广西巴马小型猪与长白猪的BMP2FGFR3基因序列及表达差异分析

Sequence and expression differences of BMP2 and FGFR3 genes in Guangxi Bama mini pig and Landrace pig

  • 摘要: 【目的】明确广西巴马小型猪与长白猪的BMP2FGFR3基因序列及表达差异,为进一步阐明大型猪与小型猪的体型形成机制提供理论依据。【方法】以1日龄的广西巴马小型猪和长白猪为研究对象,采集其四肢骨生长板软骨组织提取总RNA,反转录合成cDNA后用于扩增BMP2FGFR3基因的编码区(CDS)序列,采用PCR-RFLP检测FGFR3基因SNP位点多态性,并通过实时荧光定量PCR分析BMP2FGFR3基因在1日龄广西巴马小型猪和1日龄长白猪生长板软骨组织中的表达差异。【结果】1日龄广西巴马小型猪与1日龄长白猪的体型差异明显,其体重、体长和体高的差异均达极显著水平(P<0.01,下同),股骨长的差异达显著水平(P<0.05,下同)。广西巴马小型猪和长白猪的BMP2基因CDS序列全长1188 bp,且2个品种猪的CDS序列完全一致;广西巴马小型猪和长白猪的FGFR3基因CDS序列全长808 bp,与NCBI已公布的FGFR3基因序列(XM_005666479.1)比对发现存在8个碱基突变位点及1个碱基缺失位点。广西巴马小型猪和长白猪共有5个错义突变位点,分别为A124G、C190G、A204C、C205A和C245T,另外3个突变位点是广西巴马小型猪A438G和C549T的同义突变及长白猪A752C的错义突变;碱基缺失位点为长白猪第328~330个碱基(GCA)缺失。FGFR3基因A2374G和C2620T位点的基因型及基因频率在广西巴马小型猪与长白猪间的分布差异极显著,对应的等位基因分布均不符合Hardy-Weinberg平衡,且2个基因座均处于连锁平衡状态。1日龄广西巴马小型猪生长板软骨组织中的BMP2基因相对表达量显著低于1日龄长白猪,而FGFR3基因相对表达量极显著高于1日龄长白猪。【结论】BMP2FGFR3基因在猪的软骨和骨骼形成方面具有重要意义,其中,BMP2基因对猪骨发育起促进作用,而FGFR3基因对猪骨发育起抑制作用。广西巴马小型猪体型矮小与FGFR3基因高表达及BMP2基因低表达存在直接关联。

     

    Abstract: 【Objective】Clarify the BMP2 and FGFR3 genes sequences and expression differences between Guangxi Bama mini pig and Landrace pig, and provide theoretical basis for elucidating the formation mechanism of large and small pigs.【Method】The growth plate cartilage tissue of appendicular skeleton from one-day-old Guangxi Bama mini pig and Landrace pig was used to extract total RNA. After that, the total RNA was reverse transcribed to synthesize cDNA and then used for PCR amplification of the coding region(CDS) sequence of BMP2 and FGFR3 genes. PCR-RFLP was used to detect the polymorphism of the SNP locus of FGFR3 gene. The expression differences of FGFR3 and BMP2 in one-day-old Guangxi Bama mini pig and Landrace pig were compared by real-time fluorescence quantitative PCR.【Result】The results indicated that the body type of one-day-old Guangxi Bama mini pig and one-day-old Landrace pig were greatly different, and the differences in body weight, body length and body height were extremely significant(P<0.01, the same below), and the difference in femoral length reached a significant level(P<0.05, the same below). A full length of BMP2 gene CDS with 1188 bp was cloned from Guangxi Bama mini pig and Landrace pig, respectively, and the CDS sequence between the two breeds were completely consistent. The CDS sequence of FGFR3 gene with 808 bp cloned from Guangxi Bama mini pig and Landrace pig were found to have 8 base mutations and 1 base deletion when compared with the published FGFR3 sequence from NCBI(XM_005666479.1). Five mutations, which were A124 G, C190 G, A204 C, C205 A and C245 T missense mutations, were detected in Bama mini pig and Landrace pig, and A438 G and C549 T synonymous mutations in Guangxi Bama mini-pig and A752 C missense mutation in Landrace pig were found, respectively. There were 328-330 base(GCA) deletion in Landrace pig. The genotype and gene frequency of FGFR3 gene in A2374 G and C2620 T loci were extremely significant difference between Guangxi Bama mini pig and Landrace pig. The corresponding allele distributions were not in accordance with Hardy-Weinberg equilibrium, but the two loci were in the state of linkage disequilibrium. Compared to Landrace pig, the cartilage tissue of the growth plate of one-day-old Bama mini pig had significantly lower BMP2 expression, but the FGFR3 gene expression level was extremely significantly higher than that of Landrace pig.【Conclusion】BMP2 gene and FGFR3 gene have important significance in the formation of cartilage and bone. Among them, BMP2 promotes the development of pig bone, and FGFR3 gene inhibits the development of pig bone. It is speculated that the short size of Guangxi Bama mini-pig is directly related to the high expression of FGFR3 and the low expression of BMP2.

     

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