家蠶不同發(fā)育階段抗壞血酸含量變化及其合成關(guān)鍵酶同源基因的研究
發(fā)布時(shí)間：2018-12-24 11:54
【摘要】：抗壞血酸又稱為維生素C(VC),在生物體內(nèi)具有重要的生理功能。從低等的單細(xì)胞生物到高等的動(dòng)植物,很多生物均能從頭合成還原型抗壞血酸(As A)。但目前對(duì)于昆蟲(chóng)自身能否合成AsA尚不明確。雖然AsA對(duì)于家蠶幼蟲(chóng)是必需的營(yíng)養(yǎng)物質(zhì),但在卵期和蛹期等不取食階段,體內(nèi)AsA的含量在特定發(fā)育時(shí)期都保持相對(duì)穩(wěn)定。目前還沒(méi)有研究確定家蠶體內(nèi)的AsA僅來(lái)源于食物而自身不能合成。為此,本文測(cè)定了家蠶在不同發(fā)育階段體內(nèi)抗壞血酸含量的變化,探討了AsA合成代謝關(guān)鍵酶的同源基因及其表達(dá)變化。首先,本試驗(yàn)用分光光度法測(cè)定了家蠶在不同發(fā)育時(shí)期以及不同條件下總抗壞血酸(TAA)和AsA含量的變化。結(jié)果表明,對(duì)于取食階段的家蠶幼蟲(chóng)采用缺乏VC的人工飼料飼養(yǎng),雖然與正常飼料飼養(yǎng)的家蠶相比生長(zhǎng)發(fā)育相對(duì)緩慢,但仍能發(fā)育到4齡。滯育卵中TAA含量在產(chǎn)卵72 h內(nèi)、AsA含量在產(chǎn)卵24 h內(nèi)均有所增加,此后二者的含量有不同程度降低,并一直保持相對(duì)穩(wěn)定。正常胚胎發(fā)育的蠶卵,產(chǎn)卵24 h內(nèi)AsA含量顯著增加,產(chǎn)卵48 h~144 h,AsA含量一直保持較高水平,直到胚胎發(fā)育后期才明顯下降,而且非滯育卵的As A含量一直高于滯育卵。幼蟲(chóng)期取食桑葉或添加VC的人工飼料之后,TAA含量和AsA的含量增加,眠中、取食缺乏VC的人工飼料及饑餓一定時(shí)間后,蠶體中的TAA和AsA的含量均明顯降低,尤其是饑餓幼蟲(chóng)的As A含量下降到很低的水平。蛹期TAA含量無(wú)顯著變化,但雌蛹體內(nèi)AsA含量在化蛹0~24 h顯著升高,雄蛹體內(nèi)AsA含量總體上在0~144 h變化不顯著,一直保持較高的含量,直到蛹的后期AsA含量才有較為明顯的下降。其次,利用已知生物的AsA合成酶基因的氨基酸序列在家蠶數(shù)據(jù)庫(kù)中比對(duì)檢索AsA合成酶的同源基因、利用MEGA等生物信息學(xué)軟件對(duì)基因的氨基酸序列進(jìn)行比對(duì)和分析,獲得As A合成代謝最后一步的關(guān)鍵酶古洛糖酸內(nèi)脂氧化酶(GULO)及倒數(shù)第二步的關(guān)鍵酶醛糖內(nèi)脂酶(ALase)的同源基因分別為:BmGULO_like1、BmGULO_like2和BmALase_like1、BmALase_like2。由于從低等單細(xì)胞生物到高等的靈長(zhǎng)類,幾乎均含有完整或部分AsA合成酶基因,并且AsA具有的廣泛的生理功能,推測(cè)家蠶中應(yīng)該也存在此基因。最后,利用Primer軟件設(shè)計(jì)家蠶的4個(gè)AsA合成酶同源基因的引物,采用熒光定量PCR檢測(cè)了AsA合成相關(guān)酶同源基因在家蠶不同發(fā)育階段的表達(dá)變化。結(jié)果表明,BmGULO_like1、BmGULO_like2、BmALase_like1和BmALase_like2等4個(gè)基因在卵期及蛹期的中腸和脂肪體中的表達(dá)量總體上都顯著高于幼蟲(chóng)期的中腸和脂肪體,并且這4個(gè)基因的表達(dá)量在卵期和蛹期都隨著發(fā)育進(jìn)展呈上升趨勢(shì)。本試驗(yàn)結(jié)果提示,在胚胎發(fā)育過(guò)程中及蛹期存在As A的來(lái)源途徑,可能包括AsA自身合成途徑及由氧化型抗壞血酸(DHA)還原為AsA途徑,而在幼蟲(chóng)期主要從食物中吸收VC,缺乏其他來(lái)源。根據(jù)研究結(jié)果推測(cè),雖然家蠶中GULO的同源基因與其他已知生物的相似度較低,但家蠶仍有可能具有合成AsA的能力;具有多重功能的ALase其結(jié)構(gòu)相對(duì)保守且分布物種極其廣泛,并且在家蠶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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S881.2
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