptor proteins of clp protease （188-434_A,72-560_A and122-353_A） were uniquely detected in the adult subtracted library, and these may contribute to an active protein metabolism in chloroplast during the adult phase. The expression of several genes modulating oxidation and reduction differed between ontogenetic phases. Three transcripts were up-regulated in the juvenile phase, including peroxiredoxin I （32-408_J）, a member of the peroxiredoxin super family, glutathione S-transferase （69-453_J） and reticuline oxidase （56-439_J）. On the other hand, the expression of five redox-related genes were elevated in the adult phase, prolyl oxidoreductase （191-437_A）,2OG-Fe（II） oxygenase （204-456_A）, NADH-dependent hydroxypyruvate reductase （91-581_A）, L-ascorbate peroxidase （71-559_A） and cinnamyl/sinapyl alcohol dehydrogenase （124-355A）.Some transcripts from different subtracted libraries matched the same gene set in apple genome, but they varied significantly from each other in3’UTR sequences. Three of four rbcS transcripts （4-366_J,81-471_J and10-378_J）, encoding ribulose-1,5-bisphosphate carboxylase small subunit, were juvenile-up-regulated while the fourth transcript,45-532_A, was adult-induced. All of the four transcripts are located in3’UTR of rbcS （MDP0000731480） according to the Malus domestica genome database, but the base sequences of the3’UTR differed between each other. Both of the two transcripts （142-281_J and57-545A）, encoding glyceraldehyde-3-phosphate dehydrogenase, matched the gene set MDP0000835914of apple genome, but their length of poly （A） were much different. Four transcripts （65-449J,131-259_J,172-325_J and226-484A）, encoding metallothionein-like protein （AMT1）, were located in the3’UTR of the gene set MDP0000219584in apple genome.3. The results of SSH were confirmed by qPCR. Leaf samples of juvenile and adult phases were collected from three seedlings,02-17-042,02-17-115and02-18-081, from the same hybrid population as mentioned above, and also from three4-year-old seedlings,07-07-115,07-07-119and07-07-133, derived from an interspecific hybrid population （Zisai Pearl x Red Fuji）. Six ESTs were selected randomly from both the juvenile and the adult subtracted libraries. The qPCR data indicated that the differential expression of genes were ultimately, but not completely, uniform between seedlings within the same hybrid population. Semi-quantitative RT-PCR was performed to confirm some genes regulated by post-transcription during ontogeny. Three rbcS transcripts （4-366_J,81-471_J and10-378J） from juvenile library showed juvenile-up-regulated. One rbcS transcript （45-532A） from adult library showed adult-up-regulated.4. Phase change related genes were cloned and transfered into Tobacco and Arabidopsis. Five genes were selected from SSH library according to the results of EST analysis. These five genes were J-37-416:AP2/ERF; J-36-415:SAMs; J-160-309:small GTP-binding protein; J-52-433:histone H2B;187-430_A:phenylalanine ammonia-lyase. Full length coding sequence were cloned. Five genes were transferred into Tobacco and Arabidopsis by Agrobacterium tumefaciens. Reporter gene and target gene were detected in transgenetic plant.In conclusion, the ontogenetic variations are probably under both transcriptional and post transcriptional regulation. The expression of redox related genes differed between juvenile and adult phase, indicating that redox homeostasis may play a role in vegetative phase change and floral transition.更多还原