The RNAi Fragments Screening And And Observation Of Sex Control From Swine Zfx Gene

Zinc finger protein coupled X chromosome (zinc finger protein，X-linked, Zfx) ismember of zinc ifnger protein family，and a candidate gene of Testis Determining Factor(Testis Determining Factor, TDF). Zifc/Zjy is a pair of alleles and located on the mammalianX and Y chromosomes，respectively.^/^gene containing zinc finger structure, the structurecharacteristics of a zinc finger protein can bind selectively to a target-speciifc gene and guidethrough the nuclear membrane of the target gene，then located within the nucleus of the sperm，the sperm are formed with a certain function in the process.RNA interference (RNA interference, RNAi), a post-transcriptional gene silencing (posttranscriptional gene silence, PTGS) phenomenon, is the process of endogenous or introducedsynthetic small molecules with double-stranded RNA-speciifc degradation of homologousmRNA then formed small interfering RNA20?25nucleotides in size，block specific geneexpression in vivo in the mRNA level of phenomena.Objective: Zfx is located on the X sex chromosomes determine gender of a regional(TDF) gene with the zinc lipid structure, and sperm production related. This study hopes thatRNAi technology is combined with pig’s sex control, artificially interferences the expressionof Zifc, human gene expression zifc interference, made the X-sperm inlfuence the developmentand generation or hindered X sperm fertilization ability and activity, Resulting in higher maleoffspring.Methods: according to the design principle of siRNA, constructed the interferencevector of pig Zifc genes, atfer screening the RNAi vectors of Zfx gene of experimental in vitro,zifc gene RNAi screening experiment,the better carrier interference three times intermittentlyinjected into the pi’gs testicles, and then mating with sowes, The number of offspring wererecorded，male piglets ratios was caculated and Statiscs was used by T test.Results: l)The pSIREN for Zifc gene expression vector of A, B，C, D were constructedand the expression plasmid PLL3.7-shRNA of PLL3.7(refered to e), by enzyme digestionidentiifcation and sequencing confirmed that these vectors were correct.2) The constructed expression plasmids were transfected into swine testicular cells in vitro,48hours after transfection.Then mRNA expression level of zfx genes were detected byreal-time fluorescent quantitative PCR. By preliminary screening， the most efficient siRNArecombinant plasmid expression vector e was selected,which is downgrade the expression level of Zfygenes to54%, compared with the control group, the interference effects of e vector is significant (P<0.05).3)4boars were equally divided into2groups, The better interference effects ofrecombinant vector e was injected into the testis of boar in one group; recombinant vector with same interference fragment but different vetor d was injected into testis ofboars of other gtoup.3mg vetor was injected into each boar each time for total of3times, and1.5mg vector for each side of testicles, each time interval was10days.After the second injection, boar began to mating with sow, the sex of offspring wererecorded. Results showed that the constructed RNAi vector of Zfx gene is affected. After there times injection，the total proportion of male was56.5%and signiifcantlydifferent, compared with the normal sex ratio of1:l(/><0,05), of the first experimentgroup, the proportion of male piglets was67.5%and significant difference with thenormal sex ratio (尸<0.01), Of the second group, the proportion of male piglets was63%,it was no significant difference with the normal sex ratio (/^0.05). These resultsshowed that silenced zfx gene could influence the formation of X sperm and increasethe proportion of male piglets. At the same instructions, interference effects lentiviralvector backbone plasmid PLL3.7e better than pSIREN plasmid vector. Also, the interference vector injection requires more than three times in a row in order to play betterinterference.