Mobilized retrotransposon Tos17 of rice by alien DNA introgression transposes into genes and causes structural and methylation alterations of a flanking genomic region

Tos17 is a copia-like endogenous retrotransposon of rice, which can be activated by various stresses such as tissue culture and alien DNA introgression. To confirm element mobilization by introgression and to study possible structural and epigenetic effects of Tos17 insertion on its target sequences, we isolated all flanking regions of Tos17 in an introgressed rice line (Tong35) that contains minute amount of genomic DNA from wild rice (Zizania latifolia). It was found that there has been apparent but limited mobilization of Tos17 in this introgression line, as being reflected by increased but stable copy number of the element in progeny of the line. Three of the five activated copies of the element have transposed into genes. Based on sequence analysis and Southern blot hybridization with several double-enzyme digests, no structural change in Tos17 could be inferred in the introgression line. Cytosine methylation status at all seven CCGG sites within Tos17 was also identical between the introgression line and its rice parent (Matsumae)-all sites being heavily methylated. In contrast, changes in structure and cytosine methylation patterns were detected in one of the three low-copy genomic regions that flank newly transposed Tos17, and all changes are stably inherited through selfed generations.     

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异.在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现.发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基.序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异.但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异.而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性.甲基化变异可稳定遗传给后代.因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用.但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究.     

水稻冷胁迫诱导的甲基化差异片段CIDM7的分离和分析

文章采用基于AFLP的甲基化敏感扩增多态性的方法对5℃冷处理48小时的水稻9311叶片DNA胞嘧啶甲基化模式进行了初步研究,发现冷处理48小时后9311基因组中一些CCGG位点发生了重新甲基化或去甲基化,并获得一些与水稻cDNA高度同源的甲基化差异片段,其中CIDM7 (cold-inducing differential methylation)片段在冷胁迫后去甲基化。Northern杂交证明CIDM7在冷胁迫后增强表达。通过在日本晴全长cDNA数据库中的同源搜索获得其全长cDNA序列,该cDNA序列全长1422 bp,编码425个氨基酸,经氨基酸序列分析为一个具有F-box结构的假定蛋白。CIDM7为单拷贝,定位于日本晴10号染色体12.56 ~12.57 Mb。     

欧石楠体细胞胚发生过程中的DNA甲基化

利用甲基化敏感扩增多态性（MSAP）方法,对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对,共扩增得到506条带,统计显示,大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31．42％、27．86％和29．05％,3种试材发生甲基化变异的有175条带,变异率为34．58％。体细胞胚诱导形成胚性愈伤组织过程中,甲基化水平降低,而在再生苗中有所恢复,与大田苗接近。在外侧胞嘧啶甲基化水平上,胚性愈伤组织的甲基化水平有所增加,且在再生苗中可部分维持。另外,在175条变异带中,再生苗恢复到大田苗DNA甲基化模式的有62条,占总变异条带的35．43％,而与胚性愈伤组织维持相同DNA甲基化模式的有59条,占33．71％。回收部分甲基化变异条带,最终得到8条有效的基因组DNA序列。BLASTnI：对分析表明,在欧石楠基因组中,包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA基化修饰现象。