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朱鹮人工饲养群体6个基因SSCP检测及遗传变异研究
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摘要
朱鹮是世界极濒危鸟类,被称为我国的四大国宝之一。上世纪60~70年代,几乎在世界绝迹。1981年在陕西洋县发现的朱鹮种群数量只有7只,目前已形成陕西洋县、陕西周至、北京动物园、日本(我国赠送和繁殖)4个种群,数量达千只,是我国在濒危动物保护方面利用科学管理和现代技术取得的世界瞩目的成功范例。但是朱鹮种群仍有待扩大和复壮。本研究利用PCR-SSCP技术,首次对陕西洋县和周至人工饲养的63只朱鹮个体的糖蛋白家族FSH、TSH基因、神经营养因子家族BDNF、GDNF以及12S rRNA和c-mos基因的遗传结构、遗传变异和进化进行了研究。以期为朱鹮群体的复壮、生殖、发育以及某些疾病的发病机理、疾病防制提供理论基础。科学的指导朱鹮的保护工作。本试验研究结果如下:
     1.对BDNF基因的研究发现,其第450位点处发生1处沉默突变(G→A,编码Ala),发现AA和AB型2种基因型,其频率分别为0.7143和0.2857,等位基因A、B频率分别为:0.8571和0.1429。x~2检验表明该位点处于Hardy-Weinberg平衡状态。朱鹮群体PIC为0.2149,属于低度多态性,表明该位点遗传变异较小,提示群体内含有较低的遗传变异信息。对朱鹮 GDNF基因检测未发现多态。将朱鹮 BDNF基因序列与其它物种BDNF基因进行同源序列分析,共有820个位点,其中有405个变异位点,占总位点的49.4%,342个简并位点,占总位点的41.7%,物种间序列变异表现为转换多于颠换。朱鹮与禽类、两栖类、硬骨鱼类的系统进化研究显示:朱鹮属于硬脊椎动物的初龙次亚纲,与鳞龙次亚纲亲缘关系较近,与哺乳纲亲缘关系较远。
     2.对NTs家族BDNF、GDNF、NT3和NT5基因39种动物个体进行系统进化研究,其系统进化关系为:(GDNF,(NT5,(NT3,BDNF)))。该家族成员的同源性较低,说明在NTs进化过程中,其各个基因变异速度较快。
     3.在朱鹮FSHβ基因5′侧翼区检测到1处碱基突变(C→T),只检测到AA、AB基因型,未检测到BB型。其基因型频率分别为0.7619、0.2381。A基因频率为0.8810;B基因频率为0.1190。x~2检验表明该位点处于Hardy-Weinberg平衡状态(P>0.05),但B基因相对于A基因面临消失趋势。群体杂合度(H)为0.2097,群体遗传变异小。在FSHβ基因外显子2未发现多态。经序列测定和系统发育分析发现,朱鹮与绿头鸭、鸡、鹌鹑FSHβ基因第2外显子DNA序列具有高度的同源性,其同源性分别为:92.09%、91.37%和89.93%。系统发育分析结果显示,朱鹮与绿头鸭的亲缘关系较近,而与鸡和鹌鹑的亲缘关系较远。
     4.在TSH 5′侧翼区检测到1处碱基突变(C→T),发现AA、AB和BB型3种基因型,3种基因型频率分别为:0.3651、0.5714、0.0635,A、B基因频率分别为:0.6508和0.3492。x~2检验表明,该位点处于Hardy-Weinberg平衡状态(0.01     5.对脊椎动物糖蛋白激素β亚基51个物种的核苷酸序列分别以NJ法构建系统进化树,结果显示hCG与LH关系较近,与FSH和TSH关系较远,而氨基酸序列聚类的结果则显示为TSH和FSH、hCG和LH关系较近。二组基因的差异较大。说明FSH、TSH、LH及hCG之间具有较高的同源性,系统进化较慢。
     6.对朱鹮c-mos基因部分序列检测发现,其第282位点存在一处沉默突变(C→T),发现AA、AB和BB 3种基因型,其频率分别为:0.5873、0.2857和0.1270。基因型A和B的频率分别为:0.7302和0.2698。x~2检验表明该位点偏离Hardy-Weinberg平衡状态(0.01     7.通过6个基因5个多态位点分析结果表明,5个位点平均杂合度( H|- )为0.3436;平均多态信息含量(PIC|─)为0.2798,说明陕西省人工饲养朱鹮群体遗传变异仍较小,需要进一步较大对其保护力度,以加快其品种复壮。
Crested ibis considered as one of the four national treasures in China, is a kind of critically endangered bird. It was almost extinctive during 1960’s ? 1970’s until a population of crested ibis consisted of seven birds was found in Yangxian county of Shaanxi province. Up to now, there are four populations in Yangxian, Zhouzhi county of Shaanxi, Beijing zoo in China and Japan, and the total number is up to one thousand. Although it is a successful example in word in endangered animal protection using modern management and technologies, it is necessary to improved and amplify the population of crested ibis. In this study, Genetic variations of FSH, TSH of glycoprotein, BDNF, GDNF genes of NT family, 12S rRNA and c-mos genes in crested ibis came from Yangxian and Zhouzhi county of Shaanxi province were detected using PCR-SSCP DNA marker technique. The objective of this study was to invest the genetic characteristics of the crested ibis population, to provide information for improvement and amplification of crested ibis and the studies on reproduction, development and mechanisms of some diseases of crested ibis, so the protection of crested ibis would be carried out scientifically. The results were as follows:
     1. BDNF gene of 63 crested ibis was analyzed and one silent mutation (G→A,coding Ala) was found in the site 450. There were two genotype AA and AB were found and the frequency were 0.7143 and 0.2857, respectively. Allele frequency of A/B were 0.8571/0.1429, respectively. Chi-square test showed that this locus was in Hardy?Weinberg equilibrium. The genetic diversity indexes of H/PIC were 0.2450/0.2149 in the crested ibis population. The genetic variance of this locus was low, which indicated lower genetic variance information in these crested ibis. GDNF gene of 63 crested ibis was detected by PCR-SSCP and only 2?band haplotype was found. No polymorphism was found. Homology analyses were carried out among the BDNF sequences of crested ibis and other species. 405 mutation loci and 342 degeneracy loci were found among 820 loci and 49.4 percent and 41.7 percent, respectively. The transition was more than transversion among species sequence variant. The phyletic tree of crested ibis, amphibian and teleostei showed that crested ibis belong to archosauria of Euteleostomi and nearer to lepidosauria and farer to mammalian.
     2. Phylogenesis of NTs family BDNF、GDNF、NT3 and NT5 genes of 39 species was carried out, which the phyletic relationship was (GDNF,(NT5,(NT3,BDNF))). The lower homology of this family indicated that every gene had a rapid change speed.
     3. A SNP (C→T) was detected in FSHβ5′flanking region. Only genotype AA and AB were found in the population. Genotypic frequencies of AA and AB were 0.7619 and 0.2381, respectively. Allele frequencies of A/B were 0.8810 and 0.1190, respectively. Genotypic AA and allele A were dominant in this population. The chi-square test showed the population was at Hardy-Weinberg disequilibration (P<0.05). The genetic diversity indexes of H/PIC were 0.2097/7.7561 in the crested ibis population. So the population was low polymorphic at this SNP locus. No polymorphism was detected in exon 2 in FSHβsubunit in crested ibis population. The fragment of FSHβexon 2 was sequenced and phylogenetic analysis results showed that the high sequence similarities of exon 2 in FSHβgene of crested ibis with mallard, chicken and quail were 92.09%, 91.37% and 89.93%, respectively. Phylogenetic analysis indicated crested ibis was cluster to mallard and far from quail.
     4. A SNP (C→T) was detected in 5′flanking region of TSHβgene. There were three genotypes were found in the population and genotype AA, AB and BB frequencies were 0.3651, 0.5714 and 0.0635, respectively. Allele frequencies of A/B were 0.6508/0.3492. The chi-square test showed this population was at Hardy-Weinberg disequilibration (P<0.05). The genetic diversity indexes of H/PIC were 0.4545/0.3512 in the crested ibis population. So the locus was moderate polymorphic. A SNP (T→A) was detected in exon 2 of TSHβgene and two genotypes were found in the population. Genotype AA and AB frequencies were 0.4127, 0.5873, respectively. Allele frequencies of A/B were 0.7063/0.2937. The chi-square test showed this population was at Hardy-Weinberg critically disequilibration (P<0.01). The allele B faced to the disappear tendency. The genetic diversity indexes of H/PIC were 0.4148/0.3288 in the crested ibis population. So the locus was moderate polymorphic. The two loci indicated the genetic variation of crested ibis recovered to a certain extent.
     5. Phyletic tree of vertebrate glycoprotein hormonesβsubunit nucleotide sequences among 51 species wasconstructed. The results showed that hCG and LH had nearer relationship, and gradually farer with FSH and TSH. While the phyletic tree of nucleotide sequences showed that the difference was larger between these two groups than TSH and FSH, hCG and LH. The results indicated FSH, TSH, LH and hCG had higher homology and lower phyletic evolution speed.
     6. c-mos gene partial sequence of 63 crested ibis was analyzed and one silent mutation (C→T,coding Tyr) was found in the site 282. There were three genotype AA, AB and BB were found and the frequency were 0.5873, 0.2857 and 0.1270, respectively. Allele frequencies of A/B were 0.7302/0.2698, respectively. Chi-square test showed that this locus was in Hardy?Weinberg disequilibrium(P<0.05). The genetic diversity indexes of H/PIC were 0.3940/0.3164 in the crested ibis population, which indicated this locus was a midrange locus. Phylogenetic analysis of ciconiiformes based on analysis of the nuclear c-mos gene, mitochondrial 12S rRNA gene sequences and the combined sequences of the tow genes. The result showed that combined sequences analysis was more reliable than single sequence analysis. The Phylogenetic of ciconiiformes was (Threskiornithidae((Balaenicipitidae,Scopidae)Cathartidae, Ciconiidae, Ardeidae)). No polymorphism was detected in three loci of 12S rRNA sequence.
     7. Five polymorphic loci of 6 genes were comprehensive analyzed and the results showed that H|- and PIC|─were 0.3436 and 0.2798, respectively, which indicated that the two groups of Yangxian and Zhouzhi county of Shaanxi province still had a lower genetic variant. We should work hard to protect this critically endangered bird, and ensure this specy recovery quickly.
引文
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