Short communication: Characterization of gene expression profiles related to yak milk protein synthesis during the lactation cycle.

This research assessed the gene expression patterns related to the synthesis of milk in yak, which is characterized by high fat and protein content but low yield. The yak (Bos grunniens) is one of the most crucial domestic animals in Tibetan life; however, the genetic and molecular factors underlying yak milk protein synthesis remain understudied. Yak mammary biopsies harvested during late-pregnancy (d -15) through the end of subsequent lactation (d 1, 15, 30, 60, 180, and 240) were used to evaluate gene expression via real-time quantitative PCR. The expression pattern of 41 genes encompassing multiple pathways integral to milk protein synthesis including insulin, mammalian target of rapamycin (mTOR), 5' AMP-activated protein kinase, Jak2-Stat5 signaling, and the expression of glucose and AA transporters was evaluated. Our results confirmed that most upregulated genes increased from d -15 and peaked at d 30 or 60 and then remained relatively highly expressed. Specifically, there was an increased expression of mTOR-related amino acid transporters (SLC1A5, SLC7A5, and SLC36A1), glucose transporters (SLC2A1, SLC2A3, and SLC2A8), Jak2-Stat5 pathway (ELF5), and insulin signaling pathway components (IRS1, PDPK1, and AKT1). For activation of proteins synthesis, MTOR was significantly increased only at d 1. Among inhibitors of mTOR signaling, TSC1 and PRKAA2 were significantly upregulated during lactation. The RPL23 was downregulated among ribosomal components. In conclusion, a critical role for AA and glucose transporters and insulin signaling through mTOR for regulation of yak milk protein synthesis was revealed in this study of the yak mammary gland.

Characterisation of gene expression related to milk fat synthesis in the mammary tissue of lactating yaks.

This research communication describes the profile of gene expression related to the synthesis of yak milk as determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Significant up-regulation during lactation were observed in genes related to fatty acid (FA) uptake from blood (LPL, CD36), intracellular FA transport (FABP3), intracellular FA activation of long- and short-chain FAs (ACSS1, ACSS2, ACSL1), de novo synthesis (ACACA), desaturation (SCD), triacyglycerol (TAG) synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (PLIN2, BTN1A1, XDH), ketone body utilisation (BDH1, OXCT1), and transcription regulation (THRSP, PPARGC1A). In particular, intracellular de novo FA synthesis (ACSS2, ACACA, and FABP3) and TAG synthesis (GPAM, AGPAT6, and LPIN1), whose regulation might be orchestrated as part of the gene network under the control of SERBF1 in the milk fat synthesis process, were more activated compared to levels in dairy cows. However, the genes involved in lipid droplet formation (PLIN2, XDH, and BTN1A1) were expressed at lower levels compared to those in dairy cows, where these genes are mainly controlled by the PPARG regulator.

Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

[Identification of Regulatory Sequence of lip Genes of Phanerochaete chrysosporium under Lignin-degradation.].

Eleven subcloned DNA fragments from the 5'- upstream region of lipA, lipC and lipF of Phanerochaete chrysosporium were assayed by using the gel mobility shift assay(GMSA). The total proteins extracted from P.chrysosporium mycelia grown in Kirk medium and natural fir wood chip were used to identify the segments in these 11 DNA fragments which are controlled by some regulatory proteins. The results showed that two DNA segments LG2P3(396bp) and LG6S1-2 (738bp) in the 5'-noncoding regions of lipC and lipF were able to specifically bind total mycelial proteins of P. chrysosporium incubated in Kirk medium, separately. One DNA segment LG6S2 (226bp) from the 5'-noncoding region of lipF was found to specifically bind total mycelial proteins of this fungus on natural fir wood chip. Analysis of the sequences showed that there were many cis-regulatory elements in these DNA segments, implying that these sequences may be bound by some transcriptional regulation protein factors.