The Construction of the Goat Ovary Melatonin Signal Specific Vectors pcDNA3.1-Pro/GDF9-ASMT and pcDNA3.1-Pro/Cyp17-MTNR1A and MTNR1B

This study intended to construct an oocyte-specific expression vector (GDF9-ASMT) for melatonin (MLT) synthetase acetylserotonin methytransferase (ASMT),and granule (luteal) cell-specific expression vector (Cyp17-MTNR1A,MTNR1B) for MLT receptors MTNR1A and MTNR1B,the construction of the above vectors provided carrier materials for further production of transgenic sheep with high fertility.First,the goat GDF9 and Cyp17a1 promoter sequences were amplified separately.Then,the full-length CDS sequences of ASMT,MTNR1A and MTNR1B genes were amplified,and ASMT gene was connected to the downstream of GDF9 promoter by enzyme-cutting,with MTNR1A and MTNR1B respectively connected to the downstream of Cyp17a1 promoter to prepare Pro/GDF9-ASMT,Pro/Cyp17-MTNR1A and Pro/Cyp17-MTNR1B expression elements.Finally,the above-mentioned expression elements were integrated into the pcDNA3.1(+) vector by enzyme digestion,thereby constructing pcDNA3.1-Pro/GDF9-ASMT,pcDNA3.1-Pro/Cyp17-MTNR1A and pcDNA3.1-Pro/Cyp17-MTNR1B eukaryotic expression vectors.After sequencing verification,the GDF9 and Cyp17a1 promoters amplified in this study were 2 496 and 2 497 bp in length respectively,and they were 98% homologous to the 2 500 bp region upstream of the start codons of the goat GDF9 and Cyp17a1 genes in GenBank database.The amplified ASMT,MTNR1A and MTNR1B CDS sequence lengths were 1 037,1 100 and 1 130 bp,and the homology with the standard sequences submitted by GenBank were 98%,98% and 99%.The amino acid sequence homologies was 97%,98%,99% respectively.The construction of the above vectors provided references for further production of melatonin transgenic sheep with high fertility.