猪CAⅢ基因CDS区克隆、生物信息学及组织表达分析
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摘要
对猪碳酸酐酶Ⅲ(Carbonic anhydraseⅢ,CAⅢ)基因进行克隆及生物信息学分析,并分析该基因的时空表达特性。通过RT-PCR和克隆测序技术获得猪CAⅢ基因的CDS序列,综合利用多种生物信息学软件,对该基因编码的蛋白质生物学特性进行预测,采用qRT-PCR技术检测CAⅢ基因的组织表达谱和时序表达规律。结果表明,猪CAⅢ基因CDS区长783 bp,编码260个氨基酸;CAⅢ蛋白为亲水性碱性蛋白,无信号肽,主要在细胞质中发挥作用;CAⅢ基因在心脏、肝脏、胰脏、肺、胃、背最长肌、皮下脂肪、脾脏、盲肠等9种组织中均有表达,但在背最长肌中表达量最高,极显著地高于其他组织。从初生到5月龄,大白猪背最长肌中CAⅢ基因 mRNA表达量呈上升—下降—上升—下降的趋势,初生时最低,随后表达量逐渐升高,到3月龄时达到峰值,之后开始下降;马身猪中,从初生到5月龄,CAⅢ基因 mRNA的表达量基本呈上升趋势,在5月龄时达到峰值,极显著地高于其他各个阶段。2个不同品种相比,3月龄和4月龄时,大白猪CAⅢ mRNA表达量显著或极显著地高于马身猪;而在5月龄时,马身猪CAⅢ mRNA表达量极显著地高于大白猪,其他阶段2个品种CAⅢ mRNA表达量无显著差异。CAⅢ基因可能直接或间接参与猪骨骼肌的生长发育过程。
Carbonic anhydrase Ⅲ(CA Ⅲ) gene cloning and bioinformatics analysis were carried out, and the spatiotemporal expression characteristics of the CAⅢ gene were investigated in this study. The CDS sequence of CAⅢ gene of pig was obtained by RT-PCR and cloning and sequencing techniques, and the biological characteristics of CAⅢ protein were predicted by using various bioinformatics softwares. The mRNA expression patterns CAⅢ gene was conducted by qRT-PCR. The results showed that the CAⅢCDS sequence of pig was 783 bp, which encoded a protein with 260 amino acids. CAⅢ protein was a hydrophilic basic protein with no signal peptide, which played a major role in the cytoplasm. CAⅢ gene was expressed in nine various tissues, such as in heart, liver,pancreas, lung, stomach, longissimus dorsi, subcutaneous fat, spleen, and cecum. The highest expression was in longissimus dorsi, which was greater than that in other tissues. The mRNA expression trend of CA Ⅲ gene in Large White pig was in the trend of ascending-descending-ascending-descending from birth to five-month-old. The expression was the lowest at birth, then increased with age increase, reached the peak at three-month-old, afterwards, the mRNA expression of CAⅢ gene was decreased. In Mashen pig, the mRNA expression of CAⅢ gene was increased with age increase from birth to five-month-old, and reached the peak at five-month-age,which was the greatest than those of other stages. Compared with the expression in these two pig breeds, the expression in Large White pig was greater than that in Mashen pig at three-month-old and at four-month-old. However, the expression in Mashen pig was greater than that in Large White pig at five-month-old. There was no significant difference between Large White pig and Mashen pig at other ages.The results in this study indicated that CAⅢ gene could participate directly or indirectly the process of growth and development of skeletal muscle in pig.
Carbonic anhydrase Ⅲ(CA Ⅲ) gene cloning and bioinformatics analysis were carried out, and the spatiotemporal expression characteristics of the CAⅢ gene were investigated in this study. The CDS sequence of CAⅢ gene of pig was obtained by RT-PCR and cloning and sequencing techniques, and the biological characteristics of CAⅢ protein were predicted by using various bioinformatics softwares. The mRNA expression patterns CAⅢ gene was conducted by qRT-PCR. The results showed that the CAⅢCDS sequence of pig was 783 bp, which encoded a protein with 260 amino acids. CAⅢ protein was a hydrophilic basic protein with no signal peptide, which played a major role in the cytoplasm. CAⅢ gene was expressed in nine various tissues, such as in heart, liver,pancreas, lung, stomach, longissimus dorsi, subcutaneous fat, spleen, and cecum. The highest expression was in longissimus dorsi, which was greater than that in other tissues. The mRNA expression trend of CA Ⅲ gene in Large White pig was in the trend of ascending-descending-ascending-descending from birth to five-month-old. The expression was the lowest at birth, then increased with age increase, reached the peak at three-month-old, afterwards, the mRNA expression of CAⅢ gene was decreased. In Mashen pig, the mRNA expression of CAⅢ gene was increased with age increase from birth to five-month-old, and reached the peak at five-month-age,which was the greatest than those of other stages. Compared with the expression in these two pig breeds, the expression in Large White pig was greater than that in Mashen pig at three-month-old and at four-month-old. However, the expression in Mashen pig was greater than that in Large White pig at five-month-old. There was no significant difference between Large White pig and Mashen pig at other ages.The results in this study indicated that CAⅢ gene could participate directly or indirectly the process of growth and development of skeletal muscle in pig.
引文
[1] LOMELINO C L,ANDRING J T,MCKENNA R. Crystallography and its Impact on carbonic anhydrase research[J]. International Journal of Medicinal Chemistry,2018,2018:1-21.
[2]尚西亮,鲍苑苑,任惠民,等.碳酸酐酶Ⅲ在疾病和肌肉疲劳发生发展中的作用[J].生命科学,2011(5):429-433.
[3] WANG H L,ZHU Z M,WANG H,et al. Molecular characterization and association analysis of porcine CA3[J]. Cytogenetic and Genome Research,2006,115(2):129-133.
[4]曹景莹,杨文宇,周宇,等.碳酸酐酶与消化系统疾病关系的研究进展[J].海南医学,2018(11):1568-1570.
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[6] VULLO D,NISHIMORI I,SCOZZAFAVA A. Carbonic anhydrase activators:Activation of the human cytosolic isozyme Ⅲ and membrane-associated isoform IV with amino acids and amines[J]. Bioorganic&Medicinal Chemistry Letters,2008,18(15):4303-4307.
[7] DAI H Y,HONG C C,LIANG S C,et al. Carbonic anhydrase Ⅲ promotes transformation and invasion capability in hepatoma cells through FAK signaling pathway[J]. Molecular Carcinogenesis:Published in Cooperation with the University of Texas MD Anderson Cancer Center,2008,47(12):956-963.
[8] NISHITA T,HARADA T,SAKANOUE H,et al. Purification of swine carbonic anhydrase isoenzyme Ⅲ and measurement of its levels in tissues and plasma[J]. Journal of Animal Physiology and Animal Nutrition,2014,98(1):119-127.
[9]MC M,G K,U R,et al. Carbonic anhydrase Ⅲ regulates peroxisome proliferator-activated receptor-γ2[J]. Experimental Cell Research,2012,318(8):877-886.
[10] R?IS?NEN S R,LEHENKARI P,TASANEN M,et al. Carbonic anhydrase Ⅲ protects cells from hydrogen peroxide-induced apoptosis[J]. The FASEB Journal,1999,13(3):513-522.
[11] SHI C,UDA Y,DEDIC C,et al. Carbonic anhydrase Ⅲ protects osteocytes from oxidative stress[J]. The FASEB Journal,2017,32(1):440-452.
[12] GAILLY P,JOURET F,MARTIN D,et al. A novel renal carbonic anhydrase type Ⅲ plays a role in proximal tubule dysfunction[J].Kidney International,2008,74(1):52-61.
[13] TRIPP B C,SMITH K,FERRY J G. Carbonic anhydrase:new insights for an ancient enzyme[J]. Journal of Biological Chemistry,2001,276(52):48615-48618.
[14] ZIMMERMAN U J P,WANG P,ZHANG X,et al. Anti oxidative response of carbonic anhydrase Ⅲ in skeletal muscle[J]. IUBMB Life,2004,56(6):343-347.
[15]涂江龙,都爱莲,任惠民.碳酸酐酶Ⅲ的生物学特性及与临床疾病的关系[J].中国临床神经科学,2006,14(3):314-317.
[16]HUNTER T J C O I C B. Tyrosine phosphorylation:thirty years and counting[J]. Current Opinion in Cell Biology,2009,21(2):140-146.
[17]彭春霞,高艳明.碳酸酐酶生理功能的研究进展[J].北京大学学报(医学版),2007,39(2):210-212.
[18] LIU Y G,XIONG Y Z,DENG C Y. Isolation,sequence analysis and expression profile of a novel swine gene differentially expressed in the longissimus dorsi muscle tissues from Landrace×Large White cross combination[J]. Acta Biochimica et Biophysica Sinica,2005,37(3):186-191.
[19] LYNCH C J,JR B W,VARY T C,et al. Carbonic anhydrase Ⅲ in obese Zucker rats[J]. American Journal of Physiology-Endocrinology and Metabolism,1993,264(4):621-630.
[20] LYONS G E,BUCKINGHAM M E,TWEEDIE S,et al. Carbonic anhydrase Ⅲ,an early mesodermal marker,is expressed in embryonic mouse skeletal muscle and notochord[J]. Development,1991,111(1):233-244.
[21]PLACZEK M,JESSELL T M,DODD J. Induction of floor plate differentiation by contact-dependent,homeogenetic signals[J]. Development,1993,117(1):205-218.
[22] EDWARDS Y H,TWEEDIE S,LOWE N,et al. Carbonic anhydrase 3(CA3),a mesodermal marker[J]. Symposia of the Society for Experimental Biology,1992,46:273.
[23] JEFFERY S,EDWARDS Y,CARTER N J B G. Distribution of CAⅢ in fetal and adult human tissue[J]. Biochemical Genetics,1980,18(9/10):843-849.
[24]CARTER N,JEFFERY S,SHIELS A,et al. Characterization of human carbonic anhydrase Ⅲ from skeletal muscle[J]. Biochemical Genetics,1979,17(9/10):837-854.
[2]尚西亮,鲍苑苑,任惠民,等.碳酸酐酶Ⅲ在疾病和肌肉疲劳发生发展中的作用[J].生命科学,2011(5):429-433.
[3] WANG H L,ZHU Z M,WANG H,et al. Molecular characterization and association analysis of porcine CA3[J]. Cytogenetic and Genome Research,2006,115(2):129-133.
[4]曹景莹,杨文宇,周宇,等.碳酸酐酶与消化系统疾病关系的研究进展[J].海南医学,2018(11):1568-1570.
[5]SLY W S. Human carbonic anhydrases and carbonic anhydrase deficiencies[J]. Annual Review of Biochemistry,1995,64(1):375-401.
[6] VULLO D,NISHIMORI I,SCOZZAFAVA A. Carbonic anhydrase activators:Activation of the human cytosolic isozyme Ⅲ and membrane-associated isoform IV with amino acids and amines[J]. Bioorganic&Medicinal Chemistry Letters,2008,18(15):4303-4307.
[7] DAI H Y,HONG C C,LIANG S C,et al. Carbonic anhydrase Ⅲ promotes transformation and invasion capability in hepatoma cells through FAK signaling pathway[J]. Molecular Carcinogenesis:Published in Cooperation with the University of Texas MD Anderson Cancer Center,2008,47(12):956-963.
[8] NISHITA T,HARADA T,SAKANOUE H,et al. Purification of swine carbonic anhydrase isoenzyme Ⅲ and measurement of its levels in tissues and plasma[J]. Journal of Animal Physiology and Animal Nutrition,2014,98(1):119-127.
[9]MC M,G K,U R,et al. Carbonic anhydrase Ⅲ regulates peroxisome proliferator-activated receptor-γ2[J]. Experimental Cell Research,2012,318(8):877-886.
[10] R?IS?NEN S R,LEHENKARI P,TASANEN M,et al. Carbonic anhydrase Ⅲ protects cells from hydrogen peroxide-induced apoptosis[J]. The FASEB Journal,1999,13(3):513-522.
[11] SHI C,UDA Y,DEDIC C,et al. Carbonic anhydrase Ⅲ protects osteocytes from oxidative stress[J]. The FASEB Journal,2017,32(1):440-452.
[12] GAILLY P,JOURET F,MARTIN D,et al. A novel renal carbonic anhydrase type Ⅲ plays a role in proximal tubule dysfunction[J].Kidney International,2008,74(1):52-61.
[13] TRIPP B C,SMITH K,FERRY J G. Carbonic anhydrase:new insights for an ancient enzyme[J]. Journal of Biological Chemistry,2001,276(52):48615-48618.
[14] ZIMMERMAN U J P,WANG P,ZHANG X,et al. Anti oxidative response of carbonic anhydrase Ⅲ in skeletal muscle[J]. IUBMB Life,2004,56(6):343-347.
[15]涂江龙,都爱莲,任惠民.碳酸酐酶Ⅲ的生物学特性及与临床疾病的关系[J].中国临床神经科学,2006,14(3):314-317.
[16]HUNTER T J C O I C B. Tyrosine phosphorylation:thirty years and counting[J]. Current Opinion in Cell Biology,2009,21(2):140-146.
[17]彭春霞,高艳明.碳酸酐酶生理功能的研究进展[J].北京大学学报(医学版),2007,39(2):210-212.
[18] LIU Y G,XIONG Y Z,DENG C Y. Isolation,sequence analysis and expression profile of a novel swine gene differentially expressed in the longissimus dorsi muscle tissues from Landrace×Large White cross combination[J]. Acta Biochimica et Biophysica Sinica,2005,37(3):186-191.
[19] LYNCH C J,JR B W,VARY T C,et al. Carbonic anhydrase Ⅲ in obese Zucker rats[J]. American Journal of Physiology-Endocrinology and Metabolism,1993,264(4):621-630.
[20] LYONS G E,BUCKINGHAM M E,TWEEDIE S,et al. Carbonic anhydrase Ⅲ,an early mesodermal marker,is expressed in embryonic mouse skeletal muscle and notochord[J]. Development,1991,111(1):233-244.
[21]PLACZEK M,JESSELL T M,DODD J. Induction of floor plate differentiation by contact-dependent,homeogenetic signals[J]. Development,1993,117(1):205-218.
[22] EDWARDS Y H,TWEEDIE S,LOWE N,et al. Carbonic anhydrase 3(CA3),a mesodermal marker[J]. Symposia of the Society for Experimental Biology,1992,46:273.
[23] JEFFERY S,EDWARDS Y,CARTER N J B G. Distribution of CAⅢ in fetal and adult human tissue[J]. Biochemical Genetics,1980,18(9/10):843-849.
[24]CARTER N,JEFFERY S,SHIELS A,et al. Characterization of human carbonic anhydrase Ⅲ from skeletal muscle[J]. Biochemical Genetics,1979,17(9/10):837-854.
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