發(fā)布時間：2018-03-09 17:36

  本文選題：綿羊　切入點：角蛋白關(guān)聯(lián)蛋白　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：角蛋白關(guān)聯(lián)蛋白(keratin-associated proteins,KAPs)是決定羊毛品質(zhì)的重要結(jié)構(gòu)蛋白,由KRTAP家族基因編碼。該家族基因的變異和表達調(diào)控與羊毛性狀存在相關(guān),但對該家族基因的研究還十分有限。人類KRTAP家族基因成員已發(fā)現(xiàn)88個,在綿羊上僅27個。為了挖掘該家族新基因并解析其對羊毛性狀的影響,本研究以美利奴羊(Merino)與南丘羊(Southdown)的雜種后代和新西蘭羅姆尼羊(Romney)為研究對象,以其他物種已知KRTAP家族基因為參考,采用基因組分析、同源性搜索和進化分析方法,從綿羊基因組數(shù)據(jù)中初步篩選疑似基因,進而擴增測序,分析確定綿羊中的新基因。采用PCR-SSCP和測序方法,篩查新基因的SNPs,比較基因變異在2個類群間的差異,分析SNPs與羊毛性狀的相關(guān)性。主要結(jié)果如下:(1)鑒定出綿羊KRTAP22-1、KRTAP22-2、KRTAP21-1、KRTAP21-2和KRTAP26-1共五個KRTAPs新成員。這五個新基因定位于綿羊1號染色體上,位于KRTAP8-2和KRTAP24-1之間。(2)在390只美利奴雜種羊和75只新西蘭羅姆尼羊中,綿羊KRTAP22-1基因檢測到2個SNPs位點構(gòu)成3個等位基因(A-C)。在新西蘭羅姆尼羊群體中B為優(yōu)勢等位基因(81.3%),而在美利奴雜種羊群其中A為優(yōu)勢等位基因(51.8%)。對390份美利奴雜種羊樣品進行的相關(guān)性分析表明,等位基因B存在與較高的凈毛率(Wool Yield,Yield)和較低的平均羊毛卷曲率(Mean fibre curvature,MFC)相關(guān)。BB型和AB型綿羊的Yield較AA型高(P0.05)。在育種時,若以提高Yield或降低MFC為目標(biāo),KRTAP22-1基因可作為其分子標(biāo)記基因。(3)在美利奴雜種羊和新西蘭羅姆尼羊群體中未檢測到綿羊KRTAP22-2基因變異。但在80只柴達木黑山羊、子午嶺黑山羊、河西絨山羊和內(nèi)蒙古絨山羊中,檢測到KRTAP22-2存在3個SNPs和一處6bp長度變異構(gòu)成3個等位基因(A-C),其中一個為非同義突變,導(dǎo)致精氨酸變?yōu)楦拾彼�。等位基因C存在一個6bp的插入,插入精氨酸和半胱氨酸2個氨基酸。AA和AB為優(yōu)勢基因型,A為優(yōu)勢等位基因。KRTAP22-2基因在綿山羊中的多態(tài)性存在較大差異,這可能與該基因在兩個物種中受到的選擇不同有關(guān)。(4)在363只美利奴雜種綿羊中,KRTAP21-1基因共檢測到7個SNPs形成6個等位基因(A-F),其中3個SNPs為非同義突變。對這363份樣品中基因型頻率較高的AA和AC進行相關(guān)性分析,只有Yield在兩種基因型的綿羊間略有差異,但不顯著。其他等位基因?qū)ρ蛎�性狀的影�?還有待進一步研究。(5)在389只美利奴雜種羊中,檢測到KRTAP21-2基因4個SNPs形成5種基因型(A-E),其中1個為非同義突變。KRTAP21-2基因變異與平均羊毛長度(Mean staple length,MSL)、纖維直徑變異系數(shù)(Fibre diameter standard deviation,FDSD)和刺感指數(shù)(Prickle factor,PF)3個性狀相關(guān)�；�因型為AC的羔羊MSL比CE的要大(P0.05)�；�因型為CE的羔羊比CC和BC的羔羊有較高的FDSD(P0.05)�；�因型為CE羔羊的PF較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存在會引起MSL的降低。多變量模型分析時,等位基因A引入分析時,E對MSL的影響依舊顯著(P0.05)。等位基因E在編碼區(qū)發(fā)生了氨基酸變異,該等位基因存在會降低羊毛的MSL。可見,受該基因影響最大的羊毛性狀是MSL。在育種時,若以提高MSL為目標(biāo),KRTAP21-2基因可作為其分子標(biāo)記基因。(6)在383只美利奴雜種羊和94只新西蘭羅姆尼羊中,檢測到KRTAP26-1存在7個SNPs形成的4個等位基因,有2個為非同義突變引起了氨基酸的改變。在美利奴雜種羊中各等位基因的頻率為A:49.6%、B:25.7%、C:23.0%和D:1.7%,而在新西蘭羅姆尼羊中分別為A:40.4%、B:47.3%、C:0.5%和D:11.7%。綿羊KRTAP26-1的變異與一系列羊毛性狀相關(guān),包括Yield、平均纖維直徑(Mean fibre diameter,MFD)、FDSD、MSL、MFC和PF。然而最大、最持久的影響是對MFD、FDSD、MSL和PF4個性狀。含有等位基因C的綿羊表現(xiàn)為較低的MFD、FDSD和PF,和較高的Yield和MSL。含有等位基因B的綿羊表現(xiàn)為較高的MFD、MFC和PF�；�因型為AB和BB綿羊的MFD高于基因型AC和BC(P0.01)。基因型為AA、AB和BB的綿羊具有比AC和BC型更高的FDSD(P0.05)�；�因型為AC和BC的綿羊具有比AB和BB型高6%以上的MSL(P0.05)。對于PF,基因型為AB和BB的綿羊具有比AC和BC型更高的PF(P0.01)。提示,在育種工作中,若提高等位基因C和降低B在綿羊群體中的含量,可改良細度相關(guān)性狀。(7)在383只美利奴雜種羊和48只羅姆尼羊中,共檢測到綿羊KRTAP6-3基因存在7種基因型,包含5個SNPs位點和2處45bp的缺失突變。45bp的突變發(fā)生在一段核苷酸重復(fù)序列上,等位基因G缺失了前一個重復(fù)單元,而等位基因C缺失了后一個重復(fù)單元。基因型為AB羔羊的MFD、FDSD和PF高于基因型為AA和AG的羔羊(P0.001)。基因型為AA羔羊的MFD、FDSD和PF高于基因型為AG的羔羊(P0.001)。含有等位基因G的綿羊的羊毛MFD、FDSD和PF下降顯著,而這三個性狀值的降低符合羊毛市場需求。因此,在育種中若以上述3個性狀改良為目標(biāo),KRTAP6-3可以作為分子標(biāo)記基因。綜上所述,綿羊KRTAP22-1,KRTAP21-2,KRTAP26-1和KRTAP6-3四個基因的變異與羊毛性狀相關(guān),可作為羊毛性狀分子標(biāo)記候選基因利用。
[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%.緇電緤KRTAP26-1鐨勫彉寮備笌涓，

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