节旋藻藻胆蛋白的基因序列分析及其对果蝇生理特性的影响
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摘要
节旋藻含有丰富的蛋白质、维生素、不饱和脂肪酸及多糖等生物活性物质,已成为我国最大的微藻养殖产业。藻胆蛋白作为节旋藻中重要的捕光色素蛋白,占节旋藻干重的20%左右。藻胆蛋白不仅可作为人类优质的蛋白质来源,其特殊的生理活性如抗氧化活性、抗肿瘤活性及荧光特性等也越来越受到人们的关注。节旋藻藻胆蛋白的基因工程研究主要集中于钝顶节旋藻和极大节旋藻,对于生长在鄂尔多斯高原碱湖的钝顶节旋藻、鄂尔多斯节旋藻和方胞节旋藻的研究则较少。同时,藻胆蛋白抗氧化活性研究主要以啮齿类动物为研究对象,尚未发现以果蝇作为实验动物,研究藻胆蛋白抗氧化活性及抗衰老活性的的报道。
节旋藻藻胆蛋白包括藻蓝蛋白和别藻蓝蛋白。本实验以生长于鄂尔多斯高原碱湖的钝顶节旋藻、鄂尔多斯节旋藻及方胞节旋藻基因组DNA为模板,根据GenBank公布的节旋藻藻胆蛋白基因序列设计引物,PCR扩增获得三种节旋藻的藻胆蛋白基因,并与pMDTM19-T载体连接后转入大肠杆菌DH 5α中,挑选阳性克隆测序。结果显示：三种节旋藻的藻蓝蛋白基因全长均1119 bp,编码α亚基的基因长为519 bp,编码β亚基的基因长为489 bp,间隔序列长111 bp；鄂尔多斯钝顶节旋藻和鄂尔多斯节旋藻别藻蓝蛋白基因全长1056 bp,方胞节旋藻别藻蓝蛋白全长1054bp。三者α亚基和β亚基基因长度一致,均为486 bp。间隔序列长度有差异,鄂尔多斯钝顶节旋藻与鄂尔多斯节旋藻间隔序列长84 bp,方胞节旋藻间隔序列长82 bp。
将克隆测序后的三种节旋藻的藻胆蛋白基因与GenBank中收录的极大节旋藻、钝顶节旋藻的藻胆蛋白基因进行比对分析,结果表明：五个节旋藻藻蓝蛋白基因的同源性在99.6%以上,别藻蓝蛋白的同源性在75.4%～99.7%。GC含量分析发现,在节旋属内不同种之间,节旋藻的藻胆蛋白基因GC含量较接近,藻蓝蛋白基因GC含量在48.8%～49%之间,别藻蓝蛋白基因GC含量介于48.3%～53.6%。氨基酸组成分析表明,藻胆蛋白含有人体所需的8种必需氨基酸,其中Leu、Ile、Lys、Thr及Val含量较高。通过对编码藻胆蛋白各亚基基因内部同源性的分析,保守程度由高至低依次为：别藻蓝蛋白β亚基>别藻蓝蛋白α亚基>藻蓝蛋白α亚基>藻蓝蛋白β亚基。五个节旋藻样品的聚类分析表明,来源于鄂尔多斯高原碱湖的鄂尔多斯节旋藻、方胞节旋藻及钝顶节旋藻的亲缘关系最近,其次为钝顶节旋藻,最后为极大节旋藻。
同时,本实验还研究了藻胆蛋白对果蝇的生理生化特性的影响。果蝇寿命实验表明,藻胆蛋白能够显著延长雌果蝇寿命(p<0.01),且存在剂量依赖效应。性活力和繁殖力实验均表明,藻胆蛋白能够提高果蝇性活力,繁殖能力,且以0.50%浓度组效果最佳。藻胆蛋白还能够降低果蝇子代的雌雄比,使子代中雄果蝇多于雌果蝇。对30日龄的果蝇喂饲10 d藻胆蛋白后,果蝇体内总抗氧化能力(p<0.01)、过氧化物酶活性(p<0.01)、过氧化氢酶活性(p<0.01)明显提高,丙二醛含量显著降低(p<0.01)。藻胆蛋白对果蝇超氧化物歧化酶活性起到一定的增强作用,但未达到统计学显著水平。由于藻胆蛋白增强了果蝇体内抗氧化酶类活性,从而影响了果蝇的寿命、性活力以及繁殖能力等生理特性。本研究成果,将为鄂尔多斯高原碱湖节旋藻的研究奠定实验基础,并为节旋藻藻胆蛋白在抗氧化方面的开发与利用提供科学依据。
Arthrospira has become the biggest market for its rich composition in protein, vitamin unsaturated fatty acid and polysaccharides. Phycobiliprotein is a light-harvesting protein and its content is 20% in dry weight of Arthrospira. In recent years, the activity of anti-oxidant, anti-tumorous and the characteristics of Fluorescence in phycobiliprotein have attracted increasing attention. The research on Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri from alkaline lakes in the Ordos Plateau is still insufficient, and the study on the antioxidational activity of phycobiliprotein in Drosophila melanogaster has rarely been reported.
In this paper, phycocyanin gene and allophycocyanin gene of Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri which originated from alkaline lakes in the Ordos Plateau were cloned and sequenced. The results indicated that the length of the phycocyanin gene in three Arthrospira were all 1119 bp, which contains a-subunit gene (519 bp),β-subunit gene (489 bp), and spacer sequence (111 bp). The length of the allophycocyanin gene in both Arthrospira platensis and Arthrospira erdosensis were 1056 bp, and in Arthrospira jenneri was 1054 bp. Both of a-subunit gene andβ-subunit gene contain 486 bp among the three species, but there was 84 bp spacer sequence in Arthrospira platensis and Arthrospira erdosensis and 82 bp in Arthrospira jenneri, respectively.
The sequences of phycobiliprotein genes in three species were compared with phycobiliprotein genes of Arthrospira maxima and Arthrospira platensis publilshed in GenBank. The homology of phycocyanin gene was from 99.6% to 100% and that of allophycocyanin gene was from 75.4% to 99.7% among the five samples. The GC content in phycobiliprotein genes were similar in different species, which was 48.8%～49% in phycocyanin gene and 48.3%～ 53.6% in allophycocyanin gene. Amino acid composition analysis showed that eight essential amino acids required for human body were discovered in phycobiliprotein genes, of which the content of leucine, isoleucine, lysine, threonine and valine was higher. The homology of the sequence encoding phycobiliprotein subunits in five samples were analyzed and ordered as follows:β-subunit of allophycocyanin>a-subunit of allophycocyanin> a-subunit of phycocyanin>β-subunit of phycocyanin. The cluster analysis of five samples indicated that the relationship was closer among the Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri from alkaline lakes in the Ordos Plateau compared with Arthrospira platensis and Arthrospira jenneri.
The effects of phycobiliproteins on the characteristics of physiology and biochemistry in Drosophila melanogaster were studied. The results showed that the phycobiliproteins can significantly prolong the longevity of the female Drosophila melanogaster (p<0.01), improved sexual vitality and reproductive capacity of Drosophila melanogaster. Phycobiliprotein could also reduce the sex ratio of Drosophila melanogaster's filial generation, as a result there were more male than female. The activity of antioxidant enzymes changed after Drosophila melanogaster were fed with phycobiliprotein. Phycobiliprotein significantly increased the activity of antioxidant enzymes in Drosophila melanogaster, in which total antioxidant capacity (p<0.01), peroxides'activity (p<0.01), catalyses activity (p<0.01), and decreased malondialdehyde (p<0.01) significantly. Phycobiliproteins enhanced superoxide dismutase activity in Drosophila melanogaster, but did not reach significant level. Phycobiliprotein could increase the activity of antioxidant enzymes in Drosophila melanogaster so it also affacted Drosophila melanogaster's life span, sexual vitality and reproductive capacity.
节旋藻藻胆蛋白包括藻蓝蛋白和别藻蓝蛋白。本实验以生长于鄂尔多斯高原碱湖的钝顶节旋藻、鄂尔多斯节旋藻及方胞节旋藻基因组DNA为模板,根据GenBank公布的节旋藻藻胆蛋白基因序列设计引物,PCR扩增获得三种节旋藻的藻胆蛋白基因,并与pMDTM19-T载体连接后转入大肠杆菌DH 5α中,挑选阳性克隆测序。结果显示：三种节旋藻的藻蓝蛋白基因全长均1119 bp,编码α亚基的基因长为519 bp,编码β亚基的基因长为489 bp,间隔序列长111 bp；鄂尔多斯钝顶节旋藻和鄂尔多斯节旋藻别藻蓝蛋白基因全长1056 bp,方胞节旋藻别藻蓝蛋白全长1054bp。三者α亚基和β亚基基因长度一致,均为486 bp。间隔序列长度有差异,鄂尔多斯钝顶节旋藻与鄂尔多斯节旋藻间隔序列长84 bp,方胞节旋藻间隔序列长82 bp。
将克隆测序后的三种节旋藻的藻胆蛋白基因与GenBank中收录的极大节旋藻、钝顶节旋藻的藻胆蛋白基因进行比对分析,结果表明：五个节旋藻藻蓝蛋白基因的同源性在99.6%以上,别藻蓝蛋白的同源性在75.4%～99.7%。GC含量分析发现,在节旋属内不同种之间,节旋藻的藻胆蛋白基因GC含量较接近,藻蓝蛋白基因GC含量在48.8%～49%之间,别藻蓝蛋白基因GC含量介于48.3%～53.6%。氨基酸组成分析表明,藻胆蛋白含有人体所需的8种必需氨基酸,其中Leu、Ile、Lys、Thr及Val含量较高。通过对编码藻胆蛋白各亚基基因内部同源性的分析,保守程度由高至低依次为：别藻蓝蛋白β亚基>别藻蓝蛋白α亚基>藻蓝蛋白α亚基>藻蓝蛋白β亚基。五个节旋藻样品的聚类分析表明,来源于鄂尔多斯高原碱湖的鄂尔多斯节旋藻、方胞节旋藻及钝顶节旋藻的亲缘关系最近,其次为钝顶节旋藻,最后为极大节旋藻。
同时,本实验还研究了藻胆蛋白对果蝇的生理生化特性的影响。果蝇寿命实验表明,藻胆蛋白能够显著延长雌果蝇寿命(p<0.01),且存在剂量依赖效应。性活力和繁殖力实验均表明,藻胆蛋白能够提高果蝇性活力,繁殖能力,且以0.50%浓度组效果最佳。藻胆蛋白还能够降低果蝇子代的雌雄比,使子代中雄果蝇多于雌果蝇。对30日龄的果蝇喂饲10 d藻胆蛋白后,果蝇体内总抗氧化能力(p<0.01)、过氧化物酶活性(p<0.01)、过氧化氢酶活性(p<0.01)明显提高,丙二醛含量显著降低(p<0.01)。藻胆蛋白对果蝇超氧化物歧化酶活性起到一定的增强作用,但未达到统计学显著水平。由于藻胆蛋白增强了果蝇体内抗氧化酶类活性,从而影响了果蝇的寿命、性活力以及繁殖能力等生理特性。本研究成果,将为鄂尔多斯高原碱湖节旋藻的研究奠定实验基础,并为节旋藻藻胆蛋白在抗氧化方面的开发与利用提供科学依据。
Arthrospira has become the biggest market for its rich composition in protein, vitamin unsaturated fatty acid and polysaccharides. Phycobiliprotein is a light-harvesting protein and its content is 20% in dry weight of Arthrospira. In recent years, the activity of anti-oxidant, anti-tumorous and the characteristics of Fluorescence in phycobiliprotein have attracted increasing attention. The research on Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri from alkaline lakes in the Ordos Plateau is still insufficient, and the study on the antioxidational activity of phycobiliprotein in Drosophila melanogaster has rarely been reported.
In this paper, phycocyanin gene and allophycocyanin gene of Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri which originated from alkaline lakes in the Ordos Plateau were cloned and sequenced. The results indicated that the length of the phycocyanin gene in three Arthrospira were all 1119 bp, which contains a-subunit gene (519 bp),β-subunit gene (489 bp), and spacer sequence (111 bp). The length of the allophycocyanin gene in both Arthrospira platensis and Arthrospira erdosensis were 1056 bp, and in Arthrospira jenneri was 1054 bp. Both of a-subunit gene andβ-subunit gene contain 486 bp among the three species, but there was 84 bp spacer sequence in Arthrospira platensis and Arthrospira erdosensis and 82 bp in Arthrospira jenneri, respectively.
The sequences of phycobiliprotein genes in three species were compared with phycobiliprotein genes of Arthrospira maxima and Arthrospira platensis publilshed in GenBank. The homology of phycocyanin gene was from 99.6% to 100% and that of allophycocyanin gene was from 75.4% to 99.7% among the five samples. The GC content in phycobiliprotein genes were similar in different species, which was 48.8%～49% in phycocyanin gene and 48.3%～ 53.6% in allophycocyanin gene. Amino acid composition analysis showed that eight essential amino acids required for human body were discovered in phycobiliprotein genes, of which the content of leucine, isoleucine, lysine, threonine and valine was higher. The homology of the sequence encoding phycobiliprotein subunits in five samples were analyzed and ordered as follows:β-subunit of allophycocyanin>a-subunit of allophycocyanin> a-subunit of phycocyanin>β-subunit of phycocyanin. The cluster analysis of five samples indicated that the relationship was closer among the Arthrospira platensis, Arthrospira erdosensis and Arthrospira jenneri from alkaline lakes in the Ordos Plateau compared with Arthrospira platensis and Arthrospira jenneri.
The effects of phycobiliproteins on the characteristics of physiology and biochemistry in Drosophila melanogaster were studied. The results showed that the phycobiliproteins can significantly prolong the longevity of the female Drosophila melanogaster (p<0.01), improved sexual vitality and reproductive capacity of Drosophila melanogaster. Phycobiliprotein could also reduce the sex ratio of Drosophila melanogaster's filial generation, as a result there were more male than female. The activity of antioxidant enzymes changed after Drosophila melanogaster were fed with phycobiliprotein. Phycobiliprotein significantly increased the activity of antioxidant enzymes in Drosophila melanogaster, in which total antioxidant capacity (p<0.01), peroxides'activity (p<0.01), catalyses activity (p<0.01), and decreased malondialdehyde (p<0.01) significantly. Phycobiliproteins enhanced superoxide dismutase activity in Drosophila melanogaster, but did not reach significant level. Phycobiliprotein could increase the activity of antioxidant enzymes in Drosophila melanogaster so it also affacted Drosophila melanogaster's life span, sexual vitality and reproductive capacity.
引文
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