Cloning of porcine LPAR3 gene and its expression in endometrial cells
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摘要:目的
获得猪LPAR3基因完整mRNA及基因结构，研究其启动子活性；探究LPAR3基因在子宫内膜的转录调控及可能影响母猪产仔的机制。
方法应用5'RACE和3'RACE技术获取LPAR3基因完整mRNA序列；预测5'调控区潜在的启动子转录因子结合位点及CpG岛，构建不同长度的启动子双荧光素酶报告基因重组载体，与pRL-TK质粒共同转染至猪子宫内膜细胞，检测启动子活性；应用RT-qPCR比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的相对表达量；应用亚硫酸氢钠修饰后测序比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的甲基化状态。
结果猪LPAR3 mRNA全长为2 127 bp，其中5'UTR和3'UTR的长度分别为202和860 bp，CDS区为1 065 bp。克隆获得包括LPAR3转录起始位点上游3 080 bp (–2 430/+650 bp)的5'调控序列，分析预测显示该调控区不存在TATA box，存在GC元件、CPBP及糖皮质激素受体IR3等调控因子结合位点，且在–190/–84和–44/+651 bp处存在2个潜在CpG岛。成功构建9个不同长度的5'缺失报告重组载体并转染猪子宫内膜细胞。双荧光素酶活性检测结果显示，启动子P4(+454/+80 bp)的转录活性最高，其次是P6(–123/+80 bp)。RT-qPCR结果显示，妊娠第12天二花脸猪LPAR3基因在子宫内膜的表达量高于在其他组织的表达量，且极显著高于在妊娠第12天长大二元猪子宫内膜的表达量，LPAR3在2个猪种子宫内膜均处于低甲基化状态且差异不显著。
结论猪LPAR3mRNA全长为2 127 bp，妊娠第12天LAPR3基因在二花脸猪子宫内膜表达高于其在长大二元猪子宫内膜的表达，显示LPAR3可能参与了猪早期妊娠并影响产仔数。
Abstract:ObjectiveTo obtain the complete mRNA sequence and structure of porcine LPAR3gene, study the gene promoter activity, and explore the transcriptional regulation of LPAR3 gene in endometrium and the mechanisms which may affect sow farrowing.
MethodThe 5'RACE and 3'RACE techniques were used to obtain the complete LPAR3mRNA sequence. The potential promoter transcription factor binding sites and CpG islands in the 5' regulatory region were predicted. Recombinant vectors with different length of promoters and dual-luciferase reporter gene were constructed, then were transfected with pRL-TK plasmid into pig endometrial cells, and the promoter activities were detected. RT-qPCR was used to compare the relative expression of LPAR3 gene in Erhualian(ER) and Landrace×Large White (LL) pigs on the 12th day of gestation. Sodium bisulfite modified sequencing was used to compare the methylation status of LPAR3 gene in endometrial tissues of ER and LL pigs on the 12th day of gestation.
ResultThe full length of pigLPAR3 mRNA was 2 127 bp, the 5'UTR and 3'UTR were 202 and 860 bp respectively, and the CDS region was 1 065 bp. The 5' regulatory sequence, including 3 080 bp (–2 430/+650 bp) upstream of the LPAR3gene, was cloned. The online prediction results of the 5' regulatory region showed that there was no TATA box in the LPAR3promoter, but there were GC element and binding sites for CPBP and other regulatory factors. Two potential CpG islands were found at –190/–84 and –44/+651 bp. Nine recombinant vectors with 5' deletions of the promoter were constructed and transfected into pig endometrium cells. The dual-luciferase assay showed that the activity of promoter P4 (+454/+80 bp) was the highest, followed by P6 (–123/+80 bp). RT-qPCR results showed that the expression of LPAR3 gene in the endometrium of ER pigs on the 12th day of gestation was higher than that in other tissues, and it was extremely significantly higher than that in the endometrium of LL pigs on the 12th day of gestation. The methylation status of LPAR3 gene was low in the endometrium of two breeds and had no significant difference between two breeds.
ConclusionThe full length of pigLPAR3mRNA is 2 127 bp. The endometrial expression level of LAPR3 gene in ER pigs is higher than that in LL pigs on the 12th day of gestation, indicating that LPAR3 gene may be involved in early gestation of pigs and affects litter size.
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Keywords:
- pig /
- LPAR3 gene /
- promoter /
- endometrium /
- gestation /
- methylation
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母猪适宜的体况是保证其繁殖性能的关键，妊娠期是恢复体储的过程，泌乳期为保证泌乳量则是损失体储的过程。研究表明泌乳期体况损失过多会对下一胎次产生不利影响，当母猪采食量满足不了泌乳营养需要时, 母猪就会分解体储以满足泌乳需求，导致母猪背膘损失和体重下降，从而降低其下一胎次的繁殖性能[1]。这往往也是产生“二胎综合征”的原因之一。为研究体况损失对二胎母猪繁殖性能的影响，本研究通过测量一胎母猪的临产背膘厚及其下一胎次的开配背膘厚以计算一胎转二胎的背膘损失，来评估背膘损失对繁殖性能的影响，以期为生产应用提供参考。
1. 材料与方法
1.1 试验材料
本试验在温氏种猪公司下属的核心育种场开展，试验母猪为大白原种猪，试验从2017年7月开始，到2018年5月结束，全程测量一胎母猪的临产背膘厚及其下一胎次的开配背膘厚。
1.2 试验方法
使用超声波背膘仪(美国Renco)测量一胎母猪妊娠第110天及其下一胎次开配的P2点(最后肋骨上距背中线约6.5 cm处)背膘厚，计算母猪一胎转二胎的背膘损失，将背膘损失分为6个组，分别为掉膘≥5 mm，掉膘3~4 mm，掉膘2 mm，掉膘1 mm，不变和增膘。
1.3 数据处理
母猪分娩后收集相应的繁殖数据，包括总产仔和健仔数(初生重≥0.8 kg)，并计算无效仔率，记录初生窝重并计算初生均重。
无效仔率=(总仔数-健仔数)/总仔数×100%。
使用Excel 2013进行数据的初步整理，采用SAS 9.2统计软件对试验数据进行单因素方差分析，并用Duncan's法进行多重比较，由于母猪损耗等原因，共有666头母猪的完整数据进入后续的分析。
2. 结果与分析
2.1 母猪一胎转二胎背膘损失情况
一胎母猪临产背膘厚越高，其在产房损失的背膘就会越多，当一胎猪临产背膘厚超过16 mm时，其在产房就会出现掉膘现象，临产背膘厚达到19 mm时，背膘损失高达5 mm以上；反之，一胎母猪临产背膘越薄，其在产房往往会增膘，详情见表 1。
表 1 母猪一胎转二胎背膘损失情况mm 背膘损失/mm 数量 1胎临产
背膘厚2胎开配
背膘厚背膘厚变化 ≥5 32 19.03 13.78 -5.25 3~4 171 17.54 14.16 -3.38 2 141 16.55 14.55 -2.00 1 126 16.11 15.11 -1.00 0 105 15.97 15.97 0.00 ＜0 91 16.00 17.58 1.58 合计 666 16.67 15.16 -1.52 2.2 一胎转二胎背膘损失对母猪繁殖性能的影响
一胎转二胎背膘损失对二胎总产仔数有显著的影响，掉膘≥5和掉膘3~4 mm组总产仔数显著少于掉膘1 mm、不变和增膘组，掉膘2 mm组与其他各组差异均不显著。掉膘≥5 mm组健仔数显著少于掉膘1 mm、不变和增膘组，掉膘3~4 mm和掉膘2 mm组健仔与其他各组差异均不显著。掉膘≥5 mm组的无效仔率显著高于其他组，除掉膘≥5 mm组外，其他各组无效仔率间无显著差异。掉膘1 mm和不变组窝重显著高于掉膘3~4 mm和增膘组的，掉膘≥5 mm和掉膘2 mm组和其他各组窝重无显著差异。掉膘≥5 mm组仔猪均重显著高于掉膘3~4 mm、掉膘1 mm、不变和增膘组，掉膘2 mm组仔猪均重显著高于不变组，具体数据见表 2。
表 2 一胎转二胎背膘损失对二胎繁殖性能的影响1)背膘损失/mm 母猪/头 总仔/头 健仔/头 无效仔率/% 初生窝重/kg 初生均重/kg ≥5 32 14.59b 11.56b 21.04a 20.62ab 1.53a 3~4 171 14.89b 12.38ab 15.87b 19.11b 1.43bc 2 141 15.21ab 12.37ab 18.07ab 19.97ab 1.47ab 1 126 16.05a 12.94a 17.91ab 20.20a 1.43bc 0 105 15.70a 12.57a 18.59ab 20.95a 1.39c ＜0 91 15.97a 12.86a 18.72ab 18.92b 1.41bc 平均标准误 0.39 0.30 0.01 0.46 0.03 P 0.03 0.11 0.15 0.01 0.07 1)同列数据后凡具有一个相同小写字母者表示差异不显著(P>0.05，Duncan's法) 2.3 一胎猪临产背膘厚和背膘损失的线性回归分析
本试验数据显示，大白高产母猪在泌乳期摄入的营养物质满足不了泌乳需求，会通过损失体况来满足泌乳需要，平均背膘损失高达1.52 mm。对临产背膘(x)和背膘损失(y)进行一元线性回归分析，回归方程为y=-0.468 7x+6.279 6(图 1)。回归方程拟合度较好(R2=0.944 9)，该方程显示一胎母猪临产背膘厚与背膘损失呈高度负相关，当背膘损失为2 mm时，临产背膘厚为17.67 mm，为了避免因产房背膘损失过大而带来的不良后果，一胎母猪的临产背膘厚不宜超过18 mm。
3. 结论与讨论
经过多年的选育，大白母猪的繁殖性能已得到了极大的提高，窝产总仔和健仔数显著增多，但在母猪育种过程中，母猪泌乳期采食量并没有得到相应提高，为了满足仔猪生长发育的需要，母猪需要更多的营养物质来维持泌乳，泌乳期往往会造成体况的损失。体况包含体蛋白和体脂肪，由于体蛋白不易测量，所以通常用背膘(体脂肪)来代表体况，背膘反映的是母猪在不同生理阶段的体况和能量储备情况，母猪背膘变化与繁殖性能密切相关。研究表明母猪泌乳期体重损失很小时，不会影响母猪下一胎次的繁殖性能，但当母猪体重损失超过其体重的10%时，将会延长母猪下一胎次断奶到发情的时间间隔、阻碍卵母细胞发育和降低排卵率[2-3]。
本研究表明，一胎转二胎背膘损失对二胎母猪总产仔数有显著的影响，掉膘≥5 mm和掉膘3~4 mm组的总产仔数少于15头且显著低于掉膘1 mm、不变和增膘组，掉膘2 mm组总产仔数虽与其他各组差异不显著，但掉膘2 mm组与掉膘1 mm相比总产仔数有减少的趋势。一胎母猪在产房有一定的背膘损失是正常而且可以接受的，当背膘损失超过2 mm时，意味着在二胎开配前的母猪体况储备不足，会导致母猪排卵数减少，从而降低二胎的总产仔数。从健仔数来看，虽然掉膘3~4 mm组的总产仔数较少，但其无效仔率是最少的，说明其健仔数保持在较高的水平，可能是因为其弱仔死胎数比较少，具体原因还有待进一步的研究。初生窝重和均重的差异主要是由于总产仔数的差异导致的，掉膘1 mm、不变和增膘组仔猪重均无显著差异。
线性回归分析结果表明，为了避免因产房背膘损失过大而带来的不良后果，一胎母猪的临产背膘厚不宜超过18 mm。这提示在生产上，减少泌乳母猪体重和背膘损失是饲养母猪的重要目标，一胎母猪在妊娠期背膘增长不能过多，临产背膘厚不宜超过18 mm，同时在泌乳期要提高母猪的采食量以减少背膘损失，确保背膘损失不超过2 mm，以保证下一胎次的繁殖性能。
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图 1 猪LPAR3完整mRNA序列的RACE结果及5'调控序列扩增结果
M1为DL2000 marker，从上到下依次为：2 000、1 000、750、500、250和100 bp，M2为DL5000 marker，从上到下依次为5 000、3 000、2 000、1 500、1 000、750、500、250、100 bp；1：LPAR3基因5'UTR扩增条带；2和3分别为5'RACE Outer和Inner引物扩增特异性条带；4和5分别为3'RACE Inner和Outer引物扩增特异性条带；6:LPAR3基因5'调控区的扩增条带
Figure 1. RACE results of the complete mRNA sequence of pig LPAR3 gene and amplification results of the 5' regulatory sequence
M1: DL2000 marker，from up to bottom: 2 000, 1 000, 750, 500, 250, and 100 bp; M2: DL5000 marker，from up to bottom: 5 000, 3 000, 2 000, 1 500, 1 000, 750, 500, 250 and 100 bp; 1: Products of LPAR3 5'UTR amplification; 2 and 3: The specific bands of 5'RACE with Outer and Inner primers; 4 and 5: The specific bands of 3'RACE with Outer and Inner primers; 6: Products of LPAR3 5' regulatory sequence
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图 2 猪LPAR3基因5'调控区重要调控元件与转录因子结合位点预测结果
Figure 2. Predicted results of important regulatory elements and binding sites of transcription factors in the 5' regulatory region of pig LPAR3 gene
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图 3 猪LPAR3基因启动子各缺失片段扩增(A)及双酶切鉴定(B)
M:DL5000 marker；P1~P9片段大小分别约为1 958、1 435、1 047、534、351、203、96、218和921 bp
Figure 3. Amplification of the deletion fragments of pig LPAR3 gene promoter (A) and double enzyme digestion (B)
M: DL5000 marker; The lengths of P1-P9 were 1 958, 1 435, 1 047, 534, 351, 203, 96, 218 and 921 bp, respectively
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图 4 猪LPAR3基因各启动子缺失片段转录活性
各图中，柱子上的不同小写字母表示差异显著(P<0.05, Duncan’s 法)
Figure 4. Luciferase activities of LPAR3 promoter deletion fragments
In each gragh, different lowercase letters on the bars indicate significant difference(P<0.05, Duncan’s test)
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图 5 猪LPAR3 mRNA的组织表达谱
A图中，横坐标上的数字1~13分别表示心、肝、脾、肺、肾、大脑、小脑、肌肉、垂体、下丘脑、小肠、胃和子宫内膜；B图中，“**”表示差异显著(P<0.01，t 检验)
Figure 5. Gene expression profile of pig LPAR3 mRNA
In graph A, the numbers of 1~13 along the abscissa axis represented heart, liver, spleen, lung, kidney, brain, cerebellum, muscle, pituitary, hypothalamus, small intestine, stomach and endometrium respectively; In graph B, “**” indicates significant difference (P<0.01, t test)
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图 6 GF12 ER和GD12 LL猪LPAR3基因甲基化检测结果
A、B、C为GD12 ER LPAR3基因甲基化状态；D、E、F为GD12 LL LPAR3基因甲基化状态；每行代表1个克隆，黑色点表示甲基化的CpG位点，白色点表示未被甲基化的CpG位点，黑框表示碱基错配
Figure 6. Detection results of methylation status of LPAR3gene in GF12 ER and GD12 LL pigs
A, B, C: Methylation status of GD12 ER LPAR3; D, E, F: Methylation status of GD12 LL LPAR3; Each row represents one clone, black dots indicate methylated CpG sites, white dot indicates non-methylated CpG sites, black boxes indicate base mismatches
表 1 猪LPAR3基因mRNA克隆及启动子扩增所需引物信息
Table 1 The primers for cloning the mRNA and promoter of porcine LPAR3 gene
引物名称
Primer name引物序列(5'→3')1)
Primer sequence产物大小/bp
Product sizepLPAR3-5UTR F：TGTCCTCCACCGCTCCT R：CCATCACCGTCTTCATTAG LPAR3-P F：CCTTTTGGTGGAGAAGTGAGAC 3 080 (–2 430/+650) R：GCATGGTTCGAGCTGTTCTCT p1 F：CGACGCGTCG TGAAGTGGCTCAGCAGGTT 1 958 (–1 878/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p2 F：CGACGCGTCG TTGTATCCACCACATTGCCC 1 435 (–1 355/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p3 F：CGACGCGTCG TTTCCTGGGTTATGGTTTC 1 047 (–967/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p4 F：CGACGCGTCG GAGTTGTGTGGCTTCAGTT 534 (–454/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p5 F：CGACGCGTCG CTCTGTAGGGCTTGTGG 351 (–271/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p6 F：CGACGCGTCG TGCGTGGGAACTGTCGGT 203 (–123/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p7 F：CGACGCGTCG CTGCCAAACTTTTCTCCTGT 96 (–16/+80) R：CCGCTCGAGCGG GAGGAGCGGTGGAGGAC p8 F：CGACGCGTCG CTCTGTAGGGCTTGTGG 218 (–271/–54) R：CCGCTCGAGCGG TCCGACTTGCCCCGCCTA p9 F：CGACGCGTCG CTCTGTAGGGCTTGTGG 921 (–271/+650) R：CCGCTCGAGCGG GCATGGTTCGAGCTGTTCTCT p-GADPH F：ACGCCTGCCCTGTGTCCCAA 190 R：GAAGCACGCCCTCTCGCCTC pLPAR3 F：TGGTCGCCCCGTCACTCT 100 R：AACTGAACATCCGCTCACACT LPAR3-BSP1 F：GGTTCGGAGGTACGGTTTTGTAGG 1 006 R：AAACACCACTCCCAACAATATCCC LPAR3-BSP2 F：CGGTTATGTATTTGGGGATTAG 887 R：TTCCCGAATCTCTAAATACCTT 1) 加粗表示保护碱基，下划线表示 MluI 酶切位点 (ACGCGT) 和 XhoI 酶切位点 (CTCGAG)
1) Bold type: Protective bases; Underline type: Restriction sites for MluI(ACGCGT) and XhoI(CTCGAG)
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