绵羊角蛋白关联蛋白家族基因新成员鉴定及其与羊毛性状的相关性分析

发布时间：2018-03-09 17:36

  本文选题：绵羊　切入点：角蛋白关联蛋白　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

【摘要】：角蛋白关联蛋白(keratin-associated proteins,KAPs)是决定羊毛品质的重要结构蛋白,由KRTAP家族基因编码。该家族基因的变异和表达调控与羊毛性状存在相关,但对该家族基因的研究还十分有限。人类KRTAP家族基因成员已发现88个,在绵羊上仅27个。为了挖掘该家族新基因并解析其对羊毛性状的影响,本研究以美利奴羊(Merino)与南丘羊(Southdown)的杂种后代和新西兰罗姆尼羊(Romney)为研究对象,以其他物种已知KRTAP家族基因为参考,采用基因组分析、同源性搜索和进化分析方法,从绵羊基因组数据中初步筛选疑似基因,进而扩增测序,分析确定绵羊中的新基因。采用PCR-SSCP和测序方法,筛查新基因的SNPs,比较基因变异在2个类群间的差异,分析SNPs与羊毛性状的相关性。主要结果如下:(1)鉴定出绵羊KRTAP22-1、KRTAP22-2、KRTAP21-1、KRTAP21-2和KRTAP26-1共五个KRTAPs新成员。这五个新基因定位于绵羊1号染色体上,位于KRTAP8-2和KRTAP24-1之间。(2)在390只美利奴杂种羊和75只新西兰罗姆尼羊中,绵羊KRTAP22-1基因检测到2个SNPs位点构成3个等位基因(A-C)。在新西兰罗姆尼羊群体中B为优势等位基因(81.3%),而在美利奴杂种羊群其中A为优势等位基因(51.8%)。对390份美利奴杂种羊样品进行的相关性分析表明,等位基因B存在与较高的净毛率(Wool Yield,Yield)和较低的平均羊毛卷曲率(Mean fibre curvature,MFC)相关。BB型和AB型绵羊的Yield较AA型高(P0.05)。在育种时,若以提高Yield或降低MFC为目标,KRTAP22-1基因可作为其分子标记基因。(3)在美利奴杂种羊和新西兰罗姆尼羊群体中未检测到绵羊KRTAP22-2基因变异。但在80只柴达木黑山羊、子午岭黑山羊、河西绒山羊和内蒙古绒山羊中,检测到KRTAP22-2存在3个SNPs和一处6bp长度变异构成3个等位基因(A-C),其中一个为非同义突变,导致精氨酸变为甘氨酸。等位基因C存在一个6bp的插入,插入精氨酸和半胱氨酸2个氨基酸。AA和AB为优势基因型,A为优势等位基因。KRTAP22-2基因在绵山羊中的多态性存在较大差异,这可能与该基因在两个物种中受到的选择不同有关。(4)在363只美利奴杂种绵羊中,KRTAP21-1基因共检测到7个SNPs形成6个等位基因(A-F),其中3个SNPs为非同义突变。对这363份样品中基因型频率较高的AA和AC进行相关性分析,只有Yield在两种基因型的绵羊间略有差异,但不显著。其他等位基因对羊毛性状的影响,还有待进一步研究。(5)在389只美利奴杂种羊中,检测到KRTAP21-2基因4个SNPs形成5种基因型(A-E),其中1个为非同义突变。KRTAP21-2基因变异与平均羊毛长度(Mean staple length,MSL)、纤维直径变异系数(Fibre diameter standard deviation,FDSD)和刺感指数(Prickle factor,PF)3个性状相关。基因型为AC的羔羊MSL比CE的要大(P0.05)。基因型为CE的羔羊比CC和BC的羔羊有较高的FDSD(P0.05)。基因型为CE羔羊的PF较CC和BC的羔羊高(P0.05)。在等位基因存在缺失分析中,等位基因E存在会引起MSL的降低。多变量模型分析时,等位基因A引入分析时,E对MSL的影响依旧显著(P0.05)。等位基因E在编码区发生了氨基酸变异,该等位基因存在会降低羊毛的MSL。可见,受该基因影响最大的羊毛性状是MSL。在育种时,若以提高MSL为目标,KRTAP21-2基因可作为其分子标记基因。(6)在383只美利奴杂种羊和94只新西兰罗姆尼羊中,检测到KRTAP26-1存在7个SNPs形成的4个等位基因,有2个为非同义突变引起了氨基酸的改变。在美利奴杂种羊中各等位基因的频率为A:49.6%、B:25.7%、C:23.0%和D:1.7%,而在新西兰罗姆尼羊中分别为A:40.4%、B:47.3%、C:0.5%和D:11.7%。绵羊KRTAP26-1的变异与一系列羊毛性状相关,包括Yield、平均纤维直径(Mean fibre diameter,MFD)、FDSD、MSL、MFC和PF。然而最大、最持久的影响是对MFD、FDSD、MSL和PF4个性状。含有等位基因C的绵羊表现为较低的MFD、FDSD和PF,和较高的Yield和MSL。含有等位基因B的绵羊表现为较高的MFD、MFC和PF。基因型为AB和BB绵羊的MFD高于基因型AC和BC(P0.01)。基因型为AA、AB和BB的绵羊具有比AC和BC型更高的FDSD(P0.05)。基因型为AC和BC的绵羊具有比AB和BB型高6%以上的MSL(P0.05)。对于PF,基因型为AB和BB的绵羊具有比AC和BC型更高的PF(P0.01)。提示,在育种工作中,若提高等位基因C和降低B在绵羊群体中的含量,可改良细度相关性状。(7)在383只美利奴杂种羊和48只罗姆尼羊中,共检测到绵羊KRTAP6-3基因存在7种基因型,包含5个SNPs位点和2处45bp的缺失突变。45bp的突变发生在一段核苷酸重复序列上,等位基因G缺失了前一个重复单元,而等位基因C缺失了后一个重复单元。基因型为AB羔羊的MFD、FDSD和PF高于基因型为AA和AG的羔羊(P0.001)。基因型为AA羔羊的MFD、FDSD和PF高于基因型为AG的羔羊(P0.001)。含有等位基因G的绵羊的羊毛MFD、FDSD和PF下降显著,而这三个性状值的降低符合羊毛市场需求。因此,在育种中若以上述3个性状改良为目标,KRTAP6-3可以作为分子标记基因。综上所述,绵羊KRTAP22-1,KRTAP21-2,KRTAP26-1和KRTAP6-3四个基因的变异与羊毛性状相关,可作为羊毛性状分子标记候选基因利用。
[Abstract]:Keratin associated protein (keratin-associated, proteins, KAPs) is an important structural protein determines the wool quality, encoding by KRTAP gene family. The family variation and expression of gene regulation and wool traits are correlated, but the research on this gene family is very limited. The human KRTAP gene family members have been found in sheep only 88. 27. In order to explore the new gene family and the analysis of its effect on wool traits, the Merino sheep (Merino) and Southdown (Southdown) hybrids and New Zealand Romney (Romney) as the research object, with the other known species of KRTAP family genes as reference, using genome analysis, search and phylogenetic analysis of homologous genomic data from sheep, preliminary screening of suspected gene, and amplification and sequencing analysis to determine the new gene in sheep. Using PCR-SSCP and sequencing methods, screening new genes S NPs, comparison of gene variation in differences between the 2 groups, correlation analysis of SNPs and wool traits. The main results are as follows: (1) identification of sheep KRTAP22-1, KRTAP22-2, KRTAP21-1, KRTAP21-2 and KRTAP26-1, a total of five new members of KRTAPs. The five new genes located in sheep on chromosome 1, located between the the KRTAP8-2 and KRTAP24-1. (2) in the 390 and 75 New Zealand Merino crossbred sheep Romney, detection of sheep KRTAP22-1 gene to 2 SNPs loci constitute 3 alleles (A-C) in New Zealand. Romney group B is the dominant allele (81.3%), in which A hybrid Merino sheep the dominant allele (51.8%). Correlation analysis of 390 Merino crossbred sheep samples showed that allele B has higher rate of gross and net (Wool Yield, Yield) and lower average wool crimp ratio (Mean fibre curvature, MFC).BB type and AB type cotton Sheep Yield than type AA high (P0.05). In terms of improving the breeding, Yield or MFC decreased as the target, the KRTAP22-1 gene can be used as the molecular marker gene. (3) in Merino crossbred sheep and Romney sheep populations were not detected in sheep KRTAP22-2 gene mutation. But in 80 the Qaidam black goat Ziwuling goat, Hexi cashmere goat and Inner Mongolia cashmere goats, KRTAP22-2 was detected in 3 SNPs and a length of 6BP variation constituted 3 alleles (A-C), one non synonymous mutations, leading to arginine to glycine. Allele C is inserted into a 6BP. The insertion of arginine and cysteine 2 amino acids of.AA and AB was the dominant genotype, there is a big difference between the A allele of.KRTAP22-2 gene in sheep and goats in polymorphism, this may be different from the genes in two species selection. (4) in the 363 Merino hybrid cotton Sheep, KRTAP21-1 gene were detected in 7 SNPs 6 alleles (A-F), of which 3 SNPs non synonymous mutations. The genotypes of these 363 samples in the high frequency AA and AC correlation analysis, only Yield in two genotypes of sheep were slightly different, but not significant. Influence of other alleles of wool traits, and further research is needed. (5) in the 389 Merino crossbred sheep, KRTAP21-2 gene was detected in 4 SNPs 5 genotypes (A-E), of which 1 were non synonymous mutations in the.KRTAP21-2 gene mutation and the average length of wool (Mean staple length, MSL) the fiber diameter, coefficient of variation (Fibre diameter standard deviation FDSD (Prickle) and tingling index factor, PF) 3 traits. Genotype AC than CE to MSL lamb (P0.05). The genotype of CE was higher than CC and BC lamb lamb FDSD (P0.05). Genotype CE lamb The PF is CC and BC (P0.05). High lamb deletion analysis in alleles, allele E had lower causes MSL. Multivariate analysis model, allele A analysis is introduced, the effect of E on MSL is significant (P0.05). E allele occurred the amino acid mutation in the encoding region, the allele will reduce wool MSL. visible, wool had the greatest effect of the gene is MSL. in breeding, in terms of improving MSL as the target, the KRTAP21-2 gene can be used as the molecular marker gene. (6) in the 383 and 94 New Zealand Merino crossbred sheep Romney sheep, KRTAP26-1 was detected in 7 SNPs of 4 alleles and 2 non synonymous mutations caused amino acid change. In the hybrid Merino sheep allele frequency of A:49.6%, B:25.7%, C:23.0% and D:1.7%, and in New Zealand Romney sheep were A:40.4%, B:47.3%, C:0 .5%鍜孌:11.7%.缁电緤KRTAP26-1鐨勫彉寮備笌涓，

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