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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 790-795 |
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doi:10.1111/j.1745-7270.2008.00460.x |
Knockdown of
ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system
leads to lethal ecdysis deficiency in silkworm
Hongjiu Dai1, Li Ma2, Jue Wang1, Rongjing Jiang1,2, Zhugang Wang1, and Jian Fei1,3*
1
Shanghai Research Center
for Model Organism, Shanghai 201203, China
2
Institute of Plant Physiology
and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China
3
Tongji University,
Shanghai 200092, China
Received: May 31, 2008�������
Accepted: June 30, 2008
This work was supported by grants from the E-Institutes of Shanghai Municipal Education Commission (No. E03003) and the Science and Technology Commission of Shanghai Municipality (Nos. 06DZ19004 and 06XD14014)
*Corresponding
author: Tel, 86-21-65980334; Fax, 86-21-65982429; E-mail,
[email protected]
Ecdysis-triggering
hormone (ETH) is an integration factor in the ecdysis process of most insects,
including Bombyx mori (silkworm). To understand the function of the ETH
gene in silkworm, we developed an effective approach to knockdown� the
expression of ETH in vivo based on RNA interference (RNAi) and
a binary UAS/GAL4 expression system that has been successfully used in other
insect species. Two kinds of transgenic silkworm were established with this
method: the effector strain with the ETH RNAi sequence under the control�
of UAS and the activator strain with the GAL4 coding sequence� under the
control of Bombyx mori cytoplasmic actin3. By crossing the two strains,
double-positive transgenic silkworm was obtained, and their ETH
expression was found to be dramatically lower than that of each single positive
transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive
transgenic silkworm at the stage of pharate second instar larvae, while the
single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4
RNAi approach provides a way to study the function of endogenous� silkworm
genes at different development stages.
Keywords������� ecdysis-triggering hormone; UAS/GAL4 system; RNAi;
transgenic silkworm
RNA interference (RNAi) has been developed as a powerful� tool for gene-specific knockdown in many species including� Bombyx mori (B. mori) (silkworm). Double-stranded RNA (dsRNA) molecules can be introduced into silkworm by direct RNA injection [1-4] or virus infection [5] to achieve efficient and transient inhibition of target gene expression. Transgenesis of an RNAi expression sequence� against BmNPV in silkworm generated a heritable� transgenic silkworm line with enhanced resistance to the virus [6]. We recently reported a heat shock inducible RNAi strategy in transgenic silkworm to inhibit the expression of the endogenous ecdysis-triggering hormone gene (ETH) [7].
The binary UAS/GAL4 system was used widely to express� genes in Medaka [8], Drosophila [9], and Xenopus� [10]. In silkworm, the UAS/GAL4 system has been used for tissue specific expression of target genes in photo�receptor cells and silk gland tissue [11]; more often, the system has been used to knockdown the expression of target genes [12,13]. However, until now, RNAi based on the binary UAS/GAL4 system has not been reported in silkworm.
In this study, we developed in silkworm a new transgenic RNAi approach based on the binary UAS/GAL4 system that successfully inhibited ETH gene, an important� endogenous gene involved in regulating ecdysis behavior in insects [14-16]. In this method, piggyBac-mediated transgenesis was used to generate an effector transgenic silkworm line with an UAS element-driven DNA fragment coding for the RNAi sequences against the ETH gene and an activator transgenic silkworm line with B. mori cytoplasmic� actin3 (BmA3) promoter-controlled GAL4 sequence. Double-positive transgenic silkworms were produced� by crossing the two lines, and they showed decreased� ETH expression and ecdysis deficiency in pharate second instar larval stage.
Materials and methods
Plasmid construction
For generating the activator vector containing BmA3-GAL4, BmA3
gene promoter sequence was polymerase chain reaction� (PCR)-amplified from the
plasmid pigA3 with the primers (up) 5'-CACTCGAGTGCGCGTTACCAT�ATA�TGGTGA-3'
and (down) 5'-TAGCGGCCGCTTGAATTA�G�TCTGCAAGAAAAG-3',
which contained XholI and NotI sites respectively (underlined).
The amplified fragment� was treated with XholI and NotI and then
ligated into the vector pcDNA3.1 (Invitrogen, Shanghai, China) to form the
construct pBmA3. The open reading frame of yeast transcriptional
activator GAL4 gene was obtained from the pChs-Gal4 plasmid by HindIII
digestion, and it was ligated into the multiple cloning site of the vector
pEGFP-N1 (Takara, Dalian, China) to be the vector p-N1-GAL4.
Then, the GAL4-SV40 polyA fragment was cut off from the p-N1-GAL4
by EcoRI and AflII and was inserted downstream of BmA3
promoter into the vector pBmA3. From this vector, the BmA3-GAL4-SV40
polyA fragment was cut out and then inserted into the NheI and AflII
site of the vector pBac{3xp3-EGFPam} to generate the activator� vector
pBac{3xp3-EGFP-BmA3-GAL4af}.
To generate the effector vector containing cDNA coding� for ETH RNAi, complete ETH code sequence was first cloned from silkworm. Total RNA was extracted from the epitracheal gland of the fifth instar larvae with RNeasy mini isolation kit (Qiagen, Shanghai, China) and reverse-transcribed by SuperScript II (Takara) with oligo(dT) primer in a reaction volume of 10 ml. As the template for PCR amplification of ETH cDNA, 2 ml of reverse transcription� (RT) product was used with 30 cycles of 94 �C for 30 s, 60 �C for 40 s and 72 �C for 30 s. The primer sequences were (up1) 5'-CTGTCGACATGACTT�CA�AAATTG�ACAATGATG-3', (down1) 5'-GTCTGC��AGTTT�CT��T�CATGCTTCCCATTTTTTT-3', (up2) 5'-ACGG�GCCCA��TGACTTCAAAATTGACAATGATG-3', and (down2) 5'-GTCCGCGGTTTC�TTCATGCTTCC�C�A�T�TTTTTT-3', which contained SalI, PstI, ApaI and SacII �sites, respectively� (underlined). The two PCR fragments (with primer pair of up1/down1 or up2/down2) were treated with respective restriction enzymes and ligated tail to tail into multiple cloning site of the vector psiRNA to form the vector psiETH. An intron from fibroin light chain gene was PCR-amplified from silk gland genomic DNA with primers, (up) 5'-CACCGCGGAGCCCACCTGGT�G�TT��AAGTGGTGA-3' and (down) 5'-CACTGCAGTTA�C�T�GGTGGTAGGACCTGTTGTG-3' containing SacII and PstI sites, respectively (underlined). The amplified fragment� was treated with SacII and PstI and ligated into the vector psiETH. The double-stranded ETH (DsETH)-intron-SV40 polyA fragment from this construct was excised with SalI and PstI, and ligated into the vector pUAS to form the vector pUAS-DsETH. Then, the UAS-DsETH-SV40 polyA fragment was excised and inserted into the vector pcDNA3.1 to obtain the vector pcDNA-UAS-DsETH. Finally, the UAS-DsETH-SV40 polyA fragment was inserted� into the vector pBac{3xp3-EGFPaf} to generate the effector vector pBac{3xp3-EGFP-UAS-DsETHaf}.
The sequence of the PCR products and resulting plasmids� were confirmed by sequencing performed by a commercial service provider (Invitrogen).
B. mori strains and transgenic
silkworm production
Transgenic silkworm was constructed under piggyBac transposon introduction by way of microinjection the mixture� of the helper pigA3 plasmid and the transgenic vector [17]. The effector or the activator construct and the transposase carrying the helper plasmid pigA3 were injected mid-ventrally into the preblastoderm eggs. Afterwards, the eggs were incubated until hatching in a humidified chamber at 25 �C. Hatched larvae were transferred� onto mulberry leaves. Positive G1 larvae were selected and reared individually. The genotype of transgenic silkworm was analyzed by PCR. The sequence of primer pairs for UAS, ETH and GAL4, respectively, were (upUAS) 5'-GGTCGGAGTACTGTCCTCCG-3'; (dnETH) 5'-TC�G�A�ACGGCAAACTGTAGAC-3', (upGAL4) 5'-AA�G�A�T�GAAGCTACTGTCTTCTA-3', and (dnGAL4) 5'-TTA�C�G�ATACAGTCAACTGTCTTTGA-3'.
Real-time RT-PCR analysis
Total RNA was prepared from the 7 d embryos, first instar� and pharate second instar larvae of effector [A(-)E(+)], activator/effector [A(+)E(+)], activator [A(+)E(-)] transgenic lines and wild-type silkworm using RNeasy Mini Isolation kit (Qiagen) and treated with RNase-free DNaseI (Promega, Madison, USA) [18]. Subsequently, cDNA were synthesized. The sequence of primer pairs for ETH, DsETH and GAL4 were (upETH) 5'-CG�CTAAA�CACA�G�C�ACCGTGAAC-3', (dnETH) 5'-TC�GAACG�GCAAA�C�T�GTAGAC-3', (upDsETH) 5'-CGCGCGGA�CTCA�CAAC�AGGT-3', (dnDsETH) 5'-TATCCCATCACGTCCTCATC-3', (upGAL4) 5'-AGTGCTCCAAAGAAAAACCGA-3', and (dnGAL4) 5'-GGTCTTCTCGAGGAAAAATCAG-3'. Quantitative PCR measurement was performed with EvaGreen fluorescence dye (Biotium, Hayward, USA) on a Rotor-Gene 3000 Detection System (Biocompare, South San Francisco, USA). The RNA level for each group was measured in triplicates and normalized to an internal control� of B. mori GAPDH.
Statistical analysis
Mean values and standard error were calculated for each group, and groups were compared using Student抯 t-test. p<0.05 denotes a statistically significant difference. p<0.01 denotes a statistically very significant difference.
Results
Production of the binary UAS/GAL4
RNAi expression system in transgenic silkworm
The physical maps of the plasmids of the UAS/GAL4-mediated RNAi system are illustrated in Fig. 1. The GAL4 open reading frame was placed downstream of the BmA3 gene promoter to achieve a stable expression of GAL4 protein in silkworm [Fig. 1(A)]. The ETH sense and antisense cDNA sequences were joined tail to tail and located� downstream of the UAS promoter in order to transcribe� the DsETH RNA activated by the GAL4 protein [Fig. 1(B)].
Both plasmids contained the enhanced green fluorescent� protein gene (EGFP) driven by eye-specific expression promoter 3xp3, which served as a screening marker for transgenic silkworm. Both plasmids also contained the left and right arm of the piggyBac transposon.
Following piggyBac-mediated transgenesis, hatched larvae� (G0) were kept to develop into moths. The resulting G0 moths were allowed to intercross to produce the G1 silkworm. The insertion of a foreign gene into the silkworm� genome was confirmed in the effector transformed lines by inverse PCR using genomic DNA extracted from the silk glands of G1 larvae. The genomic junction sequences of 390, 235 and 58 bp, which flanked the 5' piggyBac inverted terminal repeat in three transgenic lines, were analyzed (Table 1). The search in Silkworm Know�ledge�base (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=insects) confirmed that these junction sequences� were derived from the B. mori genome.
RNAi inhibition of ETH
leads to the lethal ecdysis deficiency in pharate second instar larvae
Insects undergo multiple developmental stages during their life cycle, and each transition requires molting and ecdysis to produce a new epidermis and shed the old cuticle. ETH activates the ecdysis behavior by direct actions on the central neurons system [14]. Positive transgenic silkworm was screened by EGFP fluorescence signal, and moths of the effector line and the activator line were intercrossed [Fig. 2(A)]. The larvae from this crossing protocol contained� four different genotypes that were identified by PCR analysis� of the larvae genomic DNA: (1) wild type, [A(-)E(-)]; (2) activator lines, [A(+)E(-)]; (3) effector lines, [A(-)E(+)]; and (4) double-positive (activator/effector) lines, [A(+)E(+)]. The segregation ratio of the progeny of the cross was nearly 1:1:1:1, as expected, indicating that the transgenes were stably inherited in a Mendelian fashion� [Fig. 2(F)]. At the stage of pharate second instar larvae, severe ecdysis deficiency proved lethal to the double-positive� silkworm, as identified by PCR [Fig. 2(C), red arrow]. This was not observed in the control larvae [Fig. 2(B,D,E), black arrow].
ETH expression was knocked down in
the lethal pharate second instar larvae
Real-time quantitative PCR analysis showed that ETH expression� was markedly knocked down at mRNA level in the double-positive pharate second instar larvae, but no changes in ETH expression were found in the control larvae [Fig. 3(A)]. The GAL4 gene was highly expressed in the double-positive pharate second instar larvae and the activator transgenic larvae, but it was not expressed in the effector transgenic larvae and the wild-type larvae [Fig. 3(B)].
ETH and DsETH expression
patterns were analyzed at mRNA level in transgenic and wild-type silkworm
ETH gene is expressed specifically in epitracheal gland of insects [18]. The ETH expression level increases before pre-ecdysis and then declines after ecdysis stage in insects� [19]. We assayed ETH expression at different developmental stages in silkworm. Total RNA was prepared from 7 d embryos, first instar larvae, and pharate second instar larvae from double-positive silkworm and wild-type silkworm. As quantified by real-time quantitative RT-PCR, the ETH mRNA expressions in the wild-type silkworm [Fig. 4(A)] as well as the single transgenic lines (data not shown) were high at pharate second instar larval stage and relatively low at first instar stage. In contrast, only in the double-positive transgenic lines, DsETH RNA molecules, which serve as RNAi against ETH, were produced� at high levels at all developmental stages, including� the pharate second instar larval stage [Fig. 4(B)].
Discussion
In this paper, we described a new approach for efficient and specific inhibition of an endogenous gene in transgenic silkworm by RNAi based on the binary GAL4/UAS system. Tissue-specific knockdown of a gene could be achieved by selecting an appropriate promoter to drive the expression� of the activator GAL4. We believe this system will become more powerful as different types of GAL4 transgenic silkworm are generated.
The system was tested with ETH as the target gene in the current study. Two lines of transgenic silkworm, the UAS-driven ETH RNAi effector line and the BmA3-driven GAL4 activator line, were established using the piggyBac-mediated transgenesis technique. After crossing the two strains, severe ecdysis deficiency accompanied by a dramatic decrease in ETH mRNA level proved lethal to nearly all the double-positive transgenic silkworm at the stage of the pharate second instar larval. This phenomenon was consistent with the result from a recent study of Drosophila� [19] in which mutations in the ETH gene lead to a lethal ecdysis deficiency.
ETH is an integration factor for regulating the ecdysis behavior of insects [14-16,19,20]. Our study of ETH expression pattern sin silkworm showed higher ETH RNA levels at the stage of the pharate second instar larval than that at the first instar larval. This suggests ETH may play more important roles in the ecdysis process at the second instar larval stage, which may explain the lethal phenotype at the second instar larval stage observed in the study.
Although the UAS/GAL4-mediated RNAi system has been successfully used in many other species, this is the first report, to our knowledge, of its application in silkworm. The approach developed here allowed us to study the function� of silkworm genes in vivo in specific cell types and at different development stages.
Acknowledgements
We would like to thank Dr. Michael E. Adams and Dr. Sheng Li for their friendly discussions.
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