家蚕含保幼激素结合蛋白结构域的BmJHBP基因家族鉴定及BmJHBPd2的功能研究

发布时间：2018-04-23 21:50

本文选题：家蚕 + 基因家族　；参考：《西南大学》2017年硕士论文

【摘要】：家蚕(Bombyx mori)是由古代野桑蚕经长期驯化而成的泌丝昆虫,在生物学领域是经典的模式生物,在经济上具有极高的价值。由咽侧体合成的保幼激素(Juvenile hormone,JH)与前胸腺合成的蜕皮激素(20-hydroxyecdysone,20E)是调控家蚕生长发育以及变态的两种重要激素。其中,JH经过咽侧体合成并分泌至血淋巴中,与保幼激素结合蛋白(Juvenile hormone binding protein,JHBP)结合并被运送到不同的靶器官,进而调控家蚕的生长发育。家蚕的经济效益与合成蚕丝蛋白的发达丝腺密不可分。早有研究报道,对家蚕添食JH能够增加丝蛋白的产量,而家蚕JHBP(Bm JHBP)作为JH信号通路传导的第一个关键分子,与丝蛋白合成的关系是不清楚的。此前,学者已经鉴定了家蚕部分的Bm JHBP基因,发现在家蚕的多个组织器官内均有表达,其主要表达在脂肪体和中肠。本研究旨在运用生物信息学全面鉴定并分析家蚕JHBP基因家族成员,采用RT-PCR、克隆、Western blot、免疫荧光等技术手段对家蚕后部丝腺特异高量表达的Bm JHBP基因的表达模式以及功能进行了分析和研究。获得的主要研究结果如下:1、家蚕JHBP基因家族鉴定以及表达模式分析为了全面的鉴定并分析家蚕含保幼激素结合蛋白结构域的Bm JHBP基因家族,本研究基于家蚕基因组数据库和NCBI,首先利用生物信息学对家蚕Bm JHBP基因家族成员进行了鉴定。结果显示,总共鉴定到了41个Bm JHBP基因,分别分布在8条染色体上,主要位于第15和23号染色体。基因结构分析发现,家族JHBP基因结构呈现出多样化,在基因长度、外显子等方面均有较大差异。信号肽预测发现,33个Bm JHBP基因编码一个信号肽,8个Bm JHBP基因不编码信号肽。经过蛋白质结构域分析,家蚕Bm JHBP蛋白至少含有1个JHBP结构域,且发现部分Bm JHBP蛋白同时包含JHBP和Grp7_allergen结构域。将家蚕Bm JHBP与小菜蛾Px JHBP、果蝇Dm JHBP、蜜蜂Am JHBP等物种进化分析,结果表明JHBP基因家族可以分为两枝、四个亚家族。芯片数据和RTPCR实验均表明,Bm JHBP主要表达于生殖腺、头、表皮、中肠。RT-PCR分析发现,Bm JHBPd2是整个家蚕JHBP基因家族中唯一在后部丝腺高量表达基因。2、家蚕Bm JHBPd2的表达模式分析及后部丝腺JH的鉴定为了分析BmJHBPd2的表达模式,首先,利用生物信息学分析BmJHBPd2基因,结果表明该基因全长为8053bp,包括5个外显子和4个内含子,开放阅读框(Open reading frame,ORF)长度为732bp,编码243个氨基酸,前18个氨基酸编码一个信号肽,剩下的氨基酸编码一个典型的JHBP结构域,等电点为4.80,预测分子量大小为27.3k D,位于23号染色体的nscaf3027区域内。在蛋白水平上,利用Western blot方法检测Bm JHBPd2蛋白在大造5龄第3天各组织的表达情况,发现Bm JHBPd2高量表达于后部丝腺,这与芯片数据和RT-PCR结果一致。然后,通过RT-PCR分析Bm JHBPd2在大造4龄第3天到蛹期的时期表达情况,发现Bm JHBPd2在5龄1天到5天高量表达,在4龄眠期和5龄末期表达量较低。为了进一步验证Bm JHBPd2蛋白在细胞的表达部位,构建了p SLfa1180-GFP-Bm JHBPd2细胞过表达载体,转染至Bm E细胞,采用免疫荧光实验,结果表明Bm JHBPd2表达于Bm E细胞的细胞质中。利用石蜡切片和免疫荧光等方法,对Bm JHBPd2在家蚕后部丝腺组织的表达部位作了定位分析,发现该蛋白在后部丝腺的细胞质层中高量表达,这与前面的RT-PCR、Western blot、亚细胞定位结果相一致。最后,q RT-PCR分析了Bm JHBPd2在低产丝量品种大造和高丝量品种872的表达量差异,结构表明在5龄第3天和5龄第5天,Bm JHBPd2在872的表达量明显高于大造,暗示这可能与丝蛋白合成有关系。通过GC-MS技术对家蚕5龄第3天后部丝腺的JH做了定性检测,质谱结果表明,与标准品相比,在后部丝腺的同一时间检测到JH峰,说明家蚕在5龄第3天的后部丝腺具有JH。3、家蚕BmJHBPd2蛋白与JH关系的研究为了研究Bm JHBPd2与JH的关系,首先选取大造5龄第1天家蚕,沿背中线涂抹1ug的JHA。结果表明,经JHA涂抹的家蚕,其上蔟及化蛹时间均出现延迟1~2天。另外,涂抹JHA之后,家蚕的经济性状均明显提高。选取涂抹JHA后的12h、24h、48、72h家蚕,经过q RT-PCR检测Bm JHBPd2的表达水平,发现家蚕在涂抹JHA后的Bm JHBPd2的表达量呈现上调的趋势,说明外源JHA能够促进Bm JHBPd2的表达。为了进一步研究Bm JHBPd2的功能,构建了p SLfa1180-Bm JHBPd2细胞过表达载体,转染至Bm E细胞,q RT-PCR实验表明向培养基添加JHA之后,JH信号通路上的早期因子Kr-h1显著上调表达,说明JHA激活JH信号通路。单独过表达Bm JHBPd2,JH信号通路上的相关基因表达量没有明显变化,说明单独过表达Bm JHBPd2不能激活JH信号通路,对fib-H的表达也没影响。过表达Bm JHBPd2并添加JHA与单独添加JHA相比,前者的JH信号通路上的基因比起后者呈现出下调表达的趋势,说明Bm JHBPd2的过表达能够抑制JH通路信号的传递。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S881.2

【参考文献】