猪硒蛋白V（SelV）基因克隆及日粮硒水平对其组织mRNA表达丰度的影响
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摘要
硒在体内主要以硒蛋白的形式发挥生理功能。迄今为止,哺乳动物中已确定的硒蛋白基因有25种,但是大部分硒蛋白在体内的生理功能还不能确定。SelV基因是近年来报道的一个新的硒蛋白基因,在猪上,SelV基因的序列及不同硒水平日粮对其表达调控至今未有报道,因此本实验以猪为模型在克隆猪SelV基因基础上,研究了缺硒、正常和高硒日粮机体组织SelV mRNA丰度的变化规律。
试验选用了28日龄,平均体重7公斤左右的断奶公猪24头,采用单因素设计,设酵母硒添加0,0.3,3.0mg Se/kg三个处理,每个处理设8个重复,每个重复1头猪。以缺硒的玉米、豆粕配制基础日粮,含硒0.03mg Se/kg。试验期为16w,在试验0w,8w,16w清晨,前腔静脉采集试猪血样；16w后屠宰试猪并于肝、肾、肌肉、视丘下部、睾丸、心、脾、垂体和甲状腺取样。通过RACE技术克隆了包括起始密码子、UGA密码子及3’非翻译区域末端的猪SelV全长基因,并对不同日粮硒水平下试猪上述组织的SelV基因作实时定量PCR分析。
试验结果表明,克隆的猪SelV基因cDNA长度1233 bp,编码区长1029bp,编码342个氨基酸,其编码的cDNA序列与人的序列相似度达97%；并且与鼠、兔、人等氨基酸序列比对有较高的相似性。日粮添加0.3和3.0mg Se/kg硒组试猪血浆和肝脏GPX活性显著高于低硒组(P<0.05),但0.3与3.0mg之间GPX活性无显著差异(P<0.05),基础日粮组试猪处于于缺硒状态。通过实时定量PCR分析显示,各硒水平都以睾丸组织SelV mRNA丰度最高,较其他组织高出30-1000倍(P<0.01),肝脏、甲状腺、垂体、下丘脑及肾脏SelV mRNA表达丰度也较高,心脏、血液白细胞及脾脏中SelV mRNA表达丰度较低,肌肉组织中改基因表达丰度最低；随着日粮硒水平的增加,肾脏SelV mRNA丰度一直呈下降趋势(P<0.05)。低硒日粮中(0mg Se/kg)，、视丘下部和肌肉组织SelV mRNA的表达丰度高于添加硒组(0.3mg Se/kg和3.0 mgSe/kg) (P<0.05)。在三个硒水平间,只有甲状腺的SelV mRNA表达丰度在硒添加量为0.3mg Se/kg时最高,且与低、高硒组间出现差异(P<0.05),这与其他组织不同。总之,睾丸组织SelV mRNA丰度远远高于其他组织,SelV可能在睾丸中具有重要作用；不同硒水平对各组织中SelV基因调节存在较大差异。
Selenium's function is performed in the form of selenoprotein in vivo. Studies on the expression of selenoprotein to dietary Se concentrations are popular these years. Until now there are 25 selenoprotein in mammals but most of which have unidentified functions. SelV was one of them found recently. Since there are limited informations on Porcine selenoprotein V (SelV) gene sequence and effects of different levels of dietary Se on its expression. The present study was conducted to investigate the responses of SelV mRNA abundance to dietary Se concentrations based on the clone of porcine SelV gene in the swine.
In this trial, by means of RACE technology we cloned overall length porcine SelV gene at first, including initiation codon, UGA codon and 3'untranslation region. And then 24 weanling male pigs were fed a Se-deficient corn-soy diet (0.03 mg Se/kg) or the diet supplemented with 0.3 or 3.0 mg Se/kg (as Se-enriched yeast) diet (extra yeast selenium) for 16w. A set of 8 pigs. Blood sample were collected at 0w,8w and 16w. After 16w on the diet, pigs were sacrificed to collect liver, kidney, loin, hypothalamus, testis, heart, spleen, pituitary, and thyroid tissues. SelV mRNA abundance in different tissues were determined with the use of Real-time PCR.
As the results showed, we first cloned a 1,233 base pair cDNA coding for the porcine SelV gene and found that this cDNA fragment shared 97% of sequence homology in the coding region to the human SelV gene. The high similarity of porcine SelV deduced amino acid sequence with the SelV in other organisms, such as mouse, rabbit, human and so on. Dietary Se supplement resulted in higher GPX activities in blood and liver of 16-week pigs compared with the Se-deficient group (P<0.05), but showed no effect on the activities of GPX in blood of 8-week pigs fed diets with 0.3 and 3.0 mg Se/kg. Quantitative real-time RT-PCR analysis indicated that the level of the SelV mRNA was highest in testis and it higher than other analyzed tissues 30-1000 times. Besides testis, the level of the SelV mRNA was higher in liver, thyroid, pituitary, hypothalamus and kidney, and was lower in heart, blood and spleen, lowest in muscle. With the dietary selenium increasing, the SelV mRNA level decreased in kidney(P<0.05). Compared with the two dietary Se supplemental levels (0.3 and 3.0 mg Se/kg), dietary Se deficiency resulted in a higher (P<0.05) SelV mRNA levels in hypothalamus and muscle. Thyroid had the highest SelV mRNA level in pigs fed 0.3 mg Se/kg (P<0.05). In conclusion, the level of the SelV mRNA was highest in testis, the SelV mRNA level decreased in kidney, heart and liver, So effects of dietary Se on SelV gene expression varied with tissues in weanling pigs.
试验选用了28日龄,平均体重7公斤左右的断奶公猪24头,采用单因素设计,设酵母硒添加0,0.3,3.0mg Se/kg三个处理,每个处理设8个重复,每个重复1头猪。以缺硒的玉米、豆粕配制基础日粮,含硒0.03mg Se/kg。试验期为16w,在试验0w,8w,16w清晨,前腔静脉采集试猪血样；16w后屠宰试猪并于肝、肾、肌肉、视丘下部、睾丸、心、脾、垂体和甲状腺取样。通过RACE技术克隆了包括起始密码子、UGA密码子及3’非翻译区域末端的猪SelV全长基因,并对不同日粮硒水平下试猪上述组织的SelV基因作实时定量PCR分析。
试验结果表明,克隆的猪SelV基因cDNA长度1233 bp,编码区长1029bp,编码342个氨基酸,其编码的cDNA序列与人的序列相似度达97%；并且与鼠、兔、人等氨基酸序列比对有较高的相似性。日粮添加0.3和3.0mg Se/kg硒组试猪血浆和肝脏GPX活性显著高于低硒组(P<0.05),但0.3与3.0mg之间GPX活性无显著差异(P<0.05),基础日粮组试猪处于于缺硒状态。通过实时定量PCR分析显示,各硒水平都以睾丸组织SelV mRNA丰度最高,较其他组织高出30-1000倍(P<0.01),肝脏、甲状腺、垂体、下丘脑及肾脏SelV mRNA表达丰度也较高,心脏、血液白细胞及脾脏中SelV mRNA表达丰度较低,肌肉组织中改基因表达丰度最低；随着日粮硒水平的增加,肾脏SelV mRNA丰度一直呈下降趋势(P<0.05)。低硒日粮中(0mg Se/kg)，、视丘下部和肌肉组织SelV mRNA的表达丰度高于添加硒组(0.3mg Se/kg和3.0 mgSe/kg) (P<0.05)。在三个硒水平间,只有甲状腺的SelV mRNA表达丰度在硒添加量为0.3mg Se/kg时最高,且与低、高硒组间出现差异(P<0.05),这与其他组织不同。总之,睾丸组织SelV mRNA丰度远远高于其他组织,SelV可能在睾丸中具有重要作用；不同硒水平对各组织中SelV基因调节存在较大差异。
Selenium's function is performed in the form of selenoprotein in vivo. Studies on the expression of selenoprotein to dietary Se concentrations are popular these years. Until now there are 25 selenoprotein in mammals but most of which have unidentified functions. SelV was one of them found recently. Since there are limited informations on Porcine selenoprotein V (SelV) gene sequence and effects of different levels of dietary Se on its expression. The present study was conducted to investigate the responses of SelV mRNA abundance to dietary Se concentrations based on the clone of porcine SelV gene in the swine.
In this trial, by means of RACE technology we cloned overall length porcine SelV gene at first, including initiation codon, UGA codon and 3'untranslation region. And then 24 weanling male pigs were fed a Se-deficient corn-soy diet (0.03 mg Se/kg) or the diet supplemented with 0.3 or 3.0 mg Se/kg (as Se-enriched yeast) diet (extra yeast selenium) for 16w. A set of 8 pigs. Blood sample were collected at 0w,8w and 16w. After 16w on the diet, pigs were sacrificed to collect liver, kidney, loin, hypothalamus, testis, heart, spleen, pituitary, and thyroid tissues. SelV mRNA abundance in different tissues were determined with the use of Real-time PCR.
As the results showed, we first cloned a 1,233 base pair cDNA coding for the porcine SelV gene and found that this cDNA fragment shared 97% of sequence homology in the coding region to the human SelV gene. The high similarity of porcine SelV deduced amino acid sequence with the SelV in other organisms, such as mouse, rabbit, human and so on. Dietary Se supplement resulted in higher GPX activities in blood and liver of 16-week pigs compared with the Se-deficient group (P<0.05), but showed no effect on the activities of GPX in blood of 8-week pigs fed diets with 0.3 and 3.0 mg Se/kg. Quantitative real-time RT-PCR analysis indicated that the level of the SelV mRNA was highest in testis and it higher than other analyzed tissues 30-1000 times. Besides testis, the level of the SelV mRNA was higher in liver, thyroid, pituitary, hypothalamus and kidney, and was lower in heart, blood and spleen, lowest in muscle. With the dietary selenium increasing, the SelV mRNA level decreased in kidney(P<0.05). Compared with the two dietary Se supplemental levels (0.3 and 3.0 mg Se/kg), dietary Se deficiency resulted in a higher (P<0.05) SelV mRNA levels in hypothalamus and muscle. Thyroid had the highest SelV mRNA level in pigs fed 0.3 mg Se/kg (P<0.05). In conclusion, the level of the SelV mRNA was highest in testis, the SelV mRNA level decreased in kidney, heart and liver, So effects of dietary Se on SelV gene expression varied with tissues in weanling pigs.
引文
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