Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and vali...Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.展开更多
The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl oper...The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl (the gyl promoter) was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.展开更多
基金Supported by the National Natural Science Foundation of China(No.41176113)the National Basic Research Program of China(973 Program)(No.2010CB126403)+1 种基金the Changjiang Scholars Program for Innovative Research Team in Universities(No.IRT0941)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.nycytx-47)
文摘Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.
基金supported by grants from the Ministry of Science and Technology of China (2009CB118905)
文摘The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl (the gyl promoter) was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.
