對人工飼料不同攝食性家蠶品種（品系）SSR標記分析及pkg1基因的表達研究
發(fā)布時間：2018-07-27 14:11
【摘要】：家蠶作為重要的經(jīng)濟昆蟲,在人類生產(chǎn)生活及文化歷史上均有非常重要的地位。而家蠶是寡食性昆蟲,桑葉是其天然飼料,栽桑才能養(yǎng)蠶,這制約了蠶桑業(yè)的發(fā)展。用人工飼料養(yǎng)蠶是蠶業(yè)史上的一項重要的技術革新,是今后我國養(yǎng)蠶業(yè)發(fā)展的方向。但目前我國人工飼料養(yǎng)蠶技術尚未達到實用化的要求,現(xiàn)行蠶品種對人工飼料的攝食性較差是制約該技術發(fā)展的主要障礙,提高家蠶對人工飼料的攝食性和適應性成為研究的重點。前人已從生理學和遺傳學等方面對家蠶食性做了大量的研究,并取得了豐碩的成果,但家蠶食性相關的分子機理尚未完全明確,與家蠶攝食性相關的“食性基因”尚未獲得。本研究以對人工飼料攝食性差異明顯的家蠶品種(品系)為材料,利用微衛(wèi)星標記(Simple Sequence Repeats,SSR)技術對家蠶食性相關的分子標記進行分析。并采用實時熒光定量PCR方法測定了環(huán)磷酸鳥苷依賴性蛋白激酶基因1(c GMP dependent protein kinase gene 1,pkg1)在人工飼料攝食性不同的家蠶品種(品系)中的表達特征,采用藥物激活PKG的方法對pkg1基因進行功能驗證,以期探明PKG與家蠶食性的關系。主要獲得如下結(jié)果:1.以廣食性家蠶品種中廣04和低食性家蠶品種魯七、菁松A高食性品系和低食性品系、菁松B高食性品系和低食性品系3對對人工飼料攝食性存在很大差異的家蠶品種(品系)為材料,利用152對SSR分子標記引物對供試家蠶品種(品系)進行SSR-PCR擴增,共篩選出18個多態(tài)性SSR特異標記,主要位于第3、7、9、10、12、13、14、16、17、19、20和21號染色體上。20號染色體上的SSR標記最多,為3條,且多由高食性家蠶品種(品系)擴增出來,說明促進家蠶攝食人工飼料的基因可能位于20號染色體上,并且S2009為高食性家蠶品種(品系)的特異性引物,這進一步說明促進家蠶攝食的基因很可能位于20號染色體的S2009標記附近。S1902也多為高食性家蠶品種(品系)的特異引物,說明在19號染色體的S1902位點附近也有可能存在與食性相關的基因。S0911和S1603多為低食性家蠶品種(品系)的特異引物,說明抑制家蠶攝食的基因有可能位于9號染色體的S0911和16號染色體的S1603位點附近。由此推測,家蠶食性相關性狀可能受多基因控制,屬于數(shù)量性狀。并且不同家蠶品種(品系)的攝食性基因可能存在一定差別,食性的遺傳模式也將不同。2.以廣食性家蠶品種中廣04、菁松A和菁松B高食性家蠶品系及菁松A和菁松B低食性家蠶品系的3齡起蠶頭部為材料,檢測攝食性不同的家蠶品種(品系)中pkg1基因的表達特征。pkg1在高食性家蠶品種(品系)中的表達量均顯著高于低食性品種(品系)。pkg1基因在5齡96 h家蠶的組織表達特異性測定表明,該基因在家蠶頭部、中腸、脂肪體、馬氏管及絲腺中均有表達,其中在脂肪體中的表達量最高,馬氏管次之,在頭部和中腸中的表達量最低。3.對廣食性家蠶品種中廣04、低食性家蠶品種魯七、菁松A高食性和低食性家蠶品系4齡起蠶進行饑餓處理,均會導致pkg1基因的表達量顯著升高。分別用桑葉、不含桑葉粉的人工飼料和忌避劑(樟腦)對這4種家蠶品種(品系)進行氣味刺激,然后檢測家蠶頭部pkg1基因的表達變化。結(jié)果表明,桑葉、不含桑葉粉的人工飼料和忌避劑的氣味刺激對pkg1基因在不同品種(品系)中的表達影響不同,桑葉氣味刺激能使高食性和低食性家蠶品種(品系)之間的pkg1表達差異減小;不含桑葉粉的人工飼料刺激引起低食性品種(品系)pkg1的表達量顯著下調(diào),使高、低食性家蠶之間的pkg1表達差異增大;樟腦對不同品種(品系)pkg1基因的表達影響較小。本試驗結(jié)果說明,pkg1可能與家蠶的食性有密切關系,可能具有促進家蠶攝食人工飼料的功能,其具體機理有待于進一步研究。4.給4齡起蠶44 h的菁松B高食性家蠶幼蟲添食不同濃度的8-Br-c GMP,結(jié)果顯示,隨著8-Br-c GMP濃度的增加,家蠶對人工飼料的攝食性顯著降低。給5齡起蠶24 h的優(yōu)食一號家蠶幼蟲注射不同濃度的8-Br-c GMP溶液,結(jié)果表明,當注射濃度為500μM、1000μM和5000μM時,家蠶對人工飼料的攝食性隨著注射濃度的增加而顯著變差,并且當注射濃度為5000μM時,家蠶出現(xiàn)中毒現(xiàn)象,在注射48 h后恢復正常。給5齡起蠶24 h的優(yōu)食一號家蠶幼蟲注射不同濃度的c GMP溶液,家蠶對人工飼料的攝食性并無顯著變化。因此,pkg1基因與家蠶食性相關的功能需要采用其他方法進一步驗證。
[Abstract]:Silkworm, as an important economic insect, has a very important position in human production, life and culture and history. Silkworm is a oligotrophic insect. Mulberry leaves are its natural feed, planting mulberry can raise silkworm, which restricts the development of silkworm mulberry industry. But at present, the technology of artificial silkworm rearing in China has not yet reached the requirement of practical application. The poor feeding ability of the present silkworm breed to artificial feed is the main obstacle to restrict the development of the technology. It has become the key point to improve the feeding and adaptability of the silkworm to the artificial feed. A lot of research and fruitful results have been made, but the molecular mechanism related to the feeding sex of silkworm is not completely clear, and the "food gene" related to the feeding sex of the silkworm has not been obtained. This study uses the microsatellite marker (Simple Sequence Repeats, SSR) as the material of the silkworm variety (strain) which has obvious difference in the feeding ability of the artificial feed. The technique was used to analyze the molecular markers related to the diet of silkworm, and the real time fluorescence quantitative PCR method was used to determine the expression of cyclophosphate dependent protein kinase gene 1 (C GMP dependent protein kinase gene 1, pkg1) in the silkworm variety (strain) of artificial feed, and the drug activated PKG method was used for the pkg1 base. In order to verify the relationship between PKG and the diet of silkworm, the main results are as follows: 1. the varieties of silkworm, which are wide 04 and low eating silkworm variety in the broad feeding silkworm variety, the high food line of the cyanine A and the low food line, the high food line of the cyanine B and the low food line 3, have great differences in the feeding property of the artificial feed. For the material, 18 polymorphic SSR specific markers were screened by 152 pairs of SSR marker primers for SSR-PCR. The maximum number of SSR markers on chromosome.20 on chromosome 3,7,9,10,12,13,14,16,17,19,20 and 21 was the most, and 3 were amplified by the high feeding silkworm variety (strain). The genes that promote the feeding of the silkworm feeding artificial feed may be located on chromosome 20, and S2009 is a specific primer for the high feeding silkworm variety (strain). This further illustrates that the gene that promotes the feeding of the silkworm is likely to be located near the S2009 marker of chromosome 20 and the specific primers of the high feeding silkworm variety (strain). In the vicinity of the S1902 site of chromosome 19, there may be a specific primer for the gene.S0911 and S1603 associated with food, indicating that the genes that inhibit the feeding of the silkworm may be located on the S1603 site of the S0911 and the 16 chromosome of chromosome 9. Multi gene control belongs to quantitative characters, and there may be certain differences in the feeding genes of different silkworm varieties (lines), and the genetic pattern of eating sex will be different from.2. to the broad 04 of the silkworm variety, Sesbania A and cyanine B high eating silkworm, and the 3 instar silkworm heads of the Sesbania A and cyanine B low food silkworm. The expression of pkg1 gene in the silkworm variety (strain) of different sex.Pkg1 was significantly higher in the high feeding silkworm variety (strain) than that of the low food variety (strain).Pkg1 gene in the tissue expression specificity of the 5 instar 96 h silkworm, indicating that the gene was expressed in the head, midgut, fat body, martensitic tube and silk gland of the silkworm. The expression amount in the fat body is the highest, the martensitic tube is the second, the lowest expression in the head and midgut is.3. to the broad 04 silkworm variety, the low food silkworm variety Lu seven, the high food and low food silkworm of the silkworm, the silkworm, the silkworm, 4 years old silkworm is starved, all of which will lead to a significant increase in the expression of the pkg1 gene. The artificial feed and repellent (camphor) were used to stimulate the odor of the 4 species of silkworm, and then the expression of the pkg1 gene in the head of the silkworm was detected. The results showed that the effect of the odor stimulation on the pkg1 gene in the different species (strain) was different. The difference in the expression of pkg1 between high food and low feeding silkworm breeds (lines) decreased, and the artificial feed stimulation without mulberry powder caused a significant downregulation of the expression of pkg1 in the low food variety (strain), and increased the difference in the expression of pkg1 between the high and low feeding silkworms, and the camphor had less effect on the expression of the pkg1 gene of different species (strain). It is suggested that pkg1 may have a close relationship with the feeding nature of the silkworm, which may have the function of promoting the feeding of the silkworm feeding artificial feed. Its specific mechanism needs to be further studied by.4. to feed the larvae of the silkworm, Pinus Sesbania B high eating silkworm larvae of 4 years old, and 8-Br-c GMP with different concentrations. The result shows that with the increase of the concentration of 8-Br-c GMP, the silkworm's intake of artificial feed is taken. The feeding nature of the silkworm, 5 instar 24 h, was injected with different concentrations of 8-Br-c GMP solution. The results showed that when the injection concentration was 500 M, 1000 mu M and 5000 micron M, the feeding ability of the silkworm to artificial feed was significantly worse with the increase of injection concentration, and when the injection concentration was 5000 M, the Bombyx Mori was poisoned. After injection of 48 h, the larvae of silkworm, silkworm, 24 h of the silkworm, injected with different concentrations of C GMP solution, had no significant change in the feeding ability of the silkworm. Therefore, the functions of the pkg1 gene and the food of the silkworm need to be further verified by other methods.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S881.2
本文編號：2148077
[Abstract]:Silkworm, as an important economic insect, has a very important position in human production, life and culture and history. Silkworm is a oligotrophic insect. Mulberry leaves are its natural feed, planting mulberry can raise silkworm, which restricts the development of silkworm mulberry industry. But at present, the technology of artificial silkworm rearing in China has not yet reached the requirement of practical application. The poor feeding ability of the present silkworm breed to artificial feed is the main obstacle to restrict the development of the technology. It has become the key point to improve the feeding and adaptability of the silkworm to the artificial feed. A lot of research and fruitful results have been made, but the molecular mechanism related to the feeding sex of silkworm is not completely clear, and the "food gene" related to the feeding sex of the silkworm has not been obtained. This study uses the microsatellite marker (Simple Sequence Repeats, SSR) as the material of the silkworm variety (strain) which has obvious difference in the feeding ability of the artificial feed. The technique was used to analyze the molecular markers related to the diet of silkworm, and the real time fluorescence quantitative PCR method was used to determine the expression of cyclophosphate dependent protein kinase gene 1 (C GMP dependent protein kinase gene 1, pkg1) in the silkworm variety (strain) of artificial feed, and the drug activated PKG method was used for the pkg1 base. In order to verify the relationship between PKG and the diet of silkworm, the main results are as follows: 1. the varieties of silkworm, which are wide 04 and low eating silkworm variety in the broad feeding silkworm variety, the high food line of the cyanine A and the low food line, the high food line of the cyanine B and the low food line 3, have great differences in the feeding property of the artificial feed. For the material, 18 polymorphic SSR specific markers were screened by 152 pairs of SSR marker primers for SSR-PCR. The maximum number of SSR markers on chromosome.20 on chromosome 3,7,9,10,12,13,14,16,17,19,20 and 21 was the most, and 3 were amplified by the high feeding silkworm variety (strain). The genes that promote the feeding of the silkworm feeding artificial feed may be located on chromosome 20, and S2009 is a specific primer for the high feeding silkworm variety (strain). This further illustrates that the gene that promotes the feeding of the silkworm is likely to be located near the S2009 marker of chromosome 20 and the specific primers of the high feeding silkworm variety (strain). In the vicinity of the S1902 site of chromosome 19, there may be a specific primer for the gene.S0911 and S1603 associated with food, indicating that the genes that inhibit the feeding of the silkworm may be located on the S1603 site of the S0911 and the 16 chromosome of chromosome 9. Multi gene control belongs to quantitative characters, and there may be certain differences in the feeding genes of different silkworm varieties (lines), and the genetic pattern of eating sex will be different from.2. to the broad 04 of the silkworm variety, Sesbania A and cyanine B high eating silkworm, and the 3 instar silkworm heads of the Sesbania A and cyanine B low food silkworm. The expression of pkg1 gene in the silkworm variety (strain) of different sex.Pkg1 was significantly higher in the high feeding silkworm variety (strain) than that of the low food variety (strain).Pkg1 gene in the tissue expression specificity of the 5 instar 96 h silkworm, indicating that the gene was expressed in the head, midgut, fat body, martensitic tube and silk gland of the silkworm. The expression amount in the fat body is the highest, the martensitic tube is the second, the lowest expression in the head and midgut is.3. to the broad 04 silkworm variety, the low food silkworm variety Lu seven, the high food and low food silkworm of the silkworm, the silkworm, the silkworm, 4 years old silkworm is starved, all of which will lead to a significant increase in the expression of the pkg1 gene. The artificial feed and repellent (camphor) were used to stimulate the odor of the 4 species of silkworm, and then the expression of the pkg1 gene in the head of the silkworm was detected. The results showed that the effect of the odor stimulation on the pkg1 gene in the different species (strain) was different. The difference in the expression of pkg1 between high food and low feeding silkworm breeds (lines) decreased, and the artificial feed stimulation without mulberry powder caused a significant downregulation of the expression of pkg1 in the low food variety (strain), and increased the difference in the expression of pkg1 between the high and low feeding silkworms, and the camphor had less effect on the expression of the pkg1 gene of different species (strain). It is suggested that pkg1 may have a close relationship with the feeding nature of the silkworm, which may have the function of promoting the feeding of the silkworm feeding artificial feed. Its specific mechanism needs to be further studied by.4. to feed the larvae of the silkworm, Pinus Sesbania B high eating silkworm larvae of 4 years old, and 8-Br-c GMP with different concentrations. The result shows that with the increase of the concentration of 8-Br-c GMP, the silkworm's intake of artificial feed is taken. The feeding nature of the silkworm, 5 instar 24 h, was injected with different concentrations of 8-Br-c GMP solution. The results showed that when the injection concentration was 500 M, 1000 mu M and 5000 micron M, the feeding ability of the silkworm to artificial feed was significantly worse with the increase of injection concentration, and when the injection concentration was 5000 M, the Bombyx Mori was poisoned. After injection of 48 h, the larvae of silkworm, silkworm, 24 h of the silkworm, injected with different concentrations of C GMP solution, had no significant change in the feeding ability of the silkworm. Therefore, the functions of the pkg1 gene and the food of the silkworm need to be further verified by other methods.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S881.2
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