对人工饲料不同摄食性家蚕品种（品系）SSR标记分析及pkg1基因的表达研究

发布时间：2018-07-27 14:11

【摘要】：家蚕作为重要的经济昆虫,在人类生产生活及文化历史上均有非常重要的地位。而家蚕是寡食性昆虫,桑叶是其天然饲料,栽桑才能养蚕,这制约了蚕桑业的发展。用人工饲料养蚕是蚕业史上的一项重要的技术革新,是今后我国养蚕业发展的方向。但目前我国人工饲料养蚕技术尚未达到实用化的要求,现行蚕品种对人工饲料的摄食性较差是制约该技术发展的主要障碍,提高家蚕对人工饲料的摄食性和适应性成为研究的重点。前人已从生理学和遗传学等方面对家蚕食性做了大量的研究,并取得了丰硕的成果,但家蚕食性相关的分子机理尚未完全明确,与家蚕摄食性相关的“食性基因”尚未获得。本研究以对人工饲料摄食性差异明显的家蚕品种(品系)为材料,利用微卫星标记(Simple Sequence Repeats,SSR)技术对家蚕食性相关的分子标记进行分析。并采用实时荧光定量PCR方法测定了环磷酸鸟苷依赖性蛋白激酶基因1(c GMP dependent protein kinase gene 1,pkg1)在人工饲料摄食性不同的家蚕品种(品系)中的表达特征,采用药物激活PKG的方法对pkg1基因进行功能验证,以期探明PKG与家蚕食性的关系。主要获得如下结果:1.以广食性家蚕品种中广04和低食性家蚕品种鲁七、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对对人工饲料摄食性存在很大差异的家蚕品种(品系)为材料,利用152对SSR分子标记引物对供试家蚕品种(品系)进行SSR-PCR扩增,共筛选出18个多态性SSR特异标记,主要位于第3、7、9、10、12、13、14、16、17、19、20和21号染色体上。20号染色体上的SSR标记最多,为3条,且多由高食性家蚕品种(品系)扩增出来,说明促进家蚕摄食人工饲料的基因可能位于20号染色体上,并且S2009为高食性家蚕品种(品系)的特异性引物,这进一步说明促进家蚕摄食的基因很可能位于20号染色体的S2009标记附近。S1902也多为高食性家蚕品种(品系)的特异引物,说明在19号染色体的S1902位点附近也有可能存在与食性相关的基因。S0911和S1603多为低食性家蚕品种(品系)的特异引物,说明抑制家蚕摄食的基因有可能位于9号染色体的S0911和16号染色体的S1603位点附近。由此推测,家蚕食性相关性状可能受多基因控制,属于数量性状。并且不同家蚕品种(品系)的摄食性基因可能存在一定差别,食性的遗传模式也将不同。2.以广食性家蚕品种中广04、菁松A和菁松B高食性家蚕品系及菁松A和菁松B低食性家蚕品系的3龄起蚕头部为材料,检测摄食性不同的家蚕品种(品系)中pkg1基因的表达特征。pkg1在高食性家蚕品种(品系)中的表达量均显著高于低食性品种(品系)。pkg1基因在5龄96 h家蚕的组织表达特异性测定表明,该基因在家蚕头部、中肠、脂肪体、马氏管及丝腺中均有表达,其中在脂肪体中的表达量最高,马氏管次之,在头部和中肠中的表达量最低。3.对广食性家蚕品种中广04、低食性家蚕品种鲁七、菁松A高食性和低食性家蚕品系4龄起蚕进行饥饿处理,均会导致pkg1基因的表达量显著升高。分别用桑叶、不含桑叶粉的人工饲料和忌避剂(樟脑)对这4种家蚕品种(品系)进行气味刺激,然后检测家蚕头部pkg1基因的表达变化。结果表明,桑叶、不含桑叶粉的人工饲料和忌避剂的气味刺激对pkg1基因在不同品种(品系)中的表达影响不同,桑叶气味刺激能使高食性和低食性家蚕品种(品系)之间的pkg1表达差异减小;不含桑叶粉的人工饲料刺激引起低食性品种(品系)pkg1的表达量显著下调,使高、低食性家蚕之间的pkg1表达差异增大;樟脑对不同品种(品系)pkg1基因的表达影响较小。本试验结果说明,pkg1可能与家蚕的食性有密切关系,可能具有促进家蚕摄食人工饲料的功能,其具体机理有待于进一步研究。4.给4龄起蚕44 h的菁松B高食性家蚕幼虫添食不同浓度的8-Br-c GMP,结果显示,随着8-Br-c GMP浓度的增加,家蚕对人工饲料的摄食性显著降低。给5龄起蚕24 h的优食一号家蚕幼虫注射不同浓度的8-Br-c GMP溶液,结果表明,当注射浓度为500μM、1000μM和5000μM时,家蚕对人工饲料的摄食性随着注射浓度的增加而显著变差,并且当注射浓度为5000μM时,家蚕出现中毒现象,在注射48 h后恢复正常。给5龄起蚕24 h的优食一号家蚕幼虫注射不同浓度的c GMP溶液,家蚕对人工饲料的摄食性并无显著变化。因此,pkg1基因与家蚕食性相关的功能需要采用其他方法进一步验证。
[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.
【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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