【作者】 孙俊；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2005， 博士

【摘要】 转座子是真核生物基因组内重要组成成分之一，可以从一个位点转移至另一个位点并能产生一系列变异的遗传因子。根据转座方式的不同，转座子通常被分为两类：一类是以RNA为中介进行转座的逆转座子，另一类是直接以DNA到DNA的方式进行的转座子。逆转座子是其中种类最多、分布最广的一类，其特殊的转座过程使其引起的突变为稳定突变，Ty1-copia类逆转座子是植物中研究的较为深入的一类。已有研究表明Ty1-copia类逆转座子对植物基因组大小、结构、基因功能以及基因和基因组进化等都有重要影响。苹果（Malus×domestica Borka．）是世界范围内广泛栽培的果树，但目前关于苹果基因组内Ty1-copia类逆转座子的研究却很少。本试验研究了苹果基因组内Ty1-copia类逆转座子在苹果属中存在的普遍性、多样性、甲基化水平、在基因组中的作用、转录活性、以及LTRs的特性，并利用基于LTRs的SSAP技术研究了苹果属部分野生种与栽培种的遗传多样性和芽变的分子机理。1、优化了苹果Ty1-copia类逆转座子逆转录酶保守片段的PCR反应体系：Mg²⁺浓度为5mmol／L；dNTP浓度200μmol／L；引物浓度为0.5μmol／L；Taq聚合酶浓度为50μl反应体系中为1U。从苹果属12个野生种和苹果23个栽培品种中都扩增到了约270bp的目的产物，且所有试材的扩增结果一致，均无目的条带以外的产物扩增，说明Ty1-copia类逆转座子在苹果属植物中广泛存在，且存在历史可能较为久远；从扩增结果看不出该逆转座子的逆转录酶基因或附近区域在苹果基因组内有嵌套行为的发生。2、利用优化了的PCR方法从嘎拉苹果基因组克隆了45条Ty1-copia类逆转座子逆转录酶保守序列（登陆号分别为AY849580-AY849592和DQ105035-DQ105066），结果表明，同一基因组来源的这些序列存在高度的异质性。核甘酸序列长度变化范围为196bp-279bp，同源性为40％-99.2％，氨基酸序列同源性为31.3％-100％，其中有11条序列发生了移框突变，13条序列含有1-3个不等的终止密码子突变，22条序列在保守片段SLYGLKQ处发生了碱基替代突变，3条序列发生重组突变，至少有5条序列发生了碱基缺失突变，1条序列发生了插入突变。根据氨基酸序列的同源性将来自于同一祖先的45条序列对应的逆转座子分为10个家族，其中家族1-7中序列总数约占克隆总数的93％，存在有转座活性的逆转座子的可能性较大；家族8-10各只含有一条序列。比较苹果与其它物种中Tyl-copia类逆转座子逆转录酶基因保守片段同源性，发现苹果与挪威云杉、番茄和马铃薯中存在相似性很高的Tyl-copia类逆转座子。3、本试验以从嘎拉苹果基因组获得的Tyl-copia类逆转座子逆转录酶基因的总的保守序列为探针，以田间植株、组织培养植株和转基因植株的不同酶切产物为模板进行Southern杂交，结果表明苹果基因组内Tyl-copia类逆转座子拷贝数高；基因组内部分Tyl-copia类逆转座子被甲基化，这不仅在一定程度上维护了寄主基因的功能和基因组的稳定性，而且促进了寄主基因型多样性的发展与基因组进化；上述三种材料不同酶切产物的杂交信号及强度没有区别。4、研究了愈伤组织培养条件下苹果基因组内Tyl-copia类逆转座子的转录活性。在培养基MS+2，4-D 2mg／L+BA 0.2mg／L+NAA 0.5mg／L上诱导了1个月的愈伤组织呈现非正常生长状态、无再生迹象。以Tyl-copia类逆转座子逆转录酶区域的兼并引物进行RT-PCR，检测到了270bp的目的条带，而对照、继代组培苗和不含2，4-D诱导的愈伤组织及幼芽中均无该条带的扩增，说明2mg／L的2，4-D能诱导Tyl-copia类逆转座子转录活性的表达。将目的条带回收、克隆和测序后进行分析，所有序列都属于Tyl-copia类逆转座子逆转录酶的氨基酸保守序列。21条序列（登陆号分别为AY849591-AY849596和DQ105060-DQ105074）的变异性较大，核甘酸序列长度从196-277bp，同源性45.3％-99.6％；推断其氨基酸序列变异范围为36.8％-98.9％，其中有2条序列发生了移框突变，4条序列含有终止密码子突变，9条序列在保守片段SLYGLKQ处发生了碱基替代突变，2条序列发生重组突变，至少有1条序列发生了缺失突变，1条序列发生了插入突变。比较21条序列之间以及与其它物种中同类序列的相似性，发现21条序列被聚为五类，其中，第一类与甜菜、第二类与日本樱花和挪威云杉、第四类与拟南芥中含有相似性高的Tyl-copia类逆转座子。尤其是第二类的prt52与日本樱花中BAA02268.1的同源性高达91.6％。5、本文采用改进过的Pearce等方法获得了苹果Tyl-copia类逆转座子的RNase-LTR序列。所分离的20条序列的长度变化范围107-500bp；根据Tyl-copia类逆转座子的特征推断出的PPTs和IR中，有9条序列的PPTs全由嘌呤碱基构成，11条序列的PPTs中有1个嘧啶碱基的替代突变；19条序列的IR为“TG”，一条序列因重组而缺失；20条序列中有4条序列出现重组现象，重组率是20％。根据RNase氨基酸序列的同源性，20条序列可分为六类，每一类中各序列长度、组成、RNaseH的结构、PPTs的结构及起始位置等并不完全相同。在每一类中都有RNaseH核甘酸序列同源性达90％以上的序列存在，说明这些逆转座子的转座活性可能一直伴随着苹果基因组的进化历程，这增强了苹果基因组的可塑性。分析获得的Tyl-copia类逆转座子的LTRs，其中含有启动子的结构特征“CAAT box”和“TATA box”及受不同胁迫条件作用的调控元件。6、基于苹果Tyl-copia类逆转座子LTR-10的SSAP技术在苹果属8个野生种和28个栽培品种中表现出了丰富的遗传多态性，多态性片段比例为88.2％。所有供试材料被聚为三组，第一组全由国外引入的栽培种质构成；第二组由野生种质组成；槟子因其遗传背景复杂，被单独聚为第三组，第二、第三组种质都原产于中国。其中，具有相同或相近起源的种质多数能被聚在一起，说明基于Tyl-copia类逆转座子的分子标记在研究物种的遗传多样性上具有很大的发挥潜力。此外，在红星的芽变品种矮威尔中检测到了一条约180bp的特异带，表明该品种的芽变与逆转座子的插入突变可能有一定关系。更多还原

【Abstract】 Transposons are genetic elements that can move, spread and generate mutations through insertions near or within genes, and that constitute an important fraction of eukaryote genomes. Transposable elements （TEs） are usually classified in two different groups according to their mode of transposition: retrotransposons transpose through an RNA intermediate, transposons transpose directly via a DNA intermediate. Retrotransposons are the most abundant and widespread class of transposable elements and generate stable mutations. The Ty1-copia-like retrotransposons are the best studied retrotransposons group in plant. Ty1-copia-like retrotransposons have been found associated with genome size, genome structure, gene function and the evolution of plant genes and genomes.Apple （Malus×domestica Borka.） is an important fruit tree planted widely in the world. Until now studies on Ty1-copia-like retrotransposons within apple genome are little. In this study, we carried out some works: existence of Ty1-copia-like retrotransposons in the genomes of different wild and cultural species of apple genus; heterogeneity and methylation level among retrotransposon; the roles of retrotransposon in genomic and chromosomal organization; transcription activity of retrotransposons in apple calli in vitro; the characterizatics of the long terminal regions （LTRs） of the retrotransposons and their contribution to genetic diversity and bud mutations using SSAP techniques based on LTRs.1 The factors that affected the PCR results were studied, using conserved reverse transcriptase region primers of Ty1-copia-like retrotransposons. The results showed that the optimized content of Mg²⁺ was 5mmol/L; dNTP was 200μmol/L; Primer was 0.5μmol/L; Taq polymerase was lU/50ul in the PCR system. Only one fragment of about 270bp was amplified within the genome of 12 wild species and 23 cultivars of Mallus Mill., which indicated that Ty1-copia-like retrotransposons were archaic components of apple genome. The nested insertion of near and within the reverse transcriptase gene of Ty1-copia-like retrotransposons wasn’t found in apple genome.2 The reverse transcriptase conservative region of Ty1-copia-like retrotransposons were amplified from Gala apple using the optimized PCR system. The amplification product was isolated, cloned and sequenced. Forty-five clones containing reverse transcriptase（RT） conservative region were obtained （accession number: AY849580 -AY849592 and DQ105035-DQ105066）. Cluster analysis of these sequences showed a great heterogeneity among RT domains isolated from the same genotype. The nucleotide acid sequences ranged from 196bp to 279bp. The homogeneity of nucleotide acid ranged from 40% to 99.2%, amino acid from 31.3% to 100%. Eleven of the sequences contained frameshifted translations, thirteen contained stop condons mutations of one, two or three, three contained recombination mutations, one contained insertion mutation, five contained deletion mutations, and twenty-two contained substitution mutations in the conserved SLYGLKQ of plant retrotransposons. According to the result of phylogenetic analysis of conceptually translated amino acid sequences, the forty-five clones were divided into ten families. The sequence number of family 1-7 was forty-two, accounting for 93% of the total number of clones. Transposable elements could exist in the seven families. The family 8-10 contained one clone respectively. Compared to the homogeneity of RT conserved region among apple and other species, the species presenting RT sequences most similar to apple RT clones were Norway spurce（gymnosperm）, tomato and potato.3 Southern-blot hybridization using the total Gala RT PCR product as probes showed that multiple copies are integrated throughout the apple genome. The banding patterns among DNA extraction from control, tissue culture and genetic transformation showed no differences. Some Ty1-copia-like retrotransposons, presenting in apple, were found to be methylated, while no differences in methylation were observed among DNA extraction from the three materials. The methylation of retrotransposons maintained the stability of the host gene and genome, and promoted the genetic diversity of apple genotype and the evolution of apple genome.4 Transcriptionally activity of Ty1-copia-like retrotransposons were found in apple calli, propagated on MS solid medium supplemented with 2mg/L of 2,4-D, 0.2mg/L of BA and 0.5mg/L of NAA for a month. Calli, propagated on the medium, showed abnormal growth and lost the possibility of regeneration. RT-PCR was used for amplifying the conserved domain of reverse transcriptase gene using degenerated oligonucleotide primer. The 270bp of fragments were amplified from the mRNA of the above calli, while weren’t detected from the mRNA of other materials. Therfore, 2mg/L of 2,4-D could induce the transcription activity of Ty1-copia-like retrotransposons within apple genome. The 270bp of frangments were isolated, cloned and sequenced. Twenty-one aimed sequences were obtained （accession number: AY849591-AY849596 and DQ105060-DQ105074）. All 21 clones were different from each other, nucleotide acid ranged from 45.3% to 99.6%, and amino acid from 36.8% to 98.9%. Two of the sequences contained frameshifted translations, four contained stop condons mutations, three contained recombination mutations, one contained insertion mutation, one contained deletion mutations, and nine contained substitution mutations in the conserved SLYGLKQ of plant retrotransposons. A phylogenetic tree was constructed based on their predicted amino acids and twenty-one clones were divided into five families. The first, the second and the fourth family showed high homogeneity to the RT of beet, Prunus×yedoensis, Norway spruce, and A. thaliana respectively. The homogeneity of the clone prt52 in the second family and BAA02268.1 in Prunus×yedoensis was 91.6%.5 We adopted a modified version of Pearce et al.’s methodology for isolating RNase-LTR fragment of Ty1-copia-like retrotransposons in apple. The length of twenty sequences ranged from 107bp to 500bp isolated from the same genotype. Based on typical model of Ty1-copia-like retrotransposons, PPTs and IR were understood. PPTs of nine sequences consisted of purines, PPTs of eleven of continuous purines with a pyrimidine insertion. IR of nineteen sequences was the canonical "TG". One didn’t contain 3’-LTRs owing to recombination mutation. Four of twenty sequences contained recombination mutations. Based on the homogeneity of amino acid sequence of RNaseH, twenty sequences were divided into six groups. Sequence length and combination, RNaseH structure and PPTs structure and starting location were different in the same group. In every group, there existed clones with more than 90% nucleotide identity, which showed that these retrotransposons could preserve transposable activity in the past. "CAAT box" and "TATA box", potential regulatory motifs from transcription, and conserved cis-acting regulator elements were discovered within LTRs in the great number of sequences using plantCARE sortware.6 The insertional polymorphism of LTR-10 element within the genome of 8 wild species and 28 cultivars of Mallus Mill. was studied by sequence-specific amplification polymorphism（SSAP）. The result revealed high level of genetic diversity. The proportion of polymorphism products was 88.2%. Phylogenetic tree based on the SSAP data showed that SSAP marker was able to resolve different species lineages within apple genus. All species were divided into three groups. The first group consisted of foreign cultivar species. The second group included wild species. The third group only contained Malus asiatica var. rinid （Koidz.） Asami owing to complexity of genetic materials. The species in the second and third group origined from China. The 180bp amplification fragment was present in Wellspur Delicious, not in starking from which the former mutated, suggesting that retrotransposon activity might be involved in the bud mutation of Wellspur Delicious.更多还原