猪神经元蛋白3.1基因3′侧翼区多聚腺苷酸数目多态性及其与肉质性状的关系

杨云, 曾勇庆, 张哲, 徐正刚, 陈伟, 房国锋, 王守栋

(山东农业大学动物科技学院,动物遗传育种学实验室,山东 泰安 271018)

猪神经元蛋白3.1基因3′侧翼区多聚腺苷酸数目多态性及其与肉质性状的关系

杨云, 曾勇庆*, 张哲, 徐正刚, 陈伟, 房国锋, 王守栋

(山东农业大学动物科技学院,动物遗传育种学实验室,山东 泰安 271018)

选取54头莱芜猪和40头鲁莱黑猪,宰后测定肉质性状,利用聚合酶链反应及单链构象多态性(polymerase chain reaction-single strand conformation polymorphism, PCR-SSCP)技术检测其神经元蛋白3.1(neuronal protein 3.1, P311)基因3′侧翼区2个多聚腺苷酸(polyA)结构的长度多态性,测序确定连续腺苷酸的数目,并与肉质性状进行关联分析，以期发现有效多态位点.结果表明：1)在莱芜猪和鲁莱黑猪中共检测出2个连续腺苷酸数目的多态位点polyA-L1和polyA-L2,两者的polyA数目分别为18、15和15、12,2个位点的3种基因型分别记为polyA-L1位点的MM型、NN型和MN型,以及polyA-L2位点的BB型、DD型和BD型；这2个多态位点在莱芜猪和鲁莱黑猪群体中均处于哈代-温伯格平衡(P>0.05),其多态性在2个猪种间的分布差异在统计学上均不显著.2)莱芜猪polyA-L1位点的3种基因型之间在肉质性状上均未表现出统计学上的显著差异,但MN型个体各项肉质性状普遍优于2个纯合型,肉质较好,鲁莱黑猪中的结果与此相同.3)莱芜猪polyA-L2位点的3种基因型之间在肉质性状上均未表现出统计学上的显著差异,但鲁莱黑猪在该位点处的失水率和烹饪损失指标在统计学上差异显著,其中DD型个体的失水率显著高于BD型个体,BB型和DD型个体的烹饪损失显著高于BD型,其余各指标的基因型间在统计学上差异不显著.总之,在这2个位点处普遍表现为杂合型的肉质性状优于纯合型,生产中应重视2个纯合型个体间的杂交利用,以产生更多肉质优良的杂合型个体.

猪； 神经元蛋白3.1基因； 3′侧翼区； 多态性； 肉质性状

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.

神经元蛋白3.1(neuronal protein 3.1, P311)又称PTZ17,由Studler等[1]于1993年首次发现于小鼠胚胎的脑组织中.P311蛋白由定位于猪2号染色体的P311基因负责编码,现已研究证实其广泛存在于神经及肌肉组织中,且是一种细胞内蛋白质,主要存在于胞质中,在细胞核内亦有少量定位,参与肌成纤维细胞的分化及肺泡发育,与脂质合成与蓄积密切相关[2-4].P311基因全长26 252 bp,其mRNA长度为2 410 nt.P311基因mRNA序列中包含3个开放阅读框,但仅第1个开放阅读框负责编码蛋白质.Leung等[3]研究发现,P311基因能够通过上调与脂质合成相关的一系列基因的表达水平使得细胞内甘油三酯和胆固醇的水平显著提高,从而导致细胞内脂肪滴数目增多.本实验室前期通过抑制性消减杂交发现,P311基因是重要的差异表达基因,在莱芜猪中的表达量显著高于大白猪.该基因的3′侧翼区不编码蛋白质,不影响该基因所编码蛋白质的结构和功能,但其可通过调控该基因mRNA的降解速率和翻译速率等控制基因表达,进而影响性状表现[5].本研究旨在鉴定莱芜猪及鲁莱黑猪P311基因3′侧翼区的多聚腺苷酸(polyA)的结构多态性,并与其肉质性状进行关联分析,以期发现有效多态位点,为优质肉猪的培育提供科学依据.

1 材料与方法

1.1 实验猪及样品采集

实验猪均来自山东省莱芜市种猪繁育场,包括54头莱芜猪和40头鲁莱黑猪.在相同的饲养管理条件下分别饲喂至相应屠宰体质量,即莱芜猪(85.80±13.90) kg,鲁莱黑猪(93.80±10.12) kg,放血屠宰后立即剪取耳组织样置于装有75%乙醇的Eppendorf离心管中,并采集第1～5腰椎处背最长肌置于放有冰袋的保温箱中带回实验室,分别用于基因组提取及肉质测定.

1.2 实验方法

1.2.1 肉质测定[6-11]测定宰后45 min的 pH值(pH1)、宰后24 h的pH值(pHμ)、肌内脂肪含量、大理石纹、肉色、滴水损失、失水率、烹饪损失、嫩度9项指标.其中,宰后45 min直接用pH计测定pH1,肉样在0～4 ℃保存24 h后测定pHμ；肌内脂肪含量采用索氏醚浸提法测定；肉色与大理石纹对照美制的NPPC比色板(5级分制)进行分级评定；滴水损失采用吊挂处理法测定；失水率采用压力法,利用钢环式膨胀压缩仪测定；烹饪损失采用蒸煮法测定；嫩度采用剪切力测定法,利用C-LM3型数显式肌肉嫩度仪测定肌肉剪切力值,值越大则肉越粗老.

1.2.2 基因组提取 利用血液/细胞/组织基因组DNA提取试剂盒(购自天根生化科技有限公司)进行基因组提取,用1%琼脂糖凝胶电泳和核酸蛋白测定仪检测所提取DNA的浓度及纯度；将基因组稀释至100 ng/μL后于-20 ℃保存、备用.

1.2.3 样品的聚合酶链反应(polymerase chain reaction, PCR)扩增 以猪P311 DNA(NCBI登录号为NC_010444.3)序列为模板设计2对引物：polyA 1F:CCAAAAACTGACGTCTCC,polyA 1R:ATCCATTTCATTGGGAGC,polyA 2F:TTTGACAGGCGTTTTTAGGTG,polyA 2R:ACGGAGACGTCAGTTTTTGG,扩增长度分别为181 bp和155 bp,扩增位置分别为该基因的19 110～19 290 bp处和18 980～19 134 bp处.PCR扩增体系：2×PCR Master Mix(购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,加水补足至25 μL.扩增反应条件：95 ℃预变性5 min,95 ℃变性30 s,退火30 s,72 ℃延伸20 s,35个循环；最后,72 ℃延伸10 min.2对引物的退火温度分别为56.4 ℃和59.5 ℃.扩增后的产物置于4 ℃保存、备用.

1.2.4 单链构象多态性(single strand conformation polymorphism, SSCP)检测[12]向1.2.3节的扩增产物中加入8.4 μL变性缓冲液(95%甲酰胺,10 mmol/L EDTA,0.09%二甲苯青,0.09%溴酚蓝),混匀后置于PCR仪中95 ℃变性6 min.将变性产物迅速置于冰上冷却,点样于12%非变性聚丙烯酰胺凝胶中,于120 V电压、4 ℃条件下电泳13 h后银染显色并统计结果.对不同基因型个体进行抽样测序.

1.3 数据分析

多态性数据利用R软件进行统计分析；肉质测定数据利用SPSS 13.0软件进行统计分析,以平均值±标准差表示,显著性检验采用SNK(Student-Newman-Keuls)和最小显著差异(least significant difference, LSD)法.

2 结果与分析

2.1 PCR-SSCP分析

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

图1为引物对polyA 1F/1R和polyA 2F/2R的PCR-SSCP电泳图.从中可以看出,2对引物的SSCP检测结果均呈现3种带型,且带型清晰,易于分型.2个位点处的3种基因型分别记为polyA-L1位点的MM型、MN型和NN型,以及polyA-L2位点的BB型、BD型和DD型.

表1和表2分别为polyA-L1和polyA-L2位点的基因型频率、基因频率、杂合度、多态信息含量等.从中可以看出：莱芜猪(LW)与鲁莱黑猪(LL)在polyA-L1位点处的优势等位基因均为M基因；而在polyA-L2位点处莱芜猪(LW)的优势等位基因为B基因,鲁莱黑猪(LL)的2个等位基因频率相等.2个猪种在polyA-L1位点处的基因型频率为MM>MN>NN；莱芜猪在polyA-L2位点处的基因型频率为BD>BB>DD,鲁莱黑猪在该位点处的基因型频率为BD>BB=DD.2个多态位点在莱芜猪与鲁莱黑猪中均为中度多态,且polyA-L2位点在2个猪种中的杂合度均高于polyA-L1位点.卡方适合性检验表明,2个多态位点在莱芜猪和鲁莱黑猪群体中均处于哈代-温伯格平衡(P>0.05).显著性检验表明,2个位点的多态性在2个猪种间的分布差异在统计学上均不显著(P>0.05).为进一步确定各基因型个体的碱基类型,对4种纯合型个体的扩增产物进行回收测序.

表1 2个猪种polyA-L1位点的遗传组成信息

LW：莱芜猪；LL：鲁莱黑猪；PIC：多态信息含量.括号内的数据为该品种的该基因型个体数.

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个猪种polyA-L2位点的遗传组成信息

LW：莱芜猪；LL：鲁莱黑猪；PIC：多态信息含量.括号内的数据为该品种的该基因型个体数.

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 各基因型碱基类型的确定

图2为2个位点纯合型个体的测序峰图.对于位点polyA-L1,MM型个体的polyA数目为18(图2A),NN型个体的polyA数目为15(图2B),MN型个体同时存在上述2种polyA类型；对于位点polyA-L2,BB型和DD型个体的polyA数目分别为15(图2C)和12(图2D),BD型个体同时存在15和12这2种polyA类型.

2.3 polyA-L1和polyA-L2位点对肉质性状的影响

2.3.1 polyA-L1位点对莱芜猪肉质性状的影响 莱芜猪polyA-L1位点各基因型的肉质性状见表3.莱芜猪polyA-L1位点处的3种基因型之间在所检测的9项肉质性状上均未表现出统计学上的显著差异,但由表3中的数据可以看出,MN型个体的肌内脂肪含量、pH1、大理石纹评分均高于MM型和NN型个体,同时该基因型的滴水损失及烹饪损失均最低,剪切力值也较低.说明MN型个体肌内脂肪沉积能力较强,保水性能较好,肉品细嫩,肉质较好.NN型个体的肌内脂肪含量、失水率及剪切力值最低,烹饪损失最高；而MM型个体的大理石纹评分最低,滴水损失最高.

(A,B)：分别为polyA-L1位点MM型和NN型个体测序峰图；(C，D)：分别为polyA-L2位点BB型和DD型个体测序峰图. (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位点不同基因型莱芜猪的肉质性状

IMF：肌内脂肪；pH1：宰后45 min的pH值；pHμ：宰后24 h的pH值；MC：肉色评分；MB：大理石纹评分；DL：滴水损失；WL：失水率；CL：烹饪损失；SF：剪切力.同列数据后未标字母者表示在P<0.05水平差异无统计学意义.

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位点对鲁莱黑猪肉质性状的影响鲁莱黑猪polyA-L1位点各基因型的肉质性状见表4.与莱芜猪群体相同,鲁莱黑猪群体polyA-L1位点处的3种基因型之间在9项肉质指标上均未表现出统计学上的显著差异,但从表4中的数据可以看出,MN型个体的肌内脂肪含量、肉色及大理石纹评分均高于MM型和NN型个体,同时烹饪损失最低,且滴水损失、失水率及剪切力值均较低.说明MN型个体肌内脂肪沉积能力较强,保水性能较好,肉品较嫩,肉质较好,这与在莱芜猪群体中的结果一致.

表4 polyA-L1位点不同基因型鲁莱黑猪的肉质性状a)

a)各肉质性状符号的含义详见表3注.同列数据后未标字母者表示在P<0.05水平差异无统计学意义.

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位点对莱芜猪肉质性状的影响 莱芜猪polyA-L2位点各基因型的肉质性状见表5.莱芜猪群体polyA-L2位点处3种基因型之间在所检测的9项肉质性状上均未表现出统计学上的显著差异,这与莱芜猪群体在polyA-L1多态位点处的结果相同.DD型个体的肌内脂肪含量、pH1、pHμ、剪切力值最低,同时烹饪损失及滴水损失最高,且失水率也较高.说明DD型个体肌内脂肪沉积能力较弱,且保水性能较差.BB型个体肌内脂肪含量最高,且失水率最低,但滴水损失及烹饪损失较高,剪切力值也较高.BD型个体大理石纹评分最高,滴水损失及烹饪损失最低,肌内脂肪含量居中.

表5 polyA-L2位点不同基因型莱芜猪的肉质性状a)

a)各肉质性状符号的含义详见表3注.同列数据后未标字母者表示在P<0.05水平差异无统计学意义.

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位点对鲁莱黑猪肉质性状的影响鲁莱黑猪polyA-L2位点各基因型的肉质性状见表6.与莱芜猪群体内polyA-L2位点处的结果不同的是,鲁莱黑猪该位点处的3种基因型之间在失水率和烹饪损失指标上表现为统计学上的显著差异(P<0.05),且DD型个体的失水率显著高于BD型个体,BB型和DD型个体的烹饪损失显著高于BD型,其余各指标的基因型间在统计学上差异不显著.BD型个体的肌内脂肪含量、pH1、pHμ、大理石纹评分高于其他2个纯合型,且烹饪损失及失水率最低,同时剪切力值及滴水损失均较低.说明该基因型个体肌内脂肪沉积能力强,保水性能较好,肉品细嫩,肉质较好.BB型个体肌内脂肪含量、滴水损失及大理石纹评分最低,烹饪损失及失水率较高,剪切力值偏高,保水性能较差,肉质欠佳.

表6 polyA-L2位点不同基因型鲁莱黑猪的肉质性状a)

a)各肉质性状符号的含义详见表3注.同列数据后的不同小写字母表示在P<0.05水平差异有统计学意义,未标字母者表示在P<0.05水平差异无统计学意义.

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 讨论

随着经济的迅速发展,人民生活水平不断提高,人们对猪肉的需求已逐渐由数量向质量转变.莱芜猪是我国优良地方猪种,以其肉色鲜红、肌内脂肪含量高而著称,但其生长速度慢,年出栏生猪量有限.鲁莱黑猪是以莱芜猪为基础,通过导入国外瘦肉型大白猪的血液,经过近10年6个世代的选择培育而形成的新品种,是我国生产优质商品猪肉的又一优良种质资源,在很大程度上弥补了优质猪肉的市场需求.本实验在polyA-L1位点处,MN型鲁莱黑猪的肌内脂肪含量(6.00%)与NN型莱芜猪的肌内脂肪含量(7.21%)在统计学上差异不显著(P>0.05),polyA-L2位点亦表现出此规律.说明经过多年的品种选育,鲁莱黑猪的肌内脂肪含量已比较接近莱芜猪的肌内脂肪水平.

肉质性状间不是独立的,而是相互影响、相互联系的,如肌束和肌纤维间沉积一定的脂肪,肌内脂肪含量较高,肌肉断面的大理石纹评分也较高[6].肉质性状间的相关性普遍存在且多数能达到0.05的显著水平[13],因此可通过对少数肉质性状的选择实现对其他性状的间接选择.本实验鲁莱黑猪在2个位点处的大理石纹评分及肌内脂肪含量均表现为MM型

在基因组中各种多态形式广泛存在,包括单核苷酸多态性(single nucleotide polymorphism, SNP)、重复序列多态性、插入或缺失片段(1～nbp)等.姜润深[14]在研究家禽的就巢行为时发现催乳素(prolactin,PRL)基因调控区PRLpro2位点存在24 bp的插入/缺失突变且缺失型等位基因与就巢性状显著相关.本实验P311基因参考序列的2个polyA位点处的连续腺苷酸数目分别为14和11,而测序所得的2个位点处的连续腺苷酸数目分别为18、15和15、12,说明2个位点处均检测到4 bp和1 bp的插入/缺失突变,而正是这一突变,导致了该基因3′侧翼区polyA结构的多态性.基因的5′及3′侧翼区多态性与生产性状的关系已有很多报道,尤以5′侧翼区的研究报道较多,但两者的显著性关系在不同物种、不同基因上的表现不一[5,15-20].本实验所检测的2个侧翼区多态位点,鲁莱黑猪BB型、DD型和BD型之间仅失水率和烹饪损失2个指标具有统计学上的显著差异,其他指标在统计学上均不显著,莱芜猪2个位点的3种基因型之间在肉质性状上均未表现出统计学上的显著差异.因此，利用基因侧翼区寻找用于分子育种的有效分子标记,还需要进一步的科学研究.

4 结论

本实验在莱芜猪和鲁莱黑猪P311基因3′侧翼区的2个polyA位点处均检测到3种多态类型,即2种纯合型和1种杂合型,在这2个位点处莱芜猪和鲁莱黑猪的多态分布并未表现出统计学上的显著差异.在polyA-L1位点处3种基因型的肉质性状在2个猪种内均未表现出统计学上的显著差异,但均以MN型个体肌内脂肪含量及大理石纹评分最高,烹饪损失最低且剪切力值也较低.在polyA-L2位点处3种基因型的肉质性状仅鲁莱黑猪的失水率及烹饪损失2项指标具有统计学上的显著差异,但依然表现为杂合型个体的烹饪损失、滴水损失、大理石纹、肌内脂肪含量等指标优于2个纯合型.以上结果说明杂合型个体肌内脂肪沉积能力较强,肌肉保水性能较好,生产中应重视2个纯合型个体间的杂交利用,以产生更多肉质优良的杂合型个体.

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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

国家转基因重大专项(2011ZX08006-002；2013ZX08006-002)；国家高技术研究发展计划(863计划)重点项目(2008AA101008)；山东省农业良种工程重大项目(2013LZ02-015)；山东省现代农业(生猪)产业技术体系建设专项(SDAIT-06-022-02).

联系方式：杨云,E-mail:pipiyade1988@163.com

2014-04-06；接受日期(Accepted)：2014-06-19；网络出版日期(Published online)：2014-12-05

S 828.8

A

*通信作者(Corresponding author)：曾勇庆,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