Dingpei Long

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.

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.

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.

New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.

In order to investigate the role of different parts of the fibroin heavy chain (H‐chain) in the secretion of fibroin in the silk gland of the silkworm (Bombyx mori) in vivo, two enhanced green fluorescent protein (EGFP)/H‐chain fusion genes with deduced protein sequences containing an identical N‐terminal region and different C‐terminal regions of the H‐chain were introduced into the B. mori genome using a piggyBac‐mediated germline transformation. EGFP fluorescence and molecular analysis showed the products of two different EGFP/H‐chain fusion proteins were secreted into the posterior silk gland lumen and aggregated in the middle silk gland and spun into cocoons. The results revealed that only the non‐repetitive N terminus of the H‐chain is essential for secretion of the H‐chain into the posterior silk gland lumen. In addition, our results also indicated that the most likely post‐translational modification of the H‐chain is at the C‐terminal domain. Here, our results not only provide a theoretical basis for the genetic modification of silk fiber as a functional biomaterial but also are of great significance to establishing a new silk gland bioreactor to mass‐produce exogenous proteins in an active form.

An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.

De novo assembly of mulberry (Morus alba L.) transcriptome and identification of candidate unigenes related to salt stress responses

Mulberry (Morus alba L.) is a kind of plant with strong adaptation to drought, salt stress, water logging, and other environmental stresses. However, there is little knowledge on the molecular mechanism involved in its response and resistance to environmental stresses, including salt stress. In this study, a total of 101589 unigenes were obtained from 24 Morus salinity subtranscriptomes using Illumina RNA-sequencing technology, and led to 34.72% of the assembled reads being matched to known transcripts. The number of down-regulated DEGs (differentially expressed genes) under salt stress is more than that of up-regulated DEGs, and these down-regulated DEGs enriched in the process related to stress response by GO and KEGG enrichment analysis. It is notable that some genes showed diverse response patterns against salt stress in genotype- and tissue-dependent manners. The DEGs involved in signal transduction and transcription regulation were found to be more enriched in low-salt-tolerant genotypes and the majority of these responsive genes showed decreased transcript abundance, which may result in low tolerance of low-salt-tolerant genotypes. The results of this study will advance our understanding of the salt response in Morus and provide the basis for further genetic improvement of salt tolerance in Morus and other plants.

Two mulberry phytochelatin synthase genes confer zinc/cadmium tolerance and accumulation in transgenic Arabidopsis and tobacco

Phytochelatin synthase (PCS) is an enzyme involved in the synthesis of phytochelatins, cysteine-rich peptides which play a key role in heavy metal (HM) detoxification of plants. Mulberry (Morus L.), one of the most ecologically and economically important tree genera, has the potential to remediate HM-contaminated soils. However, genes involved in HM detoxification in Morus, such as the PCS genes, have not been identified and characterized. In this study, we identified two Morus notabilis PCS genes based on a genome-wide analysis of the Morus genome database. Full-length MnPCS1 and MnPCS2 cDNAs were 1509 and 1491bp long, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that, under 200μM Zn2+ or either 30 or 100μM Cd2+ stress, the relative expression of each of the two MaPCSs (from Morus alba) was induced in root, stem and leaf tissues within 24h of exposure to the metals, with Cd2+ inducing expression more strongly than did Zn2+. Based on the analysis of total root length and fresh weight of seedlings, overexpression of MnPCS1 and MnPCS2 in Arabidopsis and tobacco enhanced Zn2+/Cd2+ tolerance in most transgenic individuals. The results of transgenic Arabidopsis lines overexpressing MnPCS1and MnPCS2 suggest that MnPCS1 play a more important role in Cd detoxification than MnPCS2. Zn2+/Cd2+ concentrations in both shoots and roots of the transgenic Arabidopsis seedlings were higher than in wild type (WT) seedlings at two Zn2+/Cd2+ concentrations. In addition, there was a positive correlation between Zn accumulation and the expression level of MnPCS1 or MnPCS2. Our results indicated that the Morus PCS1 and PCS2 genes play important roles in HM stress tolerance and accumulation, providing a useful genetic resource for enhancing tolerance to HMs and for increasing the HM phytoremediation potential of these plants.

Genome-Wide Identification and Characterization of Four Gene Families Putatively Involved in Cadmium Uptake, Translocation and Sequestration in Mulberry

The zinc-regulated transporters, iron-regulated transporter-like proteins (ZIPs), the natural resistance and macrophage proteins (NRAMP), the heavy metal ATPases (HMAs) and the metal tolerance or transporter proteins (MTPs) families are involved in cadmium (Cd) uptake, translocation and sequestration in plants. Mulberry (Morus L.), one of the most ecologically and economically important (as a food plant for silkworm production) genera of perennial trees, exhibits excellent potential for remediating Cd-contaminated soils. However, there is no detailed information about the genes involved in Cd2+ transport in mulberry. In this study, we identified 31 genes based on a genome-wide analysis of the Morus notabilis genome database. According to bioinformatics analysis, the four transporter gene families in Morus were distributed in each group of the phylogenetic tree, and the gene exon/intron structure and protein motif structure were similar among members of the same group. Subcellular localization software predicted that these transporters were mainly distributed in the plasma membrane and the vacuolar membrane, with members of the same group exhibiting similar subcellular locations. Most of the gene promoters contained abiotic stress-related cis-elements. The expression patterns of these genes in different organs were determined, and the patterns identified, allowing the categorization of these genes into four groups. Under low or high-Cd2+ concentrations (30 μM or 100 μM, respectively), the transcriptional regulation of the 31 genes in root, stem and leaf tissues of M. alba seedlings differed with regard to tissue and time of peak expression. Heterologous expression of MaNRAMP1, MaHMA3, MaZIP4, and MaIRT1 in Saccharomyces cerevisiae increased the sensitivity of yeast to Cd, suggested that these transporters had Cd transport activity. Subcellular localization experiment showed that the four transporters were localized to the plasma membrane of yeast and tobacco. These results provide the basis for further understanding of the Cd tolerance mechanism in Morus, which can be exploited in Cd phytoremediation.

Identification of the genes involved in heterotrimeric G-protein signaling in mulberry and their regulation by abiotic stresses and signal molecules

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) play important roles in signal transduction and regulate responses to various stresses. Although G-protein signaling pathways have been extensively identified and characterized in model plants, there is little knowledge in non-model and especially in woody plants. Mulberry is an economically and ecologically important perennial tree, which is adaptable to many environmental stresses. In this study, we identified and cloned six G-protein genes including one Gα, one Gβ, two Gγ, one RGS (regulator of G-protein signaling protein) and one RACK1 (receptor for activated C kinase 1) involved in G-protein signaling. Sequence and phylogenetic analysis revealed that Morus G-proteins are evolutionarily conserved compared with those of other plants. A real-time quantitative reverse transcription polymerase chain reaction analysis showed that Morus G-protein signaling genes were ubiquitously but differentially expressed in various tissues. The expression of all of these genes was affected by abiotic stresses and signal molecules, which indicated that Morus G-protein signaling may be involved in environmental stress and defense responses.