【作者】 押辉远；

【导师】 霍裕平；

【作者基本信息】 郑州大学 ， 凝聚态物理， 2007， 博士

【摘要】 转座子是生物基因组上可以移动的成分，分为DNA转座子和反转录转座子，反转录转座子又可分为LTR反转录转座子和non-LTR反转录转座子。一般情况下反转录转座子在植物中并不具有活性，很少有转录转座现象，但是在一些草本植物中，反转录转座子的转录现象是普遍的。植物反转录转座子的转座可以引起植物突变，其转录影响邻近基因的表达。对低能离子束诱变的小麦突变体基因组DNA进行AFLP分析时发现，一个AFLP多态性和小麦反转录转座子有关。我们用剂量为4×10¹⁷ N⁺／cm²的低能N⁺离子束处理小麦种子后发现，在培养24小时和48小时，与对照相比，处理小麦幼苗内copia群反转录转座子的转录水平极显著提高；而在培养72小时和7天的时候，处理小麦幼苗内copia群反转录转座子的转录水平又恢复到对照水平。我们用RT-PCR的方法扩增了用低能离子束(剂量为4×10¹⁷ N⁺／cm²)处理的小麦（培养48小时）和没有用低能离子束的对照小麦（培养48小时）中的具有转录活性的Ty1-copia-反转录转座子的反转录酶（RT）的一段功能域，并对扩增片段进行了克隆，共获得107个克隆，其中42个克隆来自对照组，65个克隆来自处理组，对每个克隆进行了序列测定，根据序列用相应软件进行了核酸序列比较、编码氨基酸序列比较、系统发育树和聚类分析的分析研究，结果显示，来自对照的克隆群的异质性为81％（34／42），处理组克隆序列的异质性为97％（63／65）。对照组中共有16个克隆发生了无义突变，这些克隆不能够翻译出完整的蛋白质。对照组有20个克隆的序列中存着移码突变，占对照总克隆的59％（20／34），只有13个克隆结构上是完整的，即没有移码突变也没有无义突变，占对照组有效序列克隆的38.2％，对照组中有15个序列既存在移码突变又存在着无义突变。处理组中，有13个克隆序列存在着无义突变，占处理组中有效序列的21％（13／63），8个克隆序列中存在着移码突变，占处理组有效克隆的13％（8／63）。处理组中有48个克隆的序列中既不存在移码突变也不存在无义突变，占处理组中有效序列克隆76％（48／63），这些克隆在结构上完整的，可以翻译出结构完整的蛋白质，这个比例是对照的两倍。处理组中有6个克隆序列即存在无义突变，也存在着移码突变。这些差异说明：经低能离子束处理后的小麦中的反转录转座子的活动受到了低能离子束注入的影响并以对照组中不同的方式和功能在细胞中起作用。结果还显示，对照组中的克隆归在family4共有8个，占总数的23.5％。归在family7共9个，占对照克隆总数的26.5％。处理组中的克隆归在family4共有21个，占处理组克隆总数的33.3％，高于对照组在family4中所占比例。处理组中克隆归在family7共有23个，占处理组克隆总数的36.5％，高于对照组在family7中所占比例。对照组的反转录转座子归在family4和family7的总数占对照总克隆数的50％（23.5％+26.5％），而处理组的反转录转座子归在family4和family7的总数占处理总克隆数的69.8％（36.5％+33.3％），这说明处理样品中的反转录转座子群体上比对照样品的反转录转座子活性高，有更好地发生转座的生物物质基础。采用rea-time PCR的方法测定了不同剂量(真空；1×10¹⁷ N⁺／cm²；3×10¹⁷N⁺／cm²；5×10¹⁷ N⁺／cm²；7×10¹⁷ N⁺／cm²)的低能N⁺离子束辐照小麦后，在培养24h和48h时，小麦幼苗中copia群反转录转座子及小麦中一个很古老的反转录转座子Wis2-1A的转录水平及在基因组上的拷贝水平，结果显示，经低能离子束处理后的小麦中的反转录转座子转录水平与对照相比显著提高或极显著提高，特别是在剂量点：5×10¹⁷ N⁺／cm²辐射条件下，copia群反转录转座子总体表达水平比对照高出200多倍，Wis2-1A的转录水平比对照高出100倍左右，但其拷贝数并没有发生太大变化。7×10¹⁷ N⁺／cm²剂量点下，培养24小时时，小麦copia群反转录转座子的转录水平极显著水平低于对照，而在培养48小时时其转录水平相对于对照极显著水平提高，而其它剂量点下，无论是培养24小时或者是培养48小时，都表现出高于对照表达的现象，这说明高剂量的离子辐射对小麦存在着严重损伤。反转录转座子的转录水平与剂量存在着一定的相关关系。不同剂量下反转录转座子的转录水平与相同剂量下的小麦幼苗活力指数有着相同的趋势。实验结果还表明，某些剂量下，测得小麦copia群反转录转座子在基因组上的拷贝数在24小时有提高，但在48小时又恢复到正常水平，这可能是形成了DNA-RNA中间体，但并没有发生大规模的转座现象。所得结果显示，小麦反转录转座子在小麦应答低能离子束辐射中起重要作用。用基于反转录转座子发展起来的两种分子标记IRAP和REMAP检测低能离子束注入后小麦反转录转座子在基因组上的多态性，结果显示，低能离子束处理的小麦基因组上的几个反转录转座子与对照相比，存着IRAP和REMAP多态性，这些多态性的出现，说明反转录转座子在低能N⁺离子束辐射条件下发生了转座。这种转座效应可能在低能离子束辐射诱变产生可遗传突变体中起重要作用用转座子显示技术研究了小麦反转录转座子Wis2-1A在低能离子束处理后的转录对其邻近基因表达的影响，结果发现，在较高剂量5×10¹⁷ N⁺／cm²辐射下，由于Wis2-1A的高水平转录导致其邻近的某些基因沉默。这说明，小麦反转录转座子通过转录影响某些基因的表达，从而在低能离子束辐射当代生物效应中起重要作用。最后对所进行的研究结果进行讨论，认为低能离子束能够提高小麦中反转录转座子的转录和转座活性，反转录转座子的转录提高和转座激活对低能离子束生物当代效应和产生可遗传变异中起重要作用，这是离子束生物效应机理研究的重要发现，具有重大意义。同时在已有研究的基础上，提出了进一步要研究的几个问题。更多还原

【Abstract】 Transposons, high copy numbers especially in eukaryotes, are transposable element in genome of a broad range of organism. They are divided into two families: DNA transposons and retrotransposons based on the propagation patterns. The retrotransposons family is divided into two main categories based on the presence and absence of long terminal repeats （LTRs）: LTR-retrotransposons and non LTR-retrotransposons respectively. Retrotransposons are usually inactive in general, but retrotransposons are generally more transcriptionally active in the grasses than in other groups of plants. The activation strategy of retrotransposons offers the potential for explosive increases in copy number and leads to a concomitant genome size increase. Along the way of their propagation, retrotransposons could also cause the inserted mutagenesis in other genes, or could confer ecotopic regulatory functions upon adjacent genesWe have previously studied a wheat strain treated by low-energy N+ ion and found variations in AFLP （Amplified Fragment Length Polymorphism ） markers. One such variation is caused by re-activation of Tyl-copia-like Retrotransposons, implying that the mutagenic effects of low-energy ions might work through elevated activation of retrotransposons. The copia-retrotransposons are in the seedlings （24hr AG and 48hr AG） from the wheat seeds treated with 4×10¹⁷ N⁺/cm², transcripting in a higher level than that in the control. However, 72hr AG and 7dAG, the transcriptions of the copia-retrotransposons are the same level between the control samples and the treated sample.The sequences that obtained from the RT-PCR reactions of the treated sample have higher heterogeneity than that from the control sample;. The heterogeneity frequency （number of clones with different sequences/total number clones in control） is 81% （34/42） in the control sample, comparing to 97% （63/65）in the treated sample. Of these 34 unique control sequences, there are 16 （47%） have Nonsense mutations （table 1） and 20 （59%） have frame shift mutations. They are thus defective in coding. There are only 13 （38%） "intact" clones carrying neither frame shift nor nonsense mutations. There are 15 clones carrying both frame shift and nonsense mutations. Of these 63 unique sequences in treated samples clones, there are only 13 （21%） defective clones carrying nonsense mutations and 8 （13%） defective clones have frame shift mutations). They are thus defective in coding. There are 48 （76%） intact clones carrying neither a frame shift mutations nor nonsense mutations （table 1）. There are only 6 clones carrying both a frame shift and nonsense mutation. Of these control group clones, 10 clones are clustered into family7, comprising 29.4% of the control group clones; 7 clones are clustered into family4, comprising 20.6% of these control group clones. Of these treated group clones, 24 clones are clustered into family7, comprising 38.7% of the control group clones; 20 clones are clustered into faimy4, comprising 32.3% of the control group clones. Phylogeny and classification are computed responding to the sequences of the RT domains. All the results show that there is much difference on RT domain between the control sample and the treated sample. Especially, the RT domains from the treated group encode significantly more functional ORF （open reading frames） than those from the control sample. This observation suggests that the treated sample has higher activation of retrotransposons, possibly as a consequence of low-energy ion beam irradiation. It also suggests that retrotransposons in the two groups impact the host gene expression in two different ways and carry out different functions in wheat cell.Low-energy ion beam as a abiotic stress promote the transcriptions of the copia-retransposons in those wheat buds cultivated at 24h and 48h, the comparative expression ratio of the copia-retransposons increased by different degree （max 220 fold） in those wheat treated in different doses(vacuum; 1×10¹⁷ N⁺/cm²; 3×10¹⁷ N⁺/cm²; 5×10¹⁷ N⁺/cm²; 7×10¹⁷ N⁺/cm²) of N⁺ beam comparing to that in the controls. Use of the IRAP and REMAP to detect the DNA isolated from the 14-day leaves of the wheat treated with the low-energy N⁺ beam showed that the transposition of some copia-retransposons was activated for the amplified polymorphic bands. The increased transcripts of the copia-retransposons in wheat regulate the expression and mRNA splicing of their near genes, the new insertion in genome change primary frame of the genes in the chromosomes, which have been visualized through the phenotype of the plants. Hence, it is suggested that the mechanisms about the biological effect of low-energy ion beam is partially attributed to the activation of the retrotransposons.We test the profiles of the expression of the genes adjacent to the retrotransposons Wis2-1A in the wheat treated with 5×10¹⁷ N⁺/cm² by the transposons display. The results showed that some genes adjacent to the retrotransposons Wis2-1A have been silent because of the transcription of the Wis2-1A under the implantation in the dose 5 ×10¹⁷ N⁺/cm²According to the all the results, it suggest that low-energy ion beam promote the activities of the transcription and transposition of the copia group retrotransposons in the wheat. In other word, the activities of the transcription and transposition of the copia group retrotransposons in the wheat play important roles in the biological effect of the low-energy ion beam, not only in the temporary generation but in the heritable biological effect of the low-energy ion beam. Of course, some ideas about the future strategy in this domain are argued.更多还原