楊云, 曾勇慶, 張哲, 徐正剛, 陳偉, 房國(guó)鋒, 王守棟

(山東農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,動(dòng)物遺傳育種學(xué)實(shí)驗(yàn)室,山東 泰安 271018)

豬神經(jīng)元蛋白3.1基因3′側(cè)翼區(qū)多聚腺苷酸數(shù)目多態(tài)性及其與肉質(zhì)性狀的關(guān)系

楊云, 曾勇慶*, 張哲, 徐正剛, 陳偉, 房國(guó)鋒, 王守棟

(山東農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,動(dòng)物遺傳育種學(xué)實(shí)驗(yàn)室,山東 泰安 271018)

選取54頭萊蕪豬和40頭魯萊黑豬,宰后測(cè)定肉質(zhì)性狀,利用聚合酶鏈反應(yīng)及單鏈構(gòu)象多態(tài)性(polymerase chain reaction-single strand conformation polymorphism, PCR-SSCP)技術(shù)檢測(cè)其神經(jīng)元蛋白3.1(neuronal protein 3.1, P311)基因3′側(cè)翼區(qū)2個(gè)多聚腺苷酸(polyA)結(jié)構(gòu)的長(zhǎng)度多態(tài)性,測(cè)序確定連續(xù)腺苷酸的數(shù)目,并與肉質(zhì)性狀進(jìn)行關(guān)聯(lián)分析，以期發(fā)現(xiàn)有效多態(tài)位點(diǎn).結(jié)果表明：1)在萊蕪豬和魯萊黑豬中共檢測(cè)出2個(gè)連續(xù)腺苷酸數(shù)目的多態(tài)位點(diǎn)polyA-L1和polyA-L2,兩者的polyA數(shù)目分別為18、15和15、12,2個(gè)位點(diǎn)的3種基因型分別記為polyA-L1位點(diǎn)的MM型、NN型和MN型,以及polyA-L2位點(diǎn)的BB型、DD型和BD型；這2個(gè)多態(tài)位點(diǎn)在萊蕪豬和魯萊黑豬群體中均處于哈代-溫伯格平衡(P>0.05),其多態(tài)性在2個(gè)豬種間的分布差異在統(tǒng)計(jì)學(xué)上均不顯著.2)萊蕪豬polyA-L1位點(diǎn)的3種基因型之間在肉質(zhì)性狀上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,但MN型個(gè)體各項(xiàng)肉質(zhì)性狀普遍優(yōu)于2個(gè)純合型,肉質(zhì)較好,魯萊黑豬中的結(jié)果與此相同.3)萊蕪豬polyA-L2位點(diǎn)的3種基因型之間在肉質(zhì)性狀上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,但魯萊黑豬在該位點(diǎn)處的失水率和烹飪損失指標(biāo)在統(tǒng)計(jì)學(xué)上差異顯著,其中DD型個(gè)體的失水率顯著高于BD型個(gè)體,BB型和DD型個(gè)體的烹飪損失顯著高于BD型,其余各指標(biāo)的基因型間在統(tǒng)計(jì)學(xué)上差異不顯著.總之,在這2個(gè)位點(diǎn)處普遍表現(xiàn)為雜合型的肉質(zhì)性狀優(yōu)于純合型,生產(chǎn)中應(yīng)重視2個(gè)純合型個(gè)體間的雜交利用,以產(chǎn)生更多肉質(zhì)優(yōu)良的雜合型個(gè)體.

豬； 神經(jīng)元蛋白3.1基因； 3′側(cè)翼區(qū)； 多態(tài)性； 肉質(zhì)性狀

Summary The neuronal protein 3.1 (P311), also called PTZ17, is coded by theP311 gene, which is located in the porcine chromosome 2. The P311 is extensively distributed in the nervous and muscular systems and had the functions in myofibroblast differentiation and alveolar generation processes. It is also closely related to the fat deposition. The 3′ flanking region doesn’t code for protein, but it can affect the phenotype by some ways, such as the regulation of the degradation and translation rates of the mRNA. The research on the polymorphisms of the 3′ flanking region in the porcineP311 gene is still rare.

The aim of the study was to identify the length polymorphisms of the 3′ flanking region in the porcineP311 gene and to conduct association analysis between the polymorphisms and the meat traits in order to find some efficient polymorphic loci for the breeding of high quality pigs with good meat traits.

In this research, 54 Laiwu pigs and 40 Lulai pigs were chosen and slaughtered. The ear andLongissimusdorsisamples were collected for DNA extraction and meat traits determination. The extracted DNA was amplified by polymerase chain reaction (PCR) and the products were used to detect the polymorphic type with the method of single strand conformation polymorphism (SSCP) and sequencing. The meat traits were estimated in nine aspects including water loss, cooking loss, tenderness, pH1, pHμ, meat color score, marbling score, drip loss and intramuscular fat (IMF) content. All the data collected were analyzed using the SPSS 13.0 and R software.

The results showed that there were both two kinds of polyA numbers in the two loci of polyA-L1 and polyA-L2. The polyA numbers in the polyA-L1 and polyA-L2 loci were 18, 15 and 15, 12, respectively, and the genotypes were termed as MM, NN, MN in the polyA-L1 locus and BB, DD, BD in the polyA-L2 locus. The preponderant allele in the polyA-L1 locus in both of the two breeds was M allele, and the genotype frequency was MM>MN>NN. In the polyA-L2 locus, the preponderant allele in the Laiwu pig was B allele and the genotype frequency was BD>BB>DD, but in the Lulai pig, the gene frequencies of the two alleles were equal and the genotype frequency was BD>BB=DD. The heterozygosity index showed that the heterozygosity of the polyA-L2 locus in the two breeds was higher than that of the polyA-L1 locus. The results of chi-square test demonstrated that the two polymorphic loci in the Laiwu and Lulai populations both fitted the Hardy-Weinberg equilibrium situation (P>0.05). The polymorphic distributions between the two breeds showed no statistically significant differences at the two loci. In the polyA-L1 locus of the two breeds, none of the nine meat traits differed significantly among the three genotypes of MM, MN and NN, but the data displayed a trend that the performances of the MN genotype were better than those of the MM and NN genotypes. The IMF content, pH1and marbling score of the MN genotype were the highest and its drip loss and cooking loss were the lowest within the three genotypes. At the same time, the shear force value of MN genotype was lower than that of MM genotype. Among the three polymorphic types of BB, BD and DD in the polyA-L2 locus, significant differences were found in water loss and cooking loss in the Lulai population (P<0.05). The performances of the heterozygote were lower than those of the two homozygotes with no significant differences detected in other conditions. In the Laiwu population, the performances of DD genotype were the worst among the three genotypes, for its IMF, pH1and pHμwere the lowest while the cooking loss and drip loss were the highest. In the Lulai population, the BD genotype performed the best for its highest IMF, pH1, pHμand marbling score, and the lowest cooking loss and water loss.

It is concluded that the two breeds with different genotypes in the two loci show different meat traits and the heterozygotes display better than the homozygotes in some indexes, such as cooking loss, drip loss, marbling score and IMF content. As a result, hybridization should be widely used in practice to obtain the ideal heterozygotes with better meat traits.

神經(jīng)元蛋白3.1(neuronal protein 3.1, P311)又稱PTZ17,由Studler等[1]于1993年首次發(fā)現(xiàn)于小鼠胚胎的腦組織中.P311蛋白由定位于豬2號(hào)染色體的P311基因負(fù)責(zé)編碼,現(xiàn)已研究證實(shí)其廣泛存在于神經(jīng)及肌肉組織中,且是一種細(xì)胞內(nèi)蛋白質(zhì),主要存在于胞質(zhì)中,在細(xì)胞核內(nèi)亦有少量定位,參與肌成纖維細(xì)胞的分化及肺泡發(fā)育,與脂質(zhì)合成與蓄積密切相關(guān)[2-4].P311基因全長(zhǎng)26 252 bp,其mRNA長(zhǎng)度為2 410 nt.P311基因mRNA序列中包含3個(gè)開(kāi)放閱讀框,但僅第1個(gè)開(kāi)放閱讀框負(fù)責(zé)編碼蛋白質(zhì).Leung等[3]研究發(fā)現(xiàn),P311基因能夠通過(guò)上調(diào)與脂質(zhì)合成相關(guān)的一系列基因的表達(dá)水平使得細(xì)胞內(nèi)甘油三酯和膽固醇的水平顯著提高,從而導(dǎo)致細(xì)胞內(nèi)脂肪滴數(shù)目增多.本實(shí)驗(yàn)室前期通過(guò)抑制性消減雜交發(fā)現(xiàn),P311基因是重要的差異表達(dá)基因,在萊蕪豬中的表達(dá)量顯著高于大白豬.該基因的3′側(cè)翼區(qū)不編碼蛋白質(zhì),不影響該基因所編碼蛋白質(zhì)的結(jié)構(gòu)和功能,但其可通過(guò)調(diào)控該基因mRNA的降解速率和翻譯速率等控制基因表達(dá),進(jìn)而影響性狀表現(xiàn)[5].本研究旨在鑒定萊蕪豬及魯萊黑豬P311基因3′側(cè)翼區(qū)的多聚腺苷酸(polyA)的結(jié)構(gòu)多態(tài)性,并與其肉質(zhì)性狀進(jìn)行關(guān)聯(lián)分析,以期發(fā)現(xiàn)有效多態(tài)位點(diǎn),為優(yōu)質(zhì)肉豬的培育提供科學(xué)依據(jù).

1 材料與方法

1.1 實(shí)驗(yàn)豬及樣品采集

實(shí)驗(yàn)豬均來(lái)自山東省萊蕪市種豬繁育場(chǎng),包括54頭萊蕪豬和40頭魯萊黑豬.在相同的飼養(yǎng)管理?xiàng)l件下分別飼喂至相應(yīng)屠宰體質(zhì)量,即萊蕪豬(85.80±13.90) kg,魯萊黑豬(93.80±10.12) kg,放血屠宰后立即剪取耳組織樣置于裝有75%乙醇的Eppendorf離心管中,并采集第1～5腰椎處背最長(zhǎng)肌置于放有冰袋的保溫箱中帶回實(shí)驗(yàn)室,分別用于基因組提取及肉質(zhì)測(cè)定.

1.2 實(shí)驗(yàn)方法

1.2.1 肉質(zhì)測(cè)定[6-11]測(cè)定宰后45 min的 pH值(pH1)、宰后24 h的pH值(pHμ)、肌內(nèi)脂肪含量、大理石紋、肉色、滴水損失、失水率、烹飪損失、嫩度9項(xiàng)指標(biāo).其中,宰后45 min直接用pH計(jì)測(cè)定pH1,肉樣在0～4 ℃保存24 h后測(cè)定pHμ；肌內(nèi)脂肪含量采用索氏醚浸提法測(cè)定；肉色與大理石紋對(duì)照美制的NPPC比色板(5級(jí)分制)進(jìn)行分級(jí)評(píng)定；滴水損失采用吊掛處理法測(cè)定；失水率采用壓力法,利用鋼環(huán)式膨脹壓縮儀測(cè)定；烹飪損失采用蒸煮法測(cè)定；嫩度采用剪切力測(cè)定法,利用C-LM3型數(shù)顯式肌肉嫩度儀測(cè)定肌肉剪切力值,值越大則肉越粗老.

1.2.2 基因組提取 利用血液/細(xì)胞/組織基因組DNA提取試劑盒(購(gòu)自天根生化科技有限公司)進(jìn)行基因組提取,用1%瓊脂糖凝膠電泳和核酸蛋白測(cè)定儀檢測(cè)所提取DNA的濃度及純度；將基因組稀釋至100 ng/μL后于-20 ℃保存、備用.

1.2.3 樣品的聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)擴(kuò)增 以豬P311 DNA(NCBI登錄號(hào)為NC_010444.3)序列為模板設(shè)計(jì)2對(duì)引物：polyA 1F:CCAAAAACTGACGTCTCC,polyA 1R:ATCCATTTCATTGGGAGC,polyA 2F:TTTGACAGGCGTTTTTAGGTG,polyA 2R:ACGGAGACGTCAGTTTTTGG,擴(kuò)增長(zhǎng)度分別為181 bp和155 bp,擴(kuò)增位置分別為該基因的19 110～19 290 bp處和18 980～19 134 bp處.PCR擴(kuò)增體系：2×PCR Master Mix(購(gòu)自天根生化科技有限公司)12.5 μL,上、下游引物各1.5 μL(10 μmol/L),DNA 1 μL,加水補(bǔ)足至25 μL.擴(kuò)增反應(yīng)條件：95 ℃預(yù)變性5 min,95 ℃變性30 s,退火30 s,72 ℃延伸20 s,35個(gè)循環(huán)；最后,72 ℃延伸10 min.2對(duì)引物的退火溫度分別為56.4 ℃和59.5 ℃.擴(kuò)增后的產(chǎn)物置于4 ℃保存、備用.

1.2.4 單鏈構(gòu)象多態(tài)性(single strand conformation polymorphism, SSCP)檢測(cè)[12]向1.2.3節(jié)的擴(kuò)增產(chǎn)物中加入8.4 μL變性緩沖液(95%甲酰胺,10 mmol/L EDTA,0.09%二甲苯青,0.09%溴酚藍(lán)),混勻后置于PCR儀中95 ℃變性6 min.將變性產(chǎn)物迅速置于冰上冷卻,點(diǎn)樣于12%非變性聚丙烯酰胺凝膠中,于120 V電壓、4 ℃條件下電泳13 h后銀染顯色并統(tǒng)計(jì)結(jié)果.對(duì)不同基因型個(gè)體進(jìn)行抽樣測(cè)序.

1.3 數(shù)據(jù)分析

多態(tài)性數(shù)據(jù)利用R軟件進(jìn)行統(tǒng)計(jì)分析；肉質(zhì)測(cè)定數(shù)據(jù)利用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)分析,以平均值±標(biāo)準(zhǔn)差表示,顯著性檢驗(yàn)采用SNK(Student-Newman-Keuls)和最小顯著差異(least significant difference, LSD)法.

2 結(jié)果與分析

2.1 PCR-SSCP分析

泳道下方的字母表示該帶型的基因型.The letters below each lane mean the genotype of the band displayed.

圖1為引物對(duì)polyA 1F/1R和polyA 2F/2R的PCR-SSCP電泳圖.從中可以看出,2對(duì)引物的SSCP檢測(cè)結(jié)果均呈現(xiàn)3種帶型,且?guī)颓逦?易于分型.2個(gè)位點(diǎn)處的3種基因型分別記為polyA-L1位點(diǎn)的MM型、MN型和NN型,以及polyA-L2位點(diǎn)的BB型、BD型和DD型.

表1和表2分別為polyA-L1和polyA-L2位點(diǎn)的基因型頻率、基因頻率、雜合度、多態(tài)信息含量等.從中可以看出：萊蕪豬(LW)與魯萊黑豬(LL)在polyA-L1位點(diǎn)處的優(yōu)勢(shì)等位基因均為M基因；而在polyA-L2位點(diǎn)處萊蕪豬(LW)的優(yōu)勢(shì)等位基因?yàn)锽基因,魯萊黑豬(LL)的2個(gè)等位基因頻率相等.2個(gè)豬種在polyA-L1位點(diǎn)處的基因型頻率為MM>MN>NN；萊蕪豬在polyA-L2位點(diǎn)處的基因型頻率為BD>BB>DD,魯萊黑豬在該位點(diǎn)處的基因型頻率為BD>BB=DD.2個(gè)多態(tài)位點(diǎn)在萊蕪豬與魯萊黑豬中均為中度多態(tài),且polyA-L2位點(diǎn)在2個(gè)豬種中的雜合度均高于polyA-L1位點(diǎn).卡方適合性檢驗(yàn)表明,2個(gè)多態(tài)位點(diǎn)在萊蕪豬和魯萊黑豬群體中均處于哈代-溫伯格平衡(P>0.05).顯著性檢驗(yàn)表明,2個(gè)位點(diǎn)的多態(tài)性在2個(gè)豬種間的分布差異在統(tǒng)計(jì)學(xué)上均不顯著(P>0.05).為進(jìn)一步確定各基因型個(gè)體的堿基類型,對(duì)4種純合型個(gè)體的擴(kuò)增產(chǎn)物進(jìn)行回收測(cè)序.

表1 2個(gè)豬種polyA-L1位點(diǎn)的遺傳組成信息

LW：萊蕪豬；LL：魯萊黑豬；PIC：多態(tài)信息含量.括號(hào)內(nèi)的數(shù)據(jù)為該品種的該基因型個(gè)體數(shù).

LW: Laiwu pig; LL: Lulai pig; PIC: Polymorphism information content. The numbers in the parenthesis mean the quantities of the pigs with the same genotype in the same breed.

表2 2個(gè)豬種polyA-L2位點(diǎn)的遺傳組成信息

LW：萊蕪豬；LL：魯萊黑豬；PIC：多態(tài)信息含量.括號(hào)內(nèi)的數(shù)據(jù)為該品種的該基因型個(gè)體數(shù).

LW: Laiwu pig; LL: Lulai pig; PIC: Polymorphism information content. The numbers in the parenthesis mean the quantities of the pigs with the same genotype in the same breed.

2.2 各基因型堿基類型的確定

圖2為2個(gè)位點(diǎn)純合型個(gè)體的測(cè)序峰圖.對(duì)于位點(diǎn)polyA-L1,MM型個(gè)體的polyA數(shù)目為18(圖2A),NN型個(gè)體的polyA數(shù)目為15(圖2B),MN型個(gè)體同時(shí)存在上述2種polyA類型；對(duì)于位點(diǎn)polyA-L2,BB型和DD型個(gè)體的polyA數(shù)目分別為15(圖2C)和12(圖2D),BD型個(gè)體同時(shí)存在15和12這2種polyA類型.

2.3 polyA-L1和polyA-L2位點(diǎn)對(duì)肉質(zhì)性狀的影響

2.3.1 polyA-L1位點(diǎn)對(duì)萊蕪豬肉質(zhì)性狀的影響 萊蕪豬polyA-L1位點(diǎn)各基因型的肉質(zhì)性狀見(jiàn)表3.萊蕪豬polyA-L1位點(diǎn)處的3種基因型之間在所檢測(cè)的9項(xiàng)肉質(zhì)性狀上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,但由表3中的數(shù)據(jù)可以看出,MN型個(gè)體的肌內(nèi)脂肪含量、pH1、大理石紋評(píng)分均高于MM型和NN型個(gè)體,同時(shí)該基因型的滴水損失及烹飪損失均最低,剪切力值也較低.說(shuō)明MN型個(gè)體肌內(nèi)脂肪沉積能力較強(qiáng),保水性能較好,肉品細(xì)嫩,肉質(zhì)較好.NN型個(gè)體的肌內(nèi)脂肪含量、失水率及剪切力值最低,烹飪損失最高；而MM型個(gè)體的大理石紋評(píng)分最低,滴水損失最高.

(A,B)：分別為polyA-L1位點(diǎn)MM型和NN型個(gè)體測(cè)序峰圖；(C，D)：分別為polyA-L2位點(diǎn)BB型和DD型個(gè)體測(cè)序峰圖. (A, B): Sequencing results of the MM and NN genotypes in the polyA-L1 locus respectively; (C, D): Sequencing results of the BB and DD genotypes in the polyA-L2 locus respectively.

表3 polyA-L1位點(diǎn)不同基因型萊蕪豬的肉質(zhì)性狀

IMF：肌內(nèi)脂肪；pH1：宰后45 min的pH值；pHμ：宰后24 h的pH值；MC：肉色評(píng)分；MB：大理石紋評(píng)分；DL：滴水損失；WL：失水率；CL：烹飪損失；SF：剪切力.同列數(shù)據(jù)后未標(biāo)字母者表示在P<0.05水平差異無(wú)統(tǒng)計(jì)學(xué)意義.

IMF: Intramuscular fat; pH1: pH value at 45 min after slaughter; pHμ: pH value at 24 h after slaughter; MC: Meat color score; MB: Marbling score; DL: Drip loss; WL: Water loss; CL: Cooking loss; SF: Shear force. In the same column, the values without letters mean no significant difference at the 0.05 probability level.

2.3.2 polyA-L1位點(diǎn)對(duì)魯萊黑豬肉質(zhì)性狀的影響魯萊黑豬polyA-L1位點(diǎn)各基因型的肉質(zhì)性狀見(jiàn)表4.與萊蕪豬群體相同,魯萊黑豬群體polyA-L1位點(diǎn)處的3種基因型之間在9項(xiàng)肉質(zhì)指標(biāo)上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,但從表4中的數(shù)據(jù)可以看出,MN型個(gè)體的肌內(nèi)脂肪含量、肉色及大理石紋評(píng)分均高于MM型和NN型個(gè)體,同時(shí)烹飪損失最低,且滴水損失、失水率及剪切力值均較低.說(shuō)明MN型個(gè)體肌內(nèi)脂肪沉積能力較強(qiáng),保水性能較好,肉品較嫩,肉質(zhì)較好,這與在萊蕪豬群體中的結(jié)果一致.

表4 polyA-L1位點(diǎn)不同基因型魯萊黑豬的肉質(zhì)性狀a)

a)各肉質(zhì)性狀符號(hào)的含義詳見(jiàn)表3注.同列數(shù)據(jù)后未標(biāo)字母者表示在P<0.05水平差異無(wú)統(tǒng)計(jì)學(xué)意義.

a) Please see footnote of Table 3 for detail of each meat trait. In the same column, the values without letters mean no significant difference at the 0.05 probability level.

2.3.3 polyA-L2位點(diǎn)對(duì)萊蕪豬肉質(zhì)性狀的影響 萊蕪豬polyA-L2位點(diǎn)各基因型的肉質(zhì)性狀見(jiàn)表5.萊蕪豬群體polyA-L2位點(diǎn)處3種基因型之間在所檢測(cè)的9項(xiàng)肉質(zhì)性狀上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,這與萊蕪豬群體在polyA-L1多態(tài)位點(diǎn)處的結(jié)果相同.DD型個(gè)體的肌內(nèi)脂肪含量、pH1、pHμ、剪切力值最低,同時(shí)烹飪損失及滴水損失最高,且失水率也較高.說(shuō)明DD型個(gè)體肌內(nèi)脂肪沉積能力較弱,且保水性能較差.BB型個(gè)體肌內(nèi)脂肪含量最高,且失水率最低,但滴水損失及烹飪損失較高,剪切力值也較高.BD型個(gè)體大理石紋評(píng)分最高,滴水損失及烹飪損失最低,肌內(nèi)脂肪含量居中.

表5 polyA-L2位點(diǎn)不同基因型萊蕪豬的肉質(zhì)性狀a)

a)各肉質(zhì)性狀符號(hào)的含義詳見(jiàn)表3注.同列數(shù)據(jù)后未標(biāo)字母者表示在P<0.05水平差異無(wú)統(tǒng)計(jì)學(xué)意義.

a) Please see footnote of Table 3 for detail of each meat trait. In the same column, the values without letters mean no significant difference at the 0.05 probability level.

2.3.4 polyA-L2位點(diǎn)對(duì)魯萊黑豬肉質(zhì)性狀的影響魯萊黑豬polyA-L2位點(diǎn)各基因型的肉質(zhì)性狀見(jiàn)表6.與萊蕪豬群體內(nèi)polyA-L2位點(diǎn)處的結(jié)果不同的是,魯萊黑豬該位點(diǎn)處的3種基因型之間在失水率和烹飪損失指標(biāo)上表現(xiàn)為統(tǒng)計(jì)學(xué)上的顯著差異(P<0.05),且DD型個(gè)體的失水率顯著高于BD型個(gè)體,BB型和DD型個(gè)體的烹飪損失顯著高于BD型,其余各指標(biāo)的基因型間在統(tǒng)計(jì)學(xué)上差異不顯著.BD型個(gè)體的肌內(nèi)脂肪含量、pH1、pHμ、大理石紋評(píng)分高于其他2個(gè)純合型,且烹飪損失及失水率最低,同時(shí)剪切力值及滴水損失均較低.說(shuō)明該基因型個(gè)體肌內(nèi)脂肪沉積能力強(qiáng),保水性能較好,肉品細(xì)嫩,肉質(zhì)較好.BB型個(gè)體肌內(nèi)脂肪含量、滴水損失及大理石紋評(píng)分最低,烹飪損失及失水率較高,剪切力值偏高,保水性能較差,肉質(zhì)欠佳.

表6 polyA-L2位點(diǎn)不同基因型魯萊黑豬的肉質(zhì)性狀a)

a)各肉質(zhì)性狀符號(hào)的含義詳見(jiàn)表3注.同列數(shù)據(jù)后的不同小寫(xiě)字母表示在P<0.05水平差異有統(tǒng)計(jì)學(xué)意義,未標(biāo)字母者表示在P<0.05水平差異無(wú)統(tǒng)計(jì)學(xué)意義.

a) Please see footnote of Table 3 for detail of each meat trait. In the same column, the values followed by different lowercase letters are significantly different at the 0.05 probability level, and the values without letters mean no significant difference at the 0.05 probability level.

3 討論

隨著經(jīng)濟(jì)的迅速發(fā)展,人民生活水平不斷提高,人們對(duì)豬肉的需求已逐漸由數(shù)量向質(zhì)量轉(zhuǎn)變.萊蕪豬是我國(guó)優(yōu)良地方豬種,以其肉色鮮紅、肌內(nèi)脂肪含量高而著稱,但其生長(zhǎng)速度慢,年出欄生豬量有限.魯萊黑豬是以萊蕪豬為基礎(chǔ),通過(guò)導(dǎo)入國(guó)外瘦肉型大白豬的血液,經(jīng)過(guò)近10年6個(gè)世代的選擇培育而形成的新品種,是我國(guó)生產(chǎn)優(yōu)質(zhì)商品豬肉的又一優(yōu)良種質(zhì)資源,在很大程度上彌補(bǔ)了優(yōu)質(zhì)豬肉的市場(chǎng)需求.本實(shí)驗(yàn)在polyA-L1位點(diǎn)處,MN型魯萊黑豬的肌內(nèi)脂肪含量(6.00%)與NN型萊蕪豬的肌內(nèi)脂肪含量(7.21%)在統(tǒng)計(jì)學(xué)上差異不顯著(P>0.05),polyA-L2位點(diǎn)亦表現(xiàn)出此規(guī)律.說(shuō)明經(jīng)過(guò)多年的品種選育,魯萊黑豬的肌內(nèi)脂肪含量已比較接近萊蕪豬的肌內(nèi)脂肪水平.

肉質(zhì)性狀間不是獨(dú)立的,而是相互影響、相互聯(lián)系的,如肌束和肌纖維間沉積一定的脂肪,肌內(nèi)脂肪含量較高,肌肉斷面的大理石紋評(píng)分也較高[6].肉質(zhì)性狀間的相關(guān)性普遍存在且多數(shù)能達(dá)到0.05的顯著水平[13],因此可通過(guò)對(duì)少數(shù)肉質(zhì)性狀的選擇實(shí)現(xiàn)對(duì)其他性狀的間接選擇.本實(shí)驗(yàn)魯萊黑豬在2個(gè)位點(diǎn)處的大理石紋評(píng)分及肌內(nèi)脂肪含量均表現(xiàn)為MM型

在基因組中各種多態(tài)形式廣泛存在,包括單核苷酸多態(tài)性(single nucleotide polymorphism, SNP)、重復(fù)序列多態(tài)性、插入或缺失片段(1～nbp)等.姜潤(rùn)深[14]在研究家禽的就巢行為時(shí)發(fā)現(xiàn)催乳素(prolactin,PRL)基因調(diào)控區(qū)PRLpro2位點(diǎn)存在24 bp的插入/缺失突變且缺失型等位基因與就巢性狀顯著相關(guān).本實(shí)驗(yàn)P311基因參考序列的2個(gè)polyA位點(diǎn)處的連續(xù)腺苷酸數(shù)目分別為14和11,而測(cè)序所得的2個(gè)位點(diǎn)處的連續(xù)腺苷酸數(shù)目分別為18、15和15、12,說(shuō)明2個(gè)位點(diǎn)處均檢測(cè)到4 bp和1 bp的插入/缺失突變,而正是這一突變,導(dǎo)致了該基因3′側(cè)翼區(qū)polyA結(jié)構(gòu)的多態(tài)性.基因的5′及3′側(cè)翼區(qū)多態(tài)性與生產(chǎn)性狀的關(guān)系已有很多報(bào)道,尤以5′側(cè)翼區(qū)的研究報(bào)道較多,但兩者的顯著性關(guān)系在不同物種、不同基因上的表現(xiàn)不一[5,15-20].本實(shí)驗(yàn)所檢測(cè)的2個(gè)側(cè)翼區(qū)多態(tài)位點(diǎn),魯萊黑豬BB型、DD型和BD型之間僅失水率和烹飪損失2個(gè)指標(biāo)具有統(tǒng)計(jì)學(xué)上的顯著差異,其他指標(biāo)在統(tǒng)計(jì)學(xué)上均不顯著,萊蕪豬2個(gè)位點(diǎn)的3種基因型之間在肉質(zhì)性狀上均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異.因此，利用基因側(cè)翼區(qū)尋找用于分子育種的有效分子標(biāo)記,還需要進(jìn)一步的科學(xué)研究.

4 結(jié)論

本實(shí)驗(yàn)在萊蕪豬和魯萊黑豬P311基因3′側(cè)翼區(qū)的2個(gè)polyA位點(diǎn)處均檢測(cè)到3種多態(tài)類型,即2種純合型和1種雜合型,在這2個(gè)位點(diǎn)處萊蕪豬和魯萊黑豬的多態(tài)分布并未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異.在polyA-L1位點(diǎn)處3種基因型的肉質(zhì)性狀在2個(gè)豬種內(nèi)均未表現(xiàn)出統(tǒng)計(jì)學(xué)上的顯著差異,但均以MN型個(gè)體肌內(nèi)脂肪含量及大理石紋評(píng)分最高,烹飪損失最低且剪切力值也較低.在polyA-L2位點(diǎn)處3種基因型的肉質(zhì)性狀僅魯萊黑豬的失水率及烹飪損失2項(xiàng)指標(biāo)具有統(tǒng)計(jì)學(xué)上的顯著差異,但依然表現(xiàn)為雜合型個(gè)體的烹飪損失、滴水損失、大理石紋、肌內(nèi)脂肪含量等指標(biāo)優(yōu)于2個(gè)純合型.以上結(jié)果說(shuō)明雜合型個(gè)體肌內(nèi)脂肪沉積能力較強(qiáng),肌肉保水性能較好,生產(chǎn)中應(yīng)重視2個(gè)純合型個(gè)體間的雜交利用,以產(chǎn)生更多肉質(zhì)優(yōu)良的雜合型個(gè)體.

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PolyA polymorphisms in 3′ flanking region of pig neuronal protein 3.1 gene and their relationships with meat traits. Journal of Zhejiang University (Agric. & Life Sci.), 2015,41(2):237-244

Yang Yun, Zeng Yongqing*, Zhang Zhe, Xu Zhenggang, Chen Wei, Fang Guofeng, Wang Shoudong

(LaboratoryofAnimalGeneticsandBreeding,CollegeofAnimalScienceandTechnology,ShandongAgriculturalUniversity,Tai’an271018,Shandong,China)

pig; neuronal protein 3.1 gene; 3′ flanking region; polymorphisms; meat traits

國(guó)家轉(zhuǎn)基因重大專項(xiàng)(2011ZX08006-002；2013ZX08006-002)；國(guó)家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃)重點(diǎn)項(xiàng)目(2008AA101008)；山東省農(nóng)業(yè)良種工程重大項(xiàng)目(2013LZ02-015)；山東省現(xiàn)代農(nóng)業(yè)(生豬)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)(SDAIT-06-022-02).

聯(lián)系方式：楊云,E-mail:pipiyade1988@163.com

2014-04-06；接受日期(Accepted)：2014-06-19；網(wǎng)絡(luò)出版日期(Published online)：2014-12-05

S 828.8

A

*通信作者(Corresponding author)：曾勇慶,Tel：+86-538-8249222-8311；E-mail:yqzeng@sdau.edu.cn

URL:http://www.cnki.net/kcms/detail/33.1247.S.20141205.1444.001.html