Reciprocal Cross Differences In Growth Performance And Identification And Expression Of Microrna In The Pig

In genetics, the result of reciprocal cross is an important way to judge the genetic basis. Since it has significant differences of Large White and pure Erhualian in growth, carcass traits, meat quality, we studied reciprocal cross F1 generation differences of Large White and Erhualian in important economic traits to explore the possible mode of inheritance of these important economic traits.So far, hundreds of MicroRNA (miRNA) were found in humans, mice and rats and other mammals. Although several research groups have tried to identify more pig miRNA, but most of porcine miRNA is unknown. The complexity of miRNA gene regulatory networks (one miRNA can regulate multiple genes, a gene can also be affected by a number of regulatory miRNA) decided that the study of regulation of porcine miRNA is incomprehensive. Therefore, the study of identification and expression of new porcine miRNA is important.In this study, the reciprocal cross differences of Large White and Erhualian pigs in carcass characteristics, meat quality, fat acid and AA profiles of muscle in d 180 of finishing pigs, and the reciprocal cross differences of Large White and Erhualian pigs in growth and development in seven age stages were determined. Computational identification of new porcine microRNAs and their targets, miRNA expression profiles of porcine skeletal muscle, procine miRNA precursor cloning and sequencing methods, and real-time PCR method of procine miRNA were studied in this dissertation.1. Reciprocal cross differences of Large White and Erhualian pigs in carcass characteristics, meat quality traits, fat acid and amino acid profiles of muscleThe reciprocal cross differences of Large White and Erhualian pigs in carcass characteristics, meat quality, fat acid and AA profiles of muscle in d 180 of finishing pigs (n=52) were determined. There were no convincing differences in BW at slaughter, dressing percent, midline backfat thickness (BT) of first rib and last lumbar, skin thickness (ST), carcass length (CL), fat percent, bone percent, skin percent, meat color of LM, 45-min pH value (pH1), Water-holding capacity, tenderness and muscle fiber diam. (P> 0.05). But there were obvious differences in middle BT of 6-7th rib and last rib, LM area (LMA), hind leg percent, meat percent of hind leg, meat percent and intramuscular fat (IMF) content only in barrows (P< 0.05). Barrows from Large White sires had 57-60 mm less BT at the 6-7th and last rib (P< 0.01),4.69 cm2 larger LMA,1.71% larger hind leg percent, 4.21% larger meat percent of hind leg,2.82% larger meat percent of carcass (P<0.05) and 2.56% larger IMF content (P<0.01) than did barrows produced by Erhualian sires. There were no convincing differences in all fatty acids and AA of LM and biceps femoris (BF) muscle in gilts (P>0.05). But the LM of barrow pigs sired by Large White have higher concentration of myristic acid (14:0; P=0.039), palmitic acid (16:0; P=0.024) and oleic acid (18:1; P=0.022) compared with those sired by Erhualian. Barrow pigs sired by Erhualian had a higher (P< 0.05) concentration of linoleic acid (18:2; P=0.013) and eicosapentaenoic acid (20:5; P=0.000) in their LM compared with those sired by Large White. The concentrations of total SFA and MUFA in the LM of barrow pigs sired by Large White were higher (P<0.05) compared with those sired by Erhualian. On the other hand, the concentration of PUFA and unsaturated fatty acid in the LM of barrow pigs sired by Erhualian were higher (P<0.05) compared with those sired by Large White. In conclusion, reciprocal cross differences were detected only in barrows for middle BT of 6-7th rib and last rib, LMA, hind leg percent, meat percent of hind leg, meat percent, IMF content and fatty acid profiles of IMF from LM.2. Reciprocal cross F1 generation differences of Large White and Erhualian pigs in growth and developmentIn this study reciprocal cross F1 generation differences of Erhualian and Large White pigs at three gestational ages (50 day,70 day and 90 day) and five postpartum ages (1 day, 20 day,70 day,120 day and 180 day) in the body, tissue and organ weight and size were determined. The results show that there are differences between reciprocal cross F1 generation of Erhualian and Large White pigs in the growth and development, between boars (or barrows) and gilts. Some traits also show a different mode of inheritance. In conclusion, the offspring of female Large White pig revealed some maternal effect. At three gestational ages body weight of EL pigs were larger than LE pigs, but at postpartum ages there were no evident differences (P>0.05) between the two genotypes. 3. Computational identification of new porcine microRN As and their targetsMicroRNAs (miRNAs) represent a newly identified class of non-protein-coding～22 nt small RNAs which play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Here we present an expressed sequence tags (EST) based combined approach for the detection of novel porcine miRNAs. This was initiated by using previously known miRNA sequences from Homo sapiens (human) and Mus musculus (mouse) to blast the databases of Sus scrofa (pig) EST. A total of 65 new miRNAs were detected following a range of filtering criteria. Using these new potential miRNAs sequences, we further get the publicly available porcine mRNA database from NCBI and detected 48586 potential target hits using software RNA hybrid. So far, comparing with human and mouse, less miRNAs (only 54 miRNAs) were identified in Sus scrofa species. These new 65 miRNAs and their targets in pig have been run through miRHelper to yield data that may help us better understand of the possible role of miRNAs in regulating the growth and development of pigs. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes.4. MicroRNA expression profiles of porcine skeletal muscleMicroRNAs are endogenous,-22 nucleotide-long, non-coding RNAs that play important roles in multiple biologic processes by degrading targeted mRNAs or repressing mRNA translation. In order to evaluate the role of miRNA in porcine skeletal muscles, miRNA expression profiles in the present study were investigated by using longissimus muscle tissues sampled from 90-day post-gestation fetuses and 120-day post-partum pigs. First, we used previously known miRNA sequences from human and mouse to blast the databases of porcine-expressed sequence tags (EST), and identified 98 new miRNA candidates following a range of filtering criteria. Subsequently, these miRNA candidates and 73 known miRNAs (miRBase 13.0) from pigs were chosen in porcine miRNA microarray analysis. As a result,16 newly identified miRNAs and 31 previously known miRNAs were detected in porcine skeletal muscle tissues. During later fetal development at d 90, miR-1826, miR-26a, miR-199b and let-7 were highly expressed transcripts, while miR-1a, miR-133a, miR-26a and miR-1826 exhibited greatest abundance during fast-growing stages at d 120. Using the 47 miRNAs detected by the micoarray assay, we further investigated the publicly available porcine mRNA database from NCBI and computed potential target hits using the software "RNAhybrid". Our study has resulted in the identification of 16 new miRNA candidates, computation of potential target hits for 18 miRNA families, and determination of the expression profiles of miRNA in porcine skeletal muscle tissues at different developmental stages. Our findings provide a valuable resource for investigators interested in post-transcriptional gene regulation in the pig and related animals.5. Procine miRNA precursor cloning and sequencing methodsConstruction of endogenous small RNA by the cDNA library made cloning and sequencing approach to large-scale miRNA identification possible, but this method by other non-miRNA sequences of small RNA interference, ofter have a low efficiency of cloning and sequencing. In this study I was trying to increase the cloning and sequencing efficiency of miRNA using library of small RNA, trying to isolated small hairpin cDNA before cloning and sequencing. Adaptors were successfully added to small RNA collected from porcine skeletal muscle tissues. Their separations were separated as a single chain after PCR, and the different secondary structure characters of single-stranded small cDNA were used to separate and clone them. The cloning and sequencing results showed that this method can separate the different secondary structures of small cDNA. The cloning and sequencing results showed single-stranded cDNA isolated have different ratio of hairpin sequences with significant differences (P＜.05), while also discovered the five new candidate miRNA genes. The experimental results also showed that more accurate experiment technology of single-chain separation would have better results.6. Real-time PCR of procine miRNA to determine miR-1 and miR-1826In this study, we used the technology of adding polyA to small RNA, which made us can do more than one miRNA real-time PCR analysis using one reverse transcription sample. In brief, small RNA was first extracted, and then about 20 A was added to the small RNA by polyA polymerase, a primer with 20 T in the 3'end was used to do reverse transcription, at last a miRNA specific primer in upstream and a downstream primer were used to do real-time PCR. In this study we used 5S rRNA as control of miR-1 and miR-1826, which highly expressed in skeletal muscle of pigs. The two miRNA real-time PCR analysis showed that there were not significantly different between reciprocal cross F1 generation of Large White and Erhualian in relative expression level of miR-1 and miR-1826 (P> 0.05), but the expression level of miR-1 in gestational age at d 90 was lower than that at d 180 (P< 0.05), the expression level of miR-1826 in gestational age at d 90 was higher than that at d 180 (P<0.05). The results showed that this method is feasible, and the newly discovered miR-1826 was detected using real time quantitative PCR method, which also verified the expression of miR-1826 in skeletal muscle of pigs.