柞蚕PTTH基因的克隆及表达分析

发布时间：2018-10-31 12:48

【摘要】：柞蚕是我国特有的泌丝昆虫,也是重要的以蛹滞育的模式昆虫,通过蛹滞育以对抗恶劣环境,使种群得以延续。滞育期的昆虫发育暂时停止,解除滞育之后,昆虫会继续进行生长发育和繁殖。柞蚕的促前胸腺激素由脑分泌,通过刺激前胸腺分泌蜕皮激素来控制柞蚕的变态发育,研究柞蚕促前胸腺激素基因的表达及调控对于阐明柞蚕的滞育机理,也有助于调控柞蚕的生活环境来控制滞育的发生和解除,实现柞蚕生产的高产和稳产。本文以河南省一化性柞蚕品种“豫大1号”滞育蛹和辽宁省二化性柞蚕品种“沈黄2号”的5龄幼虫、蛹及成虫为材料,采用RT.PCR技术得到柞蚕促前胸腺激素(ApPTTH)基因,通过生物信息学分析对其进行结构和功能预测；利用原核表达技术构建了ApPTTH的原核表达载体,成功诱导ApPTTH蛋白的表达；利用半定量PCR技术,以二化性柞蚕品种“沈黄2号”5龄早期幼虫的组织为材料,分析了ApPTTH在幼虫期不同组织中的表达谱；分别以一化性和二化性柞蚕滞育蛹为材料,每天采用300 Lux照度、光照17h光照条件诱导滞育蛹解除滞育,利用实时荧光定量PCR技术检测一化性品种“豫大1号”和二化性品种“沈黄2号”滞育蛹在解除滞育过程中蛹脑组织中ApPTTH表达量,结果如下。1.利用TA克隆技术成功克隆了ApPTTH,柞蚕促前胸腺激素ApPTTH的cDNA的ORF长度为666 bp,编码221个氨基酸,蛋白质等电点PI为8.38,预测蛋白长度约为25.96kDa。柞蚕PTTH与其它几种昆虫的PTTH氨基酸序列同源性在70%以上。2.RT-PCR检测结果表明,ApPTTH除了在柞蚕5龄早期幼虫的脑组织中有表达以外,在柞蚕5龄早期幼虫的卵巢组织中也检测到ApPTTH的表达,而其它组织中则并没有检测到ApPTTH的表达。3.Real-time PCR检测结果表明,柞蚕蛹滞育期间ApPTTH在蛹脑组织中的表达量变化不大,而在解除滞育以及蛹发育将要进入成虫时ApPTTH的表达量上调而达到最大值,当柞蚕蛹基本完成成虫发育接近羽化出成虫之后ApPTTH的表达量又开始下降,说明ApPTTH基因的表达及ApPTTH的合成对滞育蛹解除滞育及成虫发育至羽化具有有重要作用。4.将克隆的ApPTTH与表达载体pEASY(?)-Blunt E1相连接,并转化菌株BL21(DE3)表达蛋白,纯化后制备抗体。从蛹脑中提取蛋白,通过Western-blot检测,在30 kD左右出现了一条很粗的条带,该大小与预测的ApPTTH分子量相同,与抗体相结合的蛋白就是柞蚕的PTTH蛋白.本研究为进一步了解 ApPTTH的作用及与滞育的关系奠定了基础。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S885.1;Q78

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