發(fā)布時(shí)間：2018-04-23 21:50

  本文選題：家蠶 + 基因家族　； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：家蠶(Bombyx mori)是由古代野桑蠶經(jīng)長期馴化而成的泌絲昆蟲,在生物學(xué)領(lǐng)域是經(jīng)典的模式生物,在經(jīng)濟(jì)上具有極高的價(jià)值。由咽側(cè)體合成的保幼激素(Juvenile hormone,JH)與前胸腺合成的蛻皮激素(20-hydroxyecdysone,20E)是調(diào)控家蠶生長發(fā)育以及變態(tài)的兩種重要激素。其中,JH經(jīng)過咽側(cè)體合成并分泌至血淋巴中,與保幼激素結(jié)合蛋白(Juvenile hormone binding protein,JHBP)結(jié)合并被運(yùn)送到不同的靶器官,進(jìn)而調(diào)控家蠶的生長發(fā)育。家蠶的經(jīng)濟(jì)效益與合成蠶絲蛋白的發(fā)達(dá)絲腺密不可分。早有研究報(bào)道,對家蠶添食JH能夠增加絲蛋白的產(chǎn)量,而家蠶JHBP(Bm JHBP)作為JH信號(hào)通路傳導(dǎo)的第一個(gè)關(guān)鍵分子,與絲蛋白合成的關(guān)系是不清楚的。此前,學(xué)者已經(jīng)鑒定了家蠶部分的Bm JHBP基因,發(fā)現(xiàn)在家蠶的多個(gè)組織器官內(nèi)均有表達(dá),其主要表達(dá)在脂肪體和中腸。本研究旨在運(yùn)用生物信息學(xué)全面鑒定并分析家蠶JHBP基因家族成員,采用RT-PCR、克隆、Western blot、免疫熒光等技術(shù)手段對家蠶后部絲腺特異高量表達(dá)的Bm JHBP基因的表達(dá)模式以及功能進(jìn)行了分析和研究。獲得的主要研究結(jié)果如下:1、家蠶JHBP基因家族鑒定以及表達(dá)模式分析為了全面的鑒定并分析家蠶含保幼激素結(jié)合蛋白結(jié)構(gòu)域的Bm JHBP基因家族,本研究基于家蠶基因組數(shù)據(jù)庫和NCBI,首先利用生物信息學(xué)對家蠶Bm JHBP基因家族成員進(jìn)行了鑒定。結(jié)果顯示,總共鑒定到了41個(gè)Bm JHBP基因,分別分布在8條染色體上,主要位于第15和23號(hào)染色體�；蚪Y(jié)構(gòu)分析發(fā)現(xiàn),家族JHBP基因結(jié)構(gòu)呈現(xiàn)出多樣化,在基因長度、外顯子等方面均有較大差異。信號(hào)肽預(yù)測發(fā)現(xiàn),33個(gè)Bm JHBP基因編碼一個(gè)信號(hào)肽,8個(gè)Bm JHBP基因不編碼信號(hào)肽。經(jīng)過蛋白質(zhì)結(jié)構(gòu)域分析,家蠶Bm JHBP蛋白至少含有1個(gè)JHBP結(jié)構(gòu)域,且發(fā)現(xiàn)部分Bm JHBP蛋白同時(shí)包含JHBP和Grp7_allergen結(jié)構(gòu)域。將家蠶Bm JHBP與小菜蛾P(guān)x JHBP、果蠅Dm JHBP、蜜蜂Am JHBP等物種進(jìn)化分析,結(jié)果表明JHBP基因家族可以分為兩枝、四個(gè)亞家族。芯片數(shù)據(jù)和RTPCR實(shí)驗(yàn)均表明,Bm JHBP主要表達(dá)于生殖腺、頭、表皮、中腸。RT-PCR分析發(fā)現(xiàn),Bm JHBPd2是整個(gè)家蠶JHBP基因家族中唯一在后部絲腺高量表達(dá)基因。2、家蠶Bm JHBPd2的表達(dá)模式分析及后部絲腺JH的鑒定為了分析BmJHBPd2的表達(dá)模式,首先,利用生物信息學(xué)分析BmJHBPd2基因,結(jié)果表明該基因全長為8053bp,包括5個(gè)外顯子和4個(gè)內(nèi)含子,開放閱讀框(Open reading frame,ORF)長度為732bp,編碼243個(gè)氨基酸,前18個(gè)氨基酸編碼一個(gè)信號(hào)肽,剩下的氨基酸編碼一個(gè)典型的JHBP結(jié)構(gòu)域,等電點(diǎn)為4.80,預(yù)測分子量大小為27.3k D,位于23號(hào)染色體的nscaf3027區(qū)域內(nèi)。在蛋白水平上,利用Western blot方法檢測Bm JHBPd2蛋白在大造5齡第3天各組織的表達(dá)情況,發(fā)現(xiàn)Bm JHBPd2高量表達(dá)于后部絲腺,這與芯片數(shù)據(jù)和RT-PCR結(jié)果一致。然后,通過RT-PCR分析Bm JHBPd2在大造4齡第3天到蛹期的時(shí)期表達(dá)情況,發(fā)現(xiàn)Bm JHBPd2在5齡1天到5天高量表達(dá),在4齡眠期和5齡末期表達(dá)量較低。為了進(jìn)一步驗(yàn)證Bm JHBPd2蛋白在細(xì)胞的表達(dá)部位,構(gòu)建了p SLfa1180-GFP-Bm JHBPd2細(xì)胞過表達(dá)載體,轉(zhuǎn)染至Bm E細(xì)胞,采用免疫熒光實(shí)驗(yàn),結(jié)果表明Bm JHBPd2表達(dá)于Bm E細(xì)胞的細(xì)胞質(zhì)中。利用石蠟切片和免疫熒光等方法,對Bm JHBPd2在家蠶后部絲腺組織的表達(dá)部位作了定位分析,發(fā)現(xiàn)該蛋白在后部絲腺的細(xì)胞質(zhì)層中高量表達(dá),這與前面的RT-PCR、Western blot、亞細(xì)胞定位結(jié)果相一致。最后,q RT-PCR分析了Bm JHBPd2在低產(chǎn)絲量品種大造和高絲量品種872的表達(dá)量差異,結(jié)構(gòu)表明在5齡第3天和5齡第5天,Bm JHBPd2在872的表達(dá)量明顯高于大造,暗示這可能與絲蛋白合成有關(guān)系。通過GC-MS技術(shù)對家蠶5齡第3天后部絲腺的JH做了定性檢測,質(zhì)譜結(jié)果表明,與標(biāo)準(zhǔn)品相比,在后部絲腺的同一時(shí)間檢測到JH峰,說明家蠶在5齡第3天的后部絲腺具有JH。3、家蠶BmJHBPd2蛋白與JH關(guān)系的研究為了研究Bm JHBPd2與JH的關(guān)系,首先選取大造5齡第1天家蠶,沿背中線涂抹1ug的JHA。結(jié)果表明,經(jīng)JHA涂抹的家蠶,其上蔟及化蛹時(shí)間均出現(xiàn)延遲1~2天。另外,涂抹JHA之后,家蠶的經(jīng)濟(jì)性狀均明顯提高。選取涂抹JHA后的12h、24h、48、72h家蠶,經(jīng)過q RT-PCR檢測Bm JHBPd2的表達(dá)水平,發(fā)現(xiàn)家蠶在涂抹JHA后的Bm JHBPd2的表達(dá)量呈現(xiàn)上調(diào)的趨勢,說明外源JHA能夠促進(jìn)Bm JHBPd2的表達(dá)。為了進(jìn)一步研究Bm JHBPd2的功能,構(gòu)建了p SLfa1180-Bm JHBPd2細(xì)胞過表達(dá)載體,轉(zhuǎn)染至Bm E細(xì)胞,q RT-PCR實(shí)驗(yàn)表明向培養(yǎng)基添加JHA之后,JH信號(hào)通路上的早期因子Kr-h1顯著上調(diào)表達(dá),說明JHA激活JH信號(hào)通路。單獨(dú)過表達(dá)Bm JHBPd2,JH信號(hào)通路上的相關(guān)基因表達(dá)量沒有明顯變化,說明單獨(dú)過表達(dá)Bm JHBPd2不能激活JH信號(hào)通路,對fib-H的表達(dá)也沒影響。過表達(dá)Bm JHBPd2并添加JHA與單獨(dú)添加JHA相比,前者的JH信號(hào)通路上的基因比起后者呈現(xiàn)出下調(diào)表達(dá)的趨勢,說明Bm JHBPd2的過表達(dá)能夠抑制JH通路信號(hào)的傳遞。
[Abstract]:The silkworm (Bombyx mori) is a silk insect that has been domesticated by the ancient silkworm for a long time. It is a classic model creature in the field of biology and is of great value in the economy. The Juvenile hormone (JH) synthesized from the pharynx and the ecdysone (20-hydroxyecdysone, 20E) synthesized from the anterior thymus (20-hydroxyecdysone, 20E) is the regulation of the growth and development of silkworm and the growth and development of silkworm. Two important hormones of metamorphosis, of which JH is synthesized and secreted into the hemolymph through the pharynx side body, combined with Juvenile hormone binding protein (JHBP) and transported to different target organs to regulate the growth and development of silkworm. The economic benefits of silkworm are inseparable from the developed silk gland of silk protein. It is reported that adding JH to silkworm can increase the yield of silk protein, while the JHBP (Bm JHBP) of silkworm (Bm JHBP) is the first key molecule of the JH signaling pathway, and the relationship with the synthesis of silk protein is not clear. After that, the scholars have identified the Bm JHBP gene in the silkworm, and found that it was expressed in many tissues and organs of the silkworm, its main table The aim of this study is to analyze and study the expression pattern and function of Bm JHBP gene in the posterior silk gland of silkworm, using RT-PCR, clone, Western blot, immunofluorescence and other technical means by using bioinformatics to comprehensively identify and analyze the family members of the JHBP gene family of silkworm. The results are as follows: 1, the identification and expression pattern analysis of the JHBP gene of silkworm in order to comprehensively identify and analyze the Bm JHBP gene family containing the silkworm containing hormone binding protein domain. This study was based on the silkworm genome database and NCBI. First, the family members of the Bm JHBP gene family of silkworm were identified by bioinformatics. The results showed that the gene family of the silkworm Bm JHBP gene family was first identified. A total of 41 Bm JHBP genes were identified and distributed on 8 chromosomes, mainly on chromosome fifteenth and twenty-third. Gene structure analysis showed that the family JHBP gene structure varied, and the gene length and exons were different. The signal peptide predicted that 33 Bm JHBP genes encode a signal peptide and 8 Bm JHBP bases. Bm JHBP protein contains at least 1 JHBP domains and some Bm JHBP proteins contain JHBP and Grp7_allergen domains at the same time by protein domain analysis. The results show that the gene family can be divided into two species: Bm JHBP, Px JHBP, Px JHBP, Drosophila melanogaster Dm. Two branches and four subfamilies. The chip data and RTPCR experiments showed that Bm JHBP was mainly expressed in the gonads, the head, the epidermis, and the midgut.RT-PCR analysis found that the Bm JHBPd2 was the only high expression gene.2 in the JHBP gene family of the whole silkworm, the expression model analysis of the Bm JHBPd2 of the silkworm and the identification of the JH of the posterior silk gland for the analysis of BmJHBPd2. First, using bioinformatics to analyze the BmJHBPd2 gene, the results show that the whole length of the gene is 8053bp, including 5 exons and 4 introns. The open reading frame (Open reading frame, ORF) is 732bp, 243 amino acids encode, the first 18 amino acids encode a signal peptide, and the remaining amino acids encode a typical JHBP structure The domain, the isoelectric point is 4.80, the predicted molecular weight is 27.3k D, located in the nscaf3027 region of chromosome 23. At the protein level, the Western blot method was used to detect the expression of Bm JHBPd2 protein in the tissues of the 5 instar third days, and the high expression of Bm JHBPd2 was found in the posterior silk gland, which was in accordance with the chip data and RT-PCR results. Then, the Bm JHBPd2 was found to be consistent with the data and RT-PCR results. The expression of Bm JHBPd2 in the period of 4 years from third days to the pupal stage was analyzed by RT-PCR. It was found that Bm JHBPd2 was expressed at a high level from 1 to 5 days in 5 years old and low in the 4 age and late 5 years. In order to further verify the expression of Bm JHBPd2 protein at the cell expression site, P SLfa1180-GFP-Bm JHBPd2 cell overexpression vector was constructed and transfected to Bm E cells. The results of immunofluorescence test showed that Bm JHBPd2 was expressed in the cytoplasm of Bm E cells. Using paraffin section and immunofluorescence, the expression of Bm JHBPd2 in the posterior silk gland tissue of the silkworm was analyzed. It was found that the protein was highly expressed in the cytoplasmic layer of the posterior silk gland, which was with the RT-PCR, Western blot and subfine in the front of the silkworm. The results of cell location were the same. Finally, Q RT-PCR analyzed the difference in the expression of Bm JHBPd2 in low yield and Kose variety 872. The structure showed that the expression of Bm JHBPd2 in 872 was obviously higher than that of the large production at 872 third days and 5 years old, suggesting that this might be related to the synthesis of silk protein. Through GC-MS technology, third days after the 5 age of silkworm Bombyx mori The JH of the silk gland was qualitatively detected. The mass spectrometry results showed that the JH peak was detected at the same time in the posterior silk gland compared with the standard, indicating that the silkworm had JH.3 in the back of the silkworm at the back of 5 and third days, and the relationship between the BmJHBPd2 protein and JH of the silkworm was studied to study the relationship between Bm JHBPd2 and JH. First, the silkworm, 5 years old and first days old, was selected and smeared along the middle line of the back. The JHA. results showed that the silkworm and the pupation time of the silkworm was delayed 1~2 days after the JHA smear. In addition, after applying the JHA, the economic characters of the silkworm were obviously improved. The 12h, 24h, and 48,72h silkworms after JHA were selected and the expression level of Bm JHBPd2 was detected by Q RT-PCR. The expression of the silkworm in the silkworm was up to be raised. The trend indicated that exogenous JHA could promote the expression of Bm JHBPd2. In order to further study the function of Bm JHBPd2, the overexpression vector of P SLfa1180-Bm JHBPd2 cells was constructed and transfected to Bm E cells. Road. There was no significant change in the expression of related genes on the JH signaling pathway alone over the expression of Bm JHBPd2, indicating that a single overexpression of Bm JHBPd2 did not activate the JH signaling pathway and did not affect the expression of fib-H. The gene on the JH letter channel of the former was down regulated by the expression of Bm JHBPd2 and the addition of JHA to the addition of JHA. The trend indicates that over expression of Bm JHBPd2 can inhibit the transmission of JH pathway signal.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S881.2

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