| The existence of biological sex dimorphism provides an important condition for the breeding and development of offspring.The most direct feature of the mammal sexual dimorphism is balance and difference of sex ratio.And Zfx(any Zinc finger-X,Zfx)gene and their family are the most important factors that affects the mammalian sex ratio balance.Zfx is being located on the X chromosome coding peculiarity of zinc finger protein gene,found in male mammals testicular tissue curve haploid X-chromosome,which can gave birth to the nuclei of sperm cells in seminiferous tubule.It has the function of transcriptional activation may be directly or indirectly participate in the development of X sperm and generated.This study refer to the Swine Zfx gene sequence characteristics,according to the designing principles of RNA interference fragment,synthetic packaging PLL3.7 plasmid carrier without virus,laboratory simulation swine testicular sperm production environment to create living sperm cells and supporting cells co-culture system,after in vitro culture in vitro transfection,carrier interference effect in the mRNA level testing,and then optimal jamming carrier,optimizing entity interference effect,with the method of scatter testis injection,divided into different doses and times gradient on the boar testis injection interference experiment.By random grouping and artificial mating,track the offspring gender and reproductive performance.Which explore interference Swine Zfx genes control effect and impact on reproductive performance.The specific research results are as follows:1.Swine Zfx gene RNA interference reconstructingCarrier construction is based on NCBI Gene bank of Swine Zfx genes of Nucleotide sequence,consulting siRNA design steps,designing 1 eligible siRNA,and then choose suitable PLL3.7 enzyme loci,synthesis of corresponding shRNA,after closed chain and enzyme digestion connection,sequencing and PCR identification,to determine the successful building of interference carrier PLL3.7 / N,can be used for cellular level test furthermore.2.Interference carrier screening on cells levelIn breeding Swine farm,gathering around the age of 30 piglets.By real-time quantitative reverse transcription and relative filtering under the cellular level,cause living sperm cells mRNA expression differences apparent interference carrier 1.PLL3.7/N Zfx genes mRNA expression quantity of 44%,compared with the control group decreased significantly(P < 0.05).3.Swine injection interference experiment in vivoWe raise the plasmid by raising the endotoxin kits,purification of enrichment of high purity and concentration of prokaryotic expression cloning PLL3.7 /N carrier interference.Choose healthy boars head,randomly divided into 6 groups,each group 3 head,with high-pressure and sterile PBS,according to the final volume of 7 mL,0.5 mg/tube,1.0 mg/tube,the dose of 1.5 mg/pipe,each set of 6 tube dilution,the control for PLL3.7 empty carrier,dose of 1.5 mg/tube.Near the injection before each tube double blender.Add 1 mL Bilateral testes,every 10 days injection needle,after successive 3 injection,recuperation after four days of pure side by side to arrange the same column under the condition of feeding,collection of semen,the testing of semen quality,and artificial insemination,breeding time for 60 days,every day,ensure that each test group 1 head boar sow mating with 2 heads above.After artificial insemination and farrowing,results showed that: the overall data show that the third time after injection of plasmid,0.5 mg/side three needle injection group male piglets,54.1% compared with the control group sex ratio difference was not significant(P > 0.05).1.0 mg/side three needle injection group male piglets,63.2% compared with control group sex ratio difference was significant(P < 0.01).1.5 mg/side three needle injection group male piglets respectively is 68.82%,compared with control group sex ratio difference was significant(P < 0.01).1.5 mg/side two needle injection group male piglets respectively is 61.72%,compared with control group sex ratio difference was significant(P < 0.01).1.5 mg/side a needle injection group male piglets,48.22% compared with the control group sex ratio difference was not significant(P > 0.05).Points and time interval,the third time after injection of plasmid,in 1,2,3 months,0.5 mg/side three needle injection group to produce the proportion of male piglets were 56.08%,53.14%,50.79%,comparedwith control group sex ratio are differences were not significant(P > 0.05);1.0 mg/side three needle injection group of sexual piglet proportion is 66.67%,63.9% and 53.13% respectively,compared with control group sex ratio were significant difference(P < 0.05)and significant difference(P < 0.05),no significant difference(P > 0.05);1.5 mg/side three needle injection group to produce the proportion of male piglets were 72.28%,70.05%,53.33%,compared with the control group sex ratio were extremely significant difference(P < 0.01),and extremely significant difference(P < 0.01),no significant difference(P > 0.05).Experiment shows that local injections of interference by RNAi technology and testicles,directional interference suppression in vivo X sperm normal development or insemination boar the testes,improve Y sperm competition advantage,so as to control the pig offspring gender technology is feasible.PLL3.7 / N plasmid interference effect of Zfx genes related to the injection time,dose,and duration,the dose of 1.5 mg/side is sex of dose for optimal control.Injection time,starting from the second needle,sex control effect is obvious,1.5 mg/side injection group,the proportion of male piglets reached 61.72%,very significant difference(P < 0.01),the third needle reached 68.82%,very significant difference(P < 0.01).Sex control valid time starts from the second needle effect.4.Injection of recombinant carrier to match the sow reproductive performance of track detection By boar after injection(0.5 mg/side three needle group,1.0 mg/side three needle group,1.5 mg/side three needle group,1.5 mg/side two needle group,1.5 mg/side stitch group)and sow mating,columns management,and tracking statistics corresponding category of artificial insemination reproductive performance,such as births,including conception rate in farrowing rate,alive rate,the healthy rate of seed rate,weak young,deformity-stillbirth-mummy rate,birth weight,weaning survival rate.The results showed that 5 kinds of injection conditions,affect and reproductive performance were not significant.In conclusion: through the testes injections,interference Zfx genes to control the gender of the pig,has no effect on the reproductive performance of sows. |
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