MicroRNA (miRNA, miR) is a kind of endogenous, non-coding small RNA which widely presents in organisms. MiRs enter and bind to the target mRNA (messenger RNA) through complementary base pairing, thus regulate the abundance and function of target mRNAs [1–3], and they play important roles in cell differentiation, proliferation and apoptosis[4, 5]. First found in Caenorhabditis elegans, miRs have been detected in a wide range of animals, plants and viruses[6, 7]. In 2002, miRs were first found in plants.
cv.Zhongmu 1 (Medicago sativa L.) is a salt-tolerant alfalfa variety developed by the Chinese Academy of Agricultural Sciences . Meanwhile, cv.Xinyan 52 is a new salt-tolerant alfalfa bred by the Agricultural School of Ningxia University. Current research of alfalfa miRs focuses on the regulation of alfalfa stem development, salt stress resistance, drought resistance, fall dormancy and biological yield improvement [10, 11]. However, there are a few reports about the cross-kingdom roles of miRs in alfalfa.
Some plant-derived miRs have been detected in human and animal sera, which were named exogenous miRNAs (xeno-miRNAs). One of the earliest mentions of xenomiRNA was reported by Zhang et al. . Studies have found that proteins, amino acids, vitamins, flavonoids and other nutrients and chemical components, as well as unknown growth promoting factors, which can improve milk yield of dairy cows are riched in alfalfa[13, 14]. Furthermore, feeding alfalfa can affect the miRs expression profile of rumen, duodenum, liver and mammary gland, and change the expression patterns of 35 miRs, which are positively correlated with N utilisation efficiency, while 10 miRs are negatively correlated with miRs in breast tissue, with significant improvements in N conversion efficiency and protein yield (P < 0.01) . miRs in mammary gland can affect N-conversion efficiency, such as miR-181a affacts milk protein content by targeting AA transport and phosphorylation gene. In addition, xenomiR-168, miR-166 and miR-156 have detected in bovine blood.
Milk is rich in nutrients, which can be easily absorbed by human body. Milk is an important source of nutrition for mammalian infants, a large number of studies have shown that milk is rich in plant food-derived miRNAs. Anna Lukasik et al. detected five plant-derived miRNAs in whole milk samples of human milk of healthy volunteers by RT-qPCR, including common miR156, miR166 and miR168 with cross-border regulation. And they detected 17 plant-derived miRNAs in pig breast milk, of which miR-168 had the highest expression. Xi Chen et al. found through bioinformatics analysis that although the abundance of plant miR in breast milk is lower than that in saliva and urine, 151 miRNAs can still be detected, of which miR166 is the most common miRs in body fluid.
Based on these prior studies, it is evident that we pay attention to whether miRNAs in alfalfa, a common cow diet, can be detected in cow body fluid and play a cross-border regulatory role. Therefore, in this study, miRNAs in two alfalfa strains were sequenced and analyzed. Three alfalfa derived miRNAs were selected to detect their expression in cow body fluid. In addition, the target gene of miR168 with high expression in normal milk was verified. The data in this study are based on a large number of RNA sequencing data and quantitative verification, strongly support the existence and role of alfalfa derive miRNA in cross-kingdom regulatory action within dairy cows.