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Identification and quantitative mRNA analysis of a novel splice variant of GPIHBP1 in dairy cattle.

Yang J, Liu X, Zhang Q, Jiang L - J Anim Sci Biotechnol (2014)

Bottom Line: However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for Animal Breeding; Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture of China; College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.

ABSTRACT

Background: Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study (GWAS). Of these, GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.

Results: In this study, we identified a novel alternatively splice transcript variant (X5) which leads to a 31 bp insertion in exon 3 and also confirmed the other four existed transcripts (X1, X2, X3 and X4) of the bovine GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.

Conclusions: Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.

No MeSH data available.


PCR analysis of theGPIHBP1coding region in samples of mammary gland using three pairs of primers. (A) With primer 1, transcript X2 (603 bp), transcript X3 (289 bp) and transcript X4 (192 bp) were observed as expected (according to the mRNA sequence of the bovine GPIHBP1 gene in NCBI database). M1: DL2000, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp. (B) With primer 2, in addition to the expected PCR band (352 bp and 344 bp), a band of 383 bp was also observed in all samples. M2: 100 bp DNA Ladder from 1,500–100 bp. (C) With primer 3, only the expected band (388 bp) was observed in all samples. M3: DL500, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp.
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Fig1: PCR analysis of theGPIHBP1coding region in samples of mammary gland using three pairs of primers. (A) With primer 1, transcript X2 (603 bp), transcript X3 (289 bp) and transcript X4 (192 bp) were observed as expected (according to the mRNA sequence of the bovine GPIHBP1 gene in NCBI database). M1: DL2000, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp. (B) With primer 2, in addition to the expected PCR band (352 bp and 344 bp), a band of 383 bp was also observed in all samples. M2: 100 bp DNA Ladder from 1,500–100 bp. (C) With primer 3, only the expected band (388 bp) was observed in all samples. M3: DL500, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp.

Mentions: Three primers (Additional file1: Table S1) were used to amplify the coding region of GPIHBP1 in samples of mammary gland. After PCR amplification with primer 1 and primer 3, we observed three (Figure 1A) and one (Figure 1C) PCR bands, respectively. These 4 bands correspond to the expected fragment size (Additional file1: Table S1) derived from the bovine GPIHBP1 sequence in the NCBI database. With primer 2, two bands with fragment lengths of 352 bp and 344 bp, respectively, would be expected according to bovine GPIHBP1 sequence. However, the PCR products showed the two bands were almost merged into one band (Figure 1B), since there is only 8 bp difference in size between the two fragments. Interestingly, we observed an additional band of 383 bp which was present in all samples (Figure 1B, only two samples are shown). To verify the results of primer 2, we purified the PCR products and cloned them in Pmd18-T (Takara Biotechnology, Tokyo, Japan). We then randomly selected twenty colonies to sequence. It turned out that 17 of them were of 352 bp length, one of them showed a deletion of 8 bp (5′-GGCCGCAG-3′, Chr14:2550837-Chr14:2550830) in exon 3 and was of length of 344 bp, and two of them showed an insertion of 31 bp (5′-TGGAGGTTTACAGGTGTCCCTGCGCGGCCAG-3′, Chr14:2551602-Chr14:2551572) in exon 3 and were of length of 383 bp. Thus, both the two expected fragments and the novel fragment were confirmed.Figure 1


Identification and quantitative mRNA analysis of a novel splice variant of GPIHBP1 in dairy cattle.

Yang J, Liu X, Zhang Q, Jiang L - J Anim Sci Biotechnol (2014)

PCR analysis of theGPIHBP1coding region in samples of mammary gland using three pairs of primers. (A) With primer 1, transcript X2 (603 bp), transcript X3 (289 bp) and transcript X4 (192 bp) were observed as expected (according to the mRNA sequence of the bovine GPIHBP1 gene in NCBI database). M1: DL2000, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp. (B) With primer 2, in addition to the expected PCR band (352 bp and 344 bp), a band of 383 bp was also observed in all samples. M2: 100 bp DNA Ladder from 1,500–100 bp. (C) With primer 3, only the expected band (388 bp) was observed in all samples. M3: DL500, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373091&req=5

Fig1: PCR analysis of theGPIHBP1coding region in samples of mammary gland using three pairs of primers. (A) With primer 1, transcript X2 (603 bp), transcript X3 (289 bp) and transcript X4 (192 bp) were observed as expected (according to the mRNA sequence of the bovine GPIHBP1 gene in NCBI database). M1: DL2000, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp. (B) With primer 2, in addition to the expected PCR band (352 bp and 344 bp), a band of 383 bp was also observed in all samples. M2: 100 bp DNA Ladder from 1,500–100 bp. (C) With primer 3, only the expected band (388 bp) was observed in all samples. M3: DL500, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp.
Mentions: Three primers (Additional file1: Table S1) were used to amplify the coding region of GPIHBP1 in samples of mammary gland. After PCR amplification with primer 1 and primer 3, we observed three (Figure 1A) and one (Figure 1C) PCR bands, respectively. These 4 bands correspond to the expected fragment size (Additional file1: Table S1) derived from the bovine GPIHBP1 sequence in the NCBI database. With primer 2, two bands with fragment lengths of 352 bp and 344 bp, respectively, would be expected according to bovine GPIHBP1 sequence. However, the PCR products showed the two bands were almost merged into one band (Figure 1B), since there is only 8 bp difference in size between the two fragments. Interestingly, we observed an additional band of 383 bp which was present in all samples (Figure 1B, only two samples are shown). To verify the results of primer 2, we purified the PCR products and cloned them in Pmd18-T (Takara Biotechnology, Tokyo, Japan). We then randomly selected twenty colonies to sequence. It turned out that 17 of them were of 352 bp length, one of them showed a deletion of 8 bp (5′-GGCCGCAG-3′, Chr14:2550837-Chr14:2550830) in exon 3 and was of length of 344 bp, and two of them showed an insertion of 31 bp (5′-TGGAGGTTTACAGGTGTCCCTGCGCGGCCAG-3′, Chr14:2551602-Chr14:2551572) in exon 3 and were of length of 383 bp. Thus, both the two expected fragments and the novel fragment were confirmed.Figure 1

Bottom Line: However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for Animal Breeding; Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture of China; College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.

ABSTRACT

Background: Identification of functional genes affecting milk production traits is very crucial for improving breeding efficiency in dairy cattle. Many potential candidate genes have been identified through our previous genome wide association study (GWAS). Of these, GPIHBP1 is an important novel candidate gene for milk production traits. However, the mRNA structure of the bovine GPIHBP1 gene is not fully determined up to now.

Results: In this study, we identified a novel alternatively splice transcript variant (X5) which leads to a 31 bp insertion in exon 3 and also confirmed the other four existed transcripts (X1, X2, X3 and X4) of the bovine GPIHBP1 gene. We showed that transcript X5 with a 31 bp insertion and transcript X1 with an 8 bp deletion might have tremendous effect on the protein function and structure of GPIHBP1, respectively. With semi-quantitative PCR and quantitative real-time RT-PCR, we found that the mRNA expression of GPIHBP1, GPIHBP1-X1 and GPIHBP1-X5 in mammary gland of lactating cows were much higher than that in other tissues.

Conclusions: Our study reports a novel alternative splicing of GPIHBP1 in bovine for the first time and provide useful information for the further functional analyses of GPIHBP1 in dairy cattle.

No MeSH data available.