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Expression Profile Analysis of Two Acetylcholinesterase Genes in Silkworm, Bombyx Mori

Author: ChenHuiJuan
Tutor: LiFei
School: Nanjing Agricultural College
Course: Agricultural Entomology and Pest Control
Keywords: Silkworm Acetylcholinesterase Bm-ace1 Bm-ace2 Expression levels Quantitative PCR Double reciprocal plot Transcription factor binding sites
CLC: S881.26
Type: Master's thesis
Year: 2009
Downloads: 23
Quote: 1
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Abstract


Insects in a variety of the acetylcholinesterase gene's existence, the the acetylcholinesterase expression profiling and functional studies to become a hot one. Silkworm there are two acetylcholinesterase gene: Bm-ace1, the Bm-ace2 because silkworm special economic value, together with its genome sequence has been published, silkworm has been used as a model organism widely used in molecular biology and biological information science study. The research projects focus mainly quantitative PCR, biochemical tests, biological informatics method of silkworm two acetylcholinesterase gene expression profiling and biochemical characteristics of the system studied. The main content and the results are as follows: 1) the use of quantitative PCR technique silkworm two acetylcholinesterase gene in different developmental stages (1-5 instar, pupae and adults, eggs) and the same age at different ages expression level was measured. The results show that, the Bm-ace2 expression level was significantly higher than in all developmental stages Bm-ace1, proportion of Bm-ace2/Bm-ace1 is 2.92.64.75 adult stage, especially males the greatest difference. This is contrary to the reported results agricultural insects, existing studies have shown that acetylcholinesterase Ⅰ expression was significantly higher than the acetylcholinesterase II in a variety of agricultural pests. Analysts believe that the existence of diversity on the evolutionary selection in the insect variety acetylcholinesterase gene. Due to the special living environment, Bombyx mori select ace2, and other pests selected ace1. Two genes in different developmental stages of each expression trend, Bm-ace1 highest expression in the larvae, the egg stage was the lowest, no significant difference of the other stages; Bm-ace2 lowest expression in the egg stage, was higher than in males larvae and females. Day old larvae in the third instar, the the Bm-ace1 expression levels peaked in the early age (1 day), middle age (three days), and the age of the end of the period (5 days); expression of Bm-ace2 trend for the first 1-3 The day-by-day rise 3-5 day-by-day decline. According to the quantitative results, Bm-ace2 expression in the third instar has been higher than Bm-ace1. The proportion of Bm-ace2/Bm-ace1 dynamic changes, the highest peak in two days, and then gradually decline, that is, two ester -by-day to reduce the difference between the amount of the basic enzyme expression. In the silkworm head, digestive tract, Malpighian tubules, silk gland, ovary, testis, two ace genes were expressed, and the two genes are expressed in different tissues amount of difference. The two enzymes are highly expressed in the head, Bm-ace1 also higher in the silk gland, Bm-ace2 highly expressed in reproductive organs (ovaries and testes). Speculated that in addition to the main hydrolysis acetylcholine function, both acetylcholinesterase there may be other non-hydrolytic enzymes. In addition, expression and not their peers during consistent, Bm-ace2 expression was significantly higher than that Bm-ace1. Testis the Bm-ace2/Bm-ace1 the ratio of the highest to the lowest ratio in the head. 2) use of silkworm 1,3 age different days insects crude enzyme solution was measured the AChE enzyme vitality and Km, Vm Determination of biochemical parameters. The results show that the third instar larvae of different ages insects enzyme ,1-3 ,3-5 age gradually increased gradually decline into a dynamic change this trend with the Bm-ace2 gene mRNA expression, and Bm-ace1 gene inconsistent, and further evidence of the dominant role in the Bm-ace2 genes in the silkworm. Calculated using double reciprocal plot Km, Vm, found in 1,3 age at different ages insects double reciprocal plot inconsistencies. This indicates that the The different ages insects of AChE protein component of the hybrid enzyme solution is significantly different, this is due to the crude enzyme solution is a mixture of the two enzymes, on the other hand, is caused by the dynamic changes of the two enzymes at different time. The test results show that the Determination of enzyme kinetics a conventional method, using the crude enzyme solution in the presence of a variety of acetylcholinesterase gene insects can not be correctly reaction enzyme and biochemical characteristics, must be various AChE enzyme were purified after determination of its biochemical characteristic parameter . 3) through bioinformatics Software TESS predicted transcription factor binding sites on the the Bm-ace1, Bm-ace2 transcription factor binding core area. Predicted 37 transcription factor binding site, and a promoter region of the Bm-ace2 33 predicted binding sites in the promoter region of the Bm-ace1. In these sites, the common binding sites of the two genes is only 15. The two genes have respective unique transcription factor binding sites, suggesting that these different binding sites on the binding of transcription factors may be caused by the silkworm two acetylcholinesterase gene expression level difference is one important reason.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Sericulture > Silkworm basic science > Silkworm physiology, genetics,ecology, biology,physics, biochemistry > Silkworm genetics
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