Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Effect of the Flanking Sequence Architecture of Translation Initiation AUG Codon on Gene Expression Level in Rice

The relationship between the codon usage bias, gene expression level and the AUG context(from -20 to +6 positions relative to the initiator AUG codon) was examined in 541unigene sequences of rice. A significant correlation for CAI values (codon adaptationindex) was observed at five nucleotide positions (-19, -18, -9, -4, +5), eight (-19, -18,-14, -9, -6, -4, -1, +5) for CPP (codon preference parameter), and seven (-18, -16, -15,-9, -7, -1, +6) for mRNA abundance in the flanking sequence of the initiator AUG codonrespectively, but a significantly positive correlation for both CAI and CPP at twopositions (-4 and +5), indicating that both those positions are evolutionally under thenatural selection constraint at the translational level. By site-directed mutagenesis atseven specific positions (-18, -16, -15, -9, -7, -1 and +6) for allergenic protein thathad the highest mRNA abundance in this study, its expression level decreased dramatically63.3 and 72.5% respectively, indicating the importance of those 7 positions for geneexpression. A highly positive correlation (r=0.625, P<0.01) between AUGCAI and GCcontent in the flanking sequence of the initiator AUG codon showed a more effectivehigher GC content on translation initiation efficiency. The strong preference for G orC at those 8 positions (-6, -5, -3, -2, -1, +4, +5 and +6) in the AUG context suggestedthat an important factor in modulation of the translation efficiency, as well assynonymous codon usage bias, particularly in highly expressed genes.

Cloning and characterization of geranylgeranyl diphosphate synthetase from Pinus massoniana and its correlation with resin productivity

Geranylgeranyl pyrophosphate synthetase(GGPPS) has gained increasing attention as a key enzyme in terpene analysis.We designed specific primers based on plant GGPPS homologs and used reverse transcription polymerase chain reaction(RT-PCR) to obtain and identify Pin GGPPS,a GGPPS gene sequence from Pinus massoniana,using bioinformatics tools.Quantitative PCR analysis of Pin GGPPS expression levels in roots,pine needles,immature stems,and semilignified stems from 6-month-old P.massoniana showed that expression levels of Pin GGPPS were highest in pine needles,followed by immature stems and semilignified stems,and lowest in roots.When we examined the correlation between Pin GGPPS gene expression levels and resin productivity in 20 adult plants for 28 successive days,Pin GGPPS expression levels presented a substantially linear distribution when plotted against their corresponding resin yields.In summary,we characterized the gene Pin GGPPS for the first time in P.massoniana,and established a correlation between Pin GGPPS gene expression levels and resin productivity,suggesting the importance of theory and production practice for P.massoniana.