本文選題：家蠶卵　切入點(diǎn)：滯育　出處：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：本文以家蠶932蠶卵為材料,采用生物信息學(xué)、PCR、qRT-PCR、DLR與ELISA等方法,通過篩查與家蠶滯育密切相關(guān)的miRNA及其靶基因,分析其在滯育卵、即時浸酸卵、赤豆色浸酸卵的表達(dá)差異及調(diào)控作用,以進(jìn)一步探索家蠶滯育發(fā)生與滯育解除的分子調(diào)控機(jī)理,取得主要結(jié)果如下:(1)利用生物信息學(xué)工具RNAhybrid等對蠶卵經(jīng)浸酸后發(fā)生顯著上調(diào)或下調(diào)的miRNA各4個進(jìn)行分析,從中篩選出2個與家蠶滯育密切關(guān)聯(lián)的bmo-miR-3384-3p與bmo-miR-2761-3p,并進(jìn)行靶標(biāo)預(yù)測,得知bmo-miR-3384-3p在候選靶基因BmNLK的3’UTR的11-64位堿基存在結(jié)合位點(diǎn),而bmo-miR-2761-3p與BmSDH基因結(jié)合位點(diǎn)在其3’UTR的396-423位堿基。(2)通過PCR擴(kuò)增技術(shù),比較候選靶基因BmNLK、BmSDH在家蠶滯育性卵、赤豆色浸酸卵中的轉(zhuǎn)錄表達(dá)情況,經(jīng)檢測分析,當(dāng)蠶卵經(jīng)鹽酸浸漬處理后,BmSDH基因和BmNLK基因發(fā)生顯著上調(diào)現(xiàn)象,而在非浸酸卵中則處于低水平的表達(dá)狀態(tài),表明兩基因在滯育性卵與浸酸卵中有明顯的差異表達(dá)。(3)用qRT-PCR技術(shù)對滯育性卵與赤豆色浸酸卵在卵齡3-7d時bmo-miR-2761-3p、bmo-miR-3384-3p、BmSDH基因和BmNLK基因的表達(dá)水平進(jìn)行定量分析,結(jié)果在浸酸卵中,bmo-miR-3384-3p、bmo-miR-2761-3p的相對表達(dá)量最低時分別比浸酸前下降了76%和78%,而BmNLK基因和BmSDH基因的相對表達(dá)量最高時則分別上調(diào)了217%和4287%。經(jīng)統(tǒng)計(jì)分析,bmo-miR-2761-3p與BmSDH基因、bmo-miR-3384-3p與BmNLK基因的表達(dá)關(guān)系呈負(fù)相關(guān)性(R12=0.94,R22=0.92)。由此表明,蠶卵處在滯育狀態(tài)時,BmSDH基因、BmNLK基因低水平表達(dá),當(dāng)蠶卵浸酸后則顯著上調(diào)。(4)為驗(yàn)證bmo-miR-2761-3與BmSDH基因、bmo-miR-3384-3p與BmNLK基因的靶標(biāo)關(guān)系,分別構(gòu)建了含有BmSDH 3’UTR、BmNLK 3’UTR的載體,并經(jīng)DLR法檢測驗(yàn)證,證實(shí)了bmo-miR-2761-3p會抑制BmSDH基因的表達(dá);bmo-miR-3384-3p會抑制BmNLK基因的表達(dá)。(5)借助ELISA方法測定了即時浸酸卵和赤豆色浸酸卵浸酸后至孵化前1d、滯育性卵于5℃冷藏0、10、20、30、40、60、80d期間的BmSDH酶和BmNLK酶的活性,結(jié)果蠶卵一經(jīng)浸酸,酶活快速上調(diào),即時浸酸卵、赤豆色浸酸卵在浸酸后經(jīng)過2d BmNLK酶的活性就到達(dá)峰值,分別為2972.24 mU/g、2800.45 mU/g,比對照分別提高了13.26倍和12.43倍。BmSDH酶則于浸酸后經(jīng)過3d時出現(xiàn)峰值,即時浸酸卵的為1003.36 mIU/g,赤豆色浸酸卵的為662.29 mIU/g,比對照分別提高了4.50倍和2.63倍。滯育卵在5℃冷庫中冷藏時,入庫初期酶活活性下降,當(dāng)冷藏經(jīng)過30d/20d時,酶活開始上調(diào),到80d時,BmNLK酶和BmSDH酶活性到達(dá)最大值。由此表明,BmNLK酶和BmSDH酶的活性變化,與蠶卵的滯育解除存在聯(lián)動關(guān)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，

本文編號：1606539