Aichun Zhao

New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm

We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins in the cocoon of transgenic silkworms. The results showed that the three different EGFP/H-chain fusion genes were all expressed selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm cocoons is up to 15% (w/w) when using the most highly efficient H-chain expression system. To our knowledge, in comparison with silkworm silk gland expression systems in the literature, the highly efficient expression system developed in this study is the most efficient silkworm silk gland expression system to date. This expression system is the best candidate for foreign gene production and for creation of novel functional silk material. The results suggested the N-terminal domain and the intron of the H-chain gene are important in the secretion of fibroin and its transcription, respectively.

Molecular cloning and expression analysis of mulberry MAPK gene family

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants. Although MAPKs have been identified and characterized in a few model plants, there is little information available for mulberry Morus sp. L., one of the most ecologically and economically important perennial trees. This study identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. Comparative analysis with MAPK gene families from other plants suggested that MnMAPKs could be divided into five subfamilies (groups A, B, C, D and E) and they could have similar functions in response to abiotic and biotic stresses. MnMAPK gene expression analysis of different stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and methyl jasmonate (MeJA)) revealed that all ten MnMAPK genes responded to high/low temperature, salt and drought stresses, and that nine of the ten MnMAPKs (MnMAPK7 excepted) could be induced by ABA, SA, H2O2 and MeJA, which suggested that MnMAPKs may play pivotal roles in signal transduction pathways. Our results indicated that almost all of the MnMAPKs may be involved in environmental stress and defense responses, which provides the basis for further characterization of the physiological functions of MnMAPKs.

FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

The molecular structures of major ampullate silk proteins of the wasp spider, Argiope bruennichi: A second blueprint for synthesizing de novo silk

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk.

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

Abstract  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 HCl 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 incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐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°C‐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.

Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry

A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.

In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange.

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84-7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species.

Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3–A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the β-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance.

Isolation and expression analysis of anthocyanin biosynthetic genes in Morus alba L.

Anthocyanins from mulberry fruits are used in medicine. However, little anthocyanin can be detected in other tissues and sometimes also mulberry fruits are colorless. The aim of this study was to investigate which gene or genes have the strongest correlation with the anthocyanin biosynthesis. The expression of several anthocyanin synthesis genes were determined in different tissues of two white and two purple fruit cultivars. Genes encoding dihydroflavonol reductase (MaDFR) and anthocyanidin synthase (MaANS) showed a high expression only in fruit tissue of purple-fruit cultivars. During the development of mulberry fruits, the anthocyanin content was well correlated with the transcripts abundance of MaDFR, MaANS, and MaCHS (encoding chalcone synthase). The skin of female mulberry flowers turns red under irradiance because of up-regulated expressions of MaCHS, MaDFR, and MaANS. These three genes may control the anthocyanin biosynthesis in mulberry and up-regulation of them may greatly increase the anthocyanin content.

Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.