【作者】 李玉梅；

【导师】 白秀娟；

【作者基本信息】 东北农业大学 ， 特种经济动物饲养， 2008， 博士

【摘要】 水貂是小型珍贵毛皮兽,水貂皮、狐皮和波斯羔皮早已成为国际裘皮贸易的三大支柱产品。当前,水貂产业正面临着良好的发展机遇和前景,因此,提高水貂的生长性能,将给水貂养殖业带来更高的经济效益。本研究以大兴安岭水貂养殖基地饲养的水貂（643只）作为研究对象,针对IGF-Ⅰ基因、GH基因、EGF基因、MSTN基因进行研究,采用克隆测序结合PCR-SSCP标记的方法,检测了这些基因部分编码区的SNPs,并分析了突变位点导致的多态性与体重和皮长性状之间的相关性。为利用与生长性状相关的标记基因进行标记辅助选择打下基础;为水貂品种的选育、提高水貂生长速度、皮张质量提供分子水平的手段和方法。同时,本研究还以大连金州水貂场的自咬水貂和健康水貂作为研究对象,采用RAPD标记技术结合SCAR标记技术对正常水貂样本（30只）和自咬水貂样本（30只）分别进行了分子水平的遗传结构分析。对各样本中出现的显著RAPD差异标记经克隆测序后,采用SCAR标记对采集的全部样本进行PCR扩增,以扩增产物中标记片段的出现与否来区分健康水貂与自咬水貂个体。此研究为今后水貂的抗病育种或免疫育种奠定了基础,如果与传统的育种技术相结合,可以进一步改良水貂品种,提高养貂业的生产效益,推动我国特种经济动物养殖的发展速度。主要研究结果如下:（1）成功克隆了水貂的GH基因的部分DNA序列,得到了1060bp的序列。从序列比较中可知该片段存在三个突变位点（分别在第269、538和539位点处）。（2）在检验的2对GH基因的引物中,我们发现2对引物的扩增片段均具有多态性。引物GH1扩增片段经PCR-SSCP检测,在水貂群体中出现3种基因型（命名为AA、BB、AB）。BB基因型个体体重及皮长均显著高于AA基因型和AB基因型个体（P<0.05）,因此可以初步判定B基因可能对水貂生长有利。引物GH2扩增片段经PCR-SSCP检测,在水貂群体中出现3种基因型（命名为CC、CD、DD）;三种基因型个体对水貂体重、皮长性状的影响均未达到显著水平（P>0.05）。（3）在检测的3对IGF-Ⅰ基因的引物中,我们发现2对引物的扩增片段具有多态性。引物IGF-Ⅰ₁扩增片段多态性经PCR-SSCP检测,在水貂群体中出现2种基因型（命名为AA、AB）。A等位基因出现的频率在整个检测群体中显著高于B等位基因。但统计结果显示,AB基因型个体体重及皮长均极显著高于AA基因型个体（P<0.01）,因此初步判定B等位基因是优势基因。引物IGF-Ⅰ₂扩增片段经PCR-SSCP检测,在水貂群体中出现3种基因型（命名为CC、CD、DD）。三种基因型个体对水貂体重、皮长性状的影响均未达到显著水平（P>0.05）。引物IGF-Ⅰ₃扩增产物经PCR-SSCP检测没有出现多态。（4）成功克隆了水貂的EGF基因的部分外显子DNA序列,得到了1002bp的序列。经同源性比较得出与小家鼠的EGF基因的同源性达到94%;与褐鼠EGF基因同源性达83%;与猕猴的EGF基因同源性为79%;与人类的EGF基因同源性为78%。从序列比较中可知该片段存在三个突变位点（分别在45、196和498位点处）。在3个突变位点两端设计引物,经PCR-SSCP检测,有2对引物扩增片段出现了多态。引物EGF1扩增片段经PCR-SSCP检测,出现3种基因型（命名为AA、AB、BB）。B等位基因出现的频率在整个检测群体中高于A等位基因。统计结果显示,BB基因型个体体重及皮长均显著高于AA基因型个体（P<0.05）,AB型个体体重也显著高于AA型个体（P<0.05）,AB型个体皮长与AA、BB型个体差异均不显著（P>0.05）。因此可以初步判定B等位基因可能是优势基因。引物EGF2扩增片段经PCR-SSCP检测,在水貂群体中出现3种基因型（命名为CC、CD、DD）。三种基因型个体对水貂体重的效应统计结果显示均未达到显著水平（P>0.05）。对皮长性状影响,CD型个体显著高于CC型（P<0.05）,二者和DD型个体均没有显著差异（P>0.05）。引物EGF3扩增产物经PCR-SSCP检测没有出现多态。（5）成功克隆了水貂的MSTN基因的外显子DNA序列,得到了1128bp的序列。经同源性比较得出与犬的MSTN基因的同源性达到94%;与狐的MSTN基因同源性达93%;与小鼠同源性为85%;与野猪的MSTN基因同源性为94%;与牛的MSTN基因同源性为86%;与人类的MSTN基因同源性为93%。从序列比较中可知该片段存在四个突变位点（分别在第186、380、616及648位点处）。在4个突变位点两端设计引物,经PCR-SSCP检测,3对引物扩增片段均出现了多态。引物MSTN₁扩增片段经PCR-SSCP检测,在水貂群体中出现3种基因型（命名为AA、AB、BB）。A等位基因出现的频率在整个检测群体中高于B等位基因。统计结果显示,BB基因型个体体重及皮长均显著高于AA基因型个体（P<0.05）,AB型个体体重显著高于AA型个体（P<0.05）,AB型个体体重和BB型个体差异不显著（P>0.05）,AB型个体皮长与AA、BB型个体差异均不显著。因此初步推测B等位基因可能是优势基因。引物MSTN₂扩增片段经PCR-SSCP检测,在水貂群体中出现2种基因型（命名为CC、CD）。C等位基因出现的频率在整个检测群体中高于D等位基因。统计结果显示,CD型个体体重及皮长均显著高于CC型个体（P<0.05）,推测D基因可能是优势基因。引物MSTN₃扩增产物经PCR-SSCP检测出现3种基因型（命名为EE、EF、FF）。E等位基因出现的频率在整个检测群体中高于F等位基因。统计结果显示,FF型个体体重及皮长均显著高于EE型个体（P<0.05）,推测F基因可能是优势基因。统计各基因型之间的组合给水貂体重及皮长性状带来的影响时,发现四种基因不同基因型之间的组合对所检测的水貂样本的体重及皮长差异显著（P<0.05）。（6）采用RAPD标记和SCAR标记技术对水貂的自咬症发病原因进行遗传分析时,100条随机引物中,筛选出26条产生清晰、稳定扩增产物的引物,其中18条引物出现多态性,多态率为26.67%。筛选出的26条随机引物,在水貂健康组与患病组以相同条件进行了3次重复扩增。扩增结果显示,有11条引物扩增的结果重复性较好、多态片段明显（分别是S103,S112,S120,S139,S144, S167,S176,S178,S356,S358,S360）。其中引物S103和S356扩增的条带共性与差异标记最明显,针对其扩增出的不同标记片段,将其回收、克隆、测序,获得了标记片段的特定序列。并对标记片段进行序列分析,重新设计4对引物,将RAPD标记成功转化成SCAR标记,在水貂的健康组与患病组个体中分别验证。结果表明水貂的自咬症发病原因与遗传基因相关,利用RAPD标记结合SCAR标记能够区分健康与自咬水貂群体。更多还原

【Abstract】 Mink is small precious fur-bearing animal,and the furs of mink, fox, and persian lamb are already the three key products in the international fur market. Now the mink industry is facing good development opportunity and prospect, so improving the growth performance of mink will bring more economic benefits to the mink industry. The minks bred in Daxinganling base were chosen as our research object, the SNPs in the coding regions of the IGF-Ⅰ, GH, EGF, and MSTN genes were studied by cloning and sequencing combined with PCR-SSCP technique, finally, the correlation between polymorphism caused by mutation sites and body weight, Mink length was analyzed. This study made foundation of marker assisted selection using marker genes related growth performance, and provided way to breed mink, improve growth rate, fur quality of mink at molecular level.Meanwhile, healthy minks（30）and those of self-biting behaviour bred（30）in Jinzhou mink base in Dalian were chosen as our research object. RAPD technique combined with SCAR was used to make analysis of genetic structure of these minks at molecular level. The RAPD markers with significant differences were cloned and sequenced, and then SCAR technique was used to amplify the markers in all the samples as to identify whether the mink was healthy or had self-biting behaviour by the appearance of marker fragments. This study made foundation of breeding for disease resistance and immunity. If combining with traditional breeding technique, it will improve mink breeding, enhance economic benefits of mink industry, and impel the development of cultivation of economic animals. The main results of this study were as follows:（1）The partial cDNA of mink GH gene was cloned successfully, and 1060bp fragment was got. There were three mutation sites in this fragment （located at 269, 538, 539, repectively）.（2）Both the fragments amplified by two pairs of primers showed polymorphism. The fragments amplified by primer GH1 was detected by PCR-SSCP, and three genetypes were found （named AA, BB, AB）. The body weight and Mink length of BB genetypes were significantly greater than those of AA and AB genetypes, so it was preliminarily considered that B gene was good for the growth of mink. The fragments amplified by primer GH2 was detected by PCR-SSCP, and three genetypes were found （named CC, CD, DD）, but there were no significant difference among these genetypes in body weight and Mink length （P>0.05）. （3）In the three pairs of primers designed for IGF-Ⅰ, two fragments amplified by two pairs of primers showed polymorphism. The fragments amplified by primer IGF-Ⅰ₁ was detected by PCR-SSCP, and two genetypes were found （named AA, AB）. The allele A frequency was significantly higher than that of allele B. But the statistical results showed that the body weight and Mink length of AB genetypes were significantly greater than those of AA genetypes （P<0.01）, so it was preliminarily considered that allele B was superior. The fragments amplified by primer IGF-Ⅰ₂ was detected by PCR-SSCP, and three genetypes were found （named CC, CD, DD）, but there were no significant differences among these genetypes in body weight and Mink length （P>0.05）. There was no polymorphism in the fragments amplified by primer IGF-Ⅰ₃.（4）The partial exon sequence of mink EGF gene was cloned successfully, and 1002bp fragment was got. The homologies of EGF gene among mink, mouse, rat, rhesus monkey and human was 94%, 83%, 79% and 78%, respectively. There were three mutation sites in this fragment （located at 45, 196, 498, repectively）. The primers designed at two ends of the mutation sites, and the fragments amplified by two pairs of primers showed polymorphism. The fragments amplified by primer EGF1 was detected by PCR-SSCP, and three genetypes were found （named AA, AB, BB）. The allele B frequency was significantly higher than that of allele A. The statistical results showed that the body weight and Mink length of BB genetypes were significantly greater than those of AA genetypes （P<0.05）,and the body weight of AB genetypes was significantly greater than that of AA genetypes （P<0.05）, but there were no significant differences among these genetypes in body Mink length（P>0.05）.So it was preliminarily considered that allele B was superior. The fragments amplified by primer EGF2 was detected by PCR-SSCP, and three genetypes were found （named CC, CD, DD）. There were no significant differences among these genetypes in body weight （P>0.05）. The Mink length of CD genetypes was significantly greater than that of CC genetypes （P<0.05）, but there was no significant difference with DD genetypes （P>0.05）. There was no polymorphism in the fragments amplified by primer EGF3.（5）The exon sequence of mink MSTN gene was cloned successfully, and 1128bp fragment was got. The homologies among mink MSTN, dog MSTN, fox MSTN, mouse MSTN, wild pig MSTN, bovine MSTN and human MSTN was 94%, 93%, 85%, 94%, 86% and 93%, respectively. There were four mutation sites in this fragment （located at 186, 380, 616, 648, repectively）. The primers designed at two ends of the mutation sites, and the fragments amplified by all three pairs of primers showed polymorphism .The fragments amplified by primer MSTN1 was detected by PCR-SSCP, and three genetypes were found （named AA, AB, BB）. The allele A frequency was significantly higher than that of allele B. The statistical results showed that the body weight and Mink length of BB genetypes were significantly greater than those of AA genetypes （P<0.05）, and the body weight of AB genetypes was significantly greater than that of AA genetypes （P<0.05）, but there was no significant difference in body weight between AB and BB genetypes（P>0.05）, and there was also no significant difference in Mink length of AB genetypes with AA and BB genetypes. So it was preliminarily considered that allele B was superior.The fragments amplified by primer MSTN₂ was detected by PCR-SSCP, and two genetypes were found （named CC, DD）. The allele C frequency was significantly higher than that of allele D. The statistical results showed that the body weight and Mink length of CD genetypes were significantly greater than those of CC genetypes （P<0.05）, and it was preliminarily considered that D gene was superior.The fragments amplified by primer MSTN3 was detected by PCR-SSCP, and three genetypes were found （named EE, EF, FF）. The allele E frequency was significantly higher than that of allele F. The statistical results showed that the body weight and Mink length of FF genetypes were significantly greater than those of EE genetypes （P<0.05）, and it was preliminarily considered that F gene was superior. The statistic influences of genotypic combination among these genes to the body weight andMink length of minks showed that the genotypic combination among four genetypes had significant influence trend to the body weight and Mink length（P<0.05）.（6）RAPD and SCAR techniques were used to made genetic analysis to the cause of self-biting behaviour, and among 100 random primers, 26 stable primers amplifying clear PCR product were selected. The fragments amplified by 18 primers showed polymorphism, and the percentage of polymorphism was 26.67%. 26 random primers were used to amplify three times under the same condition in both healthy and sick minks. The amplifying results showed that the repeatability of 11 primers was good enough, and the polymorphism was clear （S103,S112,S120,S139,S144, S167,S176,S178,S356,S358,S360, repeatability）. The fragments amplified by primer S103 and S356 had obvious generality and difference, and different marker fragments were recycled, cloned, and sequenced, and finally the sequences of the marker fragment were got. The marker fragments were analyzed, and four pairs of primers were designed, so the RAPD marker was transformed into SCAR marker to detect in both healthy and sick groups. The results showed that the cause of self-biting behaviour had direct reason with the genetic genes, and RAPD marker combined with SCAR marker could identify the healthy and sick minks well.更多还原