本文選題：家蠶 + 孵化酶基因啟動子�。� 參考：《江蘇科技大學》2017年碩士論文

【摘要】：本課題組業(yè)已報道了家蠶的兩種孵化酶基因(BmHEⅠ和BmHEII)。在家蠶胚胎發(fā)育期,BmHEⅠ和BmHEII基因轉(zhuǎn)錄本的相對表達量均隨著胚胎發(fā)育而上升并在孵化時達到頂峰,兩者的瞬時表達性基本與孵化酶基因相吻合。然而,在家蠶幼蟲期,在家蠶中腸中檢測到BmHEⅠ mRNA,提示其可能具有其他功能;在家蠶精巢組織中檢測到BmHEⅡ基因轉(zhuǎn)錄本,推測其可能與家蠶的精子發(fā)生有關。本研究對兩個家蠶孵化酶基因的啟動子進行克隆分析并對其部分轉(zhuǎn)錄因子結合位點進行初步鑒定。本研究主要獲得以下三部分結果:1.BmHEⅠ和BmHEⅡ基因啟動子的克隆與分析:PCR克隆獲得兩種家蠶孵化酶基因的上游啟動子序列,BmHEⅠp,長度為1350bp,BmHEⅡp,長度為1240bp。利用在線軟件NNPP預測出BmHEⅠp,BmHEⅡp的核心啟動子位置,BmHEⅠp上僅有一個核心啟動子,BmHEⅡp上含有多個核心啟動子;利用在線軟件Alibaba預測比較了BmHEⅠ p與BmHEⅡp上可能存在的轉(zhuǎn)錄因子結合位點,結果顯示BmHEⅠp上特有的轉(zhuǎn)錄因子結合位點有CREB、NF-1、c-Jun等13個,BmHEⅠIp上特有的轉(zhuǎn)錄因子結合位點有HNF-4α1、Pit-1a、TEF、MEB-1等17個。2.家蠶中腸、精巢和蠶卵胚胎核蛋白與Bm HE Ip和BmHEⅡp上的轉(zhuǎn)錄因子結合位點探針進行EMSA分析:首先采用細胞核蛋白質(zhì)提取試劑盒抽提家蠶中腸、精巢和蠶卵胚胎核蛋白,中腸核蛋白濃度為4.73μg/μL、精巢核蛋白濃度為1.17μg/μL,蠶卵胚胎核蛋白濃度平均為4.92μg/μL;依據(jù)前人的研究,從所預測的BmHEⅠp和BmHEⅡp上的轉(zhuǎn)錄因子結合位點中,篩選出BmHEⅠp上的Oct-1、CRE-BP1轉(zhuǎn)錄因子結合位點,BmHEⅡp上的HNF-4α1、TBP轉(zhuǎn)錄因子結合位點,利用IR700染料對上述四個轉(zhuǎn)錄因子結合位點序列進行標記,將其作為探針與上述所抽提的核蛋白進行凝膠遷移阻滯分析(EMSA)。中腸核蛋白能與Oct-1、CRE-BP1探針特異性結合,精巢核蛋白能與HNF-4α1、TBP探針特異性結合,然而,精巢核蛋白不能與Oct-1、CRE-BP1探針結合,中腸核蛋白不能與HNF-4α1探針結合,推測在幼蟲發(fā)育期,家蠶孵化酶基因表達的組織特異性可能與Oct-1、CRE-BP1、和HNF-4α1相關;催青第7~9天的蠶卵核蛋白能與TBP探針結合,催青第1~9天的蠶卵核蛋白能與HNF-4α1探針結合,推測在胚胎發(fā)育期,家蠶孵化酶基因的表達調(diào)控可能與TBP、HNF-4α1相關。3.EMSA阻滯條帶的質(zhì)譜分析:利用毛細管高效液相色譜法對上述EMSA的阻滯條帶進行質(zhì)譜鑒定,結果表明,未能直接證明凝膠阻滯條帶中存在Oct-1、CRE-BP1、HNF-4α1和TBP轉(zhuǎn)錄因子成分,然而在凝膠阻滯條帶中包含了其他轉(zhuǎn)錄因子成分。Apoptosis-related protein 1CAD、Cyclic AMP-regμLated protein和FancJ-like protein等能與CRE-BP1轉(zhuǎn)錄因子結合位點結合;14-3-3 protein zeta、actin-related 2/3 complex subunit 2和Beadex/dLMO protein等能與Oct-1轉(zhuǎn)錄因子結合位點結合;Fanconi anemia、complementation groupⅠ和HSP90等能夠與HNF-4α1轉(zhuǎn)錄因子結合位點結合;Interleukin enhancer binding factor isoform 1、p53和translation initiation factor 2 gamma subunit等能與TBP轉(zhuǎn)錄因子結合位點結合,表明家蠶孵化酶基因表達的組織特異性不僅可能與Oct-1、CRE-BP1、HNF-4α1、TBP相關,還可能與上述轉(zhuǎn)錄因子相關。上述初步結果為進一步闡明家蠶孵化酶基因表達的調(diào)控途徑提供了基礎信息。
[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.

【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S881.2;Q78

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