Recently, Molecular Therapy-Nucleic Acids (IF=7.032; Top10%) also published online at the same time that the team of Professor Wang Feng from the College of Animal Science and Technology, Nanjing Agricultural University completed the title” Long non-coding RNA lnc_3712 impedes nuclear reprogramming via repressing Kdm5b” and”LncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-534-3p/DCN/TGF-β signaling pathway in Hu sheep”. A systematic introduction to the important research progress made by the team in goat embryo cloning and the mechanism of multiple births in Hu sheep.
lncRNA regulates the epigenetic mechanism of cloned embryo reprogramming
This study used goat histone demethylase Kdm5b as the target gene and identified a new lncRNA lnc_3712 involved in regulating the reprogramming of cloned goat embryos, elucidating lnc_3712 The regulation mode with Kdm5b reveals the epigenetic mechanism of lnc_3712 regulating goat cloned embryo reprogramming, which has important guiding significance for improving the development ability of cloned embryos and the production efficiency of transgenic cloned animals.
In the past few decades, cloning technology has been successful in a variety of animals, but compared with normal fertilized embryos, the development rate of cloned embryos is still extremely high. low. Professor Wang Feng’s team obtained the first transgenic cloned goat from Nanjing Agricultural University in 2011. The preliminary research also established an lncRNA library involved in regulating early embryonic development of goats. However, there are a large number of lncRNAs and their functions are complex. What mechanism regulates the cloned embryos? Reprogramming is not yet clear.
In this study, Professor Wang Feng’s team confirmed that histone H3K4me3 is also a key obstacle to the reprogramming of goat cloned embryos. Injection of H3K4me3 demethylase Kdm5b mRNA can improve goats Cloned embryo blastocyst rate. Using high-throughput sequencing technology to analyze in detail the expression of lncRNA in the process of goat cloning embryo ZGA, and construct lncRNA-mRNA interaction network with Kdm5b and other epigenetic factors as core genes, combining interference experiments and bioinformatics analysis and screening To lncRNA lnc_3712, it was found that interfering with the expression of lnc_3712 in cloned goat embryos can increase the expression level of Kdm5b, reduce the H3K4me3 modification of cloned embryos, and then correct the abnormal transcriptome level of cloned goat embryos. More importantly, interference with lnc_3712 increased the blastocyst rate of goat cloned embryos to about 48%. This result is of great significance for improving the efficiency of cloned embryo reprogramming, and has potential application value in goat transgenic breeding.
This research work is based on the Jiangsu Province Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University. Professor Wang Feng and Professor Zhang Yanli of Nanjing Agricultural University are the co-corresponding authors of this article, and postdoctoral professor Deng Mingtian and Associate Professor Wan Yongjie are the co-first authors of this article. The project was funded by the National Natural Science Foundation of China (31672422, 31972569).
The important research progress of lncRNA regulating the mechanism of Hu sheep’s multiple births
The team accurately matched the genotype and fecundity for the first time, screened and identified a new transcript lncRNA-FDNCR in the ovaries of low-proliferation FecB+ Hu sheep, and passed in vitro A series of experiments such as functional verification and mechanism exploration have clarified the regulatory mechanism of lncRNA FDNCR-mediated DCN/TGF-β pathway involved in the follicular development of multiple fetal lake sheep. This research result will provide a basis for analyzing and perfecting the mechanism of multiple births in Hu sheep, and provide theoretical basis and practical guidance for the reproductive regulation and improvement of fecundity of sheep and other domestic animals. It has practical significance for speeding up molecular breeding of mutton sheep.
Lake sheep, as a precious genetic resource protected species in my country, is known for its”multiple births and full-year estrus”. It has been widely introduced across the country for propagation and crossbreeding Breeding is the most popular female parent breed in domestic mutton sheep production, and it is also an ideal target for studying the mechanism of multiple births. At the same time, Hu sheep is one of the few sheep breeds in the world that carries the Fecundity booroola (fecundity booroola) gene (a major gene related to ovulation), and carrying this gene has become an important symbol of fertility. However, in actual production, Professor Wang Feng’s team found that 84%of Hu sheep that gave birth to single lambs carried the homozygous FecB gene, suggesting that there may be a finer regulatory mechanism between FecB and multiple births.
Exploring the mechanism of lncRNA and miRNA regulating the follicular development of multiple fetal lake sheep is the current research hotspot of mutton sheep breeding. For the first time, our team accurately matched genotypes with fecundity. Taking different genotypes of high and low breeding Hu sheep ovary as the research object, using RNA-Seq technology to perform lncRNA sequencing on them, and found that the new transcript MSTRG.98424.7 is in FecB+ Low breeding Hu sheep. It is highly expressed in the group. The total length of the transcript was found to be 1631 nt by RACE technology. It was found that the transcript was mainly located in the granulosa cells of Huyang ovary by FISH technology, and it decreased with the proliferation of follicles. It is suggested that this transcript may be related to follicular development, so it is named FDNCR (Follicular development-associated lncRNA), and further explore the FDNCR-DCN axis, FDNCR-miR-543-3p axis, miR-543- through RIP, FISH and other technologies. The formation mechanism and function verification of the 3p-DCN axis showed that lncRNA FDNCR weakened the inhibitory effect of miR-543-3p on DCN gene expression by adsorbing miR-543-3p, thereby enhancing the effect of DCN gene on TGF-β pathway related genes The inhibitory effect ultimately promotes the apoptosis of Hu sheep granulosa cells, revealing the regulatory mechanism of lncRNA FDNCR-mediated DCN/TGF-β pathway involved in the development of multiple fetal Hu sheep follicles.
The research work is based on the Jiangsu Provincial Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, as the first unit, with post-doctoral faculty Yao Xiaolei as the first author, and Professor Wang Feng As the corresponding author, the research was funded by the National Natural Science Foundation of China (31872357; 32002174) and the Central University Basic Research Project (KYYJ202001). In recent years, the team of Professor Wang Feng has focused on the breeding of new breeds of multiple-feet Huyang sheep and the excavation of key genes for characteristic traits. In 2019, the Huyang Research Institute of Nanjing Agricultural University was established. In 3 years, 5 graduate students were sent to the national Hu sheep breeding farm in turn, and based on genetic background, production performance measurement, lambing rate, etc., a core group of high breeding Hu sheep (≥3 per litter) was established and more than 1,000. And choose to keep more than 100 low-breasted lake sheep (1 per fetus). The WGBS and RNA-Seq technologies have been used successively to conduct systematic screening and functional verification of DNA methylation and lncRNA on the Hypothalamus-Pituitary-Ovarian axis and the uterus of Hu Sheep, systematically revealing the mechanism of multiple births in Hu Sheep.
link:https://doi.org/10.1016/j.omtn.2021.02.016; https://doi.org/10.1016/j.omtn. 2021.02.030