家蚕滞育关联miRNA及其靶基因调控关系研究

发布时间：2018-03-13 13:15

  本文选题：家蚕卵　切入点：滞育　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：本文以家蚕932蚕卵为材料,采用生物信息学、PCR、qRT-PCR、DLR与ELISA等方法,通过筛查与家蚕滞育密切相关的miRNA及其靶基因,分析其在滞育卵、即时浸酸卵、赤豆色浸酸卵的表达差异及调控作用,以进一步探索家蚕滞育发生与滞育解除的分子调控机理,取得主要结果如下:(1)利用生物信息学工具RNAhybrid等对蚕卵经浸酸后发生显著上调或下调的miRNA各4个进行分析,从中筛选出2个与家蚕滞育密切关联的bmo-miR-3384-3p与bmo-miR-2761-3p,并进行靶标预测,得知bmo-miR-3384-3p在候选靶基因BmNLK的3’UTR的11-64位碱基存在结合位点,而bmo-miR-2761-3p与BmSDH基因结合位点在其3’UTR的396-423位碱基。(2)通过PCR扩增技术,比较候选靶基因BmNLK、BmSDH在家蚕滞育性卵、赤豆色浸酸卵中的转录表达情况,经检测分析,当蚕卵经盐酸浸渍处理后,BmSDH基因和BmNLK基因发生显著上调现象,而在非浸酸卵中则处于低水平的表达状态,表明两基因在滞育性卵与浸酸卵中有明显的差异表达。(3)用qRT-PCR技术对滞育性卵与赤豆色浸酸卵在卵龄3-7d时bmo-miR-2761-3p、bmo-miR-3384-3p、BmSDH基因和BmNLK基因的表达水平进行定量分析,结果在浸酸卵中,bmo-miR-3384-3p、bmo-miR-2761-3p的相对表达量最低时分别比浸酸前下降了76%和78%,而BmNLK基因和BmSDH基因的相对表达量最高时则分别上调了217%和4287%。经统计分析,bmo-miR-2761-3p与BmSDH基因、bmo-miR-3384-3p与BmNLK基因的表达关系呈负相关性(R12=0.94,R22=0.92)。由此表明,蚕卵处在滞育状态时,BmSDH基因、BmNLK基因低水平表达,当蚕卵浸酸后则显著上调。(4)为验证bmo-miR-2761-3与BmSDH基因、bmo-miR-3384-3p与BmNLK基因的靶标关系,分别构建了含有BmSDH 3’UTR、BmNLK 3’UTR的载体,并经DLR法检测验证,证实了bmo-miR-2761-3p会抑制BmSDH基因的表达;bmo-miR-3384-3p会抑制BmNLK基因的表达。(5)借助ELISA方法测定了即时浸酸卵和赤豆色浸酸卵浸酸后至孵化前1d、滞育性卵于5℃冷藏0、10、20、30、40、60、80d期间的BmSDH酶和BmNLK酶的活性,结果蚕卵一经浸酸,酶活快速上调,即时浸酸卵、赤豆色浸酸卵在浸酸后经过2d BmNLK酶的活性就到达峰值,分别为2972.24 mU/g、2800.45 mU/g,比对照分别提高了13.26倍和12.43倍。BmSDH酶则于浸酸后经过3d时出现峰值,即时浸酸卵的为1003.36 mIU/g,赤豆色浸酸卵的为662.29 mIU/g,比对照分别提高了4.50倍和2.63倍。滞育卵在5℃冷库中冷藏时,入库初期酶活活性下降,当冷藏经过30d/20d时,酶活开始上调,到80d时,BmNLK酶和BmSDH酶活性到达最大值。由此表明,BmNLK酶和BmSDH酶的活性变化,与蚕卵的滞育解除存在联动关系。
[Abstract]:In this paper, the silkworm eggs 932 were used as materials, using bioinformatics methods such as PCRX qRT-PCRN DLR and ELISA. By screening miRNA and its target genes closely related to diapause of silkworm silkworm, the diapause eggs were analyzed, and the eggs were immediately soaked in acid. In order to further explore the molecular regulation mechanism of diapause occurrence and diapause in silkworm, the difference of expression and regulation effect of red bean color pickling eggs were studied. The main results are as follows: (1) using RNAhybrid and other bioinformatics tools, we analyzed 4 miRNA which were significantly upregulated or down-regulated in silkworm eggs after soaking acid, screened out two bmo-miR-3384-3p and bmo-miR-2761-3ps closely related to diapause of silkworm silkworm, and predicted the target. The results showed that bmo-miR-3384-3p had binding sites at the 11-64 bases of the candidate target gene BmNLK, while the binding site of bmo-miR-2761-3p and BmSDH gene was 396-423 at the position 396-423 of the candidate target gene. PCR amplification technique was used to compare the candidate gene BmNLKK BmSDH in diapause eggs of silkworm Bombyx mori (Bombyx mori). The transcriptional expression of BmSDH gene and BmNLK gene were up-regulated in the eggs treated with hydrochloric acid, but the expression of BmSDH gene and BmNLK gene were low in the non-impregnated eggs. The results showed that there were significant differences between diapause eggs and acid-impregnated eggs by qRT-PCR. The expression levels of bmo-miR-2761-3pmo-miR-3384-3pmSDH and BmNLK genes in diapause eggs and red bean infected eggs at the age of 3 to 7 days were quantitatively analyzed. Results the relative expression of bmo-miR-3384-3pmo-miR-2761-3p decreased by 76% and 78 at the lowest level, respectively, while the relative expression of BmNLK gene and BmSDH gene increased by 217% and 4287when the relative expression of BmNLK gene and BmSDH gene was the highest. The statistical analysis showed that bmo-miR-2761-3p and BmSDH gene Bmo-miR-3384-3p and BmNLK gene were up-regulated by 217% and 42870.The results showed that bmo-miR-2761-3p and BmSDH gene Bmo-miR-3384-3p and BmNLK gene were up-regulated respectively. A negative correlation was found in the expression relationship of R12A0. 94 and R22. 92, which indicated that, When eggs were in diapause condition, the expression of BmSDH gene and BmNLK gene of silkworm eggs was low, and the expression of BmNLK gene was upregulated significantly when the eggs were immersed in acid. (4) to verify the target relationship between bmo-miR-2761-3 and BmSDH gene, the vector containing BmNLK3UTR of BmNLK3UTR was constructed, and the vector containing BmNLLK3UTR was constructed, and the expression of BmNLK 3UTR was verified by DLR method. It was confirmed that bmo-miR-2761-3p could inhibit the expression of BmSDH gene. The expression of BmNLK gene was inhibited by bmo-miR-2761-3p. The activities of BmSDH enzyme and BmNLK enzyme of diapause eggs were measured by ELISA method. The diapause eggs were incubated at 5 鈩，

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