家蚕孵化酶基因Ⅰ、Ⅱ启动子克隆分析及其转录因子结合位点的初步鉴定

发布时间：2018-05-02 01:57

本文选题：家蚕 + 孵化酶基因启动子　；参考：《江苏科技大学》2017年硕士论文

【摘要】：本课题组业已报道了家蚕的两种孵化酶基因(BmHEⅠ和BmHEII)。在家蚕胚胎发育期,BmHEⅠ和BmHEII基因转录本的相对表达量均随着胚胎发育而上升并在孵化时达到顶峰,两者的瞬时表达性基本与孵化酶基因相吻合。然而,在家蚕幼虫期,在家蚕中肠中检测到BmHEⅠ mRNA,提示其可能具有其他功能;在家蚕精巢组织中检测到BmHEⅡ基因转录本,推测其可能与家蚕的精子发生有关。本研究对两个家蚕孵化酶基因的启动子进行克隆分析并对其部分转录因子结合位点进行初步鉴定。本研究主要获得以下三部分结果:1.BmHEⅠ和BmHEⅡ基因启动子的克隆与分析:PCR克隆获得两种家蚕孵化酶基因的上游启动子序列,BmHEⅠp,长度为1350bp,BmHEⅡp,长度为1240bp。利用在线软件NNPP预测出BmHEⅠp,BmHEⅡp的核心启动子位置,BmHEⅠp上仅有一个核心启动子,BmHEⅡp上含有多个核心启动子;利用在线软件Alibaba预测比较了BmHEⅠ p与BmHEⅡp上可能存在的转录因子结合位点,结果显示BmHEⅠp上特有的转录因子结合位点有CREB、NF-1、c-Jun等13个,BmHEⅠIp上特有的转录因子结合位点有HNF-4α1、Pit-1a、TEF、MEB-1等17个。2.家蚕中肠、精巢和蚕卵胚胎核蛋白与Bm HE Ip和BmHEⅡp上的转录因子结合位点探针进行EMSA分析:首先采用细胞核蛋白质提取试剂盒抽提家蚕中肠、精巢和蚕卵胚胎核蛋白,中肠核蛋白浓度为4.73μg/μL、精巢核蛋白浓度为1.17μg/μL,蚕卵胚胎核蛋白浓度平均为4.92μg/μL;依据前人的研究,从所预测的BmHEⅠp和BmHEⅡp上的转录因子结合位点中,筛选出BmHEⅠp上的Oct-1、CRE-BP1转录因子结合位点,BmHEⅡp上的HNF-4α1、TBP转录因子结合位点,利用IR700染料对上述四个转录因子结合位点序列进行标记,将其作为探针与上述所抽提的核蛋白进行凝胶迁移阻滞分析(EMSA)。中肠核蛋白能与Oct-1、CRE-BP1探针特异性结合,精巢核蛋白能与HNF-4α1、TBP探针特异性结合,然而,精巢核蛋白不能与Oct-1、CRE-BP1探针结合,中肠核蛋白不能与HNF-4α1探针结合,推测在幼虫发育期,家蚕孵化酶基因表达的组织特异性可能与Oct-1、CRE-BP1、和HNF-4α1相关;催青第7~9天的蚕卵核蛋白能与TBP探针结合,催青第1~9天的蚕卵核蛋白能与HNF-4α1探针结合,推测在胚胎发育期,家蚕孵化酶基因的表达调控可能与TBP、HNF-4α1相关。3.EMSA阻滞条带的质谱分析:利用毛细管高效液相色谱法对上述EMSA的阻滞条带进行质谱鉴定,结果表明,未能直接证明凝胶阻滞条带中存在Oct-1、CRE-BP1、HNF-4α1和TBP转录因子成分,然而在凝胶阻滞条带中包含了其他转录因子成分。Apoptosis-related protein 1CAD、Cyclic AMP-regμLated protein和FancJ-like protein等能与CRE-BP1转录因子结合位点结合;14-3-3 protein zeta、actin-related 2/3 complex subunit 2和Beadex/dLMO protein等能与Oct-1转录因子结合位点结合;Fanconi anemia、complementation groupⅠ和HSP90等能够与HNF-4α1转录因子结合位点结合;Interleukin enhancer binding factor isoform 1、p53和translation initiation factor 2 gamma subunit等能与TBP转录因子结合位点结合,表明家蚕孵化酶基因表达的组织特异性不仅可能与Oct-1、CRE-BP1、HNF-4α1、TBP相关,还可能与上述转录因子相关。上述初步结果为进一步阐明家蚕孵化酶基因表达的调控途径提供了基础信息。
[Abstract]:Two kinds of hatching enzyme genes (BmHE I and BmHEII) of the silkworm (silkworm, Bombyx mori) have been reported. The relative expression of the BmHE I and BmHEII transcripts in the silkworm embryo development period increased with the development of the embryo and reached the peak at the time of hatching. The instantaneous expressiveness of the two was basically consistent with the hatching enzyme gene. However, in the larval stage of the silkworm, the silkworm larvae were at home. BmHE I mRNA was detected in the midgut of the silkworm, suggesting that it might have other functions. The BmHE II gene transcript was detected in the silkworm's spermary tissue. It is presumed that it may be related to the spermatogenesis of the silkworm. The promoter of the two silkworm hatching enzyme genes was cloned and analyzed, and the binding site of some of the transcription factor was preliminarily identified. This study mainly obtained the following three parts: cloning and analysis of the promoter of 1.BmHE I and BmHE II gene promoter: PCR clone obtained the upstream promoter sequence of the two silkworm hatching enzyme genes, BmHE I P, the length of 1350bp, BmHE II P, and the length of 1240bp. using the online software NNPP to predict the BmHE P. Only one core promoter, BmHE II P contains a number of core promoters, using online software Alibaba to predict and compare the transcription factor binding sites that may exist on BmHE I P and BmHE II P. The results show that the specific transcription factor binding sites on BmHE I P are 13, CREB, NF-1, c-Jun and other transcription factor binding sites. HNF-4 alpha 1, Pit-1a, TEF, MEB-1 and other.2. silkworm midgut, spermary and egg embryo nucleoprotein and transcription factor binding site probe on Bm HE Ip and BmHE II P for EMSA analysis. First, nuclear protein extraction kits were used to extract the midgut, spermary and egg embryo nuclear protein, and the concentration of midgut nucleoprotein was 4.73 mu g/ micron, the nucleus of the spermary The concentration of protein was 1.17 g/ mu L, and the average concentration of the egg embryo nuclear protein was 4.92 u g/ mu L. According to previous studies, the Oct-1, CRE-BP1 transcription factor binding site, the binding site of the transcriptional factor on the BmHE I P and the transcription factor binding site on the BmHE II p were screened. The above four transcription factor binding site sequences were labeled to conduct gel migration block analysis (EMSA) as a probe and the extracted nucleoprotein. The midgut nucleoprotein can specifically bind to Oct-1, CRE-BP1 probes, and the nucleoprotein can be specifically associated with HNF-4 alpha 1, TBP probe. However, the nucleoprotein of the spermary can not be detected with Oct-1, CRE-BP1 In combination, the midgut nucleoprotein can not be combined with the HNF-4 alpha 1 probe. It is speculated that the tissue specificity of the gene expression of the silkworm hatchase gene may be related to the Oct-1, CRE-BP1, and HNF-4 alpha 1 in the larval development period; the egg nucleoprotein of day 7~9 days can be combined with the TBP probe, and the egg nucleoprotein of day 1~9 can be combined with the HNF-4 alpha 1 probe to speculate on the embryo hair. During the period of incubation, the regulation of the gene expression of the hatching enzyme in the silkworm may be analyzed by the mass spectrometric analysis of the.3.EMSA blocking bands associated with TBP, HNF-4 alpha 1. The capillary high performance liquid chromatography (HPLC) was used to identify the blocking bands of the above EMSA. The results showed that the Oct-1, CRE-BP1, HNF-4 alpha 1 and TBP transcription factors were not directly found in the gel block bands, however, the components of the Oct-1, HNF-4 alpha and TBP transcription factors were not shown. The other transcription factor components,.Apoptosis-related protein 1CAD, Cyclic AMP-reg, Lated protein and FancJ-like protein, are combined with the binding site of the CRE-BP1 transcription factor in the gel block band. Fanconi anemia, complementation group I and HSP90 can bind to the binding site of HNF-4 alpha 1 transcription factor; Interleukin enhancer binding factor isoform 1, p53 and group binding sites can be combined with the binding site of the transcription factor, indicating the tissue specificity of the gene expression of the hatching enzyme in the silkworm. It is not only related to Oct-1, CRE-BP1, HNF-4 alpha 1 and TBP, but also may be related to the above transcription factors. The above preliminary results provide basic information for further clarifying the regulation pathway of the gene expression of silkworm hatching enzyme.

【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S881.2;Q78

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