本文選題：MSTN基因　切入點(diǎn)：Mdka、Mdkb重復(fù)基因　出處：《上海海洋大學(xué)》2017年碩士論文

【摘要】：團(tuán)頭魴(Megalobrama amblycephala),由于其生殖周期短、二齡即可達(dá)到性成熟,是研究肌肉生長(zhǎng)、選育新品種的良好材料。肌肉生長(zhǎng)抑制素(Myostatin,MSTN),又稱(chēng)生長(zhǎng)分化因子8(Growth differentiation factor 8,GDF 8),屬于轉(zhuǎn)化生長(zhǎng)因子β(Transforming growth factorβ,TGF-β)家族。該基因抑制骨骼肌的生長(zhǎng)、發(fā)育。本研究通過(guò)cDNA末端快速擴(kuò)增法(RACE法)克隆得到團(tuán)頭魴生長(zhǎng)抑制素(MSTN)基因全長(zhǎng)cDNA序列并分析了MSTN基因在團(tuán)頭魴胚胎、成魚(yú)組織中表達(dá)以及MSTN基因在胚胎中過(guò)表達(dá)情況。結(jié)果顯示團(tuán)頭魴MSTN基因的cDNA全長(zhǎng)為2187bp,編碼區(qū)ORF(開(kāi)放閱讀框)大小為1128bp,編碼376個(gè)氨基酸,推測(cè)的MSTN前體蛋白等電點(diǎn)為5.02,相對(duì)分子質(zhì)量為92.34KDa。MSTN基因編碼蛋白包含有兩大TGF-β蛋白結(jié)構(gòu)域、RXXR蛋白酶水解位點(diǎn)RIRR和保守的半胱氨酸殘基。組織逆轉(zhuǎn)錄PCR(RT-PCR)結(jié)果顯示,MSTN基因在肌肉、腦和精巢組織中大量表達(dá),肝臟、脾臟和卵巢組織中的表達(dá)量次之,腸、腮、心臟、眼睛和腎組織中的微量表達(dá)。胚胎RT-PCR結(jié)果顯示,在0-44hpf胚胎發(fā)育階段,MSTN基因表達(dá)量較低;而在48hpf-52hpf胚胎發(fā)育階段,MSTN基因表達(dá)量逐漸升高。整胚原位雜交(WISH)結(jié)果顯示,胚胎發(fā)育的16hpf時(shí)期MSTN基因主要在脊索中表達(dá),胚胎發(fā)育的28hpf、55hpf時(shí)期MSTN基因在腦中表達(dá)。MSTN基因過(guò)表達(dá)結(jié)果顯示,胚胎在體節(jié)發(fā)生期出現(xiàn)前-后軸伸長(zhǎng),背-腹軸縮短;脊索發(fā)生扭曲、其生長(zhǎng)發(fā)育受到抑制等現(xiàn)象。本研究為進(jìn)一步探索團(tuán)頭魴MSTN基因在生長(zhǎng)發(fā)育信號(hào)通路中的作用和育種提供了基礎(chǔ)資料。肝素結(jié)合生長(zhǎng)因子(midkine)屬于PTN家族,該家族由Mdk和PTN(pleiotrophin)兩個(gè)成員組成。Mdk是一種對(duì)細(xì)胞生長(zhǎng)、分化、遷移有重要作用的生長(zhǎng)因子。Mdk對(duì)不同組織尤其是神經(jīng)組織的生長(zhǎng)、修復(fù)同樣起著重要的作用。本研究通過(guò)RACE法克隆獲得團(tuán)頭魴Mdka/-b重復(fù)基因cDNA序列。團(tuán)頭魴Mdka基因全長(zhǎng)1409bp,編碼區(qū)ORF大小為441bp,編碼146個(gè)氨基酸,包括22個(gè)氨基酸的信號(hào)肽,124個(gè)氨基酸的成熟肽,Mdka前體蛋白等電點(diǎn)為9.57,相對(duì)分子量為15.73KD;團(tuán)頭魴Mdkb基因全長(zhǎng)1048bp,編碼區(qū)ORF大小為444bp,編碼147個(gè)氨基酸,包括21個(gè)氨基酸的信號(hào)肽,126個(gè)氨基酸的成熟肽,Mdkb前體蛋白等電點(diǎn)為9.41,相對(duì)分子量為16.23KD。與人類(lèi)、小鼠和斑馬魚(yú)一樣,團(tuán)頭魴Mdka/-b基因成熟多肽都包含10個(gè)保守的半胱氨酸殘基,一個(gè)精氨酸殘基,兩個(gè)谷氨酰胺殘基,一個(gè)高度保守的鉸鏈區(qū)和兩個(gè)堿基殘基簇。團(tuán)頭魴Mdka和Mdkb編碼區(qū)之間相似度為64%,與斑馬魚(yú)Mdka和Mdkb編碼區(qū)之間相似度分別為87%和95%,而與人類(lèi)Mdk編碼區(qū)相似度較低(分別為57%和55%)。實(shí)時(shí)熒光定量PCR(qRT-PCR)分析結(jié)果表明,成魚(yú)組織中Mdka mRNA只在腦、性腺、腸中大量表達(dá);而Mdkb mRNA除了皮膚外的所有成魚(yú)組織中都大量表達(dá)。在胚胎發(fā)育階段qRT-PCR分析結(jié)果顯示,Mdka基因mRNA首次檢測(cè)到表達(dá)在受精后12h;Mdkb基因mRNA首次檢測(cè)到表達(dá)在受精后8h。在那之后,Mdka和Mdkb mRNAs表達(dá)都穩(wěn)步上升,在受精后28h達(dá)到最大值,之后下降并逐漸保持穩(wěn)定。WISH結(jié)果表明Mdka和Mdkb mRNAs在受精后16h階段都是全身性表達(dá);在受精后28h階段Mdka mRNA依然是全身性表達(dá),Mdkb mRNA則只在眼睛、腦和尾節(jié)出表達(dá);在受精后55 h階段時(shí),Mdka和Mdkb都只在腦中表達(dá)。饑餓2、4、6天處理后,Mdka和Mdkb mRNAs表達(dá)量在腦、肝臟和腸中表達(dá)量都上升,Mdka mRNA表達(dá)量在恢復(fù)投喂6天后恢復(fù)正常水平;Mdkb mRNA在腦、腸中表達(dá)量在恢復(fù)投喂3天后恢復(fù)正常水平,肝臟中Mdkb mRNA表達(dá)量在恢復(fù)投喂6天后恢復(fù)正常水平。在注射重組人體生長(zhǎng)激素(hGH)處理過(guò)程中,與對(duì)照組相比,在注射50μg hGH肝臟、腸組織中Mdka和Mdkb mRNAs表達(dá)量迅速下降;在注射10μg hGH的腸組織中Mdka和Mdkb mRNAs表達(dá)量也都呈現(xiàn)下降趨勢(shì),但它們表達(dá)量下降程度不一樣;在注射10μg和50μg hGH的腦組織中Mdka mRNA表達(dá)量迅速上升,而Mdkb mRNA表達(dá)量沒(méi)有顯著的變化。研究結(jié)果表明Mdka和Mdkb重復(fù)基因在團(tuán)頭魴胚胎和組織發(fā)育過(guò)程中起著重要、但不同的作用。三倍體團(tuán)頭魴在生長(zhǎng)優(yōu)勢(shì)、群體產(chǎn)量和抗逆性等方面具有明顯的優(yōu)勢(shì),四倍體團(tuán)頭魴與二倍體團(tuán)頭魴雜交可以快速、高效的獲得三倍體團(tuán)頭魴,為團(tuán)頭魴育種開(kāi)創(chuàng)一種新的方法。本研究為獲得能夠穩(wěn)定遺傳的四倍體團(tuán)頭魴而進(jìn)行熱休克方法誘導(dǎo)處理團(tuán)頭魴受精卵抑制其第一次有絲分裂的研究。結(jié)果顯示,在熱休克2min條件下,40.5℃熱休克處理,團(tuán)頭魴受精卵發(fā)育30min誘導(dǎo)出苗率最高為8.33%;41℃熱休克處理,團(tuán)頭魴受精卵36min誘導(dǎo)出苗率最高為5.65%;41.5℃熱休克處理,團(tuán)頭魴受精卵24min誘導(dǎo)出苗率最高為3.76%;42℃熱休克處理,團(tuán)頭魴受精卵30min誘導(dǎo)出苗率最高為4.02%。對(duì)熱休克誘導(dǎo)的團(tuán)頭魴進(jìn)行倍性分析分析其DNA含量,結(jié)果表明熱休克誘導(dǎo)只獲得二倍體和三倍體團(tuán)頭魴,沒(méi)有得到四倍體團(tuán)頭魴。對(duì)檢測(cè)出的二倍體和三倍體團(tuán)頭魴進(jìn)行染色體分析,其結(jié)果與倍性分析結(jié)果一致,可能是由于熱休克誘導(dǎo)的團(tuán)頭魴胚胎后期發(fā)育過(guò)程中出現(xiàn)了染色體丟失所致,具體機(jī)理還需進(jìn)一步深入研究。四組溫度休克處理組中,42℃熱休克誘導(dǎo)處理團(tuán)頭魴受精卵獲得的三倍體團(tuán)頭魴率最高,為2.307%。倍性分析儀檢測(cè)DNA含量結(jié)果顯示,三倍體團(tuán)頭魴DNA含量約為正常二倍體團(tuán)頭魴DNA含量的1.5倍多。染色體分析結(jié)果顯示,正常二倍體團(tuán)頭魴的染色體48條;熱休克誘導(dǎo)的三倍體團(tuán)頭魴的染色體為72條,比二倍體團(tuán)頭魴多24條染色體。本研究采用熱休克法誘導(dǎo)處理團(tuán)頭魴受精卵為獲得四倍體團(tuán)頭魴進(jìn)行了初步研究,為后續(xù)團(tuán)頭魴多倍體育種研究提供一定的參考依舊。
[Abstract]:Bream (Megalobrama amblycephala), because of its short reproductive cycle, two age can reach sexual maturity, is the study of muscle growth, good material for breeding new varieties. Myostatin (Myostatin, MSTN), also known as growth differentiation factor 8 (Growth differentiation factor 8, GDF 8), which belongs to the transforming growth factor beta (Transforming growth factor beta, beta TGF-) family. The gene inhibits skeletal muscle growth and development. This study through rapid amplification of cDNA ends (RACE) cloned bream myostatin (MSTN) gene and the full-length cDNA sequence analysis of MSTN gene in megalobramaamblycephala embryos, adult tissues and MSTN gene expression in the embryo. The results showed that the full-length cDNA MSTN bream gene was 2187bp, encoding ORF (open reading frame) size is 1128bp, encoding 376 amino acids, putative MSTN precursor protein with an isoelectric point of 5.02, relatively The quality of sub 92.34KDa.MSTN gene encoding protein contains two TGF- beta protein domain, RXXR protease hydrolysis sites RIRR and conserved cysteine residues. Reverse transcription PCR (RT-PCR) showed that MSTN gene expressed in muscle, brain and testis tissues of liver, spleen and ovary tissue in the expression of the intestine, gills, heart, eyes and trace expression in renal tissue of the embryo. The results of RT-PCR showed that 0-44hpf in the embryonic stage, MSTN gene expression is relatively low; and the development of 48hpf-52hpf in the embryonic stage, the expression of MSTN gene was gradually increased. The whole embryo in situ hybridization (WISH) results showed that 16hpf MSTN gene in embryonic development the main expression in the notochord, the embryonic development of 28hpf, 55hpf in MSTN gene expression results showed that overexpression of.MSTN gene in the brain, in the period before the onset of embryonic somitogenesis - axle elongation, dorsal ventral axis shortening; spinal cord occurred The growth inhibition of distortion, and so on. This study to further explore the megalobramaamblycephala MSTN gene provides basic information on the role and signaling pathway in breeding. The growth of heparin binding growth factor (midkine) belongs to the PTN family, the family by Mdk and PTN (pleiotrophin) two member.Mdk is a kind of cell the growth, differentiation, migration and growth factor.Mdk an important role especially in neural tissue growth of different tissue repair, also plays an important role in this study. Mdka/-b gene cDNA sequence repeat megalobramaamblycephala was cloned using RACE. The full-length 1409bp of Mdka gene encoding region of megalobramaamblycephala, ORF size 441bp, encoding 146 amino acids. Including a signal peptide of 22 amino acids and a mature peptide of 124 amino acids, Mdka precursor protein isoelectric point is 9.57, molecular weight is 15.73KD; the full-length 1048bp bream Mdkb gene coding region ORF size 444b P, encoding 147 amino acids, including a signal peptide of 21 amino acids and a mature peptide of 126 amino acids, Mdkb precursor protein isoelectric point is 9.41, the relative molecular weight of 16.23KD. and the human, mouse and zebrafish, megalobramaamblycephala mature peptide Mdka/-b gene contains 10 conserved cysteine residues. An arginine residue, two glutamine residues, a highly conserved hinge region and two nucleotide residues cluster. The similarity between Mdka and Mdkb megalobramaamblycephala encoding region is 64%, and the similarity between the zebrafish Mdka and Mdkb encoding region were 87% and 95%, and one class of Mdk encoding region similarity low (57% and 55%). Real time fluorescence quantitative PCR (qRT-PCR) analysis showed that adult tissues Mdka mRNA only in the brain, gonad, intestine and expression; Mdkb mRNA in addition to express large amounts of all adult tissue of the skin. In the embryo stage qRT-PCR analysis The results show that for the first time, Mdka gene mRNA expression was detected in 12h after fertilization; Mdkb gene mRNA was first detected in the expression of 8h. after fertilization after that, the expression of Mdka and Mdkb mRNAs are rising steadily in 28h after fertilization and reached the maximum value, then decreased and gradually remained stable.WISH the results showed that the Mdka and Mdkb mRNAs in 16h after fertilization the stage is systemic expression; in 28h after fertilization stage Mdka mRNA systemic expression is still Mdkb, mRNA only in the eyes, brain and tail section expression; in 55 h after fertilization stage, Mdka and Mdkb were only expressed in the brain. Hunger 2,4,6 days after the treatment, the expression of mRNAs Mdka and Mdkb in the brain, the expression of liver and intestine was increased, the expression of mRNA Mdka in 6 days after refeeding returned to normal level; Mdkb mRNA in brain, intestine expression in refeeding after 3 days and returned to the normal level, the amount returned to normal in 6 days after refeeding Mdkb mRNA expression in liver The level of growth hormone. In the injection of recombinant human (hGH) process, compared with the control group, the injection of 50 g hGH and Mdkb Mdka liver mRNAs expression decreased rapidly in intestinal tissue; Mdka injection at 10 g hGH in intestinal tissue and Mdkb expression of mRNAs also showed a downward trend, but they expression level is not the same; the rapid rise in the amount of expression of Mdka in the injection of 10 g and 50 g hGH in the brain tissue of mRNA, Mdkb and mRNA expression had no significant change. The results show that Mdka and Mdkb gene during the development of bream embryo and tissue plays an important role, but different. In the triploid bream growth advantage, has obvious advantage of yield and resistance of tetraploid and diploid bream, bream hybrids can quickly and efficiently obtain triploid megalobramaamblycephala, create a new method for breeding. In this study, bream. Can the genetic stability of tetraploid bream and heat shock induced by treatment of bream fertilized eggs inhibits the first study on mitosis. The results showed that the heat shock 2min under the condition of 40.5 DEG C, heat shock treatment, bream fertilized eggs 30min induced the highest seedling rate was 8.33%; 41 degrees of heat shock treatment bream fertilized eggs, 36min induced the highest seedling rate was 5.65%; 41.5 degrees of heat shock treatment, bream fertilized eggs of 24min induced the highest seedling rate was 3.76%; 42 degrees of heat shock treatment, bream fertilized eggs of 30min induced 4.02%. had the highest rate of heat shock induced megalobramaamblycephala ploidy analysis the results show that the content of DNA, only diploid and triploid megalobramaamblycephala induced by heat shock, no tetraploid chromosome analysis of megalobramaamblycephala. Detect the diploid and triploid bream, with the results of ploidy analysis results That may be due to late embryonic development process of megalobramaamblycephala induced by heat shock in the chromosome loss caused by the specific mechanism needs further study. The four group temperature shock treatment group, 42 degrees of heat shock induced bream fertilized egg from the triploid megalobramaamblycephala rate, as a result of 2.307%. ploidy analyzer DNA the content showed that triploid Megalobrama amblycephala DNA content is about 1.5 times more than the normal diploid bream DNA content. Chromosome analysis showed that the normal diploid chromosome 48 megalobramaamblycephala; heat shock induced triploid bream with 72 chromosomes, 24 chromosomes than diploid bream. This study used heat shock induction method treatment of bream fertilized eggs were studied in order to obtain tetraploid bream, Megalobrama amblycephala for subsequent polyploid breeding research to provide certain reference still.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S917.4

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