家蚕Bmsxl基因自身剪接的调控机理研究

发布时间：2018-03-20 20:30

本文选题：Bm　切入点：Sxl　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：栽桑养蚕在中国已有五千年的历史。在几千年的家蚕养殖过程中,人们发现,家蚕的雌雄个体无论是在体型大小还是产丝量上都存在很大差异,雄蚕具有更高的经济应用价值。同时,作为重要的鳞翅目昆虫,家蚕的研究还可以作为模式,为基础科学研究提供参考。因此对家蚕性别决定的研究,不仅对提高家蚕的经济价值具有重要意义,而且也将为进一步了解昆虫的生殖繁衍及病虫害防治提供重要参考。在先前研究中发现BmSxl参与了家蚕性别决定通路,并且具有2种mRNA形式,编码2种不同形式的蛋白。为了进一步了解BmSxl的自身剪接的机理,本文设计相关实验对BmSxl自身剪接的上游调控因子和调控机理,以及与其他性别调控基因BmPSI、BmIMP和BmMasc的互作关系进行了研究。获得的主要研究结果如下:1.Bm Sxl的剪接调控因子家蚕中BmSxl有两种剪接形式,BmSxl-PA和BmSxl-PB。二者的区别在于第八外显子上的一小段序列,在BmSxl-PA中保留,而在BmSxl-PB中被剪接。为了调取到这小段序列被剪接(或保留)的上游调控因子,本实验首先设计引物,通过克隆得到在BmSxl-PB中被剪切的那一小段序列,然后进行体外转录,获得相应的RNA序列。再将该序列进行生物素标记,从5龄3天家蚕精巢中提取核蛋白,通过RNA-Protein pull down获得与该序列结合的蛋白。再通过SDS-PAGE电泳和银染比对发现特异条带,将特异条带挖胶进行质谱鉴定,共鉴定出46种蛋白。对这46种蛋白进行功能分析发现,其中heterogeneous nuclear ribonucleoprotein87F-like是一种剪接相关蛋白,并且有研究发现该蛋白在家蚕精巢中表达。考虑到BmSxl-PB只在家蚕性腺中表达,并且在精巢的表达量高于卵巢。因此,该蛋白是BmSxl剪接的重要候选调控因子。2.heterogeneous nuclear ribonucleoprotein 87F-like的原核表达纯化家蚕中heterogeneous nuclear ribonucleoprotein 87F-like在NCBI上的登录号为XP_004930561。该基因共编码317个aa,约34KD,等电点为9.2,属于碱性蛋白。根据该蛋白的基因序列设计引物,以5龄3天家蚕的精巢cDNA为模板成功克隆到目的片段,并将该序列克隆到pET-28a(+)载体质粒中,构建原核表达载体,通过转化至BL21(DE3)菌株中进行原核表达。检测发现该蛋白在诱导16℃的上清中表达量较高,并且通过亲和层析获得了较纯的目的蛋白。3.Bm Sxl对自身剪接起重要调控作用的位点选择性剪切除了需要剪接因子的参与,还与基因本身顺式元件有很大关系。为了鉴定上游调控因子结合的顺式元件,本实验设计引物,将BmSxl第八外显子内部的剪接位点附近,每10-12个碱基一组,进行替换突变。并且突变的设置避开常规剪接所需的必要位点,共获得突变17组。将17组突变的第八外显子序列克隆到1180[Hrs-BmAct4-LUC-Ser1PA]上,并转染至家蚕的卵巢细胞,48h后提取RNA进行RT-PCR分析,发现当GCCTTGGGCA/ACACGGACGA/ACGCTTCAAA/TAATCCAACA/CATCGCGGTT/GCTTAAAGGAAC这6组碱基发生突变时,BmSxl-PB形式消失。说明这6组碱基序列很可能与BmSxl的剪接调控有关。4.Bm Sxl的剪接调控序列与剪接因子的结合验证针对此6组碱基序列和剪接因子heterogeneous nuclear ribonucleoprotein87F-like之间是否存在结合这一问题,我们首先把这6组碱基序列制成RNA探针,通过EMSA进行实验验证。结果发现heterogeneous nuclear ribonucleoprotein87F-like与探针GCCUUGGGCA可以特异性结合。这些结果显示heterogeneous nuclear ribonucleoprotein 87F-like可以通过结合GCCUUGGGCA序列对BmSxl的剪接进行调控。5.BmSXL与其他性别调控基因的互作Bmdsx是家蚕性别调控网络下游的开关基因,BmSXL可以与Bmdsx的多聚U序列结合,促进Bmdsx发生雄特异性剪接。BmPSI、BmIMP、BmMasc也是调控Bmdsx发生雄特异性剪接的重要因子。为了进一步确定BmSXL与BmPSI、Bm IMP和BmMasc的关系,首先设计引物并从5龄3天的家蚕精巢中成功克隆出BmSxl、BmPSI和Bm IMP基因。通过免疫共沉淀和双分子荧光互补实验验证得出BmSXL与BmPSI之间存在相互作用关系,而与BmIMP、BmMasc不存在相互作用。所以在家蚕的性别调控通路中,BmSXL可以与BmPSI相互作用,共同参与到下游Bmdsx的剪接调控中。
[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.

【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】：Q78

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