Study On The Screening Of Zfy Interference Vector And The Effect Of Sexual Control In Cervus Elaphus's Gene

The mammalian Y chromosome plays an important role in animal gender determination and sperm production.The Zfy gene is located on the short arm of the Y chromosome and encodes a zinc finger protein.The latest study suggested us that whether or not the round sperm could eventually develop to be the sounded sperm for natural fertilization is closely related to normal occurrence of Y sperm.As a special economic animal,Cervus elaphus has many years of history in Xinjiang production and Construction Corps.Cervus elaphus breeding efficiency is the core of antler sales.Removal of motherhood and staying the male is the normal state of a weak market.With the warming of the market,through the sex ratio control of Cervus elaphus offspring,the rapid reproduction expansion of doe in a short time is an important means of reducing the cost and improving the income in the increasingly fierce market competition for the deer farm.Purpose: Design Zfy gene shRNA segment of Cervus elaphus,as well as establish and screen high-efficient Zfy gene shRNA expression vector of Cervus elaphus.Inject the screened recombined vector into the testicles of the male deer,and then disturb,restrain the Zfy gene expression in the testicles during mating,in order to influence the normal formation of Y Sperm,at the same time,provide more fertilization chance for X sperm.In this way can more female deer be reproduced.Method:(1)Conduct siRNA design and screening to Zfy gene mRNA sequence of Cervus elaphus.Connect the screened siRNA respectively to RNA interfering eukaryotic expression plasmid.Use the established recombined expression vector to conduct the external spermatogenic cells development RNAi test on Cervus elaphus.Take advantage of qPCR to detect the expression changes of mRNA,and then screen the high-efficient shRNA expression vector.(2)Select 5 healthy male deer and 67 female deer.Inject the screened recombinant plasmid which has been diluted according to certain proportion into the testicles of 5 male deer.After the injection,drive the 5 male deer randomly to 5 female deer breeding house for natural breeding(every breeding house consists of 9 to 14 female deer).During the 12 th to 14 th week gestation period of the female deer,extract the fetus plasma free DNA of the pregnant deer.Make use of the routine PCR ways to enlarge the amelogenin protein gene segment.Based on the segment features,the gender of fetus can be confirmed.Meanwhile make statistics about the natural gender proportion in the mentioned deer farm in the recent years as a contrast.After the analysis we can get the descendant gender excursion rate within this recombined plasmid after RNAi test.Result:(1)Screen 4 pair specificity siRNA sequence based on siRNA designing principle,and establish 2 shRNA expression vector of Zfy gene(recombined expression vector 1,recombined expression vector 2).After the qPCR detection,it shows that the disturbance rate of recombined expression vector 2 Zfy gene is 79%,which has an obvious difference with the non-carrier(P<0.01).While there is no obvious difference between recombined expression vector 1 and non-carrier(P>0.05).(2)Use the screened Zfy recombined expression vector 2 to conduct internal RNAi test on Cervus elaphus.After the fetus gender verification,the female rate is 70%,which presents a clear difference with the contrast group(P< 0.01).Conclusion:(1)The established recombined plasmid 2 exerts a clear reduction influence to the mRNA expression of Zfy gene,which can be used for internal RNAi test on Cervus elaphus.(2)The internal RNAi test of recombined plasmid 2 greatly influence the gender proportion of the deer descendant,which increases the female number in the later generation.