【作者】 熊家军；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2012， 博士

【摘要】 影响梅花鹿产茸量的因素很多,如何克服不良影响因素、以提高产茸量一直是人们追求的目标。了解鹿茸生长发育调控规律,可以促进提高鹿茸产量的新技术发展；开发操作简便、结果可靠的选种方法,培育产茸量高的新品种,是提高梅花鹿产茸量的基本手段。鉴于梅花鹿养殖中目前存在的选种方法落后的状况,本研究以湖北白鹿春实业有限公司梅花鹿养殖场(n=302)和吉林省东丰县东丰药业股份有限公司养鹿场人工养殖梅花鹿(n=234)种群为研究对象,通过分析生长激素(GH)、褪黑激素Ⅰ型受体a亚型CMTNR1A)基因和雄激素受体(AR)基因的单核苷酸多态性及其与产茸性状的关系,寻找SNP位点,为建立梅花鹿产茸性状分子标记辅助选择方法奠定基础；同时,应用生物信息学软件对已扩增得到的梅花鹿褪黑素受体基因进行蛋白结构与功能分析,为阐明鹿茸生长发育调控规律、开发提高产茸量的新技术奠定基础。主要研究结果如下：1.梅花鹿激素及其受体基因多态性与产茸量的关系(1)GH基因多态性及其与产茸量的关系：在2个梅花鹿种群中,发现GH基因3个突变位点,并被GeneBank收录,即A176-G176突变(登录号：GQ438251.1),A45-G45突变和C139-T139突变(登录号：GQ438253.1),A176-G176突变导致StuI限制性内切酶位点的改变。利用PCR-RFLP技术进行基因分型,分析基因的多态性及其与产茸估测值之间的关系,发现两个梅花鹿种群各基因型之间的产茸估测值差异不显著(P>0.05)；A45-G45突变未引起限制性内切酶位点的改变,因此用AS-PCR引物,进行基因分型。而后将基因的多态性与产茸估测值之间进行关联分析,发现两个梅花鹿种群各基因型的产茸估测值都没有差异(P>0.05)；C139-T139突变也未引起限制性内切酶位点的改变,用AS-PCR引物进行基因分型,并分析基因多态性与产茸估测值之间的关系,发现在五三梅花鹿种群中各基因型之间的产茸估测值没有差异(P>0.05),而在东丰梅花鹿种群中,CT基因型和CC基因型的产茸量估测值显著高于TT基因型(P<0.05)。(2)MTNR1A基因多态性及其与产茸量的关系：在2个梅花鹿种群中,MTNR1A基因外显子2出现3个突变,测定其分子结构后提交并被GeneBank收录,即C518-T518、G629-C629和C635-T635突变(登录号：JN038179)。C518-T518突变引起Ecol881酶切位点的改变,利用PCR-RFLP方法进行基因分型,然后分析基因多态性及其与产茸估测值之间的关系,发现在两个梅花鹿种群中各基因型之间的产茸估测值都没有差异(P>0.05)；在G629_C629处突变能引起Mval酶切位点的改变。利用PCR-RFLP方法进行基因分型,并分析基因多态性与产茸估测值之间的关系,发现在五三梅花鹿种群中,CC基因型的产茸估测值显著高于GC基因型(P<0.05),GC基因型的产茸估测值显著高于GG基因型(P<0.05),而CC基因型与GC基因型之间没有达到统计学差异水平(P>0.05)。C635_T635突变未引起限制性内切酶位点的改变,所以用AS-PCR引物进行基因分型,然后分析基因多态性与产茸估测值之间的关系,发现在两个梅花鹿种群的各基因型之间,产茸估测值都没有差异(P>0.05)。(3)AR基因多态性及其与产茸量的关系：在2个梅花鹿种群中,AR基因存在2个突变位点,测序后提交并被GeneBank收录,外显子3的C75_T75和外显子8的C256-T256(登录号：JF719040)位点。C75-T75突变引起RsaⅠ酶切位点改变,利用PCR-RFLP方法进行基因分型,然后分析基因多态性与产茸估测值之间的关系,发现在五三梅花鹿群体中,CT基因型的产茸量估测值高于CC基因型(P<0.05),而CT基因型与TT基因型以及TT基因型与CC基因型之间没有差异(P>0.05)。在东丰梅花鹿群体中,CT基因型的产茸量估测值高于CC基因型(P<0.05),而CT基因型与TT基因型以及TT基因型与CC基因型之间没有差异(P>0.05)；C256-T256突变通过SSCP-PAGE技术进行基因分型,分析基因多态性及其与产茸估测值之间的关系。发现该位点仅在五三梅花鹿种群中存在多态性,但各基因型之间的产茸估测值没有达到统计学差异(P>0.05)。2.梅花鹿雄激素受体和褪黑素受体结构分析与功能预测(1)应用Clustal W多序列比对模式对MTNR1A氨基酸序列与9种其他脊椎动物比对,发现梅花鹿中该基因编码的氨基酸序列与其他动物一样保守,在MTNR1A编码序列864位的核苷酸由G突变为C,导致氨基酸突变。比较G突变前、后所编码蛋白的理化特性,证明该蛋白非常稳定。(2)分析梅花鹿MTNR1A基因的分子进化树,发现MTNR1A基因的进化与其他物种的进化非常一致,梅花鹿和牛属于反刍动物,故分歧时间晚于鸟类前于高等动物,说明在GPCR进化历程中,梅花鹿同样遵循物种分化的规律。(3)应用SMART蛋白功能区域预测软件,预测MTNR1A编码序列有7个跨膜区段,为7次跨膜蛋白,在864bp位点的R288S突变体位于NRF蛋白保守功能区域。应用神经网络算法和Hidden Markov models模型,得到了两种可能的信号肽剪接位点,分别是33-34位和61-62位。(4)分析MTNR1A基因编码蛋白的二级结构特征,发现864bp处唯一引起氨基酸改变的突变,位于第6次跨膜和第7次跨膜之间的细胞外信号分子结合位点区域的LOOP区,该区域暴露于细胞膜之外,是可能参与下游信号分子传导的功能区。(5)分析MTNR1A家族成员,发现其属于GPCR家族A成员,与视紫红质有55%的同源性,突变氨基酸位于细胞外的袢环位置,可能对褪黑素配体的结合以及激活受体密切相关,说明梅花鹿的褪黑素受体可能存在物种特异性的结合及其激活机制,提示该位点可能是活性位点。(6)采用IPA蛋白互作网络分析,发现MTNR1A除了与其配体褪黑素结合外,还与众多基因、转录因子、接头蛋白等相互作用,形成复杂的调控网络。MTNR1A是一类重要的GPCR受体,与生物体诸多生理功能有关。该互作调控网络显示MTNR1A可能与MTNR1B结合,形成异二聚体发挥作用。该受体与CREB、ELK1/2等相互作用,决定细胞的增殖与分化功能。更多还原

【Abstract】 Many researchers have proved that Sika deer antler production was affected by many factors. In the present, the hot spot in Sika Deer Farming was to get high-yield by improving the unfavorable factors. If we could understand regulation mechanism of antler growth and development, antler production could be promoted by the development of new technologies.The basic means to improve sika deer antler production is to develop a method that is easy to operate, by which the results of selective breeding is reliable and the new Sika Deer would be high-yield antler production. Based on the backwardness deer selective breeding, for establishment the marker-assisted selection method, two sika deer populations of the Hubei BaiLuChun Industrial Corporation (n=302) and Jilin DongFeng farms(n=234)were selected to search single nucleotide polymorphisms (SNP) of Growth hormone (GH), melatonin receptor a (MTNR1A) and androgen receptor (AR) and analyze the correlation analysis between the SNPs in candidate genes and antler production traits. Meanwhile, protein structural and structural analysis of melatonin receptor gene was predicted by the bioinformatics software; the structure of MTNR protein can also be analyzed for confirming the functional units or domain and providing a target for genetic manipulation.The main results were showed as follows:1. SNPs screeningof GH, MTNR1A, and AR genes and their association with antler production of sika deer.(1)Polymorphism of GH gene and its association with antler production of sika deer: There are three mutations were found, including A176-G176(accession numbers: GQ438251.1), A45-G45, C139-T139(accession number:GQ438253.1) in growth hormone (GH) of two sika deer populations. The mutation A176-G176can lead to the change of restriction endonuclease recognition site StuⅠ, but the results of genotypes using PCR-RFLP analysis and correlation analysis between the gene polymorphism and the antler estimation value showed that there was no significant difference in two sika deer populations of the Five-three and Dongfeng farm (P>0.05); The mutation A45-G45and cannot lead to the change of restriction endonuclease recognition site, and there was no significant difference in two sika deer populations in the Five-three and Dongfeng farms (P>0.05); The mutation C139-T139cannot lead to the change of restriction endonuclease recognition site, and there was no difference in the sika deer population in the Five-three. But in Dongfeng farm, the antler production of sike deer with CT and CC genotypes were more than that with TT genotype (P<0.05).(2) Polymorphism of MTNR1A gene and its association with antler production of sika deer:There are three mutations of the MTNR1A genes exon2were found:c518-T518, G629-C629and C635-T635(accession numbers:JN038179) in these two sika deer populations. The mutation of C518-T518can change the restriction sites Ecol881, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value demonstrated that there was no significant difference in these two sika deer populations (P>0.05); the mutation of G629-C629can change the restriction endonuclease sites Mval, in the Five-three Farm, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value showed that the antler production of sika deer with CC genotype was more than that with GG genotype, and the difference was significant (P<0.05), and GC genotype was more than GC genotype, and the difference was significant (P<0.05); GC genotype was more than GG genotype, but the difference was not significant (P>0.05). The mutation of C635-T635can change the restriction sites. The correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value demonstrated that there was no significant difference in these two sika deer populations of the Five-three and Dongfeng farms (P>0.05).(3) Polymorphism of AR gene and its association with antler production of sika deer: There are two mutations of the androgen receptor gene (AR) were found:C75-T75in exon3and C256-T256in exon8(accession number:JF719040) in the two sika deer populations. The mutation of C75-T75can change the restriction endonuclease sites RsaⅠ In the Five-three Farm, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value showed the antler production of sika deer with CT genotype was significant more than that with CC genotype (P<0.05); TC genotype was more than TT genotype (P>0.05), and TT genotype was more than CC genotype (P>0.05). In the Dongfeng Farm, TC genotype was significant more than CC genotype (P<0.05), TC genotype was more than TT genotype (P>0.05), and TT genotype was more than CC genotype (P>0.05). Correlation analysis between the gene polymorphism C256-T256mutation genotyping by SSCP-PAGE and the antler production showed that the polymorphism site was only found in the sika deer populations of Five-three Farm. And there was no significant difference between CC genotype and the TT genotype in Shuangyang sika deer antler production (P>0.05).2. Bioinformatic analysis of androgen receptor and melatonin receptor gene in sika deer(1) MTNR1A amino acid sequences of nine kinds of other vertebrates were analyzed using ClustalW multiple sequence model. The results showed that the amino acid of MTNR1A in sika deer was highly conserved sequence, just like other animals. The mutation of864G-C could change the animo acid. The predicted results of the physical and chemical characteristics of MTNR1A demonstrated that the change of animo acid could not change the stable feature of MTNR1A protein.(2) Phylogenetic analysis showed that MTNR1A gene in sika deer was consistency with the evolution of other species. The divergence time of sika deer which is ruminants as cattle is later than the higher animals before the birds. This study demonstrated that the sika deer follow the law of evolution of species during the course of evolution.(3) The results of protein prediction by SMART software found that there are seven transmembrane regionin MTNR1A protein sequence. The mutation of R288S was located in the864bp sites of conserved region of the NRF. Based on neural network algorithms and Hidden Markov models, we found two possible signal peptides,33-34and61-62, respectively.(4) Secondary structure features of MTNR1A protein showed that the changed protein by the864bp mutation was located between the sixth and seven transmembrane domain, the LOOP areas which was extracellular signaling molecule binding sites area. The region was exposed to the extracellular region and may be involved in downstream signaling pathway.(5) MTNR1A belongs to members of the GPCR family A. The results of homology modeling found that MTNR1A has55%homology with rhodopsin. The mutation of amino acids located in the extracellular loop ring position, which may be closely related with the combination of melatonin ligands and receptor. These results indicated that the melatonin receptor of the sika deer may be species-specific and special activation mechanism, which suggested that this locus could be the active site.(6) The results of IPA protein interaction network analysis showed that the melatonin receptor could interplay with many genes, transcription factors, adapter proteins, and form a complex regulatory network. MTNR1A is an important receptor of GPCR family, which could affect many physiological functions of the organism. The interaction network demonstrated that the heterodimers including MTNR1A and MTNR1B could perform its function together. Furthermore, MTNR1A could interact with CREB, ELK1/2and others to direct cell proliferation and differentiation.更多还原