Construction of Eukaryotic Co-expression Plasmid Carrying pGH and IGF-Ⅰ Gene and It's Transformation in Landrace

doi:10.7668/hbnxb.2016.03.014

Abstract

Abstract: In order to explore the effects of pGH and IGF-Ⅰ on the growth and development of Landrace,eukaryotic co-expression plasmid containing pGH and IGF-Ⅰ was constructed and transfected into Landrace,which will lay the foundation for the cultivation of new breed of feed-saving grain pigs.Total RNA was extracted from the ear tissue of Landrace pig,and the coding sequence of pGH without termination codon and complete coding sequence of IGF-Ⅰ were amplified by RT-PCR and cloned into the pcDNA3.1(+) eukaryotic expression vector after verified by sequencing.The recombinant plasmid was verified by sequencing and enzyme digestion and then transfected into PK15 cells.Expression of the two genes in PK15 cells was detected by Q-PCR.Transgenic pigs were prepared with sperm-mediated transformation after the sperm were encapsulated by nanometer material.Transgenic pigs were identified by PCR and sequencing.The expression of the two target genes were detected by Q-PCR.Stability of transgene was detected by PCR and sequencing aged from 1 month to 7 month.RT-PCR and sequencing results showed that the coding sequences of pGH and IGF-Ⅰ of Landrace were successfully cloned.Digestion and sequencing analysis showed the eukaryotic co-expression vector containing pGH and IGF-Ⅰ was successfully constructed,and Q-PCR analysis showed that pGH and IGF-Ⅰ were successfully expressed at the mRNA level after transfected into PK15 cells.As a result,13 young piglets were born,and 4 of them were positive for both two genes detected by PCR and sequencing.As a result,the positive rate was 30.76%.Q-PCR results showed that exogenous pGH and IGF-Ⅰ were successful expressed in transgenic pigs.Exogenous pGH and IGF-Ⅰ could be detected in transgenic positive individuals from aged 1 month to 7 month,which proved the two exogenous genes were stable in transgenic pigs and were not lost in growth process.Exogenous pGH and IGF-Ⅰ could be detected in sperm of transgenic boars showed that exogenous pGH and IGF-Ⅰ may be passaged stable.

Key words: pGH, IGF-Ⅰ, Co-expression, Transgene, Landrace

CLC Number:

Q78
S828

YAO Yanzhu, WU Mingming, SUN Jinhai. Construction of Eukaryotic Co-expression Plasmid Carrying pGH and IGF-Ⅰ Gene and It's Transformation in Landrace[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(3): 94-100. doi: 10.7668/hbnxb.2016.03.014.

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