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Insect Chitin Metabolism and Plant Protection ZHANG Jian-Zhen Scientia Agricultura Sinica    2014, 47 (7): 1301-1302.   DOI: 10.3864/j.issn.0578-1752.2014.07.006 Abstract （429）   HTML （3）    PDF （216KB）（724）       Knowledge map    Save Reference | Related Articles | Metrics

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Research Progresses in Insect Glycosyl Hydrolyase Family 20 β-N-acetylhexosamindase QU Ming-Bo, LIU Tian, CHEN Lei, CHEN Qi, YANG Qing Scientia Agricultura Sinica    2014, 47 (7): 1303-1312.   DOI: 10.3864/j.issn.0578-1752.2014.07.007 Abstract （468）   HTML （13）    PDF （674KB）（807）       Knowledge map    Save β-N-acetylhexosaminidase (Hex), a family 20 glycosyl hydrolyases (GH20), is an essential enzyme for the metabolism of N-acetylhexosamine by catalyzing the removal of β-linked N-acetylhexosamine from the non-reducing ends of glycans, glycoproteins and glycolipids. Insect GH20 β-N-acetylhexosaminidase involves in multiple physiological processes, including chitin degradation, protein N-glycan modification, glycoconjugates degradation and sperm-egg recognition. Because of its important functions, β-N-acetylhexosaminidase may serve as a potential target for designing eco-friendly pesticides. The insect family 20 β-N-acetylhexosaminidase could be grouped into 4 groups according to their phylogenetic relationship and physiological functions, that are Hex1, Hex2, Hex3 and Hex4. Hex1 is mainly expressed in epidermis during insect molting. The RNAi of Hex1 would cause lethal phenotype during insect molting. The old cuticle of the dsHex1 injected larvae could not be shed off. The study of the enzymatic properties of Hex1 demonstrates that it could efficiently hydrolyze β-1,4 linked chitin oligosaccharide with very high specificity but could not act on β-1,3 or β-1,2 linked substrates. It is a special enzyme for chitin degradation. So far, the only crystal structure of insect β-N-acetylhexosaminidase is OfHex1 from Ostrinia furnacalis. The structure at subsite “+1” in OfHex1 explains the high efficiency and specificity of OfHex1 towards chitin oligosaccharides. Hex2 could not be found in insects belong to diptera. It shows highly similarity towards human β-N-acetylhexosaminidase than the other insect β-N-acetylhexosaminidases. The RNAi of Hex2 will cause abnormalities of larval abdomen, pupa and adult appendages. The enzymatic properties of Hex2 show that it is an enzyme with broad substrate-spectrum by hydrolyzing β-N-acetylhexosamine from chitin oligosaccharides, N-glycan and glycolipids. Hex2 may be involved in the degradation of glycoconjugates like the function of human β-N-acetylhexosaminidase. Hex3 is not well studied so far. The RNAi of Hex3 could cause abnormalities during insect molting. It is proved to be in molting fluid and interact with Hex1. The enzymatic activity analysis indicates that OfHex3 is able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Besides, Hex3, together with Hex1 and Hex4, are also found to be in the plasma membrane of spermatozoa in dipteran insects, suggesting that it may be involved in sperm-egg recognition. Hex4 is also called FDL because the mutant of this gene will cause fused lobes phenotype of the mushroom body in Drosophila melanogaster. It is another β-N-acetylhexosaminidase with strict substrate specificity. It could exclusively hydrolyze the terminal β-1, 2-GlcNAc residue from the α-1,3 branch instead of the α-1,6 branch of the substrate GnGn. The subcellular localization of Hex4 indicates that it locates in the Golgi apparatus. All these suggest that Hex4 is a β-N-acetylhexosaminidase involved in the modification of N-glycans. So far, although big progress has been achieved on insect β-N-acetylhexosaminidase, only Hex1 has been well studied, including its physiological functions, enzymatic properties and crystal structures. It is proved to be a potential target for designing eco-friendly pesticides. How the other three β-N-acetylhexosaminidases function during insect development and the structure basis of the different enzymatic properties among the four groups of β-N-acetylhexosaminidases are still uncover. This review focuses on the recent progresses on phylogenetic relationship, crystal structure, enzymatic properties and physiological significance of insect β-N-acetylhexosaminidases. Reference | Related Articles | Metrics

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Molecular Characterization and Function of Chitinase 10 Gene (OcCht10) from Oxya chinensis LI Da-Qi-1, WANG Yan-1, ZHANG Jian-Qin-1, LI Tao-1, SUN Yi-2, ZHANG Jian-Zhen-1 Scientia Agricultura Sinica    2014, 47 (7): 1313-1320.   DOI: 10.3864/j.issn.0578-1752.2014.07.008 Abstract （484）   HTML （2）    PDF （642KB）（9517）       Knowledge map    Save 【Objective】 The objectives of this study are to obtain cDNA sequence of chitinase 10 gene (OcCht10) from Oxya chinensis, analyze its functional domain and phylogenetic relationship with chitinases from other known insect species, investigate its expression patterns and biological function during molting process, and to provide a new candidate gene for pest control.【Method】 cDNA fragments of OcCht10 were searched from O. chinensis’ transcriptome database. After blast analysis, the cDNA sequence of OcCht10 was assembled and translated, the functional domains of OcCht10 were predicted by bioinformatics methods. Phylogenetic analysis was performed with other insect chitinase 10 amino acid sequences. The first-stranded cDNAs were synthesized by using RNA isolated from integument of each day of 5th instar nymphs and various tissues of the 6th day in 5th instar nymphs. Reverse transcription quantitative PCR (qPCR) was carried out to analyze the gene expression patterns. Biological function of OcCht10 was studied by RNA interference method. The dsRNA primers were designed for dsOcCht10 synthesis in vitro. The dsRNAs were injected into the 2nd day of 5th instar nymphs for RNA interference, integument was dissected for silencing efficiency detection at 24 h after injection by using qPCR method. The phenotype was carefully observed and mortality was calculated till control insects molted to adults.【Result】 The obtained cDNA (9 318 bp) of OcCht10 contained an open reading frame of 8 613 bp, encoding 2 870 amino acid residues and a non-coding region of 705 bp at 3′ end. There were about 500 bp lost in 5′ end. The deduced amino acid sequence included five chitinase catalytic domains and six chitin binding domains. Phylogenetic analysis showed that OcCht10 belonged to chitinase group Ⅱ, the genes from this group were crucial for insect molting based on references. Tissue specific expression analysis of OcCht10 showed that it was predominately expressed in the integument, foregut and hindgut, which developed from ectoderm. The results suggested that OcCht10 may be involved in chitin metabolism of insect integument. Developmental expression patterns showed that OcCht10 was highly expressed before and after molting stages, lower in middle stages of 5th instar nymphs, which implied that OcCht10 could digest chitin of integument during molting process. RNA interference results indicated that the corresponding transcript level was silenced by 70% after OcCht10 dsRNA injection. Compared with the dsGFP injected control group, the nymphs injected with OcCht10 dsRNA displayed slow development and failed to detach old cuticle during molting, the mortality reached 100%.【Conclusion】 The partial cDNA sequence of OcCht10 was obtained from O. chinensis, the mRNA expression of OcCht10 was higher in the integument before molting; OcCht10 is involved in O. chinensis molting process, and dsOcCht10 injection can effectively silence mRNA expression of this gene and result in the block of ecdysis and even death of O. chinensis. Reference | Related Articles | Metrics Cited: Baidu(2)

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Molecular Characterization and Functional Analysis of Chitin Deacetylase 2 Gene in Oxya chinensis YU Rong-Rong, DING Guo-Wei, GUO Ya-Ping, MA 恩Bo, ZHANG Jian-Zhen Scientia Agricultura Sinica    2014, 47 (7): 1321-1329.   DOI: 10.3864/j.issn.0578-1752.2014.07.009 Abstract （464）   HTML （1）    PDF （627KB）（447）       Knowledge map    Save 【Objective】 In order to provide a theoretical basis for selecting novel target for pest control, molecular characteristics and biological function of chitin deacetylase 2 gene (OcCDA2) from Oxya chinensis were studied.【Method】The cDNA sequences of OcCDA2 were searched from transcriptome of O. chinensis by bioinformatics method, and the conserved domain was anaylzed, the homologous sequences from Tribolium castaneum, Drosophila melanogaster; Anopheles gambiae, Bombyx mori and Choristoneura fumiferana were selected to construct phylogenetic tree. The real-time PCR was applied to detect the relative expression of OcCDA2 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference (RNAi) was performed to study the biological function of the OcCDA2 during molting and development of O. chinensis. 【Result】The full length cDNA sequences of two chitin deacetylase genes were identified. The amino acid analysis showed that they possessed the signal peptide, the open reading frame contained three conserved domains: chitin binding domain (ChBD), low-density lipoprotein receptor chass A domain (LDLa) and chitin deacetylase catalytic domain (CDA). The phylogenetic analysis showed that two variants of OcCDA2 clustered with the CDA2s of five insect species, and the two variants differed only in one exon consisting of about 120 nucleotides, the alternatively spliced regions gathered with CDA2a or CDA2b, respectively. Based on sequence alignment and phylogenetic analysis, two variants of OcCDA2 were named as OcCDA2a and OcCDA2b. Tissue-specific expression analysis indicated that both OcCDA2a and OcCDA2b were mainly expressed in the integument, foregut and hindgut. Developmental expression patterns showed that the relative expressions of OcCDA2a and OcCDA2b were higher in the 1st day of 5th instar nymphs, then decreased gradually, and began to ascend before molting. RNAi results showed that the expression of target genes was significantly reduced in 24 h after dsOcCDA2a or dsOcCDA2b was injected into 1st day of 5th instar nymphs, the silence efficiency of genes was 96.72% and 80.43%, respectively. Compared with dsGFP injected controls, the majority of insects injected with dsOcCDA2 and dsOcCDA2a were unable to shed the old cuticle and led to body warping, finally to death. A minority of nymphs died before molting without obvious phenotypes. A few nymphs could develop to the adults, however, the legs appeared strengthless and the movement became slowly. The mortality rates were 87.5% and 94.4%, respectively. While insects injected with dsOcCDA2b alone could successfully molt to adults, and no phenotype was observed.【Conclusion】The OcCDA2 has two alternatively spliced variants, namely OcCDA2a and OcCDA2b. OcCDA2a plays a key role in the development of O. chinensis. The silence of OcCDA2a leads to the ecdysis failure and death of O. chinensis. However, OcCDA2b is not essential to molting process of O. chinensis. Reference | Related Articles | Metrics Cited: Baidu(3)

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Expression, Function and Regulation of Chitin Synthase 2 Gene in Locusta migratoria LIU Xiao-Jian, CUI Miao, LI Da-Qi, ZHANG Huan-Huan, YANG Mei-Ling, ZHANG Jian-Zhen Scientia Agricultura Sinica    2014, 47 (7): 1330-1340.   DOI: 10.3864/j.issn.0578-1752.2014.07.010 Abstract （552）   HTML （15）    PDF （903KB）（11074）       Knowledge map    Save 【Objective】Chitin synthase is one of the key enzymes responsible for chitin synthesis in insects. As this enzyme is absent in higher animals, it could be served as a potential target for developing safe and effective insecticides. In our earlier research, the cDNA of chitin synthase 2 gene (LmCHS2, GenBank accession number: GU067731) in Locusta migratoria was cloned. The objectives of this paper are to further study the expression, function and regulation of LmCHS2, and to provide a scientific basis for effective pest control using RNAi methods.【Method】Based on the nucleotide sequence of LmCHS2, a pair of specific expression primers was designed, the expression patterns of LmCHS2 were studied in eggs, nymphs and adults by RT-qPCR. The dsRNA of LmCHS2 was synthesized in vitro, and then injected into the female or male adults on day 1, respectively. The midguts dissected from the injected insects on day 5 were pooled for each RNA extraction. cDNA synthesis and RT-qPCR were performed to determine the down-regulation of LmCHS2. After dissected the whole gut, the midgut changes and integrity of peritrophic matrix (PM) were observed to explore the biological functions of this gene in L. migratoria adults. Locusts were maintained with no food in different times, and feeding again, to observe the changes of guts. Then the transcript levels of LmCHS2 were detected by RT-qPCR. 【Result】 LmCHS2 was almost undetectable during the early and middle embryogenesis, but dramatically up-regulated in late eggs. It was consistently expressed throughout the nymphal and adult stages. After dsCHS2 was injected into the female or male adults on day 1, significantly reduced transcript of LmCHS2 was observed as compared with that of the controls, and resulted in a decreased feeding and a high mortality of insects (78% for female and 85% for male adults). After dissection, it was found that there was virtually no food contained in dsCHS2-injected insects and the average length of midguts and gastric caeca was shorter than that of the control. Furthermore, histological observation of midguts showed that the control locusts contained a fully developed PM, however, locusts injected with dsCHS2 exhibited a disrupted PM or even absence of the PM. Locusts were treated under starvation for 48 h, the midguts hardly contained food and the average length of midguts was significantly shorter than that of the control midguts. From the H & E stained results, it was found that the PM was almost absent in non-fed midguts while the PM of control midguts was well-structured, which was very similar with the RNAi. But after fed again, the insects contained a fully developed PM. When locusts were maintained with no food for 24 h and 48 h, the transcript levels of LmCHS2 were suppressed significantly. When locusts were fed for another 0.5 h period, the transcript levels increased to the control level rapidly, which suggested that feeding affected the expression of LmCHS2. 【Conclusion】LmCHS2 is responsible for chitin biosynthesis of peritrophic matrix of the midgut and plays a key role for the development of L. migratoria. The decreased expression of this gene affected the integrity of the PM, thus hindered the food absorption and led to the mortality of the locusts. In addition, feeding regulated the expression of LmCHS2. Reference | Related Articles | Metrics

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Cloning and Expression of Two Laccase Genes OfLac1 and OfLac2 from the Insect Ostrinia furnacalis CHEN Peng, QU Ming-Bo, YANG Jun, YANG Qing Scientia Agricultura Sinica    2014, 47 (7): 1341-1350.   DOI: 10.3864/j.issn.0578-1752.2014.07.011 Abstract （491）   HTML （2）    PDF （536KB）（691）       Knowledge map    Save 【Objective】 The objectives of this study are to clone two genes encoding OfLac1 and OfLac2 from Ostrinia furnacalis, and to predict physiological functions of these two genes based on the stage-specific and tissue-specific expression patterns of the two genes during development as well as their responses to the ecdysone (20E) treatment. 【Method】 The first fragment of OfLac1 or OfLac2 was amplified by RT-PCR using degenerated primers designed according to the conserved amino acid sequences of the other insects’ Lac1 and Lac2, respectively, and cDNAs generated from RNA isolated from O. furnacalis at pupa stage as template. The 3′ and 5′ regions of OfLac1 or OfLac2 were obtained by RACE using gene specific primers designed according to the first fragment of OfLac1 or OfLac2, respectively. Seven different tissues including epidermis, fat body, trachea, midgut, Malpighian tubule, silk gland and testes were collected from the fifth-instar day-4 larvae of O. furnacalis and the expression levels of OfLac1 and OfLac2 in these tissues were analyzed through semi-quantitative PCR. Twenty-nine samples were collected at different growth periods of O. furnacalis from egg to adult and the expression levels of OfLac1 and OfLac2 were analyzed by using real-time PCR. The fifth-instar day-2 larvae of O. furnacalis were treated with ecdysone (20E) and samples were collected at 1, 4, 8, 12 and 24 h post treatment. The expression levels of OfLac1 and OfLac2 were then analyzed by using real-time PCR. 【Result】 The full-length cDNA of OfLac1 and OfLac2 from O. furnacalis were obtained. The cDNA sequence of OfLac1 gene was 3 065 bp, containing a 5′-untranslated region (5′-UTR) of 222 bp and a 3′-untranslated region (3′-UTR) of 440 bp. It contained an ORF of 2 403 bp encoding 800 amino acid residues with a predicted molecular weight of 90.6 kD and an isoeletric point of 5.34. OfLac2 was 3 405 bp in length, containing a 5′-UTR of 162 bp and a 3′-UTR of 960 bp. The ORF of OfLac2 was 2 283 bp, encoding 760 amino acid residues with a predicted molecular weight of 84.0 kD and an isoelectric point of 6.43. OfLac1 was predicted to contain an N-terminal signal peptide with 22 amino acids in length and OfLac2 was predicted to contain an N-terminal signal peptide with 23 amino acids. The expression pattern analysis revealed that OfLac1 was mainly expressed in midgut and Malpighian tubule, and low amount of OfLac1 transcripts could also be detected in trachea and epidermis. During development, OfLac1 was mainly expressed in adult, and the expression level of OfLac1 during larval and pupal stages was low. OfLac2 was mainly expressed in midgut and silk gland, low amount of OfLac2 transcripts could also be detected in trachea and epidermis. During development, OfLac2 was mainly expressed at prepupa stage during the larval-pupal molting. The expression of OfLac2 was slightly up-regulated in the last day of each instar. The expression levels of OfLac1 and OfLac2 were significantly increased at 24 h after 20E treatment. 【Conclusion】 Two genes encoding OfLac1 and OfLac2 were cloned from O. furnacalis. The expression patterns of OfLac1 and OfLac2 were different, but they were both up-regulated by 20E. OfLac2 was related with the cuticle tanning during molting. Reference | Related Articles | Metrics Cited: Baidu(4)

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Molecular Cloning, Expression Patterns and RNAi of UDP-N-acetylglucosamine Pyrophosphorylase in Spodoptera exigua CHEN Jie, CHEN Hong-Xin, YAO Qiong, ZHANG Wen-Qing Scientia Agricultura Sinica    2014, 47 (7): 1351-1361.   DOI: 10.3864/j.issn.0578-1752.2014.07.012 Abstract （384）   HTML （2）    PDF （790KB）（563）       Knowledge map    Save 【Objective】 In insects, chitin biosynthesis is a key process for their growth and development. Although it has been studied deeply on fungi, little is known for other genes in the chitin biosynthesis pathway of insects, because most of researches were focused in trehalase and chitin synthase. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is one of the enzymes in the chitin biosynthesis pathway of insects. This paper aims to clarify its effect on the expression of chitin synthase genes and survival rates of Spodoptera exigua. 【Method】 With the technique of molecular biology, such as the polymerase chain reaction (PCR) and RACE technique, the SeUAP from S. exigua was cloned and sequenced. Also, in order to clarify the expression pattern of SeUAP, the total RNA of several tissues of the 2nd day of 5th instars of S. exigua and total RNA of everyday of its life cycle were extracted to detect the expression levels of SeUAP by RT-PCR. Finally, dsRNA was injected into the 1st day of 5th instars of S. exigua to survey the survival rate and detect the expression levels of CHSA and CHSB, which are the last enzymes in the pathway. 【Result】 The complete cDNA sequence of SeUAP was 2 098 bp encoding a protein of 491 amino acid residues, which has a close evolutionary relationship with orthologues in Culex quinquefasciatus, Aedes aegypti, Drosophila melanogaster and Anopheles gambiae. RT-PCR results showed that SeUAP was highly expressed in integument and ovaries. The expression was also detected in trachea and midgut but virtually not in Malpighian tubules and fat body. In addition, RT-PCR results showed that SeUAP was highly expressed at egg stage, L22 (2nd day 2nd instars), L31, L42, L51, P0, P5, P7 and A3, which appeared to be related to the high demanding of chitin synthesis. Further research of RNAi showed that the injection of dsRNA of SeUAP into the larvae with 5 μg caused lethal effect and abnormal phenotypes of pupae, which could be classified into two major groups: (1) Abdomen puparium was formed while head puparium was not and the pupa had difficulty in breaking the old cuticle and getting out; (2) The head puparium was formed and the pupa already broke the old cuticle, but the pupation process stopped and never finished. Silencing of target gene could down-regulate the mRNA levels of chitin synthase gene A (CHSA) and CHSB, especially CHSB. 【Conclusion】 UAP influences gene expression in the chitin biosynthesis pathway and play a role in growth and development of S. exigua. Reference | Related Articles | Metrics Cited: Baidu(7)

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