本文選題：Bm　切入點：Sxl　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：栽桑養(yǎng)蠶在中國已有五千年的歷史。在幾千年的家蠶養(yǎng)殖過程中,人們發(fā)現(xiàn),家蠶的雌雄個體無論是在體型大小還是產(chǎn)絲量上都存在很大差異,雄蠶具有更高的經(jīng)濟(jì)應(yīng)用價值。同時,作為重要的鱗翅目昆蟲,家蠶的研究還可以作為模式,為基礎(chǔ)科學(xué)研究提供參考。因此對家蠶性別決定的研究,不僅對提高家蠶的經(jīng)濟(jì)價值具有重要意義,而且也將為進(jìn)一步了解昆蟲的生殖繁衍及病蟲害防治提供重要參考。在先前研究中發(fā)現(xiàn)BmSxl參與了家蠶性別決定通路,并且具有2種mRNA形式,編碼2種不同形式的蛋白。為了進(jìn)一步了解BmSxl的自身剪接的機理,本文設(shè)計相關(guān)實驗對BmSxl自身剪接的上游調(diào)控因子和調(diào)控機理,以及與其他性別調(diào)控基因BmPSI、BmIMP和BmMasc的互作關(guān)系進(jìn)行了研究。獲得的主要研究結(jié)果如下:1.Bm Sxl的剪接調(diào)控因子家蠶中BmSxl有兩種剪接形式,BmSxl-PA和BmSxl-PB。二者的區(qū)別在于第八外顯子上的一小段序列,在BmSxl-PA中保留,而在BmSxl-PB中被剪接。為了調(diào)取到這小段序列被剪接(或保留)的上游調(diào)控因子,本實驗首先設(shè)計引物,通過克隆得到在BmSxl-PB中被剪切的那一小段序列,然后進(jìn)行體外轉(zhuǎn)錄,獲得相應(yīng)的RNA序列。再將該序列進(jìn)行生物素標(biāo)記,從5齡3天家蠶精巢中提取核蛋白,通過RNA-Protein pull down獲得與該序列結(jié)合的蛋白。再通過SDS-PAGE電泳和銀染比對發(fā)現(xiàn)特異條帶,將特異條帶挖膠進(jìn)行質(zhì)譜鑒定,共鑒定出46種蛋白。對這46種蛋白進(jìn)行功能分析發(fā)現(xiàn),其中heterogeneous nuclear ribonucleoprotein87F-like是一種剪接相關(guān)蛋白,并且有研究發(fā)現(xiàn)該蛋白在家蠶精巢中表達(dá)�？紤]到BmSxl-PB只在家蠶性腺中表達(dá),并且在精巢的表達(dá)量高于卵巢。因此,該蛋白是BmSxl剪接的重要候選調(diào)控因子。2.heterogeneous nuclear ribonucleoprotein 87F-like的原核表達(dá)純化家蠶中heterogeneous nuclear ribonucleoprotein 87F-like在NCBI上的登錄號為XP_004930561。該基因共編碼317個aa,約34KD,等電點為9.2,屬于堿性蛋白。根據(jù)該蛋白的基因序列設(shè)計引物,以5齡3天家蠶的精巢cDNA為模板成功克隆到目的片段,并將該序列克隆到pET-28a(+)載體質(zhì)粒中,構(gòu)建原核表達(dá)載體,通過轉(zhuǎn)化至BL21(DE3)菌株中進(jìn)行原核表達(dá)。檢測發(fā)現(xiàn)該蛋白在誘導(dǎo)16℃的上清中表達(dá)量較高,并且通過親和層析獲得了較純的目的蛋白。3.Bm Sxl對自身剪接起重要調(diào)控作用的位點選擇性剪切除了需要剪接因子的參與,還與基因本身順式元件有很大關(guān)系。為了鑒定上游調(diào)控因子結(jié)合的順式元件,本實驗設(shè)計引物,將BmSxl第八外顯子內(nèi)部的剪接位點附近,每10-12個堿基一組,進(jìn)行替換突變。并且突變的設(shè)置避開常規(guī)剪接所需的必要位點,共獲得突變17組。將17組突變的第八外顯子序列克隆到1180[Hrs-BmAct4-LUC-Ser1PA]上,并轉(zhuǎn)染至家蠶的卵巢細(xì)胞,48h后提取RNA進(jìn)行RT-PCR分析,發(fā)現(xiàn)當(dāng)GCCTTGGGCA/ACACGGACGA/ACGCTTCAAA/TAATCCAACA/CATCGCGGTT/GCTTAAAGGAAC這6組堿基發(fā)生突變時,BmSxl-PB形式消失。說明這6組堿基序列很可能與BmSxl的剪接調(diào)控有關(guān)。4.Bm Sxl的剪接調(diào)控序列與剪接因子的結(jié)合驗證針對此6組堿基序列和剪接因子heterogeneous nuclear ribonucleoprotein87F-like之間是否存在結(jié)合這一問題,我們首先把這6組堿基序列制成RNA探針,通過EMSA進(jìn)行實驗驗證。結(jié)果發(fā)現(xiàn)heterogeneous nuclear ribonucleoprotein87F-like與探針GCCUUGGGCA可以特異性結(jié)合。這些結(jié)果顯示heterogeneous nuclear ribonucleoprotein 87F-like可以通過結(jié)合GCCUUGGGCA序列對BmSxl的剪接進(jìn)行調(diào)控。5.BmSXL與其他性別調(diào)控基因的互作Bmdsx是家蠶性別調(diào)控網(wǎng)絡(luò)下游的開關(guān)基因,BmSXL可以與Bmdsx的多聚U序列結(jié)合,促進(jìn)Bmdsx發(fā)生雄特異性剪接。BmPSI、BmIMP、BmMasc也是調(diào)控Bmdsx發(fā)生雄特異性剪接的重要因子。為了進(jìn)一步確定BmSXL與BmPSI、Bm IMP和BmMasc的關(guān)系,首先設(shè)計引物并從5齡3天的家蠶精巢中成功克隆出BmSxl、BmPSI和Bm IMP基因。通過免疫共沉淀和雙分子熒光互補實驗驗證得出BmSXL與BmPSI之間存在相互作用關(guān)系,而與BmIMP、BmMasc不存在相互作用。所以在家蠶的性別調(diào)控通路中,BmSXL可以與BmPSI相互作用,共同參與到下游Bmdsx的剪接調(diào)控中。
[Abstract]:Sericulture in Chinese has five thousand years of history. People found in the silkworm breeding process for thousands of years, both male and female silkworm, the great differences in size or amount of silk production, male silkworm has higher economic value. At the same time, as an important scale insect, Bombyx mori research can also be used as a model, to provide reference for basic scientific research. It was decided on Silkworm Sex Research, not only has important significance to improve the economic value of silkworm, but also to further understand the reproductive and pest insect pest prevention provides important reference. In the previous study found that BmSxl in Silkworm Sex determination pathway, and has 2 mRNA, encoding 2 different forms of the protein. In order to further understand the mechanism of BmSxl splicing, this paper designed the experiments on BmSxl splicing upstream regulation factor And the regulation mechanism, and other sex control genes BmPSI, BmIMP and BmMasc of the interaction are studied. The main results are as follows: BmSxl splicing factor 1.Bm of silkworm Sxl in two different splicing variants, the difference between BmSxl-PA and BmSxl-PB. two in exon eighth in a short sequence on the reservation, in the BmSxl-PA, and in BmSxl-PB by splicing. In order to obtain the short sequence by splicing (or retention) of the upstream regulatory factor, this paper designed primers, cloned by the short sequence shear in BmSxl-PB, and then in vitro transcription, obtained the corresponding RNA sequence. Then the sequence. Biotin, nuclear proteins were extracted from 5 3 day old testis was obtained, combined with the sequence of pull protein by RNA-Protein down. Through SDS-PAGE electrophoresis and silver staining was found on the specific bands, the specific. Dig with glue were identified, 46 proteins were identified. Of these 46 kinds of protein function analysis found that the heterogeneous nuclear ribonucleoprotein87F-like is a splicing related protein, and studies have found that the expression of the protein in silkworm testis. Considering that BmSxl-PB only expressed in silkworm gonad, and higher expression in the testis ovary volume. Therefore, the prokaryotic expression of BmSxl protein is an important candidate splicing regulatory factor.2.heterogeneous nuclear ribonucleoprotein 87F-like heterogeneous nuclear ribonucleoprotein 87F-like in the purification of silkworm in the NCBI accession number is XP_004930561. the gene encoding 317 AA, about 34KD, isoelectric point was 9.2, which belongs to the basic protein. The primers were designed according to the gene sequence of the protein, 5 to 3 days old silkworm testis cDNA as template and cloned the target fragment, and the sequence of cloning to pET-28 A (+) plasmid, prokaryotic expression vectors were constructed by the conversion to BL21 (DE3) for prokaryotic expression strains. Results showed that the protein is highly expressed in the induction of 16 DEG C in the supernatant by affinity chromatography, and obtain relatively pure.3.Bm protein Sxl plays an important role in the regulation of alternative splicing sites in addition to the participation of self splicing factors and gene splicing, is itself a great relationship type element CIS cis element binding. In order to identify upstream regulatory factors, the experimental design of primers, BmSxl exon eighth splice sites near the internal sub base of every 10-12, a group of loci and mutations necessary substitution mutations. The settings needed to avoid conventional splicing, received a total of 17 groups. The 17 group mutation mutation of exon eighth sequence was cloned into 1180[Hrs-BmAct4-LUC-Ser1PA], and transfected into silkworm ovary cells, after 48h extraction of RNA RT- PCR analysis found that the GCCTTGGGCA/ACACGGACGA/ACGCTTCAAA/TAATCCAACA/CATCGCGGTT/GCTTAAAGGAAC of these 6 groups of mutations, BmSxl-PB disappeared. The 6 group sequence is likely to exist whether this combination between BmSxl and Sxl on.4.Bm splicing regulation of splicing regulatory sequences and splicing factor combination verification of the 6 group base sequence and splicing factor heterogeneous nuclear ribonucleoprotein87F-like first of all, we put the 6 sets of sequences into RNA probe, were verified by EMSA. The results showed that heterogeneous nuclear and ribonucleoprotein87F-like GCCUUGGGCA probe specific binding. These results show that heterogeneous nuclear ribonucleoprotein 87F-like.5.BmSXL can be regulated with other sex control gene interaction Bmdsx silkworm by binding to the GCCUUGGGCA sequence of BmSxl splicing Switch gene regulatory network downstream of the gender, BmSXL and Bmdsx poly U sequence combined with Bmdsx, promote the occurrence of male specific splicing of.BmPSI, BmIMP, BmMasc is also an important factor in the regulation of Bmdsx occurrence of male specific splicing. In order to further determine the relationship between Bm BmSXL and BmPSI, IMP and BmMasc, were designed and from age 5 the 3 day of the testis was successfully cloned by BmSxl, BmPSI and Bm. IMP gene and bimolecular fluorescence complementation experiments show that the presence of interactions between BmSXL and BmPSI by CO immunoprecipitation and BmIMP, BmMasc does not exist interaction. So in the sex control of silkworm pathway, BmSXL can interact with BmPSI and to participate in the regulation of splicing downstream of Bmdsx.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

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本文編號：1640678