Jhdm1d論文：小鼠新基因Jhdm1d的印記鑒定和表達(dá)譜分析

1、jhdmld論文：小鼠新基因jhdmld的印記鑒定和表達(dá)譜分析【中文摘要】jhdmld命名為組蛋白去甲基化酶1同系物d,定位 于小鼠6號(hào)染色體,編碼由940個(gè)氨基酸構(gòu)成的組蛋白去甲基化酶， 屬于包含jmjc結(jié)構(gòu)域的jumonji蛋白家族。jmjc結(jié)構(gòu)域被認(rèn)為可能 是一類新的組蛋白去甲基化酶共同基序，對(duì)h3k9me2和h3k27me2有去 甲基化作用，研究發(fā)現(xiàn)jumonji家族中的有些蛋白參與轉(zhuǎn)錄抑制和染 色質(zhì)調(diào)節(jié)過(guò)程。為了探索jhdmld基因在小鼠胚胎不同發(fā)育階段的表 達(dá)與組織發(fā)育的關(guān)系，本課題對(duì)jhdmld基因在mrna水平上的時(shí)空表 達(dá)譜進(jìn)行了初步分析，為進(jìn)一步探索該基因編碼的組蛋白去甲基

2、化酶 在小鼠發(fā)育過(guò)程中的功能奠定理論基礎(chǔ)o木課題選取小鼠胚胎為實(shí)驗(yàn) 材料,利用原位雜交技術(shù)對(duì)jhdmld基因進(jìn)行表達(dá)譜分析。全胚胎原位 雜交數(shù)據(jù)表明該基因在e (embryonic day) 9. 5> e10. 5和ell. 5天 的小鼠全胚胎腦組織屮持續(xù)高表達(dá)，此外，在e9. 5天胚胎的心臟和 e11. 5天胚胎的體節(jié)中都有較強(qiáng)的信號(hào)表達(dá);胚胎組織切片的原位雜 交數(shù)據(jù)表明,jhdmld基因在e12.5天小鼠的腦、肝臟、心臟、脊椎軟 骨原基、嗅上皮、舌等器官和組織中均有不同水平的表達(dá)，其中腦、 脊椎軟骨原基、肝中表達(dá)水平較高;在e15. 5天胚胎組織切片中，該基 因在腦、肝、脊柱軟骨原

3、基、腎、腎上腺、肺、嗅上皮、舌、肋軟骨、 心等器官和組織中均有不同水平的表達(dá),其中肝臟組織里表達(dá)信號(hào)最 強(qiáng),腦次之，脊柱軟骨原基、肋軟骨和腎上腺中也有較強(qiáng)表達(dá)。此外， 選用 e15. 5 天 bff1 (c57b l/6j 早 xfvb £ )腦組織 dna 和 cdna 為材 料，利用 snp (single nucleotide polymorphism)位點(diǎn)直接測(cè)序法初 步對(duì)jhdmld基因進(jìn)行印記分析，結(jié)果表明jhdmld在e15. 5天bff1小 鼠腦組織中為非印記基因。研究結(jié)果表明jhdmld是參與小鼠胚胎發(fā) 育過(guò)程的一個(gè)重要基因,在小鼠胚胎早期腦的發(fā)育、骨骼生長(zhǎng)、多組

4、織形成等過(guò)程中可能起重要的作用。初步分析jhdmld基因的表達(dá)譜 為該基因在功能方面的研究提供一定的理論依據(jù)?！居⑽恼縯he jhdmld gene which is named jumonjic domain-containing of the histone demethylase 1 homologues d is located on mouse chromosome 6. it encodes a 940 - amino-acid histone demethylase protein that belongs to jumonji family containing the j

5、mjc domain jmjc domain is considered to be a common motif in a novel class of histone demethylase, and can demethylate di-methylated lysine 9 and 27 on histone h3 (h3k9me2, h3k27me2) some proteins in the jumonji family may regulate chromatin and transcript!on inhibition. tn order to explore the corr

6、elations between the gene expression and tissue development during mouse embryonic development, we made an initiatory analysis to study the expression of the jhdmld gene in mrna level at diverse developmental embryo stages, and analyzed the function of the histone demethylase during mouse developmen

7、t to establish the theory foundation. we analyzed the expression pattern of the jhdmld gene using in situ hybridization with mouse embryo as the experimental materials whole mount in situ hybridization data showed that high and continuous expression of jhdmld appeared in brain tissue of e9.5, e10.5

8、and ell.5 mouse whole embryos in addition, the heart in e9. 5 embryo and somite in ell. 5 embryo had strong expression. section in situ hybridization data showed that in e12 5 tissue sections, different expression levels presented in brain, liver, heart, spinal cartilage primordium, olfactory epithe

9、lium and tongue, with higher expression level in brain, spinal cartilage primordium and 1iver tn e15.5 embryo, different levels of expression displayed in brain, liver, spinal cartilage primordium, kidney, adrenal gland, lung, olfactory epithelium, tongue, costal cartilage and heart it demonstrated

10、that the strongest expression revealed in liver, brain, followed by spinal cartilage primordial , costal cartilage and adrenal gland we used direct sequencing basic on snp site in brain tissue dna and cdna of bff1 (c57bl/6j 早 x fvb $ ) at e15 5 to determine jhdmld gene imprinting status. the results

11、 showed double allelic expression, indicating that the jhdmld gene in mouse bff1 brain at e15 5 was a non-imprinted gene.the preliminary analysis data showed that jhdmld is an important gene during mouse embryonic development and plays animportant role in nervous system, bone development and develop

12、ment of multiple tissues in early mouse embryo. this initiatory analysis of jhdmld expression provides a theory support of jhdmld function in development 【關(guān)鍵詞】jhdmld組蛋白去甲基化酶 原位雜交 小鼠【英文關(guān)鍵詞】jhdmldhistone demethylaseinsitu hybridizationmouse【目錄】小鼠新基因jhdmld的印記鑒定和表達(dá)譜分析摘要4-5 abstract 5 笫1章 緒論 8-141. 1課題背景

13、及研究目的和意義8-912相關(guān)基礎(chǔ)知識(shí)和國(guó)內(nèi)外研究進(jìn)展9-131.2.1孤雌生殖9-101.2.2基因組印記10-111.2.3組蛋白去甲基化酶11-131.3研究?jī)?nèi)容13-14 第2章 實(shí)驗(yàn)材料與方法14-262. 1實(shí)驗(yàn)材料和儀器14-152. 1. 1實(shí)驗(yàn)動(dòng)物142. 1. 2實(shí)驗(yàn)材料14 2. 1. 3實(shí)驗(yàn)試劑14-152. 1. 4實(shí)驗(yàn)儀器15 2. 2實(shí)驗(yàn)方法與步驟15-262. 2. 1探針的合成15-192. 2. 2組織切片原位雜交19-222. 2. 3全胚胎原位雜交22-252. 2. 4印記鑒定方法25-26 第3章實(shí)驗(yàn)結(jié)果與分析26-403. 1 jhdmld基因生物

14、信息學(xué)分析26-293. 1. 1 jhdmld 基因結(jié)構(gòu) 263. 1. 2 jhdmld 基因編27-29碼的蛋口 26-273. 1. 3小鼠jhdmld蛋口的生物信息學(xué)分析3. 2 jhdmld基因在小鼠e9. 5-e11. 5天全胚胎中的表達(dá)結(jié)果29-333.2. 1地高辛標(biāo)記的反義rna探針的制備29-303. 2. 2 jhdmld基因在小鼠e9. 5-e11.5天全胚胎中的表達(dá)分析30-333. 3 jhdmld在小鼠e12. 5和e15. 5天胚胎組織切片中的表達(dá)結(jié)果33-353.4 jhdmld基因的印記分析35-363.4. 1尋找snp位點(diǎn) 35-363.4.2印記鑒定結(jié)果363.5討論36-3