Electroporation can be used to explore the gene function in the silkworm, Bombyx mori

ZHOU Wen-Lin, Haruhiko FUJIWARA, Nozomi UEMURA, YE Ai-Hong,WU Xue-Hui, CHEN Xue-Dong, ZHANG Ting-Ting, CAO Jin-Ru, *

(1.Institute of Sericulture and Tea, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;2.Department of Integrated Biosciences, Graduate School of Frontier Sciences,The University of Tokyo, Kashiwa, Chiba 277-8562, Japan)

Abstract:【Aim】To confirm the effectiveness of electroporation-mediated functional analysis system in the silkworm, Bombyx mori.【Methods】siRNAs were synthesized for the target gene Wnt1(Wingless), which is known to be involved in larval melanin coloration in B. mori.The day-3 4th instar larvae of B. mori were injected with Wnt1 siRNAs and subjected to electroporation as the treatment group(ERFA-RNAi)and those injected with Wnt1 siRNAs but without subjected to electroporation were used as the negative control group, the epidermis of the corresponding speckled area of the 5th instar larvae was dissected, and the relative expression level of Wnt1 in the epidermis was detected with real-time quantitative RT-PCR(qRT-PCR)to verify the effect of electroporation-mediated RNAi.The transposon vector pPIG-A3GR with the enhanced green fluorescent protein reporter gene(EGFP)and the red florescence protein(RFP)reporter gene(DsRed2)expression cassettes, was introduced into the 2nd instar larvae of B. mori by electroporation.After 72 h of normal rearing, the expression of EGFP and DsRed2 in the larvae was observed under a fluorescent stereo microscope, to verify the somatic transgenesis of the silkworm.【Results】After the introduction of Wnt1 siRNAs into the day-3 4th instar larvae of B. mori, the formation of a speckle pattern of the 5th instar larvae was prevented on the larval body surface, and the qRT-PCR analysis showed that the expression level of Wnt1 in the epidermis of the 5th instar larvae was significantly decreased.The positive rate of somatic transgenic silkworm was 56.60%, and two fluorescent reporter genes EGFP and DsRed2 were continuously expressed in larval, pupal and adult stages.【Conclusion】Electroporation is an efficient technology for exploration of gene function in vivo, by efficiently introducing exogenous RNA or DNA into silkworm.

Key words: Bombyx mori; electroporation; transgenesis; Wnt1; RNAi; gene function

1 INTRODUCTION

Analyzing gene function is important for basic biology and the field of life sciences.Analysis of gene functions is typically carried out with model organisms such asDrosophilaand mice.RNA interference(RNAi)(Bumcrotetal., 2006), zinc-finger nucleases(ZFNs)(Bibikovaetal., 2002), transcription activator-like effector nucleases(TALENs)(Joung and Sander, 2013; Yodaetal., 2014)and CRISPR/Cas(clustered regularly interspaced short palindromic repeats, CRISPR; CRISPR-associated gene, Cas)genome editing technology(Zhangetal., 2015)have been successfully applied to many organisms.Therefore, gene function analysis can now be carried out directly for non-model organisms.

The domesticated silkworm,Bombyxmori, is important for both basic biological research and applied science(Zhouetal., 2012).Completion of the framework of theB.morigenomic map in 2004 has brought about the era of functional genomics ofB.mori(Xiaetal., 2004; Zhouetal., 2012).

To analyze and verifyinvivogene functions, expression of the target gene is usually knocked-down using RNAi, or the gene is over-expressed via transgenic techniques.However, in most lepidopteran insects, such asB.mori, it is difficult for double-stranded RNA(dsRNA)to enter cells efficiently(Tomoyasuetal., 2008).Therefore, it is difficult to perform RNA interference efficiently by simply injecting dsRNA.By contrast, gene function analysis can be performed by classicB.moritransgenic technology(Tamuraetal., 2000; Tanetal., 2005)or by genome editing technology(Liuetal., 2017).However, both approaches are technically difficult and time consuming.Additionally, transgenic or genome editing technology also requires substantial manpower, and it is costly and time consuming to raise the silkworms.To overcome these difficulties, the electroporation-mediated rapid functional analysis system for insects has been used with substantial efficacy(Ando and Fujiwara, 2013; Yamaguchietal., 2013; Yodaetal., 2014; KonDoetal., 2017; Andoetal., 2018; Iijimaetal., 2019; Jinetal., 2019).

B.morilarva has three body parts: head, thorax, and abdomen(Lü, 1991).In addition to the head, it has 13 somite sections that include 3 thoracic segments and 10 abdominal segments(Fig.1: A).Wild typeB.morilarva in traditional sericulture is generally white and the surface markings are either normal(+p)or plain(p)(Luetal., 1981).The normal marked silkworm has one pair of eye spots in the 2nd thoracic segment, one pair of semilunar markings in the 2nd abdominal segment, and one pair of star spots in the 5th abdominal segment(Yodaetal., 2014).The plain silkworm has no speckled patterns, but there are non-pigmented normal markings(Fig.1: B)(Luetal., 1981).There are many types of natural mutations, such asMultilunar(L),Stripedmarking(pS), andZebra(Ze).A mutant strain ofB.mori,Multilunar(L), has pairs of round and yellow-brown markings on the dorsal side of successive multiple segments starting at the 2nd segment(Fig.1: B)and is a good choice for analyzing the genetic phenotype of the speckle patterns(Yamaguchietal., 2013; Edayoshietal., 2015).

Fig.1 Typical patterns of mutant larvae of Bombyx mori

In this study, we used electroporation to introduce small interfering RNA(siRNA)intoB.moriand to verify gene functioninvivoby knocking down the related geneWnt1(wingless)that affects the speckle pattern formation of larvae(Yamaguchietal., 2013).In addition, we introduced plasmid DNA(transgenic vector)to specific positions on theB.moribody, and realized somatic transgenesis.

2 MATERIALS AND METHODS

2.1 Silkworm strain and feeding

The natural mutant strain ofB.morilarva,Multilunar(L), has distinct pairs of markings on the dorsal side of the 2nd thoracic segment to the 6th abdominal segment(Fig.1: B).TheLmutant strain ofB.mori, g03, was obtained from Kyushu University(Japan)and raised on artificial diet(NIHON NOSAN, Yokohama, Japan)that contained mulberry leaf powder at 25℃(Ando and Fujiwara, 2013).

2.2 Synthesis of siRNAs

Based on theWnt1 mRNA sequence ofB.mori(GenBank ID: D14169)and a previous study(Ando and Fujiwara, 2013), the online tool siDirect ver.2.0(http:∥sidirect2.rnai.jp/)was applied to design double-stranded siRNA sequences with different nucleotide lengths for targeting different loci(Table 1).The siRNAs were synthesized by FASMAC(Kanagawa, Japan).

2.3 Real-time quantitative RT-PCR

Using the housekeeping gene ribosomal protein L3 gene(RpL3)(GenBank ID: AY769270.1)as an internal reference(KonDoetal., 2017), real-time quantitative RT-PCR(qRT-PCR)primers forWnt1 andRpL3 were designed(Table 2)and synthesized by Sigma-Aldrich(Tokyo, Japan).

Table 1 Sequences of the siRNAs

Table 2 Primers for real-time quantitative RT-PCR

Total RNA was extracted with TRI reagent(Sigma, St.Louis, MO, USA).After treatment with DNase I(TaKaRa, Kusatsu, Shiga Prefecture, Japan)to remove genomic DNA, RNA was extracted with phenol-chloroform and precipitated with alcohol and re-dissolved in deionized water.After reverse transcription with a cDNA synthesis kit(GE HealthCare, Chicago, IL, USA), qRT-PCR amplification was performed on a StepOne system(Applied Biosystems, Foster City, CA, USA)using a SYBR green PCR master mix kit.The reaction conditions were as follows: 95℃ for 10 min; 95℃ for 15 s and 60℃ for 60 s for 40 cycles.

The relative expression level of the target gene was calculated by 2-ΔΔCtmethod, based on the expression level of internal reference gene.The expression level of each sample was determined by three biological repeats.We set the target gene level of the right(-)(the right speckled area connected to the cathode)as 1.0 for comparisons with that of the left(+)(the left speckled area connected to the anode).Student’st-test was used for statistical comparison.

2.4 Construction of plasmid DNA vectors

A plasmid DNA vector was constructed based on pPIGA3GFP as previously described(Tamuraetal., 2000).The vector pPIG-A3GR contains the expression cassettes of the enhanced green fluorescent protein(EGFP)reporter geneEGFPand the red florescence protein(RFP)reporter geneDsRed2, which are driven by theactinA3 promoter(Fig.2: B)(Ando and Fujiwara, 2013).

Fig.2 Schematic diagram of in vivo electroporation and the structure of the transgenic vector

2.5 In vivo electroporation of silkworm larvae

The key period affecting larval melanin formation inB.morilarvae occurs 60-72 h after the molt(Kiguchi and Agui, 1981; Yamaguchietal., 2013).To ensure RNA interference efficacy, two double-stranded siRNAs, Wnt1-a and Wnt1-b(Table 1), targetingWnt1 were mixed in equal amounts with a molar ratio of 1∶1 and then dissolved in the injection buffer[100 mmol/L KOAc, 2 mmol/L Mg(OAc)2, 30 mmol/L HEPES-KOH, pH 7.4]and then adjusted to a final concentration of 250 μmol/L.The day-3 4th instar larvae ofLmutant strains ofB.mori, were fixed with tape, and the siRNAs mixture was aspirated with a glass needle(GD-1; Narishige, Japan), passed through a microinjection system(FemtoJet, Eppendorf, Germany), and injected into the body cavity in the inter-segmental membrane between the 3rd and 4th abdominal segments(1.0 μL/larva).Immediately following injection, the positive electrode was placed on the left side of the 3rd abdominal segment while the negative electrode was placed on the right side of the 3rd abdominal segment.Phosphate buffer(PBS, pH 7.4)was added dropwise to connect the electrode to the larval body wall.The markings on the corresponding side were connected to the corresponding positive and negative electrodes(Fig.2: A).The electrode was connected to an electric pulse generator(CG-KA-001, Cellproduce, Japan), and the pulse electric excitation(five square pulses of 20 V, 280 ms width)was performed.

The plasmid pPIG-A3GR carrying theEGFPandDsRed2 expression cassettes was mixed with the helper plasmid pHA3PIG(Fig.2: B)which encodes a transposase at a molar ratio of 1∶1; the final DNA concentration was adjusted to 1.0 μg/μL.After the 2nd instar larva was fixed with tape, the mixed DNA solution was injected into the body cavity(1.0 μL/larva)through the intersegmental membrane between the 4th and 5th larval abdominal segments.The positive electrode was placed on the left side of the 4th abdominal segment while the negative electrode was placed on the right side of the 4th abdominal segment.Drops of PBS were added to connect the electrode to the larval body wall, and pulsed electroporation(five square pulses of 20 V, 280 ms width)was applied.

2.6 Image acquisition

All images were acquired from living larvae using a fluorescent stereo microscope(Leica M165FC, Germany)and a digital imaging system(AxioCam MRc5, Carl Zeiss, Germany).SPSS 16.0(IBM, Endicott, NY, USA)was used for statistical analysis.

2.7 Data analysis

Student’st-test was used for the variable-difference analysis.

3 RESUILTS

3.1 Effect of electroporation on RNAi efficacy

ERFA was used to study the effects of RNA interference againstWnt1.The day-3 4th instar larvae ofB.moriwere injected with siRNAs and subjected to electroporation as the treatment group(ERFA-RNAi)(n=7).The 4th instar larvae injected with siRNAs but not subjected to electroporation were used as the negative control group(n=7).

After electroporation, the larvae continued to feed and grow.In the 5th instar newly molted larvae, the 3rd abdominal segment of larvae of the ERFA-RNAi group showed obvious changes compared to the negative control larvae(Fig.3).Five larvae survived in the ERFA-RNAi group, and three of these larvae showed a reduction in the speckle patterns in the anode treated area of the 3rd abdominal segment.The results showed that the color of the treated area on the anode was faded, and the pattern size appeared to be smaller.By contrast, the patterns on the cathode area remained unchanged(Fig.3: A).There was no obvious difference in the left(+)and right(-)speckle patterns of the negative control larvae.

Fig.3 Pigmentation and Wnt1 expression in the 5th instar larvae of Bombyx mori associated with electroporation-mediated RNAi

3.2 Analysis of RNAi efficacy by qRT-PCR

To verify the effect of ERFA-RNAi treatment, the relative expression level ofWnt1 was detected with qRT-PCR.In the 5th instar larvae of the ERFA-RNAi treatment and negative control, the epidermis of the corresponding speckled area was dissected, and qRT-PCR analysis was performed(Fig.3: B).

In the ERFA-RNAi group, the relative expression level ofWnt1 in the left(+)speckled area of the 3rd abdominal segment was significantly reduced as compared to that in the control group, and showed extremely significant difference from that in the right(-)speckled area(P<0.01).There was no significant difference in the relative expression level ofWnt1 in the speckled area of the negative control(n=3, two-tailedt-test).These results indicated that electroporation significantly improved RNAi efficacy.

3.3 Somatic transgenesis of the silkworm

After the plasmid pPIG-A3GR and the helper plasmid pHA3PIG were mixed, the 2nd instar newly molted larvae were injected for electroporation.After 72 h of normal rearing, we observed the larvae under a fluorescent stereo microscope, and several larvae showed green fluorescence(EGFP)and red fluorescence(RFP)at the same time on the left side of the 4th abdominal segment(i.e., the position of the positive electrode)(Table 3).The expression of the two fluorescent reporter genes continued throughout the larval(Fig.4: A), pupal(Fig.4: B), and adult(Fig.4: C)stages.The expression of EGFP and RFP is caused by the transgene in local body wall cells mediated by thepiggyBactransposon vectors, and is not lost during molting and metamorphosis.

Table 3 Positive rate of electroporation-mediated somatic transgenesis

Fig.4 Fluorescence observation of the silkworm Bombyx mori using ERFA somatic transgenesis

The DNA vector carries both theEGFPandDsRed2 expression cassettes.Therefore, the positive individuals all expressed two different fluorescent proteins simultaneously.A total of 34 silkworm larvae were treated in three repeated experiments, among which 19 showed fluorescence.The proportion of positive individuals showing fluorescence was 56.60%.No positive individual was observed in the control group injected with the same vehicle but without electroporation(Table 3).

4 DISCUSSION

Gene function analysis of living organisms usually involves a process to achieve a specific gain-of-function or loss-of-function.However, this process may be labor and material intensive.For non-model insects, such asB.mori, there are additional technical obstacles, such as the lower entry efficiency of siRNA or transgenic vector DNA，the silkworm eggs being damaged by microinjection thus being unable to be diapause-terminated by hydrochlorizing(Zhouetal., 2012).These challenges may be circumvented by using ERFA.

Through transgenic overexpression ofWnt1, Yamaguchietal.(2013)and Yodaetal.(2014)achieved redundant spotting patterns on the body surfaces of silkworm larvae, demonstrating thatWnt1 participates in the regulatory network of melanin synthesis inB.mori.Their results suggest thatWnt1 is related to the formation of speckle patterns on the larval body surface.By applying ERFA, we successfully introducedWnt1 siRNAs intoB.morilarvae and significantly inhibited the expression ofWnt1(P<0.01).In the anode-treated area, color of the larval body surface speckle pattern was faded(Fig.3).These results show thatWnt1 is related to melanin production on the body surface, the introduction of exogenous siRNA by electroporation can cause RNA interference inB.mori.

Ando and Fujiwara(2013)successfully introduced plasmid DNA intoB.mori,PapilioxuthusandTriboliumcastaneumby using electroporation.In this study, using electroporation, the plasmid pPIG-A3GR carryingEGFPandDsRed2 was introduced into the larval silkworm to achieve expression of EGFP and RFP(Fig.4).After electroporation of the 2nd instar larva, the continuous and stable expression of foreign genes in the larva through the adult stage was achieved(Fig.4).

Invivoelectroporation allows plasmid DNA to penetrate the cell membrane and enter the nucleus; thus, somatic transgenic mice can be quickly obtained byinsitutransgenesis(Kimetal., 2019; Ortiz-A′lvarezetal., 2019).For non-model organisms, such asB.mori, analysis of gene function by conventional transgenic methods is technically difficult, time consuming, and produces a low rate of positive individuals(Zhouetal., 2012).In this study, electroporation was used to obtain a somatic transgenic chimera silkworm.We introduced the transgenic vector plasmid pPIG-A3GR into the 2nd instar larvae, and expression of the exogenous fluorescent reporter genes was observed after 72 h.Expression continued throughout the entire life cycle(Fig.4), with a positive rate of 56.60%(Table 3).

Electroporation is a common and effective technique used to promote exogenous siRNA or DNA into cells.RNA and DNA are usually negatively charged.Therefore, we conclude that during ERFA treatment ofB.mori, the exogenous siRNA or DNA will move toward and be accumulated in the area where the positive electrode is located, and then enter the silkworm somatic cells by electroporation.

Electroporation technology has the advantages of low cost, relatively simple operation, and produces formation of a chimera of local body tissue after processing.It can be used for gene function analysis in specific tissues and organs.In addition to theB.morilarval speckle patterns in this study, electroporation can also be used in tissues and organs such as adult wings and antennae(Ando and Fujiwara, 2013; Andoetal., 2018).Other insects, such asPapiliopolytes,P.xuthusandT.castaneum, had also been studied using this technique(Ando and Fujiwara, 2013; Iijimaetal., 2019; Jinetal., 2019).

The stability of the technical system directly affects the reliability of its application.The electroporation technique has good stability and reliability and is also fast and efficient.The influence of DNA or RNA on the phenotype can be analyzed from 2 to 7 d following introduction(Ando and Fujiwara, 2013; Andoetal., 2018).Therefore, electroporation is suitable for rapid gene function analysis by introducing the exogenous RNA in RNAi or the exogenous DNA in somatic transgenesis.The method has the advantages of being quick and highly efficient, and thus has broad application prospects in the field of gene function analysis as applied to the silkworm and other insects.

ACKNOWLEDGEMENTSThis research was mainly performed in the Laboratory of Innovational Biology of Tokyo University, Japan.We are especially grateful to Drs.Kojima T, Narisu N, Jin H, KonDo Y and Yoda S, and Ms.Chagi K for their assistance and valuable suggestions.