Construction and Functional Verification of siRNA Eukaryotic Expression Vector Directed at the Follicular Inhibin Alpha Gene in Ye Mule Sheep
Construction and Functional Verification of siRNA Eukaryotic Expression Vector Directed at the Follicular Inhibin Alpha Gene in Ye Mule Sheep
Zengwen Huang1,2, WuReliHazi Hazihan2*, Baheti Bodai2, Kadyken Rizabek3, Nuralieva Ulzhan3, Omarova Karlygash4, Juan Zhang1 and Yaling Gu1*
ABSTRACT
The Ye Mule Aries (YM) Sheep is a valuable local sheep breed from Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. The effect of small interfering RNA (siRNA) on the expression of follistatin inhibin alpha gene (INHα) in YM sheep was analyzed, and the INHα gene was amplified from total RNA of the ovary tissue of YM sheep and was successfully cloned. The eukaryotic siRNA expression vector was constructed with the cloned gene as the target and validated at the cellular level.Three RNA interference fragments at different sites were designed based on obtained cDNA of the target gene and the siRNA expression vector pGenesil 10-3p-siRNA was constructed. The siRNA expression vector was digested, identified and sequenced, and then transfected into YM sheep granulosa cells. The sequence of YM sheep INHα gene was obtained and uploaded on the NCBI database (accession number KP-113678.1). The INHα gene from YM sheep is 1109 bp long and translates to approximately 40 KDa protein. Q-PCR analysis showed that the plasmid pGenesil 10-3p-siRNA could interfere with the expression of INHα in granulosa cells to an efficiency of 83%, which was also confirmed through western Blot assay.This study successfully constructed the eukaryotic expression plasmid pGenesil 10-3p-siRNA, and confirmed that the plasmid interfered significantly with the expression of INHα gene in YM sheep. Our current findings can serve as a model for studying the effect of INHα on the breeding performance of YM sheep, providing a novel strategy to improve their reproductive capacities.
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December 2020
Vol. 52, Iss. 6, Pages 2027-2426
