Papers by Author: Wei De Shen

Abstract: The Bombyx mori serves as model organism among the Lepidoptera insects. In the post-genomic era, in order to study gene function, the profiling of mRNA transcription has become a popular research field. Real-time quantitative RT-PCR (qRT-PCR) has become established as the sensitive method for detecting the expression level of low abundance mRNA, and it usually chooses one or several reference genes to standardize the expression level of target gene. Since the changes in amplification of reference gene can reflect the changes of RNA production, quality or cDNA synthesis efficiency. So choosing an appropriate reference gene can reduce the differences between tested samples. Based on the comparison of Standardization of three frequently-used reference genes (GAPDH, Actin-3, 28srRNA), and decide which is the best way to study gene expression level in silkworm, Bombyx mori.

Abstract: The present study was undertaken to clarify the change of induction of CYP4M5 and CYP4M9 expression level by rutin. In this study, we used dual-spike-in qPCR to examine expression profiles of the silkworm Bombyx mori CYP4M5 and CYP4M9 genes in the larval midgut and fatbody after exposure to rutin. In organization-course study, rutin at middle concentration (5×10-2ng/µL) caused significant upregulation of CYP4M5 and CYP4M9 genes at early time point (2h) in fatbody, higher concentration (5×10-1ng/µL) did secondly, while lower concentration (5×10-3ng/µL) caused little change. In the midgut of silkworm, rutin in all concentrations didn't affect the expression of the two genes. These findings showed that induction of CYP4M5 and CYP4M9 expression level by rutin are concentration depended and tissue-specific.Collectively, CYP4M5 and CYP4M9 genes may have some relationships with metabolism of rutin in silkworm.

Abstract: In order to explore the roles of Bombyx mori glutathione S-transferase gene (BmGST) in detoxification and resistance to insecticides, we used real-time PCR method to detect the transcription levels of five BmGST genes in different tissues of the 5th instar larvae feeding on sodium fluoride treated mulberry leaves. The detection results were normalized by using 3 internal reference genes. The results indicated that the transcription levels of BmGSTs were different in various tissues. Transcription of BmGST genes could be induced by NaF, The normalized data with the above 3 internal reference genes indicated that, it is very important to choose adequate internal reference genes so as to ensure the reliability of the detection results.

Abstract: Acetylcholinesterase (AChE), which contains two subfamilies, ace1 and ace2 in insects, was identified to be the target of organophosphorous and carbamate insecticides. To research the sequences and tissues expressions of two aces, full length cDNAs encoding two ace genes were cloned, designated as Bmm-ace1 and Bmm-ace2 from larvae of the Bombyx mandarina. The amino acid sequence of Bmm-ace1 shared 99.71 % homology with its homolog, Bm-ace1, in silkworm, Bombyx mori, with two mutations (G664S and S307P), and the amino acid sequence of Bmm-ace2 shared 99.37 % homology with Bm-ace2, in B. mori , with four mutations (M18I, N233S, I310V and G621S). Tissue expression analysis showed that ace1 gene expressed only in the brains and fat bodies of B. mandarina, while ace2 genes expressed in all the tissues tested. ace1 and ace2 expressed highly in brains and fat bodies. The present results are significant to the study of resistance evolution of Lepidorptera as well as the understanding of the mechanism of pesticide resistance of insects.

Abstract: The cytochrome P450-dependent monooxygenases play an extremely important role in metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. According to the predicted P450 sequences from the genome of Bombyx mori, a pair of primers was designed and a novel gene named CYP6AE22 was successfully cloned from the midgut mRNA of Bombyx mandarina by RT-PCR (GenBank accession number: FJ843077). Sequence analysis revealed that this gene contains a 1551 bp ORF, encoding a protein of 516 amino acids. The predicted molecular weight and isoelectric point of this protein was 60 kD and 9.0, respectively. The results of semi-quantitative RT-PCR showed that this gene was highly expressed in fat body and brain. And the expression level could be increased by induction with cypermethrin. Treatment with 5ng/uL cypermethrin could increase the expression level in midgut and fat body of the larvae of 1.5 fold and 2.5 fold, respectively. It is inferred that CYP6AE22 gene may be involved in detoxification of insecticide in Bombyx mandarina.

Abstract: Serpins can block different steps in the activation cascade of prophenoloxidase (proPO) system, and play an important role in immunity of insect. In this paper, Haemolymph was collected from the 4th molting, newly moulted 5th instar and day-3 fifth instar larval challenged by lipopolysaccharide (LPS) or Bacillus thuringiensis, respectively. The results revealed that the transcriptional level of Bmserpin-6 in different developmental stages showed a trend of rise first, then fall. Bmserpin-6 of the 4th molting larva expressed highest at 6h post-infection with LPS and 3h post-infection with Bacillus thuringiensis. Bmserpin-6 of newly moulted 5th instar larva expressed highest at 9h post-infection with LPS and 6h post-infection with Bacillus thuringiensis. Bmserpin-6 of day-3 fifth instar larva expressed highest at 9h post-infection with Bacillus thuringiensis. Bmserpin-6 was all highly induced and highly expressed in haemolymph of larval at different developmental stages. But The time to arrive the highest transcriptional level was different. This is inferred that the Serpin gene may play an important role in immunity of Bombyx mori.

Abstract: Acetylcholinesterase (AChE, 2 EC 3.1.1.7), encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. In this study, AChE genes (Bm-ace1, Bm-ace2) were cloned from domesticated silkworm Bombyx mori (Dazao strain) through RT-PCR. Sequence analysis showed that the ORF of Bm-ace1 gene contained 2 025 bp nucleotides, encoding 683 amino acid residues. The predicted protein has a molecular weight (MW) of 76.96 kD and an isoelectric point (pI) of 6.36; The ORF of Bm-ace2 gene contained 1 917 bp nucleotides, encoding 638 AA’s. The predicted protein has a MW of 71.68 kD and a pI of 5.49. These two acetylcholinesterase genes both contain conserved motifs including a catalytic triad, a choline-binding site and an acyl picket. A clustering analysis showed that Bm-ace1 (ABY50088)shared highest similarity with Bmm-ace1 (ABM66370) from Chinese wild silkworm (B. mandarina), Bm-ace2 (ABY50089) shared highest similarity with Bm-ace2 (NP_001037366) from B. mori. Using semi-quantitative RT-PCR, expression analyses in insect tissues and in development period demonstrated that Bm-ace1and Bm-ace2 were expressed highly in head and fat bodies; Bm-ace1 and Bm-ace2 were expressed firstly higher, then lower and higher again from 1st instar to 5th instar stages. Bm-ace1 was expressed higher than that of Bm-ace2 in all the stages. This result will help understanding of the resistance mechanism of B. mori to organophosphosphorous insecticides.

Abstract: With the development of the PCR technology, especially the improvement of its reagent and a method of pebrine detection by PCR in infected Bombyx mori eggs was established.With the 16sRNA gene of Nosema bombycis as target sequence, the results of extraction of genomic DNA from purified microspores showed that 1.3×10-7µg DNA can be extracted from each spore. The sensitivity detection showed the detection limit of Nosema bombycis DNA was 3.25×10-2pg, i.e. 4 spores. (PCR system volume is 25µl). The method of total DNA extraction from pebrine infected silkworm eggs just before hatching was created. The result showed that extracting total DNA from silkworm eggs after the eggs had been treated with 30% KOH met the PCR detection requirements. The result of application study showed the spores in the pebrine infected egg just before hatching can sensitively be found with PCR. The result of a group of eggs just before hatching detection showed that the maximum PCR detection level was of a pebrine infected eggs just before hatching in 300 healthy eggs when the total DNA extraction had been purified with Agarose electrophoresis. The probability of identifying groups of one pebrine spore in infected eggs just before hatching mixed with 100 healthy ones was about 80%.