伴性赤蚁（sch）蚕胚胎期温敏性的DDRT-PCR研究

【作者】 梁应霞；

【导师】 鲁成； 周泽扬；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2001， 硕士

【摘要】 Peng.Liang等于1992年报道的mRNA差别显示技术（DDRT-PCR），其简单易行、高效灵敏、能同时进行多个样本的差异分析，有效地解决了其它同类方法成本高、方法繁琐、可比较样本数仅为2个且目的性差等问题，为差异表达基因的分离与克隆提供了强有力的工具。 sch蚕胚胎期温敏性的特点是：在催青后期高温干燥处理即不能孵化而致死，但正常条件下催青，则正常孵化，这表明sch蚕的温敏性与环境调控导致基因差异表达有关。因此，本研究首先建立了一套适用于蚕卵基因差异表达研究的DDRT-PCR方法，并用此方法对不同催青处理的夏sch蚕卵及其对照夏芳蚕卵的基因差异表达进行了研究，结果如下： 1、一步法提取的总RNA经DNase处理后完全适用于DDRT-PCR。其A260/A280为1.8～2.0，说明用此方法抽提的蚕卵RNA纯度较高，无DNA、蛋白质等杂质污染；其变性琼脂糖凝胶电泳显示，用此方法抽提的蚕卵RNA质量较好，为无降解的完整的RNA。 2、建立了一种适用于蚕卵mRNA差异显示的方法。其用购买和合成的试剂取代了RNAimage kit，大大地降低了实验成本；同时用银染法代替了同位素标记-放射自显影，使操作起来更加安全易行。该方法每次扩增可以获得大约60条长度在200～600bP的清晰带，克服了RNAimage kit与银染结合起来用于蚕卵研究时所得带谱少、小、弱等弱点。尽管呈现的差异带中非特异扩增带很多，但由于我们仅对重复出现的差异带进行研究，假阳性反而得到了很好的控制，比率下降为50％。 3、从己₂胚子开始高温干燥（35℃，60％RH）处理了36小时的夏sch卵（后简称夏sch/高）、常温常湿（25℃，80％RH）处理的夏sch卵（后简称夏sch/常）、高温干燥处理的夏芳卵（后简称夏芳/高）和常温常湿处理的夏芳卵（后简称夏芳/常）四个样本的DDRT-PCR研究结果表明，同一组引物扩增出来的谱带，四者之间绝大多数是相同的，少数差异带表现为以下6类：（a）夏芳蚕品种特有带；（b）夏sch蚕品种特有带；（c）高温特异带；（d）夏sch/常缺失带；（e）夏sch/高缺失带；（f）夏sch/高特异带。仅出现在夏sch/常、夏芳/常和夏芳/高中的（e）类或夏sch/高中的（f）类差异带与温敏致死性状相匹配。 4、经分析，在三次重复操作中共狄得了 24条（e）类和（t”）类差异带，其中能稳 定重复的有2条，即片段HAP。280、HA卜200，这正是我们研究SCh蚕的温敏致死机制所 要寻找的目的带。 5、Northern杂交证明，2条目标带中仅HAP。280为真实的，其为夏sob／高的特异 表达产物。另外，山于取材时尽可能地排除了个体差异和发育进程差异，保证了取得的 材料除了表达与不表达与温敏致死有关的基因外，其它的遗传背景几乎充全一致；而在 进行DD！订十“分析时，采用夏芳／高、夏拼常作为对照，对存在于夏sc”高与夏sc” 常之问的与温敏性无关的基因表达差异带予以排除，山此，我们有理山相信HAP上80参 与了SCh蚕胚胎期温敏致死反应。因此有必要把HAP。280向5端延伸，获取基因全序列， 以进－步确定其结构和功能。 6、秆川T－八兑隆策略成功地将IIA‘上HO克隆主 r－pUC19载休上。 7、IIAP8280测序后汲行序列分析得知：HAP。280长度为 273bn，含有一段 PO（A）． 其AI’含量高达61％，符合通过差异显示技术得到片段的AT含量；HAP8280与Genbank 中的巳知基因同源的碱基区域最长不超过25hP，仅占HAP。280全长的9％，故考虑其为新 序列；出HA巳280的椎定的氨基酸序列有三种可能，需尽量向5延长，找到起始密码后 确定其真汇的氨基酸编码及其在sCh蚕温敏致死反应中可能扮演的角色。更多还原

【Abstract】 Differential display reverse-transcriptase PCR (DDRT-PCR), reported first by Peng.Liang cf at in 1992. has the characteristics of convenience, high sensitivity and is suitable to many samples. DDRT-PCR effectively overcomes the drawbacks of the other similar methods, such as high cost, laboriousness and only two comparative samples can be analyzed etc. So DDRT-PCR is a powerful protocol to identify and c lone differentially expressed genes. The ch~ractet of sc/i of temperature-sensitive lethality during embryonic period suggests that temperature-sensitive lethality of sc/i is related with differential expression of genes controlled by environment. So an effective method of DDRT-PCR for silkworm eggs was developed in this investigation and then eggs of Xia sc/i incubated under 60% RI-I at 35~C for 36h (Abbreviate as:Xia sch/high) ,eggs of Xia sc/i treated in normal temperature and humidity (Abbreviate as:Xia sch /normal), eggs of Xia fang under 60% RH at 35扖 for 36h (Abbreviate as :Xia fang/high) eggs of Xia fang treated in normal temperature and humidity (Abbreviate as :Xia fang/normal) were used to research. The gene expression of them 憊ere analyzed by DDRT-PCR.The results are as the following: I Total RNA extracted by one-step-method and treated with DNase free of RNase was found to be applicable to DDRT-PCR. Its absorbance ratio A260/A280 and results of the electrophosis showed that the RNA samples thus-obtained were in good integrity?and free of degradation of RNA and contamination of DNA. 2.An effective method of DDRT-PCR for silkworm eggs was developed in this investigation. Reagents in RNA image kit \vere replaced by those purchased seperately and primers synthesized commercially; autoradiography was replaced by silver staining, so its cost was considerably declined and safety and convenience were improved. This method overcomes the general defects of less, small and weak bands when RNA image kit and silver staining are used together. Using this method, one pair of primers can obtain 60 clear hands range from 200bp to 600hp. Although there were some non-specifically amplified bands in differentially expressed bands, false-positive was obviously controlled and its ratio was down to VI 50% because only differentially expressed bands that could be replicated were further analyzed. 3. The results of DDRT-PCR on Xia sch/high and Xia sc/i/normal, Xia fang/high, Xia fang/normal indicated that the great majority of bands amplified by every pair of primers were the same among the four samples, the small minority of differentially expressed bands included 6 types as the following: (a) bands specific to Xia fang; (b) bands specific to Xia sch; (c) bands sepecific to high temperature treatment; (d)deftciency bands in Xia sc/i/normal; (e) bands specific to Xia sch/normal, Xia fang/high, Xia fang/normal; (f)bands specific to Xia sc/i/high. Type (e) and type (f) were found to be in good accordance with temperature sensitivity of sc/i. 4. 24 differentially expressed bands of type(e) and (0 were obtained in the triplication. But only HA P8280 and I-1AP5200 which could complicate steadily were used as the bands of interest for we study the machanism of temperature sensitivity of sc/i. 5.The results of Norhern blot indicated that only HAP8280, the band specific to Xia schlhigh, was true.更多还原