【作者】 王红艳；

【导师】 刘宝；

【作者基本信息】 东北师范大学 ， 遗传学， 2009， 博士

【摘要】 远缘杂交在植物界中频繁发生,在基因组进化和新种形成中具有创造性作用。由远缘杂交所产生的杂交种,异源多倍体以及渐渗杂交种与其亲本相比往往会出现大量遗传和表观遗传上的变异。但是我们仍然不知道的是来自有性上不亲和物种的花粉授粉在没有发生异缘渐渗的情况下,是否会导致一种“胁迫”,从而引起受体基因组的遗传和表观遗传不稳定性。为了探讨这一问题,本文研究了来自于有性上不亲和的两个远缘物种一水稻和稗草杂交所产生的后代,并且获得了一个在表型上有明显变异的水稻突变体后代(称之为Rb0)。首先,通过荧光显微镜技术,对这种水稻与稗草远缘渐渗杂交的受精行为进行了观察,发现稗草花粉可以在水稻柱头上萌发,并且花粉管可以生长至水稻子房部位,甚至到达珠孔。随后,通过对Rb0进行供体基因渐渗分析,结果显示没有稗草DNA渗入的证据。对Rb0及其自交后代S1代的鉴定可以排除由于花粉或其他品种种子污染或者多倍化等诱因造成上述表型变异的可能。虽然没有检测到稗草基因渐渗的证据,但是这种来自远缘物种花粉的授粉却引起了水稻突变体Rb0及S1代转座子的激活,其中包括MITE类转座子mPing及其转座酶供体Pong,LTR类反转座子Tos17和Osr42;以及一些反转座子的甲基化变异,包括Tos17和Osr36。利用AFLP和SSR分子标记技术对水稻突变体Rb0及S1代进行基因组遗传稳定性分析,结果显示稗草花粉胁迫诱导产生了遗传变异,包括碱基突变、碱基的缺失与插入以及微卫星序列重复次数的改变。同时,利用MSAP技术对突变体Rb0及其S1代进行了基因组范围表观遗传稳定的分析,结果显示稗草花粉胁迫同样导致了广泛的表观遗传变异,主要包括基因组整体DNA甲基化水平的提高和位点特异的模式改变。并且,这些在Rb0中所表现出来的表型变异以及遗传和表观遗传变异不仅能够以较高的频率传递给子代(S1代),而且在子代中又出现了Rb0所没有的新变异,暗示具有“跨代基因组不稳定性”的特点。为了探究上述遗传变异的原因,运用实时定量PCR技术,对选定的维持基因组稳定性的错配修复基因(mismatch repair,MMR)进行了研究,分析结果显示,无亲缘关系物种间的相互作用可以导致后代Rb0及其连续2个自交后代的维持基因组稳定性的错配修复基因表达的可遗传的改变。同时,为了探究上述表观遗传变异的原因,对维持染色质表观修饰状态的基因的表达进行了同样的分析,结果显示无亲缘关系物种间的相互作用也可以导致维持染色质结构和状态的表观修饰基因表达可遗传的改变。最后为了验证这类基因表达变异在这种“胁迫”过程的普遍性,我们对以同样方法获得的月见草花粉胁迫的水稻表型突变体及其自交后代做了同样的分析,结果显示这些基因的表达也发生了改变,而且大多数变化是可遗传的。以上结果暗示着这些与维持染色质结构相关的基因表达的变异是远缘物种花粉胁迫所导致的遗传和表观遗传不稳定性的一个诱因。综上可知,来自远缘物种花粉的授粉可能对受体基因组造成一种胁迫,从而引起遗传和表观遗传的不稳定。鉴于这项杂交实验的简单性,不难想象类似的事件可以发生在自然情况下。同时本实验结果为“不亲和远缘杂交”在育种上的应用提供了初步的理论依据。更多还原

【Abstract】 Wide hybridization occurs frequently in plants,which may promote genome evolution and induce genetic and epigenetic instabilities in the resultant hybrids, allopolyploids and introgressants.It remains unclear however whether pollination by alien pollens of an incompatible species may impose a "stress" even in the absence of genome-merger or genetic introgression,whereby genetic and/or epigenetic instability of the maternal recipient genome might be provoked.In this study,we uncovered a rice mutator-phenotype(named Rb0) from a set of rice plants derived from a crossing experiment involving two remote and apparently incompatible species,Oryza sativa L. and Echinochloa crusgalli L..We observed the fertilization behavior of barnyard grass pollens on the rice stigma via microscopic observation.We found that barnyard grass pollen tube can geminate and grow on the stigma of rice and the pollen tube can enter into rice micropyle after pollination.However,gel-blotting by total genomic DNA of the pollen-donor showed no evidence for introgression in the resultant mutant plant(Rb0). Characterization of Rb0 and its selfed progenies(S1) ruled out contamination(via seed or pollen) or polyploidy as a cause for its dramatic phenotypic changes,but revealed mobilization of several previously characterized transposable elements(TEs),including a MITE(mPing) and its putative transposase-encoding partner Pong and two LTR retrotransposons(Osr42 and Tos17) and methylation variation of two LTR retrotransposons(Tos17 and Osr36).Genome-wide analysis by the AFLP and SSR fingerprinting markers revealed extensive,transgenerational alterations in genetic variation in Rb0 and/or its immediate progenies S1 by alien pollens of barnyard grass,including base mutation,deletion and insertion,as well as the number of microsatellite repeat sequence changes.Meanwhile, genome-wide analysis by MSAP revealed an enhanced DNA methylation level and changing of site-specific patterns.Notebly,the transgenerational continuous occurrence of multiple phenotypic,genetic and epigenetic variations from a single individual(Rb0) is characteristic of a "transgenerational genomic instability".To further explore the possible reasons for the genetic variations described above, assay by q-RT-PCR of the steady-state transcript abundance of a set of genes encoding for the various putative mismatch repair(MMR) genes showed inheritance of the altered expression of these genes in Rb0,S1 sefled progenies and S2 sefled progenies relative to the rice parental line.At the same time,we analyzed the expression-state of genes which involved in chromatin regulation in order to explore the possible causes for the epigenetic variations. We also found expression of these genes changed and inherited.Finally,in oder to detect whether perturbed homeostatic expression-state of genes is prevalent in this "stress" or not,we investigated these genes in another rice mutator-phenotype (Tong211-LP) that produced by pollinating the rice stigma with pollens of O.biennis L. Strikingly,the expression-state of these genes were also found heritably changed in Tong211-LP and its selfed progenies as well as in S1 that produced by pollinating the rice stigma with pollens of barnyard grass.Perturbed homeostatic expression-state of genes which involved in maintenance of chromatin structure is likely an underlying cause for the alien pollination-induced epigenetic/genetic instability,and which occurred apparently without entailing genome merger or genetic introgression.Our results suggest that pollination by unrelated alien pollens in plants might impose a stress condition and induce genetic and epigenetic instabilities in the maternal genome.Given simplicity of this cross-pollination experiment,it is not difficult to imagine that similar incidents may occur under natural conditions.At the same time,the results of this study have provided a theoretical basis for the possible utility of "incompatible wide-hybridization" in crop breeding.更多还原