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Proteomic analysis of the immune response of the silkworm infected by Escherichia coli and Bacillus bombyseptieus

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Insect Science 2012年第5期19卷 559-569页

作者： Xiao-Wu zhong Ping Zhao Yong Zou Hong-Yi Nie Qi-Ying Yi Qing-You Xia zhong-huai xiang The Key Sericultural Laboratory of Agriculture Ministry Southwest University Chongqing China

he silkworm, Bombyx mori, is an economically important insect with a 5 000-year history of domestication. During evolution, the silkworm has developed highly effective defenses against invasion and parasitization by microorganisms. In this study, two microorganisms Escherichia coli and Bacillus bombyseptieus were orally infected to silk- worm larvae. After infection with E. coli and B. bombyseptieus for 24 h, we investigated the polypeptide changes in the hemolymph, midgut and integument using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spec- trometry. Forty-seven differentially expressed proteins were identified in these tissues. They belonged to a variety of functional classes, including immune proteins, metabolic proteins and structural proteins. Compared with controls, E. coli-infected silkworms showed 21 up- regulated proteins, 25 down-regulated proteins and lost one protein. After infection with B. bombyseptieus, silkworms showed 15 up-regulated proteins, 27 down-regulated pro- teins, lost three proteins and retained two proteins unchanged. We speculate that all these proteins may play a role in the silkworm immune response, although it is unclear why and how the two kinds of bacteria can so markedly alter expression of these proteins. These re- sults offer valuable insights for measuring the proteomic responses of the silkworm innate immune mechanism.

关键词： Bacillus bombyseptieus Escherichia coli immune response proteomics silkworm

The silkworm homolog of Methoprene-tolerant （Met） gene reveals sequence conservation but function divergence

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Insect Science 2010年第4期17卷 313-324页

作者： Zhi-Qing Li Dao-Jun Cheng Ling Wei Ping Zhao Xu Shu Lin Tang zhong-huai xiang Qing-You Xia The Key Sericultural Laboratory of Agricultural Ministry Southwest University Chongqing School of Life Science Southwest University Chongqing The Institute of Agronomy and Life Sciences Chongqing University Chongqing China

Methoprene-tolerant （Met） gene has been found to be involved in juvenile hormone （JH） action in insects. Herein, we isolated a silkworm （Bombyx mori） homolog of Met gene from Drosophila melanogaster using bio-informatics analysis and rapid amplification of cDNA ends - polymerase chain reaction method, and defined it as BmMet. The full-length cDNA ofBmMet gene consists of 1 917 nucleotides and includes a 1 368 bp of open reading frame for a deduced protein of 455 amino acids. All deduced protein sequences from Met genes in B. mori and other surveyed insects contain four typical domains ofbHLH, PAS-A, PAS-B and PAC, highlighting a high sequence conservation of Met genes during insect evolution. Also, genomic structure and phylogenic analysis suggested that Met in both B. mori and Drosophila species may originate from an ancestor gene with gce, another member of bHLH-PAS family, via gene duplication. In addition, BmMet was detected in all surveyed tissues and throughout the whole life of silkworm at transcriptional levels. Furthermore, silkworm individuals with RNAi silencing of BmMet gene in the early stage of the fourth instar larvae could molt normally and pupate successfully. This result was different from the observation in T. castaneum but similar to that in D. melanogaster after Met knockdown, revealing that the action mode of Met in B. mori and D. melanogaster should be divergent with that in other insect species.

关键词： Bombyx mori expression pattern genomic structure Methoprene-tolerantgene RNAi

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

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Insect Science 2012年第2期19卷 172-182页

作者： Ai-Chun Zhao Ding-Pei Long San-Yuan Ma Long-Xia Xu Mei-Rong Zhang Fang-Yin Dai Qing-You Xia Cheng Lu zhong-huai xiang The Key Sericultural Laboratory of Agricultural Ministry Institute of Sericulture and Systems BiologySouthwest UniversityChongqingChina

To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time-consuming to maintain non-diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HC1 treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incu- bating developing mother eggs from Chinese lineage diapause silkworm strains at 15℃ （15℃-IME）, we were able to prevent the diapause of their daughter eggs; a similar strategy （15℃-IMES） for the germline transformation of the same strains was that the mother eggs were incubated at 15℃, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non-diapause eggs. By combining tempera- ture and light controls, the improved 15℃-IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15℃-IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.

关键词： Bombyx mori diapause functional genomics gerrnline transformation insect piggyBac

Global expression profile of silkworm genes from larval to pupal stages： Toward a comprehensive understanding of sexual differences

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Insect Science 2011年第6期18卷 607-618页

作者： Min Zhao Xing-Fu Zha Jin Liu Wen-Ji Zhang Ning-Jia He Dao-Jun Cheng Ya Dai zhong-huai xiang Qing-You Xia Institute of Sericulture and Systems Biology College of Biotechnology Southwest University Chongqing Institute of Agronomy and Life Science Chongqing University Chongqing China Tobacco Chuanyu Industrial Corporation Chengdu China

Sexual dimorphism is a widespread phenomenon in many higher animals. The genes and gene networks that underlie sex differences are poorly understood. Using microarray data we analyzed sex-related differences in the global expression profiles of silkworm genes from larval to pupal stages. Sex-biased genes could be divided into three clusters. Cluster 1 contained 932 genes that showed a female-biased expression trend at first and a male-biased trend afterward. Cluster 2 included 283 male-biased genes. Cluster 3 was comprised of 497 female-biased genes that were expressed during the late pupal stage. Cluster 1 genes were found to be related closely to cuticle proteins, hormones, binding proteins, enzyme regulators, structural proteins, transcription regulators and so on. Several genes in clusters 2 and 3 were associated with spermatogenesis and oogenesis, respectively. The chromosomal distribution of sex-biased genes showed evidence of chromosomal enrichment. In particular a large number of the silkworms＇ male-biased genes are located on the Z chromosome. These results provide new insights into the molecular differences that dictate sexual dimorphism in the silkworm.

关键词： Bombyx mori microarray sex-biased gene sexual dimorphism

Abnormal expressions of Ultrabithorax and abdominal-A genes are responsible for an extra thoracic prolegs mutant(EKh) in the silkworm, Bomybx mori

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Abnormal expressions of Ultrabithorax and abdominal-A genes ...

中国蚕学会第八届青年学术研讨会

作者： Ming-Yue Fu Peng Chen Xiao-Ling Tong Lei Chen zhong-huai xiang Cheng Lu Fang-Yin Dai State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

In insects, there are many sets of serially homologous appendages. Nevertheless, they show great divergences in phenotype, function, identity and so on. The gain-of-function mutation of Hox gene Antenpedia(Antp) transformed the antenna into second thoracic legs in Drosophila melanogaster. However, it is still unknown which factors determine the differences between the thoracic and abdominal prolegs. Here we show that a 55kb region including Bombyx mori Ultrabithorax(Bm Ubx) and abdominal-A(Bmabd-A) was responsible for the silkworm mutant Kh-extra-crescents(E), which displayed additional thoracic leg-like prolegs and extra markings on the ventral and dorsal side of the first abdominal segment(A1), respectively. On one hand, we found that Bm Ubx gene was down-regulated not only at the level of m RNA, but also at the protein level. Furthermore, the Bm Ubx protein expression was reduced on the A1 segment probably correlated with the phenotype of the extra thoracic leg-like prolegs. On the other hand, Bmabd-A was up-regulated at both levels and ectopically over-expressed on the segments of the supernumerary prolegs, A1 and A2. In conclusion, our results suggest that the Bmabd-A gene promotes the development of abdominal limbs, while Bm Ubx gene modifies the identity of the thoracic legs.

关键词： Bombyx mori thoracic legs Ultrabithorax abdominal-A

ap locus encodes a C2H2-type transcription factor that functions in the body plan development of the silkworm, Bombyx mori

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ap locus encodes a C2H2-type transcription factor that funct...

中国蚕学会第八届青年学术研讨会

作者： Peng Chen Ming-Yue Fu Xiao-Ling Tong Dan-Dan Li Lei Chen Hai Hu zhong-huai xiang Fang-Yin Dai Cheng Lu State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

The morphological diversity of insects plays an important role in their survival, and in essence it results from the differential expression of genes during the insect body development. In the silkworm Bombyx mori, the ap（apodal） mutant exhibited degraded thoracic legs and that made their crawling and eating mulberry leaves difficult, and also showed female sterility, which is an ideal subject for the study of molecular mechanisms for morphogenesis. In this study, we confirmed that the infertility of ap female moths was due to the degradation of the bursa copulatrix, and report the identification and characterization of the gene responsible for the ap *** mapped the ap locus using 384 F2 individuals with ap mutant phenotype and identified the candidate gene of the ap locus, Bombyx mori sister of odd and bowl（Bmsob）. Analysis of the spatio-temporal expression pattern of Bmsob gene revealed strong expression in the silkworm larval genital gland and in the third day of wandering stage and the first day of pupa. This period is the key stage from larvae to pupae metamorphosis and also the important stage of genital gland development, implying that Bmsob gene plays an important role in the development of silkworm genital gland. Quantitative RT-PCR（qRT-PCR） revealed that the expression of Bmsob was both significantly reduced in the ap mutant compared with the wild-type in the embryo and 1st day pupa. We found that there were sequence differences in the Bmsob promoter region between Dazao and ap mutant. Then we detected the promoter activity in wild-type and ap mutant using the dual luciferase report system. The results showed that the activity of the Bmsob promoter in ap mutant was decreased significantly. Hence, we speculated that the sequence variation of Bmsob promoter region may be a significant reason for the decreased expression level of Bmsob gene. In addition, q RT-PCR showed that the expression levels of Bm Antp, Bm Ubx and Bmabd-A genes were all up-regulate

关键词： Bombyx mori apodal sister of odd and bowl female sterility

Antennapedia is involved in the development of thoracic legs and segmentation in the silkworm,Bombyx mori

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Antennapedia is involved in the development of thoracic legs...

第十届家（柞）蚕遗传育种及良种繁育学术研讨会

作者： Peng Chen Xiao Ling Tong Dan Dan Li Ming Yue Fu Song Zhen He Hai Hu zhong huai xiang Cheng Lu Fang Yin Dai State Key Laboratory of Silkworm Genome Biology Southwest University College of Biotechnology Southwest University

Homeotic genes, which are associated with body patterning of various species, specify segment identity. The Wedge eye-spot（Wes） is a new homeotic mutant located on the sixth linkage group. Homozygous Wes/Wes embryos are lethal and display a pair of antenna-like appendages under the mouthparts as well as fused thoracic segments. These mutants also exhibit a narrower eye-spot at the larval stage compared with the wild type. By positional cloning, we identified the candidate gene of the Wes locus, Bombyx mori Antennapedia（BmAntp）. Two BmAntp transcripts were identified in the homozygote of the Wes mutant,including a normal form and an abnormal form with a 1570-bp insertion. Our data showed that the insertion element was a long interspersed nuclear element（LINE）-like transposon that destroyed the original open reading frame of BmAntp. Quantitative RT-PCR analysis showed that the expression levels of normal BmAntp transcripts were increased markedly in the Wes heterozygous larvae compared with the wild type. Furthermore, we performed RNAi of BmAntp and observed fused thoracic segments and defective thoracic legs in the developing embryos. Our results indicated that BmAntp is responsible for the Wes mutant and has an important role in determining the proper development of the thoracic *** identification of a homeotic mutation in the silkworm is an important contribution to our understanding of the regulation of Hox genes at different levels of expression.

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Fine mapping of a supernumerary proleg mutant (ECs-l) and comparative expression analysis of abdominal-A gene in silkworm,Bombyx mori

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Fine mapping of a supernumerary proleg mutant (ECs-l) and co...

第十届家（柞）蚕遗传育种及良种繁育学术研讨会

作者： Peng Chen Xiao-Ling Tong Dan-Dan Li Ping-Feng Liang Ming-Yue Fu Chun-Feng Li Hai Hu zhong-huai xiang Cheng Lu Fang-Yin Dai State Key Laboratory of Silkworm Genome Biology Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural MinistrySouthwest University College of Biotechnology Southwest University

Patterning and phenotypic variations of appendages in insects provide important clues on developmental genetics. In the silkworm, Bombyx mori, morphological variations associated with the E complex, an analogue of the Drosophila melanogaster bithorax complex, mainly determine the shape and number of prolegs on abdominal segments. Here, we have reported the identification and characterization of the allele responsible for the ECs-l mutant, a model derived from spontaneous mutation of the E complex, with supernumerary legs and extra crescents. Fine mapping with 1605 individuals revealed a 8 kb sequence in the upstream intergenic region of B. mori abdominal-A(Bmabd-A) clustered with the ECs-l *** RT-PCR and Western blotting analyses disclosed a marked increase in Bmabd-A expression in the ECs-l mutant at both the transcriptional and translational levels,compared to wild-type Dazao. Furthermore, we observed ectopic expression of the Bmabd-A protein in the second abdominal segment(A2) of the ECs-l mutant. Our results collectively suggest that the 68 kb region contains important regulatory elements of the Bmabd-A gene, and provide evidence that the gene is required for limb development in abdominal segments in the silkworm.

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The UDP-glucosyltransferase multigene family in Bombyx mori

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The UDP-glucosyltransferase multigene family in Bombyx mori

中国蚕学会第六届青年学术研讨会

作者： Fei-Fei Huang,Chun-Li Chai,Ze Zhang,Zeng-Hu Liu,Fang-Yin Dai,Cheng Lu and zhong-huai xiang (Institute of Sericulture and Systems Biology,Southwest University,Chongqing 400716)

糖基化作用在抑制和排除一系列内生和外源化合物的毒性方面起重要作用。一组尿苷二磷酸-糖基转移酶基因参与催化糖基化反应。昆虫的糖基转移酶在多种生理学过程中起重要作用,包括使酶作用物解毒,例如一些植物中的有毒化合物,以及角质层...

糖基化作用在抑制和排除一系列内生和外源化合物的毒性方面起重要作用。一组尿苷二磷酸-糖基转移酶基因参与催化糖基化反应。昆虫的糖基转移酶在多种生理学过程中起重要作用,包括使酶作用物解毒,例如一些植物中的有毒化合物,以及角质层形成,色素沉着和嗅觉作用等过程。对家蚕糖基转移酶基因进行鉴别和分析可以为解释这一重要家族在昆虫的解毒机制及其它过程中的作用提供有价值的信息。利用完成的基因组数据,我们对家蚕的糖基转移酶基因进行了分析。根据糖基转移酶的特征序列以及与其它物种的同源性,我们在家蚕基因组中共鉴别出了42个糖基转移酶基因。大部分基因成簇分布于染色体上,主要有两大基因群分别位于7号和28号染色体上。系统发生分析表明,这42个基因主要分为5大类群。对家蚕的糖基转移酶基因与其它已进行基因组测序的昆虫的同源基因进行比较发现,其中一些基因是家蚕特有的糖基转移酶基因。利用EST数据,基因芯片数据和RT-PCR对家蚕的糖基转移酶基因进行了表达模式的研究,总共有36个基因在组织中有表达证据,很多基因表现不同的表达模式,表明这些糖基转移酶基因可能具有不同的功能。家蚕基因组中存在42个糖基转移酶基因,这是迄今为止报道的拥有这一家族基因数目最多的昆虫。系统发生分析,基因组结构和表达模式的研究为以后研究这一基因家族的功能提供了重要基础。

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