【作者】 吉文林；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 特种经济动物饲养， 2013， 博士

【摘要】 近年来,我国大部分地区所饲养的肉用型鸭主要是樱桃谷鸭、北京鸭,虽然生长速度快,饲料报酬高,但存在脂肪含量高、肉质不够理想等缺点,难以满足已经实现小康的消费者需求。为了满足人们对小型优质肉鸭的需求,我们利用国内外番鸭资源进行杂交培育,开展了瘦肉率高、肉质好、体型适中、羽色纯黑的优质番鸭新品系---黑羽番鸭的培育工作,目前该品系遗传特性已基本稳定。本研究以黑羽番鸭为试验素材,测定了不同周龄体重,13周龄体尺、屠宰性能、常规肉品质、肌肉营养成分等指标,同时对黑羽番鸭生长激素(GH)和脂蛋白脂酶(LPL)基因进行了克隆、序列分析及多态性检测,分析了生长激素基因多态性与生长、屠宰性能的关联性及脂蛋白脂酶基因多态性与肉质性能的关联性,检测了2个基因在肌肉组织中mRNA发育性表达。主要研究结果如下：1、对黑羽番鸭生长发育性能测定结果表明,初生重在公母之间没有显著差异,随着周龄的上升,公鸭生长速度明显高于母鸭(P<0.05),13周龄时,公鸭体重为3149.6g,母鸭体重为1916.9g,公鸭是母鸭体重的1.64倍,公鸭日增重极显著大于母鸭(P<0.01)；13周龄体尺测定结果发现,公鸭体斜长、胸深、胸宽、龙骨长、胫长、胫围、半潜水长等7个体尺指标均极显著大于母鸭(P<0.01)；体重与体尺呈极显著正相关(P<0.01)。2、对黑羽番鸭早期体重生长曲线进行了拟合,结果显示Logistic、Gompertz、Von Bertalanffy3种曲线模型均能较好拟合黑羽番鸭的生长曲线,其中Gompertz模型在估计黑羽番鸭早期体重效果更优。Gompertz模型估计公鸭拐点体重和时间分别为1309.9g、5.3w,母鸭拐点体重和时间分别为754.7g、4.2w。Gompertz曲线模型结果可以为黑羽番鸭培育提供参考。3、黑羽番鸭屠宰性能测定结果表明,除腹脂重外,黑羽番鸭公鸭所有屠宰指标及比率均极显著高于母鸭(P<0.01)。肉品质测定结果发现,公鸭胸肌剪切力和腿肌失水率显著高于母鸭(P<0.05)；公鸭胸肌铁、铜含量显著高于母鸭,腿肌4种矿物元素含量均显著高于母鸭(P<0.05)；胸肌中铁、铜、镁含量显著高于腿肌,锌含量显著低于腿肌(P<0.05)。母鸭腿肌中肌苷酸含量显著高于公鸭(P<0.05)。肌肉中部分氨基酸含量在不同性别的组织中有显著差异(P<0.05)。这些结果显示不同组织、不同性别会影响肌肉肉品质。4、利用同源克隆策略,获得了黑羽番鸭生长激素基因(GH)761bp的cDNA序列,包括651bp的编码区,编码216个氨基酸。与禽类GH基因核苷酸和氨基酸序列同源性均在90%以上,与人和哺乳动物同源性在55%-77%之间。实时荧光定量PCR检测结果显示,GH基因在第2周龄肌肉中mRNA表达量最高；第4周龄发生了大幅度下降；在第6周龄时胸肌中表达量有所回升,腿肌仍然处于较低水平；在以后的周龄中维持在一定水平。5、利用同源克隆策略,获得了黑羽番鸭脂蛋白脂酶基因(LPL)1515bp的cDNA序列,包括1485bp的编码区,编码494个氨基酸。与禽类LPL基因核苷酸和氨基酸序列同源性均在90%以上,与人和哺乳动物同源性在70%-75%之间。实时荧光定量PCR检测结果显示,LPL基因在第2周龄肌肉组织中mRNA表达量较低,在第4周龄出现大幅度上升；在第6周龄公鸭胸、腿肌中表达量继续上升,然后下降并维持在较低水平；母鸭腿肌中LPL基因mRNA表达量起伏变化,胸肌维持在较低水平。6、利用PCR-SSCP技术,在GH基因第1内含子上发现了A1251C、A1322G、T1378C、 G1440A、G945A,T1039G共6个SNP,群体平均纯合度为0.5030,多态信息含量为0.3735。前4个点突变形成了AA、AB、BB3种基因型,在番鸭早期生长发育过程中,BB型公鸭体重在不同周龄时处于最高值；在13周龄屠宰时,公鸭BB型的宰前活重、屠体重、半净膛重、全净膛重显著高于AA型和AB型(P<0.05)；在内脏组织中,BB型腹脂重、心重显著高于AB型(P<0.05)；AA型、AB型、BB型3种基因型间屠宰指标百分比无显著差异(P>0.05)；母鸭所有指标在3种基因型间均无显著差异(P>0.05)。后2个点突变形成了CC、CD、DD3种基因型,在番鸭早期生长发育过程中,CC型公鸭体重在不同周龄时处于最高值；在13周龄屠宰时。公鸭CC型的宰前活重、屠体重、半净膛重、全净膛重、腹脂重、腺肌胃重显著高于CD型和DD型(P<0.05)；CC型心重显著高于CD型(P<0.05)；CC型、CD型、DD型3种基因型间屠宰指标百分比无显著差异(P>0.05)。母鸭所有指标在3种基因型间均无显著差异(P>0.05)。这些关联分析表明B、C基因可能是公鸭早期增重的有利基因,在母鸭上还需进一步验证。7、利用PCR-SSCP技术,在LPL基因第3内含子上发现了A4G, G120A、A128G共3个SNP,群体平均纯合度为0.5028,多态信息含量为0.3736。前1个点突变形成了EE、EF、FF3种基因型,EF型母鸭腿肌失水率显著高于FF型(P<0.05,下同)。EF型公鸭腿肌中镁含量显著高于FF型。EE型公鸭胸肌中Ala含量显著性高于EF型、FF型；FF型母鸭胸肌中Asp、His含量显著性高于EF型；FF型公鸭腿肌中Ser、Pro含量显著高于EE型；EE型母鸭腿肌Lys含量显著高于EF型,EF型Cys、Met含量显著性高于FF型(P<0.05)。后2个点突变形成了HH、HI、Ⅱ3种基因型,Ⅱ型公鸭胸肌中Pro含量显著高于HI型,Ⅱ型Val、Met、Phe含量显著高于HH型；HI型母鸭胸肌中His含量显著性高于Ⅱ型；Ⅱ型公鸭腿肌中Asp含量显著高于HI型,HH型His含量显著高于Ⅱ型,Ⅱ型Pro含量显著高于HH型,Ⅱ型Ile、Phe含量显著高于HH型、HI型；HI型母鸭腿肌中Gly含量显著高于Ⅱ型,HI型Arg含量显著高于HH型(P<0.05)。这些结果推断LPL可作为公鸭胸肌中Ala、Pro、Val、Met、Phe含量,公鸭腿肌中镁、Ser、Pro、Asp、His、Ile、Phe含量,母鸭胸肌中Asp、His含量,母鸭腿肌中失水率、Lys、Cys、Met、Gly、Arg含量的辅助选择标记。更多还原

【Abstract】 In recent years, most parts of China meat type duck were Cherry Valley duck and Pekin duck. Although these ducks grow quickly with high feed reward, they have high fat content and their meat quality is not ideal, which leads to these ducks can not satisfy the consumer’needs, who hadawell-offlife. Small and fine quality meat duck, such as black Muscovy duck, could be satisfied goods to people. Black Muscovy duck was hybridized by domestic and exotic Muscovy ducks. The new stock has high lean meat rate, fine meat quality, middle body type and black feather. Now, the emphytic character of stock is basic stabilization. Black Muscovy ducks were used for this study. Their body weight at different weeks,13-week-old body size, slaughtering, routine meat quality, chest and leg muscle nutrients were measured. Meanwhile, the growth hormone (GH) and lipoprotein lipase (LPL) genes of the black Muscovy duck were cloned, sequenced and their SNPs were detected. This paper analyzed the correlation between the growth hormone gene polymorphism and growth, slaughter performance; the correlation between lipoprotein lipase and meat quality. The mRNA expression of the two genes was analyzed in muscle at different ages. The main results were listed as follows:1. The result of growth and development performance measure about black Muscovy duck indicated that there was not significant difference in birth body weight between the male and the female ducks. However, the growth velocity of male black Muscovy duck was obviously faster than that of female black Muscovy duck in the next several weeks (P<0.05). At the age of13weeks, the weight of the male reached3149.6g while the female was only1916.9g. That was to say, the weight of male duck was1.64times as much as that of female duck. The daily weight gain of the male black Muscovy duck was significantly larger than that of the female duck’s (P<0.01). The result of body size measure at13-week-old indicated that the body slope length, breast depth, breast width, fossil bone length, shank length, shank circumference, half-diving depth of the male ducks were significant greater than those of the female duck’s (P<0.01). There was a very significant positive correlation between the body weight and the body size (P<0.01).2. The growth curve fitting of early body weight was studied in black Muscovy duck. The results indicated that the three curve models, Logistic, Gompertz and Von Bertalanffy can all be fitted with the growth curve of the black Muscovy ducks. The Gompertz curve model was the best choice to estimate the black Muscovy duck’s weight in the early period. According to Gompertz curve model, the inflection weight and age of growth were1309.9g and5.3weeks in male duck, and754.7g and4.2weeks in female duck, respectively. The Gompertz curve model could be given information to black Muscovy duck breeding.3. The results of slaughter performance measure in black Muscovy duck indicated that, except for the abdominal fat weight, all slaughter indexes and ratios were significantly higher than those of female ducks (P<0.01). The results of meat quality measure acquired that the shear force of chest muscle and water loss rate of leg muscle in male ducks were obviously higher than those of the females (P<0.05). The Fe, Cu content in the male duck’s chest muscle was significantly higher than that of the female ducks (P<0.05). The content of the four mineral elements in the male duck’s leg muscles was obviously higher than the female’s. The Fe, Cu, Mg content in the chest muscles was significantly higher than that in leg muscles while the Zn content in chest muscles was significantly lower than that in the leg muscles (P<0.05). The IMP content in the female duck’s leg muscles was significantly higher than that in the male ducks (P<0.05). Part of the amino acid content in the muscles had significant difference in different genders (P<0.05). The result indicated that the meat quality could be influence by different tissues and sexes.4. By homology cloning strategy, the cDNA of black Muscovy duck GH gene were761bp which contained651bp CDS, encoding216amino acids. When compared to poultry, the nucleotide and amino acid sequence homology were over90%, about55%-77%homologous to human and mammals. The result of Real-Time PCR displayed that the mRNA expression level of GH gene in muscle reached the highest point at2-week-old; the expression level dropped greatly at4-week-old; the expression level in the chest muscles rose gradually at6-week-old, and in the leg muscles remained a low level. In the later weeks, the expression level began to decline and maintained at a certain level.5. By homology cloning strategy, the cDNA of black Muscovy duck LPL gene were1515bp which contained1485CDS, encoding494amino acids. When compared to poultry LPL gene, the nucleotide and amino acid sequence homology of black Muscovy duck were over90%, about70%-75%homologous to human and mammals. The results of Real-Time PCR showed that the mRNA expression level of LPL gene was relatively lower at2-week-old. The expression level rose greatly at4-week-old. At6-week-old, the expression level in the male duck’s chest and leg muscles still rose and then declined and maintained at a low level. The mRNA expression level of LPL gene in the female duck’s leg muscles fluctuated constantly, and it remained at a low level in the female duck’s chest muscle.6. In intron1of GH gene, six SNPs were discovered by technique of PCR-SSCP. These SNPs included A1251C, A1322G, T1378C, G1440A, G945A and T1039G. The average homozygosity was0.5030in the whole population. The polymorphic information content was0.3735. The first4mutations formed AA, AB and BB genotypes. The weight of BB male was highest at different weeks in process of black Muscovy duck early growth. When the ducks were slaughtered at13-week-old, the live weight, dressed weight, half-eviscerated weight and eviscerated weight of BB male ducks was significantly higher than that of AA and AB genotypes in male ducks (P<0.05). In the visceral tissues, the weight of BB duck abdominal fat and heart was significantly higher than that of AB duck (P<0.05). There was no obvious difference in slaughter index percentage among three genotypes (AA, AB and BB)(P>0.05). All indexes of female black Muscovy ducks had no significant difference among the three genotypes (P>0.05). The follow2mutations formed CC, CD and DD genotype. The weight of CC male was highest at different weeks in process of black Muscovy duck early growth (P<0.05). When the ducks were slaughtered at13-week-old, the live weight, dressed weight, half-eviscerated weight, eviscerated weight, abdominal fat weight and glandular muscle stomach weight of CC male ducks was significantly higher than that of CD and DD male ducks (P<0.05). The heart weight of CC male ducks were significantly higher than those of CD male ducks (P<0.05). There was not significant difference in the slaughter percentage indexes among the three genotypes (AA, AB and BB)(P>0.05). All of the female duck’s indexes had no obvious difference among the three genotypes (P>0.05). The correlation analysis implied that allele B and C maybe were the favorable genes for body weight, but that regularity should be probation in female duck. 7. In intron3of LPL gene, three SNPs were discovered by technique of PCR-SSCP. These SNPs included A4G, G120A and A128G. The average homozygosity was0.5028in the whole population. The polymorphic information content was0.3736. The first mutations formed EE, EF and FF genotype. The rate of water loss of EF female duck was significant higher that of FF female duck. The Mg content in leg muscle of the EF male duck was significantly higher than that in leg muscle of the FF male duck (P<0.05, The same as follows). The Ala content in chest muscle of the EE male duck was significant higher than that in chest muscle of the EF and FF male duck. The Asp and His content in chest muscles of the EF female duck were significantly higher than that in chest muscles of EF female duck. The content of Ser and Pro in leg muscles of the EF male duck was significantly higher than that in leg muscle of EE male duck. The Lys content in leg muscles of the EE female duck was significantly higher than that in leg muscles of EF female duck, and the Cys and Met was significantly higher than that in FF. The follow2mutations formed HH, HI and II genotype. The Pro content in the II male duck chest muscles was significantly higher than that in HI male duck chest muscles. The Val, Met and Phe content were significantly higher than those in HH ducks. The His content in chest muscle of the HI female duck was significantly higher than that of chest muscle in the II female duck. The Asp content in leg muscle of the II male duck was also significantly higher than that in the HI male duck. The His content in the HH male duck was significantly higher than that in the II male duck. The Pro content in the II male duck was notably higher than that in the HH male duck. The lie and Phe content in the II male duck were significantly higher than those in the HH and HI male duck. The Gly content of leg muscle in the HI female duck was greatly higher than that in the II female duck. The Arg content in the HI duck was significantly higher than that in the HH duck, too. The result indicated that LPL gene could be an assistant marker for several meat quality indexes, which include content of Ala, Pro, Val, Met, Phe in male duck’s breast muscle, and Mg, Ser, Pro, Asp, His, Ile, Phe in male duck’s leg muscle; and Asp, His in female duck’s breast muscle, and rate of water loss, Lys, Cys, Met, Gly, Arg in female duck’s leg muscle.更多还原