Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 790-795
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, 2008This 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,
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 photoreceptor 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‘-CACTCGAGTGCGCGTTACCATATATGGTGA-3‘
and (down) 5‘–TAGCGGCCGCTTGAATTAGTCTGCAAGAAAAG-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‘-CTGTCGACATGACTTCAAAATTGACAATGATG-3‘,
(down1) 5‘-GTCTGCAGTTTCTTCATGCTTCCCATTTTTTT-3‘,
(up2) 5‘-ACGGGCCCATGACTTCAAAATTGACAATGATG-3‘, and
(down2) 5‘-GTCCGCGGTTTCTTCATGCTTCCCATTTTTTT-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‘-CACCGCGGAGCCCACCTGGTGTTAAGTGGTGA-3‘
and (down) 5‘-CACTGCAGTTACTGGTGGTAGGACCTGTTGTG-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‘-TCGAACGGCAAACTGTAGAC-3‘,
(upGAL4) 5‘-AAGATGAAGCTACTGTCTTCTA-3‘, and (dnGAL4)
5‘–TTACGATACAGTCAACTGTCTTTGA-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‘-CGCTAAACACAGCACCGTGAAC-3‘,
(dnETH) 5‘-TCGAACGGCAAACTGTAGAC-3‘, (upDsETH)
5‘-CGCGCGGACTCACAACAGGT-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?
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 Knowledgebase (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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