Cloning And Expression Analysis Of PLCζ Gene From Chinese Merino Sheep

Objective:Chinese Merino sheep is one of the typical meat and wool sheep breeds in Sinkiang. The amount of sheep slaughter and processing is very large, however the recycling rate of ovarian tissue is not very high, so the cloned animals or transgenic animals generated by nuclear transfer were even fewer in Sinkiang. The great disadvantage of using the protein kinase inhibitors or the protein synthesis inhibitors is that these inhibitors do not specifically inhibit the activity of a particular kinase or the synthesis of a specific protein that control cell-cycle progression. However, they inhibit the activity of several kinases or the synthesis of several proteins that may be involved in other cell functions, whose inhibition may have a deleterious effect on the subsequent cellular events after oocyte activation. Therefore, a new activation regimen without using either protein synthesis or protein phosphorylation inhibitors but just through single calcium increase effectively improved blastocyst yield need to find. The purpose of our experiment is to explore whether PLCζ can be used as a new activation regimen for activating oocytes.Methods:(1) Chinese Merino sheep testicular tissue RNA was extracted with TRIzol lysis method and the cDNA obtained by reverse transcription, then PLCζ gene was amplificated by PCR with the cDNA as a template;(2) PLCζ Gene was connected to T vector, named pMD18-T-PLCζ, then the PLCζ gene was linked to pCznl vector and pEGFP-N1vector by double digestion, finally proved by sequencing and restriction enzyme digestion.(3) pCznl-PLCζ was transfected into E.coli Arctic Express TM (DE3) for production of PLCζ protein.(4) pEGFP-N1-PLCζ vector was transfected into293T cells by LipofectamineTM2000;(5) Merino sheep fetal fibroblasts isolated by tissue-sticking method, and then transfected pEGFP-N1-PLCζ by LipofectamineTM2000;(6) Collection of the sheep ovaries from a local slaughterhouse, and then obtain the sheep oocytes. microinjection of pEGFP-N1-PLCζ plasmid into them for research of parthenogenetic activation.Results:(1) Successfully cloned the merino sheep PLCζ gene, sequencing found that it has the highest homology with cattle PLCζ gene sequence published on GenBank;(2) Successfully constructed the eukaryotic expression vector (pCznl-PLCζ) and produced purified protein;(3) Successfully constructed the eukaryotic expression vector contained PLCζ gene (pEGFP-N1-PLCζ) was detected by digestion and sequencing;(4) pEGFP-N1-PLCζ vector has been successfully expressed in293T cells and merino sheep fetal fibroblasts;(5) The oocytes injected with pEGFP-N1-PLCζ plasmid can carry out parthenogenetic development.Conclusions:(1) PLCζ gene can expresse in somatic Cell lines, successfully.(2) Oocytes in M stage injected with the recombinant plasmid of PLCζ can carry out parthenogenetic development.