【作者】 王彦；

【导师】 朱庆；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 长链脂肪酸如同信号分子一样,也是重要的代谢产物。研究发现在长链脂肪酸摄取中一些候选蛋白如脂肪酸易位酶(FAT/CD36),脂肪酸合成蛋白(FABP),长链脂肪酰基辅酶A合成酶(ACSL)和脂肪酸转运蛋白(FATP)对长链脂肪酸的摄取和代谢具有重要作用,但都不如FATP对脂质代谢和脂肪相关疾病的影响,为此,当前对FATP基因家族的研究也逐渐成为了热点。本研究以四川山地乌骨鸡为试验材料,通过生物信息学分析,获取四川山地乌骨鸡FATP4蛋白的部分序列,以pMD-18T为载体,在DH5α菌株中进行FATP4蛋白CDS区的分子克隆,并对其质粒进行了序列测定。运用蛋白质组学分析工具,对CDS区的基本性质、糖基化位点、磷酸化位点、二级结构等进行了预测,确定该序列具有FATP家族的结构特点,是鸡FATP家族成员蛋白。本研究以地方品种四川山地乌骨鸡和培育优质鸡“大恒”S01系为试验材料,采用SYBR greenⅠ定量PCR分析FATP1基因和FATP4基因在10周龄这个时间点两品种间的表达差异,同时分析了四川山地乌骨鸡0周龄,2周龄,4周龄,6周龄,8周龄,10周龄和12周龄等这7个时间点FATP1和FATP4基因表达的组织差异,以及在胸肌,腿肌,心脏,肝脏等组织中FATP1和FATP4基因表达量的发育性变化。结果表明:FATP1和FATP4在两个品种间的表达差异不显著(P＞0.05)。FATP1和FATP4在不同阶段各有优势表达组织,同一组织中也有表达的优势时间点。本研究运用PCR-SSCP及测序技术检测突变位点。根据FATP1基因组序列共设计10对引物,在8个大恒优质肉鸡群体中,只有4对引物检测到多态,共检测到5处突变,分别为49360bp(SNP1,G/A),48195 bp(SNP2,G/A),46847bp(SNP3,A/G),46818bp(SNP4,A/G)和46555bp(SNP5,A/G)。检测到的突变分别位于外显子3、内含子5、内含子7、内含子7和外显子8内。由于SNP4变异位点的个体数少,为此我们没有对该位点进行后续的分析。FATP1基因突变位点与屠宰性状关联分析结果显示:SNP1位点:在70d鸡群中,GG型个体的胸肌重,腿肌重和胸肌率均显著高于AA和AG型个体(P＜0.05);在91d鸡群中,AG型个体的活重,屠体重,半净膛重,全净膛重,腿肌重和腿肌率显著高于GG型个体(P＜0.05);在所有鸡群中,AA基因型个体的活重,屠体重,半净膛重和全净膛重显著高于AG和GG型个体(P＜0.05),GG型个体的胸肌率显著高于AA和AG型个体(P＜0.05)。SNP2位点:在70d鸡群中,NN型个体的胸肌重,腿肌重,胸肌率和腿肌率显著高于MM和MN型个体(P＜0.05);在91d鸡群中,NN型个体的活重,屠体重,半净膛重和全净膛重显著高于MM和MN型个体(P＜0.05),而MN型个体的皮脂厚和腹脂率显著高于NN型个体(P＜0.05);在所有鸡群中,NN型个体的活重,屠体重,半净膛重,全净膛重,腿肌重,胸肌率和腿肌率显著高于MM和MN型个体(P＜0.05)。SNP3位点:在70d和91d鸡群中,3种基因型个体与所有屠体性状之间都没有显著差异(P＞0.05);在所有鸡群中,AA型个体的活重和屠体重显著高于AG型个体(P＜0.05)。SNP5位点:在70d鸡群中,MN型个体的活重,屠体重和全净膛重显著高于MM型个体(P＜0.05),而NN型个体的腿肌率显著高于MN型个体(P＜0.05);在91d鸡群中,NN型个体的活重,屠体重,半净膛重,全净膛重和腿肌率显著高于MM和MN型个体(P＜0.05),同时MN型个体的胸肌率显著高于NN型个体(P＜0.05);在所有鸡群中,NN型个体的腿肌率显著高于MN和NN型个体(P＜0.05)。单倍型与8个群体的屠宰性状关联分析结果显示:在70d鸡群中,单倍型组合对活重,屠体重,全净膛重,胸肌重,腿肌重,皮脂厚,胸肌率和腿肌率的遗传效应达到显著水平(P＜0.05);在91d鸡群中,单倍型组合对活重,屠体重,全净膛重,半净膛重,皮脂厚,腿肌率和腹脂率的遗传效应达到显著水平(P＜0.05);在所有鸡群中,单倍型组合对活重,屠体重,全净膛重,半净膛重,皮脂厚,腿肌率和胸肌率的遗传效应达到显著水平(P＜0.05)。同样运用PCR-SSCP及测序技术检测突变位点,根据FATP4基因组序列共设计15对引物,在8个鸡群中,有6对引物检测到多态,共检测到8处突变,分别为5108778bp(G/A),5108695(G/A),5108287(C/-),5107545(T/C),5106740(C/T),5106005(A/G),5105481(G/A)和5105454(C/T)。在FATP4基因的8个突变中,等位基因A、C、C和A依次为SNP2,SNP3,SNP5和SNP7的优势等位基因。FATP4基因单突变位点与屠体性状关联分析结果显示,SNP1位点(G5108778A):在70d鸡群中,3种不同基因型与胸肌率和腿肌率显著相关(P＜0.05);在90d鸡群中,SNP1对胸肌率和腹脂率的遗传效应达到显著水平(P＜0.05);在所有鸡群中,3种基因型与活重,屠体重,半净膛重,全净膛重,腹脂重,胸肌率,腿肌率和皮脂厚显著相关(P＜0.05)。SNP2位点(G5108695A):在70d和91d鸡群中,3种基因型与所有屠体性状都没有达到显著相关水平(P＞0.05),在所有鸡群中,GG型个体的腹脂重和皮脂厚显著高于AG型个体(P＜0.05)。SNP3位点(C5108287-):在70d鸡群中,SNP3对腹脂重,腿肌率和腹脂率的遗传效应达到显著水平(P＜0.05);在91d鸡群中,TT型个体的胸肌率显著高于CT型个体(P＜0.05);在所有鸡群中,TT型个体的腹脂重,胸肌率,腿肌率和腹脂率显著高于CT和CC型个体(P＜0.05)。SNP4位点(T5107545C):在70d鸡群中,TT型个体的胸肌重,腿肌重和腿肌率显著高于CC型个体(P＜0.05),而TC型个体的皮脂厚显著高于CC型个体(P＜0.05);在91d鸡群中,TT型个体的屠体重,活重,半净膛重和全净膛重显著高于CC型个体(P＜0.05);在所有鸡群中,TC型个体的活重,活重和全净膛重显著高于TT型个体,而TT型个体的腿肌率显著高于CC型个体(P＜0.05)。SNP5位点(C5107640T):在70d鸡群中,不同基因型只与胸肌率显著相关(P＜0.05);在所有鸡群中,SNP5对应的不同基因型间的皮脂厚性状差异显著(P＜0.05)。SNP6位点(A5106005G):在70d鸡群中,AG型个体的胸肌重和胸肌率显著高于AA和GG型个体(P＜0.05);在91d和所有鸡群中,GG型个体的活重,屠体重,半净膛重,全净膛重和腿肌重显著高于AA型个体(P＜0.05)。SNP7位点(G5105481A):在70d和90d鸡群中,GG型个体的活重和全净膛重显著高于AG型个体(P＜0.05);在所有鸡群中,AG型个体的胸肌率显著高于AA型个体(P＜0.05)。SNP8位点(C5105454T):在3个鸡群中不同基因型个体的所有屠宰性状之间差异不显著(P＞0.05)。单倍型与8个群体的屠宰性状关联分析结果显示:在70d鸡群中,单倍型组合对活重,屠体重,全净膛重,胸肌重,腿肌重,腹脂率,胸肌率和腿肌率的遗传效应达到显著水平(P＜0.05);在91d鸡群中,单倍型组合对活重,屠体重,全净膛重,半净膛重,腹脂重,皮脂厚,胸肌率,腿肌率和腹脂率的遗传效应达到显著水平(P＜0.05);在所有鸡群中,单倍型组合对活重,屠体重,全净膛重,半净膛重,腹脂重,腿肌重,皮脂厚,胸肌率,腿肌率和腹脂率的遗传效应达到显著水平(P＜0.05)。更多还原

【Abstract】 Long-chain fatty acids(LCFA) are both important metabolites and signaling molecules.At present,several candidate proteins have been proposed to be involved in the uptake process of LCFAs including fatty acid translocase(FAT/CD36),fatty acid binding protein(FABPs),long-chain fatty acyl-CoA synthetases(ACSL) and fatty acid transport proteins(FABPs).Although many of the above-mentioned proteins have important role in the proceeding of LCFAs uptake and metabolism,recent in vivo studies have particularly highlighted the contribution of FATPs to lipid metabolism and fatty acid-associated disorders.So,the studies about FATP are being focused in today because of FATP special character and biological function.In the studies,we adopted Sichuan Mountains Black-Bone chicken as materials. The code sequences of FATP4 protein were gained by bioinformatics analysis.The sequences were cloned with pMD18-T vector in E.coli DH5a and sequenced.Then, the basic physical parameters,glycosylation sites,phosphorylation sites, hydrophobicity and second structure were predicted by proteome analysis tools. Through analysis,we concluded that it was the protein of chicken FATP4.The present study also took Sichuan Mountainous Black-Bone chicken and S01 of Daheng chicken as materials.We used the real-time PCR(SYBR greenⅠ) method to test the expression quantity of FATP1 and FATP4 gene in different breed of above-mentioned at 10 week.And we tested the expression quantity of FATP1 and FATP4 gene in different tissues and the expression quantity at different growth points. The results showed that the expression quantity of FATP1 and FATP4 had no significant difference between the two breeds.Moreover,there was advantageous tissue for gene expression at each growth point and there was also advantageous growth point for gene expression in a certain tissue. The current study adopted eight different populations of Daheng high-quality chicken in Sichuan province as samples.Five SNPs in FATP1 gene were identified using PCR-SSCP method,including 49360bp(SNP1,G/A),48195 bp(SNP2,G/A), 46847bp(SNP3,A/G),46818bp(SNP4,A/G) and 46555bp(SNP5,A/G), respectively.Because the sample number of A46818G was few,we didn’t analyze this mutation.The results of single locus analysis showed that at SNP1(G 49360A) locus, GG genotype had significantly higher(P＜0.05) breast muscle weight(BMW),leg muscle weight(LMW) and the percentage of breast muscle weight(BMWP) than AA and AG genotypes in 70d chicken populations;AG genotype had significantly higher live weight(LW),carcass weight(CW),semi-eviscerated weight(SEW), eviscerated weight(EW),LMW and the percentage of leg muscle weight(LMWP) than GG genotype in 91d chicken samples;AA genotype had significantly higher (P＜0.05) LW,CW,SEW and EW than AG and GG genotype,moreover,GG genotype had significantly higher BMWP than AA and AG(P＜0.05) in all examined samples.At SNP2(G48195A) locus,NN genotype had significantly higher(P＜0.05) BMW,LMW, BMWP and LMWP than MM and MN genotypes in 70d chicken populations;NN genotype had significantly higher LW,CW,SEW and EW than MM and MN genotype, while the NN genotype had significantly lower subcutaneous fat thickness(SFT) and the percentage of abdominal fat weight(AP) than that of MN genotype(P＜0.05) in 91d chicken samples;NN genotype had significantly higher(P＜0.05) LW,CW,SEW, EW,LMW,BMWP and LMWP than MM and MN genotype in all samples.At SNP 3 (A46847G) locus,in 70d and 91d chicken populations,there was no significantly association between genotype and carcass traits(P＞0.05);while AA genotype had significantly higher LW and CW than AG genotypes(P＜0.05) in all samples.At SNP5 (A46555G) locus,in 70d chicken populations,MN genotype had significantly higher (P＜0.05) LW,CW and EW than MM genotypes,but had lower LMWP when compared with NN genotype;in 91d chicken populations,NN genotype had significantly higher LW,CW,SEW,EW and LMWP than MM and MN genotypes, while the NN genotype had significantly lower BMWP than that of MN genotype (P＜0.05);in all chicken populations,NN genotype had significantly higher(P＜0.05) LMWP than MM and MN genotypes.The haplotype analysis indicated that in 70d chicken populations,the haplotype combinations had significant genetic effects on LW, CW,EW,BMW,LMW,SFT,BMWP and LMWP(P＜0.05);in 91d chicken populations,the haplotype had significant genetic effects on LW,CW,SEW,EW,SFT, LMWP and AP(P＜0.05);in all chicken populations,the haplotype had significant genetic effects on LW,CW,SEW,EW,SFT,BMWP and LMWP.Eight SNPs were detected at 5108778bp(G/A),5108695bp(G/A),5108287bp (C/-),5107545bp(T/C),5106740bp(C/T),5106005bp(A/G),5105481bp(G/A) and 5105454bp(C/T) in FATP4 gene.The allele A,C,C and A were the predominate allele in SNP2,SNP3,SNP5 and SNP6,respectively.The single locus analysis indicated that single nucleotide polymorphisms at SNP1(G5108778A) locus were significantly associated with BMWP and LMWP(P＜0.05) in 70d chicken populations;SNP1 was associated significantly with BMWP and AP(P＜0.05) in 91d chicken populations; SNP 1 was associated significantly with LW,CW,SEW,EW,AW,BMWP,LMWP and SFT(P＜0.05) in all samples.At SNP2(G5108695A) locus,there was no significantly association between genotypes and carcass traits(P＞0.05) in 70d and 91d chicken populations;while AW and SFT of the GG genotype were significantly higher than those with the AG genotype(P＜0.05) in all samples.At SNP3(C5108287-) locus, genotypes at this locus were significantly associated with AW,LMW-P and AP(P＜0.05) in 70d chicken populations;while,the TT genotype had higher BMWP than that of CT (P＜0.05) in 91d chicken populations;TT genotype had significantly higher AW, BMWP,LMWP and AP than CT and CC genotype(P＜0.05) in all chicken populations. At SNP4(T5107545C) locus,TT genotype had significantly higher(P＜0.05) BMW, LMW and LMWP than CC genotypes in 70d chicken populations,moreover,the SFT of chicken with the TC genotype was significantly higher than that of CC genotype; TT genotype had significantly higher LW,CW,SEW and EW than CC genotype in 91d chicken populations;TC genotype had significantly higher(P＜0.05) LW,CW and EW than TT genotype,moreover,TT genotype had significantly higher LMWP than CC(P＜0.05) in all chicken populations.At SNP5(C5106740T) locus,SNP at this locus was only significantly associated with BMWP(p＜0.05) in 70d chicken populations;in all chicken populations,single nucleotide polymorphisms were significantly associated with SFT(p＜0.05).At SNP 6(A5106005G) locus,AG genotype had significantly higher BMW and BMWP than AA and AG genotype (P＜0.05) in 70d chicken populations;GG genotype had significantly higher LW,CW, SEW,EW and LMW than AA genotypes(P＜0.05) in 91d chicken populations.At SNP7(G5105481A) locus,GG genotype had significantly higher(P＜0.05) LW and EW than AG genotypes in both 70d and 91d chicken populations;in all chicken populations,AG genotype had significantly higher(P＜0.05) BMWP than AA genotype. At SNP8(C5105454T) locus,there were no significant difference among all samples (P＞0.05).The haplotype analysis indicated that in 70d chicken populations,the haplotype combinations had significant genetic effects on LW,CW,EW,BMW,LMW, AP,BMWP and LMWP(P＜0.05);in 91d chicken populations,the haplotype had significant genetic effects on LW,CW,SEW,EW,AW,SFT,BMWP,LMWP and AP (P＜0.05);in all chicken populations,the haplotype had significant genetic effects on LW,CW,SEW,EW,LMW,AW,SFT,BMWP,LMWP and AP(P＜0.05).更多还原