Enhancement of polysaccharides production using microparticle enhanced technology by Paraisaria dubia.

BACKGROUND: Polysaccharides are important active ingredients in Ophiocordyceps gracilis with many physiological functions. It can be obtained from the submerged fermentation by the anamorph (Paraisaria dubia) of Ophiocordyceps gracilis. However, it was found that the mycelial pellets of Paraisaria dubia were dense and increased in volume in the process of fermentation, and the center of the pellets was autolysis due to the lack of nutrient delivery, which extremely reduced the yield of polysaccharides. Therefore, it is necessary to excavate a fermentation strategy based on morphological regulation for Paraisaria dubia to promote polysaccharides accumulation. RESULTS: In this study, we developed a method for enhancing polysaccharides production by Paraisaria dubia using microparticle enhanced technology, talc microparticle as morphological inducer, and investigated the enhancement mechanisms by transcriptomics. The optimal size and dose of talc were found to be 2000 mesh and 15 g/L, which resulted in a high polysaccharides yield. It was found that the efficient synthesis of polysaccharides requires an appropriate mycelial morphology through morphological analysis of mycelial pellets. And, the polysaccharides synthesis was found to mainly rely on the ABC transporter-dependent pathway revealed by transcriptomics. This method was also showed excellent robustness in 5-L bioreactor, the maximum yields of intracellular polysaccharide and exopolysaccharides were 83.23 ± 1.4 and 518.50 ± 4.1 mg/L, respectively. And, the fermented polysaccharides were stable and showed excellent biological activity. CONCLUSIONS: This study provides a feasible strategy for the efficient preparation of cordyceps polysaccharides via submerged fermentation with talc microparticles, which may also be applicable to similar macrofungi.

The complete mitochondrial genome of Bos taurus coreanae (Korean native cattle).

Korean native cattle is one of the famous native breeds in Korean. In the present work, we report the complete mitochondrial genome sequence of Korean native cattle for the first time. The total length of the mitogenome was 16,339 bp with the base composition of 33.4% for A, 27.2% for T, 26.0% for C, and 13.4% for G, and an A-T (60.6%)-rich feature was detected. It harbored 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding control region (D-loop region). The arrangement of all genes was identical to the typical mitochondrial genomes of cattle. The complete mitochondrial genome sequence of Korean native cattle would serve as an important data set of the germplasm resources for further study.

Haplotype analysis of TLR4 gene and its effects on milk somatic cell score in Chinese commercial cattle.

Bovine mastitis is a very complex and common disease of dairy cattle and a major source of economic losses to the dairy industry worldwide. In this study, the bovine TLR4 was taken as a candidate gene for mastitis resistance. This study aimed to analyze the associations of single nucleotide polymorphisms (SNP) or haplotype and somatic cell score (SCS) in 404 Chinese commercial dairy cattle including Chinese Holstein, Sanhe cattle and Chinese Simmental breeds. The polymerase chain reaction and sequencing methods were used for detecting genotype and allele frequency distribution of the two SNPs (rs8193062, rs8193064), statistical results showed that T allele at rs8193062 and C allele at rs8193064 were the predominate alleles. Moreover, six SNPs, including two SNPs (rs8193062, rs8193064) and four SNPs (rs8193060, rs8193069, rs29017188, rs8193046) which were chosen according the polymorphism level for the same cattle populations in previous studies, were used for haplotype analysis, the results revealed that twenty-one haplotypes were found in the mentioned animals, of which, Hap1 (30.5 %) and Hap2 (30.4 %) were the most common haplotypes. Hap2, Hap4 and Hap12 might negatively effect on milk SCS, whereas Hap13 might positively effect on milk SCS. The results in this study might assist in marker assisted selection and provided some reference to be implemented in breeding programs to improve the mastitis resistance of dairy cattle.

[Integration sites and their characteristic analysis of piggyBac transposon in cattle genome].

As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon.

[Artificial selection for cattle based on high-density SNP markers].

With the implementation of genetic improvement in recent years, artificial selection has greatly improved beef cattle production performance and its genetic basis has been dramatically changed. In this study, based on the Illumina BovineSNP50 (54K) and BovineHD (770K) BeadChip and the FST value, we analyzed the genetic differentiation of cattle and screened the imprints of selection in bovine genome. Finally, we found 47104 OUTLIER SNP loci and 3064 candidate genes, for example, CLIC5, TG, CACNA2D1, and FSHR etc. The biological processes and molecular functions of genes were analyzed through gene annotation.The results of this study established a genome-wide map of selection footprints in beef cattle genome and a clue for in-depth study of artificial selection and understanding of biological evolution.Our results indicate that artificial selection has played an important role in cattle breed genetic improvement.

Effect of castration on carcass quality and differential gene expression of longissimus muscle between steer and bull.

The effect of castration on carcass quality was investigated by ten Chinese Simmental calves. Five calves were castrated randomly at 2 months old and the others were retained as normal intact bulls. All animals were slaughtered at 22 months old. The results showed that bulls carcass had higher weight (P < 0.05), dressing percentages and bigger longissimus muscle areas (P < 0.05) than steers. But steer meat had lower shear force values and was fatter (P < 0.05) than bull. Furthermore, in order to discover genes that were involved in determining steer meat quality, we compared related candidate gene expression in longissimus muscle between steer (tester) and bull (driver) using suppressive subtractive hybridization. Ten genes were identified as preferentially expressed in longissimus muscle of steer. The expression of four selected differentially expressed genes was confirmed by quantitative real-time PCR. Overall, a 1.96, 2.41, 2.89, 2.41-fold increase in expression level was observed in steer compared with bull for actin, gamma 2, smooth muscle, tropomyosin-2, insulin like growth factor 1 and hormone-sensitive lipase, respectively. These results implied that these differentially expressed genes could play an important role in the regulation of steer meat quality.

Single nucleotide polymorphism of CACNA2D1 gene and its association with milk somatic cell score in cattle.

The objective of the present study was to identify polymorphisms of the CACNA2D1 gene, and to analyze associations between these polymorphisms and mastitis in several cattle breeds. Through PCR-RFLP methods and DNA sequencing, an allelic variant corresponding to the A→G mutations and Aspartic (Asp) to Glycine (Gly) amino acid replacement at positions 526745 in the exon 25 of bovine CACNA2D1 gene could be detected. Two alleles, A and G, and three genotypes, AA, AG and GG were defined. Genetic character in the studied populations indicated that the A526745G loci of CACNA2D1 gene was moderate polymorphism and fitted with Hardy-Weinberg equilibrium (P > 0.05). The effects of CACNA2D1 polymorphisms on somatic cell score (SCS) were analyzed and significant association was found between A526745G and SCS. The mean of genotype GG was significantly lower than those of genotype AG and AA (P = 0.0469). Information provided in this research could be useful in further studies to determine the role of CACNA2D1 gene in the mastitis resistance.

Association of thyroglobulin gene variants with carcass and meat quality traits in beef cattle.

Thyroid hormones play an important role in regulating metabolism and can affect homeostasis of fat deposition. The gene encoding thyroglobulin (TG), producing the precursor for thyroid hormones, has been proposed as a positional and functional candidate gene for a QTL with an effect on fat deposition. In the present study, we identified 6 novel SNPs at the 3' flanking region of the TG gene. The SNP marker association analysis indicated that the T354C, G392A, A430G and T433G SNP markers were significantly associated with marbling score (P < 0.05). Animals with the new homozygote genotype had higher marbling score than those with the other genotypes. Otherwise, the linkage disequilibrium analysis indicated that these four SNPs were completely linked (r (2) = 1). Results from this study suggest that TG gene-specific SNP may be a useful marker for meat quality traits in future marker assisted selection programs in beef cattle.

Genetic polymorphisms of the CACNA2D1 gene and their association with carcass and meat quality traits in cattle.

The objective of this study was to identify genetic polymorphisms of the CACNA2D1 gene and to analyze associations between SNPs and carcass and meat quality traits in cattle. Through PCR-RFLP and DNA sequencing methods, a new allelic variant corresponding to the A --> G mutation (aspartic to glycine amino acid replacement) of the bovine CACNA2D1 gene was detected. Two alleles and three genotypes (AA, AG, and GG) were defined. Genetic character indicated that the A526745G locus showed moderate polymorphism and was in Hardy-Weinberg equilibrium. Gene-specific SNP marker association analysis showed that the A526745G mutant was significantly associated with carcass weight, dressing percentage, meat percentage, and backfat thickness. The results add new evidence that CACNA2D1 is an important candidate gene for the selection of carcass and meat quality traits in the cattle industry.

Polymorphisms in MC4R gene and correlations with economic traits in cattle.

MC4R contributes to the control of food intake and energy expenditure, and single nucleotide polymorphisms (SNPs) in the MC4R gene have clearly been associated with backfat depth, feed intake and growth rate in pig. Our objectives were to scan the complete coding region by sequencing in samples from eight cattle breeds, to estimate the frequency of the SNPs in the MC4R gene and to determine if individual genotypes were associated with several economic traits. Five polymorphisms were detected at position 19 (C/A), 20 (A/T), 83(T/C), 128 (G/A), and 1069 (G/C), and the last one was significantly associated with backfat thickness value (P < 0.01, n = 245). The linkage disequilibrium analysis indicated that the SNP markers C19A, A20T, T83C and G128A were completely linked (r (2) = 1).

[Effects of Chinese medicine for regulating "sea of blood in brain" combined with bone marrow stromal stem cell transplantation on angiogenesis in ischemic brain tissue of rats].

OBJECTIVE: To observe whether Naomai Yihao (NM) Capsule, a compound traditional Chinese herbal medicine for regulating the "sea of blood in brain", and bone marrow stromal stem cell (BMSC) transplantation could improve angiogenesis in focal cerebral ischemia in rats. METHODS: A rat model of middle cerebral artery occlusion (MCAO) was established. The rats were divided into untreated group, NM group, BMSC group and combination of NM and BMSC group (combined treatment group). Another 8 normal rats were selected as sham-operated group. After 3-, 7- and 14-day reperfusion, behavioral rating scale (BRS) of the rats and histopathology of the cerebral tissue were evaluated. Expression of CD31 in the brain tissue was also measured by immunohistochemical method. RESULTS: Compared with the untreated group, BRSs of the NM group, BMSC group and combined treatment group were decreased significantly (P=0.000), but there was no interacting effect between NM and BMSC transplantation. Compared with the untreated group, the numbers of CD31 positive cells in NM group, BMSC group and combined treatment group were increased significantly (P=0.000), and there were interacting effects among NM, BMSC transplantation, and the observation time (P<0.01). After 14-day reperfusion, combination of NM and BMSC transplantation could largely increase the number of CD31 positive cells. CONCLUSION: NM is able to promote the angiogenesis and neurological impairment improvement in focal cerebral ischemia of rats which are administered with BMSC transplantation, and the effect is reinforced with the extension of treatment time.

Association analysis of thyroglobulin gene variants with carcass and meat quality traits in beef cattle.

Thyroid hormones play an important role in regulating the metabolism and can affect the homeostasis of fat deposition. The gene encoding thyroglobulin (TG), producing the precursor for thyroid hormones, has been proposed as a positional and functional candidate gene for a QTL with an effect on fat deposition. In the present study, we identified 6 novel SNPs at the 3' flanking region of the TG gene. The SNP marker association analysis indicated that the SNP markers G133C, G156A, C220T and A506C were significantly associated with marbling score (P<0.05, N=271). Animals with the new homozygote genotype had higher marbling scores than those with the other genotypes. Besides, the linkage disequilibrium analysis indicated that these 4 SNPs were completely linked (r2 = 1). Results of this study suggest that the TG-gene-specific SNP may be a useful marker for meat quality traits in future marker-assisted selection programmes in beef cattle.

[Relationship between genotypes at MyoD locus and carcass traits in cattle].

A 261 bp sequence of the bovine MyoD gene intron 2 was cloned and was found to bear no similarities to the human MyoD gene sequence. Polymorphisms of the Myod gene in cattle including three Chinese breeds (Luxi cattle, Jinnan cattle and Qinchuan cattle) and four cross-breeding populations (Limousin x Luxi cattle, Simmental x Luxi cattle, Charolais x Luxi cattle and Angus x Luxi cattle) were detected by PCR-SSCP. Two SSCP alleles (A and B) were detected, which caused by a C-->T at 39 bp and a C-->G transition at 112 bp. Only two genotypes AA and AB occurred in the population. In Chinese local cattle, B allele was dominant, and this locus was at Hardy-Weinberg equilibrium except for the Charolais x Luxi cattle and Angus x Luxi cattle. The association of these polymorphisms with cattle carcass traits was analyzed using the general linear model (GLM). Statistical analysis revealed a higher value of living weight, carcass weight and loin eye area for individuals with genotype AA than AB (P < 0.05). Further studies on a bigger population size are needed to confirm the observed effect of MyoD genotypes on carcass quality traits.