The Research of Mus Sex Control by Zfy Gene Interference
|Course||Basic Veterinary Science|
|Keywords||RNAi Mus musculus gonepoiesis sex control|
Testis is the capital genital gland of masculinity. It secretes androgenic hormone and the place where is spermatogenesis. Zfy locates in short arm of Y chromosome and is related with spermatogenesis. Zfy is activating transcription factor, lead the target gene through the nuclear membrane and orientation in the inner of spennatid nucleus. According to the character of Zfy, the present study designed shRNA fragment, carried out the RNAi experiment about Zfy. Expression level of Zfy mRNA was determined by RT-PCR, and statistics the sex proportion of F2 generation after interference. The concrete finding as following:1. According to Zfy mRNA sequence of Mus provided by Gen Bank, the interference fragment was designed and synthesized from the coding region. The expression vectors pSilencer5.1/Zfy215 and pSilencer5.1/Zfy2102 was constructed by two shRNA fragments of Zfy and vector pSilencer5.1-H1 Retro which is from Ambion company. After the recombination expression vectors were verified by enzyme incise and 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 two shRNA expression vectors were constructed successfully, and they could be used in Zfy interference experiment of Zfy.2. The two recombination expression vectors were researched by RNAi experiment.64 male KunMing Mus were divided into four groups randomly and averagely. The two recombination expression vectors were respectively injected into two groups through testis. The other two groups were injected with the same volume of physiological saline and empty vector pSilencer5.1-H1 Retro, respectively. They were injected every ten days and four times in all.17d after the fourth injection,8 males of each group mated with 8 female Mus. The testis tissue of the other 8 male Mus of each group was collected, and the expression level of Zfy mRNA was determined by fluorescence quantitation real time PCR (qRT-PCR). The result showed that the expression of Zfy mRNA decreased significantly after injection of pSilencer5.1/Zfy2102 (P<0.01), and that 72.3% of the offspring was female, a number significantly higher than in the control group (P<0.01). In pSilencer5.1/Zfy215 group, the expression of Zfy mRNA was significantly lower than in control (P<0.05), but the female rate of offspring was not.This research silenced the expression of Zfy, and it affected the form of Y sperm. It deserved to point out that the number and body weight of second filial generation in test group was not significantly difference with control. That means the spheroid which was produced after interfered with Zfy didn’t influence the breeding performance of later generations. This paper may establish a basis for sex control of livestock before fertilization.