家蚕不同发育阶段抗坏血酸含量变化及其合成关键酶同源基因的研究
发布时间：2018-12-24 11:54
【摘要】：抗坏血酸又称为维生素C(VC),在生物体内具有重要的生理功能。从低等的单细胞生物到高等的动植物,很多生物均能从头合成还原型抗坏血酸(As A)。但目前对于昆虫自身能否合成AsA尚不明确。虽然AsA对于家蚕幼虫是必需的营养物质,但在卵期和蛹期等不取食阶段,体内AsA的含量在特定发育时期都保持相对稳定。目前还没有研究确定家蚕体内的AsA仅来源于食物而自身不能合成。为此,本文测定了家蚕在不同发育阶段体内抗坏血酸含量的变化,探讨了AsA合成代谢关键酶的同源基因及其表达变化。首先,本试验用分光光度法测定了家蚕在不同发育时期以及不同条件下总抗坏血酸(TAA)和AsA含量的变化。结果表明,对于取食阶段的家蚕幼虫采用缺乏VC的人工饲料饲养,虽然与正常饲料饲养的家蚕相比生长发育相对缓慢,但仍能发育到4龄。滞育卵中TAA含量在产卵72 h内、AsA含量在产卵24 h内均有所增加,此后二者的含量有不同程度降低,并一直保持相对稳定。正常胚胎发育的蚕卵,产卵24 h内AsA含量显著增加,产卵48 h~144 h,AsA含量一直保持较高水平,直到胚胎发育后期才明显下降,而且非滞育卵的As A含量一直高于滞育卵。幼虫期取食桑叶或添加VC的人工饲料之后,TAA含量和AsA的含量增加,眠中、取食缺乏VC的人工饲料及饥饿一定时间后,蚕体中的TAA和AsA的含量均明显降低,尤其是饥饿幼虫的As A含量下降到很低的水平。蛹期TAA含量无显著变化,但雌蛹体内AsA含量在化蛹0~24 h显著升高,雄蛹体内AsA含量总体上在0~144 h变化不显著,一直保持较高的含量,直到蛹的后期AsA含量才有较为明显的下降。其次,利用已知生物的AsA合成酶基因的氨基酸序列在家蚕数据库中比对检索AsA合成酶的同源基因、利用MEGA等生物信息学软件对基因的氨基酸序列进行比对和分析,获得As A合成代谢最后一步的关键酶古洛糖酸内脂氧化酶(GULO)及倒数第二步的关键酶醛糖内脂酶(ALase)的同源基因分别为:BmGULO_like1、BmGULO_like2和BmALase_like1、BmALase_like2。由于从低等单细胞生物到高等的灵长类,几乎均含有完整或部分AsA合成酶基因,并且AsA具有的广泛的生理功能,推测家蚕中应该也存在此基因。最后,利用Primer软件设计家蚕的4个AsA合成酶同源基因的引物,采用荧光定量PCR检测了AsA合成相关酶同源基因在家蚕不同发育阶段的表达变化。结果表明,BmGULO_like1、BmGULO_like2、BmALase_like1和BmALase_like2等4个基因在卵期及蛹期的中肠和脂肪体中的表达量总体上都显著高于幼虫期的中肠和脂肪体,并且这4个基因的表达量在卵期和蛹期都随着发育进展呈上升趋势。本试验结果提示,在胚胎发育过程中及蛹期存在As A的来源途径,可能包括AsA自身合成途径及由氧化型抗坏血酸(DHA)还原为AsA途径,而在幼虫期主要从食物中吸收VC,缺乏其他来源。根据研究结果推测,虽然家蚕中GULO的同源基因与其他已知生物的相似度较低,但家蚕仍有可能具有合成AsA的能力;具有多重功能的ALase其结构相对保守且分布物种极其广泛,并且在家蚕中的同源基因也具有高度保守性。
[Abstract]:Ascorbic acid is also called vitamin C (VC), and has important physiological function in the organism. From low-grade, single-celled organisms to higher plants and plants, many organisms can start from the synthesis of reduced ascorbic acid (As A). But it is unclear whether or not the insect itself can be synthesized by AsA. Although AsA is a necessary nutrient for the larvae of the silkworm, the content of AsA in the body remains relatively stable during the period of non-feeding, such as the egg period and the period of time. At present, there is no research to determine that the AsA in the silkworm body is only from the food and cannot be synthesized itself. In this paper, the changes of the content of ascorbic acid in silkworm in different developmental stages were determined, and the homologous genes and their expression changes of the key enzymes of AsA synthesis were discussed. First, the changes of total ascorbic acid (TAA) and AsA content of the silkworm in different developmental stages and under different conditions were determined by spectrophotometry. The results showed that the silkworm larvae fed on the feeding stage were fed with artificial feed without VC, although the growth and development of the silkworm was relatively slow compared with that of the normal feed, but it could still be developed to the age of 4. The content of TAA in diapause eggs increased during the laying period of 72 h, and the content of AsA increased in the laying period of 24 h, and the contents of the two were reduced to a certain extent and remained relatively stable. In the normal embryo, the content of AsA was increased significantly in 24 h, and the content of AsA was kept at a high level for 48 h ~ 144 h, and the content of As A in non-diapause eggs was higher than that of diapause eggs. The contents of TAA and AsA in the silkworm body decreased significantly after the larvae feed into the mulberry leaf or the artificial feed supplemented with VC, and the contents of TAA and AsA in the silkworm body decreased significantly, especially when the content of As A of the hungry larvae decreased to a very low level. The content of AsA in the female pupae increased significantly from 0 to 24 hours, and the content of AsA in the male pupae was not significantly changed from 0 to 144h, and the content of AsA in the pupal was kept higher than that in the later stage of the pupae. secondly, using the amino acid sequence of the AsA synthetase gene of the known organism in the silkworm database to compare and analyze the amino acid sequence of the gene by using the bioinformatics software such as MEGA in the silkworm database, The homologous genes of the key enzyme gulonic acid internal lipoxygenase (GULO) and the key enzyme aldolase (ALase) in the second step are: BmGULO _ like1, BmGUO _ like2 and BmALase _ like1, BmALase _ like2, respectively. As a result of the fact that from low-to-low-celled organisms to higher primates, almost all of the AsA synthase gene is completely or partially contained, and AsA has a wide range of physiological functions, it is presumed that this gene should be present in the silkworm. In the end, four AsA synthase homologous genes of the silkworm were designed by Primer software, and the expression of the homologous gene of AsA was detected by fluorescence quantitative PCR. The results showed that the expression of BmGULO _ like1, BmGULO _ like2, BmALase _ like1 and BmALase _ like2 was significantly higher than that of the midgut and the fat body in the stage of the larvae, and the expression of these four genes increased with the development of the egg and fat. As a result of this test, as the source of As A in the development of the embryo and during the stage of the development, the pathway of AsA's own synthesis and the reduction of the oxidized ascorbic acid (DHA) to the AsA pathway may be included, while the VC is mainly absorbed from the food during the larval stage, and there is no other source. According to the results of the study, although the homolog of GULO in the silkworm is lower than that of other known organisms, it is still possible for the silkworm to have the ability to synthesize AsA; the structure of the alase with multiple functions is relatively conservative and the distributed species are extremely wide, and the homologous genes in the silkworm have high conservation.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S881.2
[Abstract]:Ascorbic acid is also called vitamin C (VC), and has important physiological function in the organism. From low-grade, single-celled organisms to higher plants and plants, many organisms can start from the synthesis of reduced ascorbic acid (As A). But it is unclear whether or not the insect itself can be synthesized by AsA. Although AsA is a necessary nutrient for the larvae of the silkworm, the content of AsA in the body remains relatively stable during the period of non-feeding, such as the egg period and the period of time. At present, there is no research to determine that the AsA in the silkworm body is only from the food and cannot be synthesized itself. In this paper, the changes of the content of ascorbic acid in silkworm in different developmental stages were determined, and the homologous genes and their expression changes of the key enzymes of AsA synthesis were discussed. First, the changes of total ascorbic acid (TAA) and AsA content of the silkworm in different developmental stages and under different conditions were determined by spectrophotometry. The results showed that the silkworm larvae fed on the feeding stage were fed with artificial feed without VC, although the growth and development of the silkworm was relatively slow compared with that of the normal feed, but it could still be developed to the age of 4. The content of TAA in diapause eggs increased during the laying period of 72 h, and the content of AsA increased in the laying period of 24 h, and the contents of the two were reduced to a certain extent and remained relatively stable. In the normal embryo, the content of AsA was increased significantly in 24 h, and the content of AsA was kept at a high level for 48 h ~ 144 h, and the content of As A in non-diapause eggs was higher than that of diapause eggs. The contents of TAA and AsA in the silkworm body decreased significantly after the larvae feed into the mulberry leaf or the artificial feed supplemented with VC, and the contents of TAA and AsA in the silkworm body decreased significantly, especially when the content of As A of the hungry larvae decreased to a very low level. The content of AsA in the female pupae increased significantly from 0 to 24 hours, and the content of AsA in the male pupae was not significantly changed from 0 to 144h, and the content of AsA in the pupal was kept higher than that in the later stage of the pupae. secondly, using the amino acid sequence of the AsA synthetase gene of the known organism in the silkworm database to compare and analyze the amino acid sequence of the gene by using the bioinformatics software such as MEGA in the silkworm database, The homologous genes of the key enzyme gulonic acid internal lipoxygenase (GULO) and the key enzyme aldolase (ALase) in the second step are: BmGULO _ like1, BmGUO _ like2 and BmALase _ like1, BmALase _ like2, respectively. As a result of the fact that from low-to-low-celled organisms to higher primates, almost all of the AsA synthase gene is completely or partially contained, and AsA has a wide range of physiological functions, it is presumed that this gene should be present in the silkworm. In the end, four AsA synthase homologous genes of the silkworm were designed by Primer software, and the expression of the homologous gene of AsA was detected by fluorescence quantitative PCR. The results showed that the expression of BmGULO _ like1, BmGULO _ like2, BmALase _ like1 and BmALase _ like2 was significantly higher than that of the midgut and the fat body in the stage of the larvae, and the expression of these four genes increased with the development of the egg and fat. As a result of this test, as the source of As A in the development of the embryo and during the stage of the development, the pathway of AsA's own synthesis and the reduction of the oxidized ascorbic acid (DHA) to the AsA pathway may be included, while the VC is mainly absorbed from the food during the larval stage, and there is no other source. According to the results of the study, although the homolog of GULO in the silkworm is lower than that of other known organisms, it is still possible for the silkworm to have the ability to synthesize AsA; the structure of the alase with multiple functions is relatively conservative and the distributed species are extremely wide, and the homologous genes in the silkworm have high conservation.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S881.2
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