【摘要】：柞蠶是我國(guó)特有的泌絲昆蟲,也是重要的以蛹滯育的模式昆蟲,通過蛹滯育以對(duì)抗惡劣環(huán)境,使種群得以延續(xù)。滯育期的昆蟲發(fā)育暫時(shí)停止,解除滯育之后,昆蟲會(huì)繼續(xù)進(jìn)行生長(zhǎng)發(fā)育和繁殖。柞蠶的促前胸腺激素由腦分泌,通過刺激前胸腺分泌蛻皮激素來控制柞蠶的變態(tài)發(fā)育,研究柞蠶促前胸腺激素基因的表達(dá)及調(diào)控對(duì)于闡明柞蠶的滯育機(jī)理,也有助于調(diào)控柞蠶的生活環(huán)境來控制滯育的發(fā)生和解除,實(shí)現(xiàn)柞蠶生產(chǎn)的高產(chǎn)和穩(wěn)產(chǎn)。本文以河南省一化性柞蠶品種“豫大1號(hào)”滯育蛹和遼寧省二化性柞蠶品種“沈黃2號(hào)”的5齡幼蟲、蛹及成蟲為材料,采用RT.PCR技術(shù)得到柞蠶促前胸腺激素(ApPTTH)基因,通過生物信息學(xué)分析對(duì)其進(jìn)行結(jié)構(gòu)和功能預(yù)測(cè)；利用原核表達(dá)技術(shù)構(gòu)建了ApPTTH的原核表達(dá)載體,成功誘導(dǎo)ApPTTH蛋白的表達(dá)；利用半定量PCR技術(shù),以二化性柞蠶品種“沈黃2號(hào)”5齡早期幼蟲的組織為材料,分析了ApPTTH在幼蟲期不同組織中的表達(dá)譜；分別以一化性和二化性柞蠶滯育蛹為材料,每天采用300 Lux照度、光照17h光照條件誘導(dǎo)滯育蛹解除滯育,利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)一化性品種“豫大1號(hào)”和二化性品種“沈黃2號(hào)”滯育蛹在解除滯育過程中蛹腦組織中ApPTTH表達(dá)量,結(jié)果如下。1.利用TA克隆技術(shù)成功克隆了ApPTTH,柞蠶促前胸腺激素ApPTTH的cDNA的ORF長(zhǎng)度為666 bp,編碼221個(gè)氨基酸,蛋白質(zhì)等電點(diǎn)PI為8.38,預(yù)測(cè)蛋白長(zhǎng)度約為25.96kDa。柞蠶PTTH與其它幾種昆蟲的PTTH氨基酸序列同源性在70%以上。2.RT-PCR檢測(cè)結(jié)果表明,ApPTTH除了在柞蠶5齡早期幼蟲的腦組織中有表達(dá)以外,在柞蠶5齡早期幼蟲的卵巢組織中也檢測(cè)到ApPTTH的表達(dá),而其它組織中則并沒有檢測(cè)到ApPTTH的表達(dá)。3.Real-time PCR檢測(cè)結(jié)果表明,柞蠶蛹滯育期間ApPTTH在蛹腦組織中的表達(dá)量變化不大,而在解除滯育以及蛹發(fā)育將要進(jìn)入成蟲時(shí)ApPTTH的表達(dá)量上調(diào)而達(dá)到最大值,當(dāng)柞蠶蛹基本完成成蟲發(fā)育接近羽化出成蟲之后ApPTTH的表達(dá)量又開始下降,說明ApPTTH基因的表達(dá)及ApPTTH的合成對(duì)滯育蛹解除滯育及成蟲發(fā)育至羽化具有有重要作用。4.將克隆的ApPTTH與表達(dá)載體pEASY(?)-Blunt E1相連接,并轉(zhuǎn)化菌株BL21(DE3)表達(dá)蛋白,純化后制備抗體。從蛹腦中提取蛋白,通過Western-blot檢測(cè),在30 kD左右出現(xiàn)了一條很粗的條帶,該大小與預(yù)測(cè)的ApPTTH分子量相同,與抗體相結(jié)合的蛋白就是柞蠶的PTTH蛋白.本研究為進(jìn)一步了解 ApPTTH的作用及與滯育的關(guān)系奠定了基礎(chǔ)。
[Abstract]:Antheraea pernyi is a unique silk insect in China and an important model insect with pupae diapause. The development of diapause is suspended and the insect continues to grow and reproduce after diapause is released. The prothymic hormone of Antheraea pernyi is secreted by the brain, and the metamorphosis and development of Antheraea pernyi are controlled by stimulating the ecdysone of the prethymic gland to study the gene expression of prothymic hormone and its regulation to clarify the diapause mechanism of Antheraea pernyi. It also helps to regulate the living environment of tussah to control the occurrence and release of diapause and to realize the high and stable production of tussah. Based on the diapause pupae of Henan Province Antheraea pernyi cultivar "Yuda 1" and the 5th instar larva, pupae and adult of Liaoning Province, the (ApPTTH) gene of prothymic hormone was obtained by RT.PCR technique. The structure and function were predicted by bioinformatics analysis. The prokaryotic expression vector of ApPTTH was constructed by prokaryotic expression technique, and the expression of ApPTTH protein was induced successfully. Using semi-quantitative PCR technique, the expression profiles of ApPTTH in different larval tissues were analyzed by using the tissue of the 5th instar larva of "Shenhuang No. 2", a diploid Antheraea pernyi cultivar "Shenhuang No. 2". Diapause pupae of Antheraea pernyi were treated with 300 Lux illumination and 17 h light for 17 h, respectively. The expression of ApPTTH in brain tissue of diapause pupae of "Yuda 1" and "Shenhuang 2" diapause pupae was detected by real-time fluorescence quantitative PCR. The results were as follows. 1. Using TA cloning technique, the ORF length of ApPTTH cDNA of ApPTTH, was 666 bp, encoding 221 amino acids, the protein isoelectric point PI was 8.38, and the predicted protein length was about 25.96 kDa. The homology of PTTH amino acid sequence between tussah PTTH and other insects was more than 70%. The results of 2.RT-PCR showed that ApPTTH was expressed in the brain of early 5th instar larvae of tussah. The expression of ApPTTH was also detected in ovary tissues of early 5th instar larvae of tussah, but no expression of ApPTTH was detected in other tissues. During diapause, the expression of ApPTTH in pupae brain of tussah did not change much, but the expression of ApPTTH increased to the maximum when diapause was removed and pupae development was about to enter the adult. The expression of ApPTTH began to decrease after the adult development of tussah silkworm pupae was close to the emergence of adult, indicating that the expression of ApPTTH gene and the synthesis of ApPTTH played an important role in relieving diapause and adult development to emergence. 4. The cloned ApPTTH was linked to the expression vector pEASY (?)-Blunt E1 and transformed into the expressed protein of the strain BL21 (DE3). The antibody was prepared after purification. The protein extracted from pupa brain was detected by Western-blot and a coarse band appeared at about 30 kD. The size of the protein was the same as the predicted molecular weight of ApPTTH. The protein bound to the antibody was the PTTH protein of Antheraea pernyi (Antheraea pernyi). This study laid a foundation for further understanding the role of ApPTTH and its relationship with diapause.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S885.1;Q78

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