团头鲂生长相关基因（MSTN,Mdk）的克隆及热休克诱导四倍体初步研究

发布时间：2018-04-09 15:41

本文选题：MSTN基因　切入点：Mdka、Mdkb重复基因　出处：《上海海洋大学》2017年硕士论文

【摘要】：团头鲂(Megalobrama amblycephala),由于其生殖周期短、二龄即可达到性成熟,是研究肌肉生长、选育新品种的良好材料。肌肉生长抑制素(Myostatin,MSTN),又称生长分化因子8(Growth differentiation factor 8,GDF 8),属于转化生长因子β(Transforming growth factorβ,TGF-β)家族。该基因抑制骨骼肌的生长、发育。本研究通过cDNA末端快速扩增法(RACE法)克隆得到团头鲂生长抑制素(MSTN)基因全长cDNA序列并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果显示团头鲂MSTN基因的cDNA全长为2187bp,编码区ORF(开放阅读框)大小为1128bp,编码376个氨基酸,推测的MSTN前体蛋白等电点为5.02,相对分子质量为92.34KDa。MSTN基因编码蛋白包含有两大TGF-β蛋白结构域、RXXR蛋白酶水解位点RIRR和保守的半胱氨酸残基。组织逆转录PCR(RT-PCR)结果显示,MSTN基因在肌肉、脑和精巢组织中大量表达,肝脏、脾脏和卵巢组织中的表达量次之,肠、腮、心脏、眼睛和肾组织中的微量表达。胚胎RT-PCR结果显示,在0-44hpf胚胎发育阶段,MSTN基因表达量较低;而在48hpf-52hpf胚胎发育阶段,MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示,胚胎发育的16hpf时期MSTN基因主要在脊索中表达,胚胎发育的28hpf、55hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示,胚胎在体节发生期出现前-后轴伸长,背-腹轴缩短;脊索发生扭曲、其生长发育受到抑制等现象。本研究为进一步探索团头鲂MSTN基因在生长发育信号通路中的作用和育种提供了基础资料。肝素结合生长因子(midkine)属于PTN家族,该家族由Mdk和PTN(pleiotrophin)两个成员组成。Mdk是一种对细胞生长、分化、迁移有重要作用的生长因子。Mdk对不同组织尤其是神经组织的生长、修复同样起着重要的作用。本研究通过RACE法克隆获得团头鲂Mdka/-b重复基因cDNA序列。团头鲂Mdka基因全长1409bp,编码区ORF大小为441bp,编码146个氨基酸,包括22个氨基酸的信号肽,124个氨基酸的成熟肽,Mdka前体蛋白等电点为9.57,相对分子量为15.73KD;团头鲂Mdkb基因全长1048bp,编码区ORF大小为444bp,编码147个氨基酸,包括21个氨基酸的信号肽,126个氨基酸的成熟肽,Mdkb前体蛋白等电点为9.41,相对分子量为16.23KD。与人类、小鼠和斑马鱼一样,团头鲂Mdka/-b基因成熟多肽都包含10个保守的半胱氨酸残基,一个精氨酸残基,两个谷氨酰胺残基,一个高度保守的铰链区和两个碱基残基簇。团头鲂Mdka和Mdkb编码区之间相似度为64%,与斑马鱼Mdka和Mdkb编码区之间相似度分别为87%和95%,而与人类Mdk编码区相似度较低(分别为57%和55%)。实时荧光定量PCR(qRT-PCR)分析结果表明,成鱼组织中Mdka mRNA只在脑、性腺、肠中大量表达;而Mdkb mRNA除了皮肤外的所有成鱼组织中都大量表达。在胚胎发育阶段qRT-PCR分析结果显示,Mdka基因mRNA首次检测到表达在受精后12h;Mdkb基因mRNA首次检测到表达在受精后8h。在那之后,Mdka和Mdkb mRNAs表达都稳步上升,在受精后28h达到最大值,之后下降并逐渐保持稳定。WISH结果表明Mdka和Mdkb mRNAs在受精后16h阶段都是全身性表达;在受精后28h阶段Mdka mRNA依然是全身性表达,Mdkb mRNA则只在眼睛、脑和尾节出表达;在受精后55 h阶段时,Mdka和Mdkb都只在脑中表达。饥饿2、4、6天处理后,Mdka和Mdkb mRNAs表达量在脑、肝脏和肠中表达量都上升,Mdka mRNA表达量在恢复投喂6天后恢复正常水平;Mdkb mRNA在脑、肠中表达量在恢复投喂3天后恢复正常水平,肝脏中Mdkb mRNA表达量在恢复投喂6天后恢复正常水平。在注射重组人体生长激素(hGH)处理过程中,与对照组相比,在注射50μg hGH肝脏、肠组织中Mdka和Mdkb mRNAs表达量迅速下降;在注射10μg hGH的肠组织中Mdka和Mdkb mRNAs表达量也都呈现下降趋势,但它们表达量下降程度不一样;在注射10μg和50μg hGH的脑组织中Mdka mRNA表达量迅速上升,而Mdkb mRNA表达量没有显著的变化。研究结果表明Mdka和Mdkb重复基因在团头鲂胚胎和组织发育过程中起着重要、但不同的作用。三倍体团头鲂在生长优势、群体产量和抗逆性等方面具有明显的优势,四倍体团头鲂与二倍体团头鲂杂交可以快速、高效的获得三倍体团头鲂,为团头鲂育种开创一种新的方法。本研究为获得能够稳定遗传的四倍体团头鲂而进行热休克方法诱导处理团头鲂受精卵抑制其第一次有丝分裂的研究。结果显示,在热休克2min条件下,40.5℃热休克处理,团头鲂受精卵发育30min诱导出苗率最高为8.33%;41℃热休克处理,团头鲂受精卵36min诱导出苗率最高为5.65%;41.5℃热休克处理,团头鲂受精卵24min诱导出苗率最高为3.76%;42℃热休克处理,团头鲂受精卵30min诱导出苗率最高为4.02%。对热休克诱导的团头鲂进行倍性分析分析其DNA含量,结果表明热休克诱导只获得二倍体和三倍体团头鲂,没有得到四倍体团头鲂。对检测出的二倍体和三倍体团头鲂进行染色体分析,其结果与倍性分析结果一致,可能是由于热休克诱导的团头鲂胚胎后期发育过程中出现了染色体丢失所致,具体机理还需进一步深入研究。四组温度休克处理组中,42℃热休克诱导处理团头鲂受精卵获得的三倍体团头鲂率最高,为2.307%。倍性分析仪检测DNA含量结果显示,三倍体团头鲂DNA含量约为正常二倍体团头鲂DNA含量的1.5倍多。染色体分析结果显示,正常二倍体团头鲂的染色体48条;热休克诱导的三倍体团头鲂的染色体为72条,比二倍体团头鲂多24条染色体。本研究采用热休克法诱导处理团头鲂受精卵为获得四倍体团头鲂进行了初步研究,为后续团头鲂多倍体育种研究提供一定的参考依旧。
[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.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

【参考文献】