菰DNA渐渗及组织培养诱发水稻基因组表观遗传变异的研究

【作者】 刘振兰；

【导师】 郝水； 刘宝；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2002， 博士

【摘要】 表观遗传学(epigenetics)是指在DNA序列没有发生变异的情况下，基因表达的可遗传改变；它主要通过对DNA和／或组蛋白的共价修饰来实现。DNA甲基化的可遗传改变是表观遗传学的重要内容。 DNA甲基化在真核生物中普遍存在。虽然DNA甲基化的确切功能尚不十分清楚，但最新的观点认为，胞嘧啶甲基化至少在调控基因表达和基因组防御两个方面具有重要作用。 McClintock(1984)曾经预言，远缘杂交对植物基因组是一种强烈冲击，在这种情况下，基因组内处于沉默状态的转座子(包括反转座子)可能被激活，从而对这一冲击发生应答并会引起基因组的重建。远缘杂交是许多天然植物种群的一个显著特性，其结果往往是通过多倍体化或者在二倍体水平上形成新物种。考虑到反转座子在植物基因组内存在的普遍性，可以推测至少在某些情况下，反转座子的激活及其对基因表达的影响促进了杂交后的物种形成和新物种对环境的适应性。然而，目前还没有直接的证据表明适应性或物种形成与反转座子活性之间存在必然联系。 菰为禾本科稻族的野生种，与水稻为同族异属。我们实验室早期的工作发现，将菰的基因组DNA导入水稻能够诱发水稻基因组产生表观遗传变异，包括DNA甲基化变异和反转座子的激活。本论文在我们实验室已有工作的基础上，对菰DNA渐渗诱导的水稻表观遗传变异进行了更为深入的研究，包括：①利用TAIL-PCR方法克隆了渐渗系RZ-2和RZ-35中Tos17反转座子插入位点的侧翼序列，并利用多种甲基化敏感内切酶消化和Southern杂交研究了这些侧翼序列的DNA甲基化变异。结果表明，在所得的8个序列中，除一个序列的功能未知外，其余7个序列均与已知功能基因同源，这说明由于外源基因导入激活的Tos17反转座子与组织培养激活的相似，即Tos17反转座子优先插入基因组中低拷贝的基因区域；Southern杂交结果表明，菰DNA渐渗诱发了Tos17插入位点的侧翼序列发生DNA甲基化变异。对从组织培养的RZ-2和RZ-35的再生苗中克隆到的8个Tos17插入位点侧翼序列的分析表明，Ji 4“1、为向－人’列，且与未培养渐渗系中的、个侧翼序列相同（均为permease人 说明组织培养过程可能没有重新激活RZ士和 RZ刁5中己沉默的Tosl7反转座子，所克降的侧翼序列。帕g均为山于揽 DNA渐渗惭天的『fOS 17的插入；②利用多种甲 ：l士化敏感内＊吓饲讹和＊CR扩增灯次系纹地川I穴了前＊N八渐参秀发的几s17反转座’厂目身的 DNA小兑化变汁，结果表明梳 DNA渐惨激活的 Ah。17反转座广的胞屹归人牛了对称及不对称的＊NA甲丛化”过异，巾J 上L腺咄11》匕发十了HI4化变X；③组织培养可诱发水稻基囚纠中gyPSy类］二转座厂发卜DNA ql％化变X，而未检测主扼DNA渐渗诱发gyPSy殆反转座了的DNA小－’；［化变歼；④攸DNA渐渗和组织培并能够诱人水们丛山组中棱抛仆KN八汁山发’I：＊N八中基化艾汗。 迎过对1l问地的m陀，形J。厂狐＊＊A渐渗和组织培养均能够润发水创基冈组发生扒队迟传变斤，11f二首的作用有所个同；灭观巡传业汁介人然远缘杂交介川的物f。Z。形成和组川培养中的什细胞尤什系变汁十。啊沾趴X人啦公仆川更多还原

【Abstract】 Epigenetics refers to changes in gene expression that do not entail a change in DNA sequence. Among the several mechanisms responsible for epigenetic phenomena, the most important appear to be DNA methylation and histone deacetylation.In eukaryotes, DNA methylation is very intensive. Although the precise role of DNA methylation is not yet well-known, recent studies have implicated that cytosine methylation in eukaryotes plays important roles in at least two different cellular processes, namely, control of the states of gene activity and to serve as a host genome defense system.McClintock predicted decades ago that inter-species hybridization is a traumatic experience to the plant genome (called "genomic shock"), under which dormant transposable elements (TEs) could be activated to respond and to restructure the genome. Hybridization is a prominent feature of natural plant populations, and one that often leads to speciation, either through polyploidization or at the diploid level. Considering retrotransposons are ancient and ubiquitous constituents of eukaryotic genomes, it is tantalizing to speculate that, in at least some cases, speciation and (or) adaptation have been facilitated by retroelement activation and its effects on gene expression. Nevertheless, at present no data causally connect adaptation and (or) speciation with retroelement mobilization.Zizania latifolia (Griseb.) Turcz ex Stapf is a wild perennial grass in the tribe of Oryzeae, and thus related to cultivated rice (Oryza saliva L.). We have reported that Zizania latifolia DNA introgression could induce epigenetic changes of the rice genome, including DNA methylation changes and activation of both copia-like and gypsy-like retrotransposons of rice. In this thesis we studied the following questions: To examine the target-site specificity of Tosl transposition in the introgression linesby cloning and characterization of sequences flanking transposed Tosl 7 copies. It was found that at least seven out of eight target sites examined are coding regions. These results indicate that the activation of Tosl? induced by introgression is analogous to that by tissue culture, i.e., Tosl7 prefers low-copy-number sequences as integration targets. DNA methylation changes in the flanking sequences of Tosl7 insertions were observed by Southern analysis using several kinds of methylation-sensitive enzymes. Characterization of flanking sequences of Tosl7 insertions in regenerated plants of two introgression lines indicate that the repressed Tosl7 retrotransposons were probably not reactived by tissue culture; (2) Studies on methylation assay on LTR and RT regions of Tosl7 of two introgression lines and their rice parent using methylation-sensitive enzyme digestion and PCR amplification with specific primers indicates that methylation changes occurred in both LTR and RT regions of Tosl 7 in the two introgression lines, and methylation changes were detected at both symmetrical and asymmetrical cytosines, as well as at adenine bases; DNA methylation changes of gypsy-like retrotransposons were detected in rice genome under tissue culture but not under Zizania latifolia DNA introgression; Zizania latifolia DNA introgression and tissue culture both could induce DNA methylation changes in ribosomal RNA genes of rice.To conclude, we have demonstrated that both Zizania latifolia DNA introgression and tissue culture could induce epigenetic changes in the rice genome, but there are some differences in these two conditions. Our results suggest that epigenetic mechanisms may play important roles in wide hybridization-mediated speciation as well as in somaclonal variations of plant tissue cultures.更多还原