Identification, Selection And Functional Research Of MiRNAs In Dairy Goat Mammary Gland

Dairy goats(Capra hircus) is an important economic species, whose milk has higher nutrition value than cow milk and is easily to absorb, so it is appropriate for people to drink and become an important raw material for the modern dairy industry. It is extremely necessary to study mammary gland development, lactation and milk fat metabolism because that the characteristics of goat milk are closely related to lipid metabolism in mammary gland. However, there are few studies about lipid metabolism in goat milk, and most of them mainly focused on exploration and analysis of gene function, lack of studies on a post-transcriptional level, such as microRNAs(miRNAs) level.In recent years, with the development of high-throughput sequencing and bioinformatics, a series of miRNAs are identified and submitted to miRBase, while there is no goat miRNA sequence available in miRBase 20.0(June 2013). In previous studies, the solexa sequencing results of goats are generally mapped to its related species, such as cattle, sheep to identified its miRNA transcriptome because of no goat genome is available. In this study, a more reliable goat miRNA transcriptome in lactation period is obtained by mapping the solexa sequencing results with goat genome, and the characteristics of goat mi RNA transcriptome were identified by analyzing miRNA sequences and its location on goat genome, then some miRNAs which may play a vital role in regulating goat mammary gland development, lactation and fat metabolism can be found by screening and validating differentially expressed miRNAs in different lactation periods. The main results are as follows:1. 24,479,196 raw data were detected in mid-lactation period of goat mammary gland by solexa sequencing technology, then 21,517,296 clean reads which accounted for 87.9% of raw data were finally selected after a series of mass filter. By analyzing the length of clean read, a conclusion can be drawed that the length of clean read is mainly between 21-23 nt, with 22 nt get the most reads. By merging clean reads with the same sequence into a unique reads and mapping it with annotated mammalian pre-miRNAs, mature miRNAs in miRBase 20.0 and the goat genome, 821 pre-miRNAs and 1059 mature miRNAs were finally got, which includes 796 conserved miRNAs and 263 new miRNAs.2. The analysis about the first base preference of 1059 mature miRNAs, revealed that the first base of miRNAs showed a strong preference for U base, but have some resistance to G. And the sequence analysis for 796 conserved mi RNAs showed that compared with annotated sequences, most of goats miRNAs sequences are changed in the 3’ end and result in isomiR, which means most miRNAs in goat miRNA transcriptome are present in the form of isomiR.3. Compared 796 conserved miRNAs with 24 species of mammals miRNAs submitted in miRBase 20.0 database, we can find that goats and cow have the largest number of same miRNAs, which is about 404, while the goats and sheep have fewer homologous miRNAs for only 102 miRNAs.4. 796 pre-miRNAs can mapped to goat chromosome sequences when compared 821 pre-miRNAs to goat genome, while the remaining 25 pre-miRNAs are not matched. Further analysis of pre-miRNA density on each chromosomes showed that the density of pre-miRNAs on goat chromosomes are between 1.1-0.11 / Mbp, among which the density on chromosome 9 is the minimum, while the density on chromosome 21 is the maximum with 1.1/Mbp.5. miRNAs are clustered in the genome arrangement, so the maximum inter-distance are defined as 3Kb, 5Kb, 10 Kb and 50 Kb respectively for the mi RNA cluster analysis. The results showed that when MID is 3Kb, 429 miRNAs can form 86 clusters, and when MID is 5Kb, 10 Kb and 50 Kb, 442, 460, 501 miRNAs can form 87, 90, 99 clusters respectively.6. MiRNAs which contain the identical seed sequence are defined as a miRNA family. By analyzing the miRNA family of 1059 miRNA, we can found that there are 479 mi RNAs can form a total of 164 miRNA families, and the let-7 family was found to be the largest one which including 12 members, while the remaining 573 miRNAs can not form a miRNA family because they do not contain the same seed with other sequences.7. Reverse transcription primers and quantitative primers for U2, 18 s rRNA, 5s rRNA gene were designed, then RT-qPCR and identification the amplified products were used to test the primers, after all, 5s rRNA were ultimately determined to be the internal control for miRNA S-Poly(T) quantitative detection.8. 37 differentially expressed miRNAs during lactation of goat mammary gland were obtained by comparing the sequencing results with the reported findings, then RT-qPCR of these miRNAs was performed in different lactation period of mammary gland, including early lactation, peak lactation, middle lactation and dry period. Results showed that all miRNAs, except for miR-222_R + 2, have significant difference in the expression among four different lactation periods.9. After overexpressed miR-145 in goat mammary epithelial cells, oil red O staining and total cellular triglyceride content assay were perform to found that miR-145 can not only significantly increased the content of lipid droplets in cells, but also promote the intracellular triglyceride accumulation.Conclusion: identification and characteristic analysis of the goat miRNA transcriptome in lactation period were accomplished, meanwhile 36 miRNAs were screened and preliminary validated which may involve in goat mammary gland development, lactation and milk fat metabolism.