| Animal body size mainly depends on the development of bone and muscle tissue. Most bone tissue develops from endochondral ossification, while the development of epiphyseal growth plate is the most important process for endochondral bone formation. BMP2 plays an important role in postnatal skeletal homeostasis, and the osteogenic signal from BMP2 is required for generation and repairment of bone. It was also found that three human inherited dwarfism syndromes, hypochondroplasia, achondroplasia, and thanatophoric dysplasia, are caused by missense mutations in the FGFR3 gene. However, no report had been found about FGFR3 sequence cloning and the correlation between FGFR3 and skeletal development in pig. cDNA amplified fragment length polymorphism (cDNA-RFLP) has advantages of good repeatablility, low flase positive rate, and less amount of samples need, and can be used to screen the differential expression genes without knowing the sequence information in advance.In order to screen the genetic markers for the breeding of experimental Guangxi Bama mini-pig, we cloned the BMP2 and FGFR3 CDS sequences of Guangxi Bama mini-pig and Landrace, analyzed the expression levels of limb bone in new born Guangxi Bama mini-pig and Landrace, obtained the full length CDS of FGFR3 using SMARTer RACE technology, and screened the genes differential display in limb bone from one-day old Guangxi Bama mini-pig and Landrace using cDNA-AFLP technology as well. The results were as follow:1. A full length of BMP2 cDNA with 1188bp was cloned from Guangxi Bama mini-pig and Landrace, respectively. Two mutations were detected when BMP2 CDS was compared with that released in NCBI, but the BMP2 CDS between the two breeds were completely consistent.2. We successfully cloned a part of FGFR3 CDS with 808bp from Guangxi Bama mini-pig and Landrace. Compared to the derived FGFR3 sequence in NCBI,8 mutations and 1 deletion were found. Five mutations, the A124G, C190G, A204C, C205A and C245T missense mutations, were detected in Guangxi Bama mini-pig and Landrace, and A438G and C549T synonymous mutations in Guangxi Bama mini-pig and A752C missense mutation in Landrace were found, respectively.3. The genotype and gene frequency of FGFR3 gene in A2374G and C2620T locies were significant difference between Guangxi Bama mini-pig and Landrace (P<0.01). The two locies were not in a state of linkage disequilibrium.4. The body weight, length, height and femur length between one-day old Guangxi Bama mini-pig and Landrace were significant difference (P<0.05). Compared to Landrace, the BMP2 expression was significantly lower (P<0.05), while the FGFR3 was significantly higher (P<0.01) in epiphyseal growth plate of Guangxi Bama mini-pig.5. One 5’end and three 3’end sequences of FGFR3 gene were obtained by SMARTer RACE technology and the start codon of pig FGFR3 gene was determined after three FGFR3 CDS sequences were contiged.6. Eighteen differential display TDFs between Guangxi Bama mini-pig and Landrace by cDNA-AFLP technology were found. Five fragments with annotated functions may be invoved in the regulation of cell cycle, but the functions of 10 fragments homologous with those sequences in NCBI were not annotated, the left 3 fragments didn’t match with any homologous sequence in NCBI.In general, BMP2 and FGFR3 are involved in the regulation of skeletal development in Guangxi Bama mini-pig. Further reseaches are need to verify the function of differential display genes from cDNA-AFLP in skeletal development in Guangxi Bama mini-pig. |
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