The RNAi Vecter Screening And Verification Of Theeffects Of Sexual Control From Swine Zfx Gene

Testis is the place of generating sperm and secreting hormonal.The Zfx(Zinc finger-X)gene is a member of zinc lipoprotein,located in the X chromosome short wall gender decided to area(TDF) inside. Zfx is related in X sperm formation,can serve as a strong transcription activation factor, and guide the target genes through the nuclear membrane positioning in X sperm nuclei, it may to some genes in X spermatogenesis process with transcription activation function. According to the character of Zfx, the present study designed sh RNA fragment, carried out the RNAi experiment about Zfx. Expression level of Zfx m RNA was determined by q PCR, and statistics the sex proportion of offspring after interference. The concrete finding as following:1. Cloning Zfx gene m RNA sequenee in Swine:Four pairs of primers were designed according to bovine Zfx gene sequenee, combined PCR technique to amplify the Zfx gene of swine, sequencing and splicing.The results showed that m RNA sequence of the swine Zfx gene was 2154 bp.2. According to Zfx m RNA sequence by cloning, the four sh RNA fragments were designed and synthesized from the coding region. The linear vector p LL3.7 was linked to four sh RNA fragments(p LL3.7/d、p LL3.7/e、p LL3.7/f、p LL3.7/g). After recombination, expression vectors were verified by sequencing, it was found that the location and direction of interference sequence was inserted correctly, and the expression regulatory element of upstream and downstream was integrity. That means the four sh RNA expression vectors were constructed successfully, and they could be used in Zfx interference experiment in vitro.3. The 4 sh RNA interference vector was used in vitro research of RNAi. We take the testicle of 27~32 day swine,and the sperm cells of testicle tissue was separated by two enzyme digestion method and cultured with sertoli cells. The 4 interference vector were transfer into the sperm cells with HET, 48 hours after transfection, q PCR to detect Zfx genes m RNA expression level.Plasmid p LL3.7/d、p LL3.7/e、p LL3.7/f were selected, which the expression level of Zfx genes were 55%、56%、44%, significant difference compared with the control group(P<0.05).4. The Zfx gene interference vector was tested in vivo of swine. 6 large white were divided into 3 groups, the interference vector was injected into 3 groups through testis, respectively. They were injected every 9 days and 3 times in all. After three times injected plasmid p LL3.7/d,sow was breeding. Every injected p LL3.7/e and p LL3.7/f, boar began to mating with sow, the sex of offspring were recorded.Results showed : Compared with normal sex ratio of 1:1, p LL3.7/d group had 69.9% male piglet ratio and significantly different(P<0.05), male piglet ratio of p LL3.7/e and p LL3.7/f group had no significant difference after every injected; A total of there times injected, p LL3.7/e group had 59% male piglet ratio and significantly different,while p LL3.7/f group was 49%, which is no significant difference with the normal sex ratio(P>0.05).The results showed that silenced Zfx gene could influence the fertilization ability of sperm X.