- Email: [email protected]
An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli
Journal of Virological Methods 113 (2003) 95–101
An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli Wuwei Wu, Jinwen Wang, Riqiang Deng, Xunzhang Wang∗ , XiongLei He, Qingxin Long State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China Received 15 April 2003; received in revised form 14 July 2003; accepted 15 July 2003
Abstract The RecA-mediated homologous recombination method was improved and used to direct gene replacement in baculoviruses. With this method, the p74 gene in the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was substituted precisely by the p74 gene of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV). In the recombinant bacmid, the AcMNPV p74 gene promoter controlled directly the expression of SpltMNPV p74 gene. Results of RT-PCR showed transcription of SpltMNPV p74 gene in the recombinant, implying the potential use of this easy and efficient method in baculovirus gene function research. © 2003 Published by Elsevier B.V. Keywords: Gene substitution; Baculovirus; p74 gene; RecA-mediated homologous recombination
1. Introduction Insect baculovirus is a double-stranded DNA virus used widely as a biological pesticide and a eukaryotic gene expression vector (Jones and Morikawa, 1996). Two forms of viral particles, budded virus (BV) and occlusion-derived virus (ODV), are generated during its life cycle. An ODV envelope protein encoded by the p74 gene was found to be essential to baculovirus ODV infectivity (Faulkner et al., 1997). Exposure of P74 protein on the surface of ODV suggests the function of this protein in attachment of ODV to the midgut cell membranes of insect larvae. Is this protein species-specific? May it be replaced by P74 from different baculovirus species such as SpltMNPV without infectivity loss? Construction of recombinant AcMNPV with the target gene replaced precisely by homologous gene from other species may help to answer these questions. Due to the large size of baculoviral genomes, it is almost impossible to modify baculovirus by simple restriction enzyme digestion and ligation. The conventional method of obtaining recombinant baculovirus is to construct a plasmid containing the desired alteration flanked by viral sequences of the target site, and then to co-transfect cultured insect ∗ Corresponding author. Tel.: +86-20-84112504; fax: +86-20-84113964. E-mail address: [email protected] (X. Wang).
0166-0934/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/S0166-0934(03)00225-8
cells with this plasmid and wild-type viral DNA. Thus, the recombinant virus is generated through homologous recombination between transfer plasmid and viral DNA in the insect cells. Although this method has its advantages, it is very time consuming. In addition to this method, some improved approaches were also developed, such as linearized virus (Kitts and Possee, 1993), Cre-Lox recombination system (Peakman et al., 1992), homologous recombination in yeast (Patel et al., 1992), Bac-to-Bac® system (Luckow et al., 1993), and direct insert of the exogenous gene into the single restriction sites of baculovirus (Lu and Miller, 1996). Unfortunately, most of these strategies can only apply to the polyhedrin gene locus when used in constructing recombinant virus. Modification of other genes still requires the traditional co-transfection method, thus making functional research of these genes very laborious. Recently, some alternative approaches for constructing recombinant baculovirus were described (Bideshi and Federici, 2000; Hou et al., 2002; Lin and Blissard, 2002; Pijlman et al., 2002). All of these approaches used the baculovirus bacmid instead of the wild-type baculovirus. A bacmid is a baculovirus that has an F-factor-based plasmid backbone inserted into the polh gene locus, and can propagate in both insect cells and E. coli. This feature is very similar to the bacterial artificial chromosome (BAC) that had been developed in the past decades (Shizuya et al., 1992) and which is used extensively. BAC can carry DNA
96
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
fragment as large as 300 kb, and many new techniques for modifying such large plasmids were also described recently (Court et al., 2002; Lalioti and Heath, 2001; Yang et al., 1997; Zhang et al., 1998). Bideshi and Federici (2000) first used one of these new techniques—E. coli BJ5183 system on the AcMNPV bacmid. Other systems, ET-recombination and phage lambda red system were used to modify the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) bacmid (Pijlman et al., 2002) and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) bacmid (Hou et al., 2002). However, all the above studies only resulted in the knockout of certain baculovirus genes, and offered a repaired or substituted gene at the polh gene locus using Bac-to-Bac system. In this paper, we describe the use and modification of a technique called RecA-mediated homologous recombination which was reported to carry out easily gene substitution in those baculovirus that had been cloned as a bacmid (Deng et al., 2000; Luckow et al., 1993; Pijlman et al., 2002; Zhu et al., 1997). This technique was described originally by Yang et al. (1997) and improved by Lalioti and Heath (2001).
2. Materials and methods 2.1. Viruses, plasmids, and primers AcMNPV strain accompanying the Bac-to-Bac® system from Invitrogen was used in this study. The SpltMNPV G2 strain was isolated from suburban of Guangzhou and kept in our laboratory, and the viral genome was sequenced recently (Pang et al., 2001). The AcMNPV bacmid-polh containing gentamicin (Gm)-resistant gene was constructed by introducing polyhedrin gene from pFastBac1 into the commercial AcMNPV bacmid of the Invitrogen Bac-to-Bac® system using the method described by Luckow et al. (1993). The plasmid pDF25 (Lalioti and Heath, 2001) contains a temperature-sensitive replicon RepA, a recA gene and a tetracycline (Tet)-resistant gene. The temperature-sensitive replicon RepA makes pDF25 replicate at the permissive temperature (30 ◦ C) and to stop replication at the non-permissive temperature (43 ◦ C). pfu DNA polymerase (Bioasia Biotech, Shanghai, PR China) was used to amplify DNA fragments from the virus genomic DNA. The AcMNPV p74 gene upstream fragment from genome position of 121661–121073 was amplified using the primers ACP74R1 and ACP74R2 and then digested with BamH1. SpltMNPV p74 gene fragment from genome position of 19508–21679 was amplified using the primers SLP741 and SLP742 and then digested with Sac1. The AcMNPV p74 gene downstream fragment from genome position of 118729–119268 was amplified using the primers ACP74L1 and ACP74L2 and then digested
with Sac1 and Not1. The resulted fragments were then ligated into the plasmid pKOV-Kan (Lalioti and Heath, 2001) between the BamH1 and Not1 sites, producing plasmid pKOV-S74I which contains the temperature-sensitive replicon RepA, a chloramphenicol (Cm)-resistant gene, and a counter selection marker sacB gene. The cloned fragments were confirmed by sequencing using an ABI PRISM® 377 automated DNA sequencer (PE Biosystems). Two pairs of PCR primers were designed to distinguish the p74 gene of AcMNPV and SpltMNPV: primer AT1 and AT2 amplify the AcMNPV p74 gene fragment from position 119713–120335, primer ST1 and ST2 amplify the SpltMNPV p74 gene fragment from position 20365–21224. The position of primers used and the structure of plasmid pKOV-S74I are shown in Fig. 1. Sequences of the primers are shown in Table 1. 2.2. Substitution of the p74 gene The pKOV-S74I DNA (0.5 g) and pDF25 DNA (0.5 g) were co-transformed into competent E. coli strain DH10B (100 l) harboring the AcMNPV bacmid-polh. The transformed bacteria were incubated at 30 ◦ C overnight on an LB plate containing 7 g/ml Gm, 20 g/ml Cm and 10 g/ml Tet. Three to five colonies were then picked and washed into 1 ml LB media and 100 l of this were spread onto a LB plate containing 7 g/ml Gm. After incubation at 43 ◦ C overnight, colonies were again washed and spread onto a LB plate containing 20 g/ml Cm and incubated at 43 ◦ C overnight. Survived co-integrant colonies should appear on the Cm plate and three to five colonies were picked and cultured in the LB media containing 20 g/ml Cm at 43 ◦ C for 2 h. The bacteria were harvested and made competent with 100 mM CaCl2 , and then transformed with plasmid pDF25 (0.5 g). The transformed cells were plated on LB plates containing 7 g/ml Gm and 10 g/ml Tet and left to resolve at 30 ◦ C overnight. Three to five colonies were picked and washed into 1 ml LB media and then diluted 100-fold to obtain single colonies after aliquots of 100 l of the diluted cells were spread on an LB plate containing 20 g/ml Gm and 7% sucrose and grew at 43 ◦ C overnight. Ten to 20 larger colonies were then selected and streaked on Gm and Cm simultaneously and left at 30 ◦ C overnight. Those colonies that only grew on Gm were selected for further analysis. The procedure for p74 gene substitution is shown in Fig. 2. 2.3. PCR and RT-PCR confirmation of substitution and transcription of p74 gene Bacmid DNA was isolated from each selected colony. For the recombinant bacmid-polh-slp74, PCR amplification with primer pair ST1 and ST2 would expect an 860 bp product whereas primer pair AT1 and AT2 would produce nothing.
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
97
Fig. 1. The primers position and structure of plasmid pKOV-S74I. (A) Three pairs of primers were designed to amplify AcMNPV p74 gene upand downstream fragments and the SpltMNPV p74 gene fragment. Two pairs of primers, AT1, AT2, ST1, and ST2, were designed for p74 gene confirmation. (B) Structure of plasmid pKOV-S74I. Showing the SpltMNPV p74 gene flanked by AcMNPV p74 gene up- and downstream fragments. Cm: chloramphenicol-resistant gene; RepA: a temperature-sensitive replicon; and SacB: a counter selection marker that blocks the growth of colony on the medium containing sucrose.
DNA of the recombinant bacmid was transfected into the insect cell line Tn-5B1-4 (Hi5) using lipofectin (Gibco BRL). The supernatant was collected at 72 h posttransfection and used to infect the cells at 5 MOI (multiplic-
ity of infection). Total RNA was extracted from about 106 cells at 20 h post-infection using TriPure isolation reagent (Boehringer Mannheim). One unit of RQ1 RNase-free DNase (Promega) was added to 5 g total RNA to digest
Table 1 Primers used in this study Primer
Sequencea
Virus
Positionb
ACP74L1 ACP74L2 ACP74R1 ACP74R2 AT1 AT2 SLP741 SLP742 ST1 ST2
5 -AGGCGGCCGCGACCTTTAATTCAACCC-3 5 -CGAGCTCTGGCGTTTACAGCATTTGTT-3 5 -GTTATATAGGACTTAAAATAAAC-3 5 -TCGGATCCAGGGAAACATACAC-3 5 -AAACCAAATCTGCCAGCGTCAAT-3 5 -TTCCAGCATACTACCGCCACGAC-3 5 -CAGAGCTCGATTGTCCGGGTGGCG-3 5 -ATGAGCGCGCACGTAAATTCTCCGA-3 5 -CTGAATTGCCATCCGAGTCAGTG-3 5 -TCTAAATTCCCCGTACCTACGCA-3
AcMNPV AcMNPV AcMNPV AcMNPV AcMNPV AcMNPV SpltMNPV SpltMNPV SpltMNPV SpltMNPV
118729 119268 121073 121661 119713 120335 19508 21679 20365 21224
a b
The restriction sites are underlined and the real viral sequences are shown in bold letters. Genome position of the 5 nucleotide of real viral sequence of the corresponding primer.
98
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
Fig. 2. Substituting the p74 gene of AcMNPV bacmid-polh. The substitution begins with the integration of the plasmid pKOV-S741 containing SpltMNPV p74 gene into the AcMNPV bacmid-polh through homologous recombination. The co-integrant may then resolve into either the original or the recombinant bacmid-polh. The diagram shows only the recombinant one.
for 5 min. The DNase was inactivated by heating at 80 ◦ C and the resulting RNA was used for reversed transcription (RT) using OligdT and 1 l of the RT product was used as the PCR template using primers ST1 and ST2. A control PCR was also undertaken with DNase treated RNA as the template.
3. Results
cloned fragment in the plasmid pKOV-S74I was sequenced and confirmed to be correct. Partial results are shown in Fig. 3, and we can see the ATG codon of SpltMNPV p74 gene follows immediately the upstream sequence of AcMNPV p74 gene. A recombinant bacmid AcMNPV bacmid-polhSL74 was produced by integration of plasmid pKOV-S74I into AcMNPV bacmid-polh forming the co-integrant at the first step and resolution of the co-integrant into either the original or the recombinant bacmid at the second step.
3.1. p74 gene substitution 3.2. Analysis of the recombinant bacmid SpltMNPV and AcMNPV p74 gene up- and downstream fragments were PCR amplified, ligated, and inserted into the plasmid pKOV-Kan (Lalioti and Heath, 2001) between the BamH1 and Not1 sites to construct plasmid pKOV-S74I. The
Recombination was confirmed by PCR test. In PCR experiments, an expected 860 bp band was observed when amplified with the SpltMNPV p74 gene-specific primers ST1
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
99
Fig. 3. Partial sequence results of plasmid pKOV-S74I. Showing the sequence upstreaming ATG codon is exactly the upstream sequence of AcMNPV p74 gene and there was no nucleotide insertion or deletion found in the sequence.
and ST2 while no band appeared when using the AcMNPV p74 gene-specific primers AT1 And AT2, showing the successful replacement of the AcMNPV p74 gene by SpltMNPV p74 gene. The PCR result is shown in Fig. 4. The SpltMNPV p74 gene-specific primers ST1 and ST2 were also used to perform RT-PCR to determine expression of SpltMNPV p74 gene in the recombinant AcMNPV bacmid. The RT-PCR result was shown in Fig. 4, showing that the substitute gene was able to express in the recombinant bacmid. 3.3. Methodology improvement
Fig. 4. PCR and RT-PCR test of the recombinant bacmid-polhSL74. (A) PCR identification of SpltMNPV p74 gene. Only the SpltMNPV p74 gene-specific primers ST1 and ST2 produced an 860 bp PCR product, whereas with the AcMNPV p74 gene-specific primer AT1 and AT2, no product was obtained. Lane M: marker; lane 1: with primers ST1 and ST2; and lane 2, with primers AT1 and AT2. (B) RT-PCR identification of the transcription of the SpltMNPV p74 gene in the Tn-5B1-4 cell infected by AcMNPV bacmid-polhSL74. Using the SpltMNPV p74 gene-specific primers ST1 and ST2, an expected 860 bp RT-PCR product was obtained in lane 1. Lane M: marker; lane 1: cDNA template; and lane 2: control with RNA template.
The method described by Lalioti and Heath (2001) was modified to facilitate the selection of the co-integrant colonies from un-integrated pKOV-S74I colonies. Since temperature selection accompanied with Cm selection pressure was not able to efficiently suppress the growth of the un-integrated colonies, a step with temperature selection but without Cm selection pressure was added to eliminate pKOV-S74I colonies before selection of the co-integrant colonies. This modification resulted in big co-integrant colonies at clear background. The result was shown in Fig. 5.
Fig. 5. Comparison of the co-integrant selection plate obtained by Lalioti method (B) and the modified method (A). Co-integrant colonies are indicated by black arrows. The co-integrant colonies obtained by the modified method are big and clear compared to the relative smaller ones at the background of tiny un-integrant colonies obtained by Lalioti’s original method.
100
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
4. Discussions p74 gene is an essential gene for ODV infectivity of baculovirus, and therefore gene function should be studied with a polh-positive baculovirus. The commercial AcMNPV bacmid is a polh-negative protein expression tool with an F-factor-based plasmid backbone at the polyhedrin locus (Luckow et al., 1993). Therefore, we began the study by constructing AcMNPV bacmid-polh, a polyhedrin-positive bacmid, by transposing the polyhedrin gene, which had been cloned into the plasmid pFastBac1 (Invitrogen), into the commercial AcMNPV bacmid using Bac-to-Bac® protein expression system (data not shown). Many other methods had been used for gene modification of bacmid (Bideshi and Federici, 2000; Hou et al., 2002; Pijlman et al., 2002), and all use linear DNA as the gene target tool. Comparing to our method of RecA-mediated recombination, those methods are less convenient at in situ gene substitution: When undertaking in situ gene substitution, all those methods, including the E. coli BJ5183 system, ET-recombination and the phage lambda red system, require two steps. One step is to co-insert a selection and anti-selection marker into the target site; and the second step is to replace the marker by the modified gene. Unfortunately, the lack of good counter selection marker makes the second step very inefficient (Nefedov et al., 2000; Zhang et al., 1998). Therefore, it is essential to confirm that there are no mutations in anti-selection marker when the marker is amplified by PCR to produce the linear insertion DNA used in the first co-insert step. However, the RecA-mediated recombination does not require sequencing of the anti-selection marker, and this save a lot of time. Moreover, further improvement of the RecA-mediated recombination method in this study makes the selection of the modified bacmid much easier. In plasmid pKOVSL74I, the translation start codon of SpltMNPV p74 gene follows immediately the upstream sequence of AcMNPV p74 gene, so the AcMNPV p74 promoter directly controls the expression of SpltMNPV p74 gene. Fig. 2 shows the flowchart of p74 gene substitution procedure containing two steps, co-integration and resolution. Each step may have one recombination event taking place between the homologous sequences of AcMNPV bacmid-polh and pKOVSL74I. For example, if the first recombination event took place at the downstream sequence of AcMNPV p74 gene and the second recombination event took place at the upstream sequence of AcMNPV p74 gene, the recombinant AcMNPV bacmid-polh-slp74 was produced. At the step when selecting the first co-integration recombinant, the protocol described by Dr. Lalioti was modified. After obtaining the colonies containing three plasmids (AcMNPV bacmid-polhSL74, pDF25 and pKOV-S74I), the Lalioti method was to select four to six colonies and wash them into 1 ml LB, then spread them onto the plate containing Cm and Gm, and grown at 43 ◦ C overnight. Tens
to hundreds larger colonies will appear on a thick lawn of non-recombinant small colonies, and these larger colonies were selected for testing of the co-integrants by PCR or Southern blots (Lalioti and Heath, 2001). Due to the persisting existence of selection pressure (chloramphenicol), the un-integrated pKOV-S74I will not be lost from the colony even when the colony was grown at 43 ◦ C. Therefore, using the Lalioti method co-integration recombinants must be selected from a thick lawn of non-recombinant small colonies background, thus performing PCR or Southern blots to confirm the co-integrants is necessary. In order to simplify the procedures and decrease the non-recombinant background, we first pool those recombinants from the three antibiotics plate and spread them on the Gm plate and incubate at 43 ◦ C overnight. This treatment will make the recombinants lose the un-integrated pKOV-S74I. As the selection pressure (chloramphenicol) is withdrawn at this step, the un-integrated pKOV-S74I will be lost quickly from the colony. Then, the Gm-resisted colonies were pooled and spread on the Cm plate at 43 ◦ C to select the co-integration colony. After this treatment, the background of the co-integration step decreases remarkably comparing to the former protocol, so that the step of testing the co-integration is no longer required. Using the SpltMNPV p74-specific primers (ST1 and ST2), an expected 860 bp fragment can be amplified from the recombinant AcMNPV while using the AcMNPV p74-specific primers (AT1 And AT2), no PCR product was obtained, thus demonstrating that the new bacmid only has the SpltMNPV p74 gene. RT-PCR result also showed that the SpltMNPV p74 gene in the recombinant AcMNPV bacmid was expressed. It was also found that the SpltMNPV p74 gene of the recombinant bacmid was transcripted at the same time phase as that of the wild-type bacmid (data not shown). This result is consistent with the fact that the 5 UTR and 3 UTR of p74 gene were all un-touched in the recombinant AcMNPV bacmid. The successful substitution of p74 gene using AcMNPV bacmid provides an easy method for further research of gene function of the p74 gene and other genes in AcMNPV genome or even in other types of large viral genome that had been cloned into the BAC or PAC plasmid.
Acknowledgements We thank Dr. Maria Lalioti for providing the plasmids pDF25 and pKOV-Kan, Ms. Xiaoxin Zhang for kindly reviewing the draft. This work was supported by National Natural Science Foundation, No. 30170039.
References Bideshi, D.K., Federici, B.A., 2000. The Trichoplusia ni granulovirus helicase is unable to support replication of Autographa californica
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101 multicapsid nucleopolyhedrovirus in cells and larvae of T. ni. J. Gen. Virol. 81, 1593–1599. Court, D.L., Sawitzke, J.A., Thomason, L.C., 2002. Genetic engineering using homologous recombination. Annu. Rev. Genet. 36, 361–388. Deng, X., Zhu, Y., Diao, Z., Qi, Y., Zhou, Z., 2000. Construction of a novel Bm NPV Bac to Bac system. Acta Microbiol. Sin. 40, 155–160. Faulkner, P., Kuzio, J., Williams, G.V., Wilson, J.A., 1997. Analysis of p74, a PDV envelope protein of Autographa californica multicapsid nucleopolyhedrovirus required for occlusion body infectivity in vivo. J. Gen. Virol. 78, 3091–3100. Hou, S., Chen, X., Wang, H., Tao, M., Hu, Z., 2002. Efficient method to generate homologous recombinant baculovirus genomes in E. coli. Biotechniques 32 (4), 783–788. Jones, I., Morikawa, Y., 1996. Baculovirus vectors for expression in insect cells. Curr. Opin. Biotechnol. 7 (5), 512–516. Kitts, P.A., Possee, R.D., 1993. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14 (5), 810–817. Lalioti, M., Heath, J., 2001. A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli. Nucleic Acids Res. 29 (3), E14. Lin, G., Blissard, G.W., 2002. Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication. J. Virol. 76 (6), 2770–2779. Lu, A., Miller, L.K., 1996. Generation of recombinant baculoviruses by direct cloning. Biotechniques 21 (1), 63–68. Luckow, V.A., Lee, S.C., Barry, G.F., Olins, P.O., 1993. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579.
101
Nefedov, M., Williamson, R., Ioannou, P.A., 2000. Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli. Nucleic Acids Res. 28 (17) E79. Pang, Y., Yu, J., Wang, L., Hu, X., Bao, W., Li, G., Chen, C., et al., 2001. Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287 (2), 391–404. Patel, G., Nasmyth, K., Jones, N., 1992. A new method for the isolation of recombinant baculovirus. Nucleic Acids Res. 20 (1), 97–104. Peakman, T.C., Harris, R.A., Gewert, D.R., 1992. Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination. Nucleic Acids Res. 20 (3), 495– 500. Pijlman, G.P., Dortmans, J.C., Vermeesch, A.M., Yang, K., Martens, D.E., Goldbach, R.W., Vlak, J.M., 2002. Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J. Virol. 76 (11), 5605–5611. Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., Simon, M., 1992. Cloning and stable maintenance of 300 kilobase pair fragments of human DNA in Escherichia coli using an F factor based vector. Proc. Natl. Acad. Sci. U.S.A. 89, 8794–8797. Yang, X.W., Model, P., Heintz, N., 1997. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865. Zhang, Y., Buchholz, F., Muyrers, J.P., Stewart, A.F., 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20 (2), 123–128. Zhu, Y., Qi, Y.P., Liu, D.L., 1997. Construction of a shuttle vector using HaNPV and expression of GFP gene in insect cell and larvae. J. Wuhan Univ. (Nat. Sci. Ed.) 43 (6), 819–820.
An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli Wuwei Wu, Jinwen Wang, Riqiang Deng, Xunzhang Wang∗ , XiongLei He, Qingxin Long State Key Laboratory for Biocontrol, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China Received 15 April 2003; received in revised form 14 July 2003; accepted 15 July 2003
Abstract The RecA-mediated homologous recombination method was improved and used to direct gene replacement in baculoviruses. With this method, the p74 gene in the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was substituted precisely by the p74 gene of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV). In the recombinant bacmid, the AcMNPV p74 gene promoter controlled directly the expression of SpltMNPV p74 gene. Results of RT-PCR showed transcription of SpltMNPV p74 gene in the recombinant, implying the potential use of this easy and efficient method in baculovirus gene function research. © 2003 Published by Elsevier B.V. Keywords: Gene substitution; Baculovirus; p74 gene; RecA-mediated homologous recombination
1. Introduction Insect baculovirus is a double-stranded DNA virus used widely as a biological pesticide and a eukaryotic gene expression vector (Jones and Morikawa, 1996). Two forms of viral particles, budded virus (BV) and occlusion-derived virus (ODV), are generated during its life cycle. An ODV envelope protein encoded by the p74 gene was found to be essential to baculovirus ODV infectivity (Faulkner et al., 1997). Exposure of P74 protein on the surface of ODV suggests the function of this protein in attachment of ODV to the midgut cell membranes of insect larvae. Is this protein species-specific? May it be replaced by P74 from different baculovirus species such as SpltMNPV without infectivity loss? Construction of recombinant AcMNPV with the target gene replaced precisely by homologous gene from other species may help to answer these questions. Due to the large size of baculoviral genomes, it is almost impossible to modify baculovirus by simple restriction enzyme digestion and ligation. The conventional method of obtaining recombinant baculovirus is to construct a plasmid containing the desired alteration flanked by viral sequences of the target site, and then to co-transfect cultured insect ∗ Corresponding author. Tel.: +86-20-84112504; fax: +86-20-84113964. E-mail address: [email protected] (X. Wang).
0166-0934/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/S0166-0934(03)00225-8
cells with this plasmid and wild-type viral DNA. Thus, the recombinant virus is generated through homologous recombination between transfer plasmid and viral DNA in the insect cells. Although this method has its advantages, it is very time consuming. In addition to this method, some improved approaches were also developed, such as linearized virus (Kitts and Possee, 1993), Cre-Lox recombination system (Peakman et al., 1992), homologous recombination in yeast (Patel et al., 1992), Bac-to-Bac® system (Luckow et al., 1993), and direct insert of the exogenous gene into the single restriction sites of baculovirus (Lu and Miller, 1996). Unfortunately, most of these strategies can only apply to the polyhedrin gene locus when used in constructing recombinant virus. Modification of other genes still requires the traditional co-transfection method, thus making functional research of these genes very laborious. Recently, some alternative approaches for constructing recombinant baculovirus were described (Bideshi and Federici, 2000; Hou et al., 2002; Lin and Blissard, 2002; Pijlman et al., 2002). All of these approaches used the baculovirus bacmid instead of the wild-type baculovirus. A bacmid is a baculovirus that has an F-factor-based plasmid backbone inserted into the polh gene locus, and can propagate in both insect cells and E. coli. This feature is very similar to the bacterial artificial chromosome (BAC) that had been developed in the past decades (Shizuya et al., 1992) and which is used extensively. BAC can carry DNA
96
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
fragment as large as 300 kb, and many new techniques for modifying such large plasmids were also described recently (Court et al., 2002; Lalioti and Heath, 2001; Yang et al., 1997; Zhang et al., 1998). Bideshi and Federici (2000) first used one of these new techniques—E. coli BJ5183 system on the AcMNPV bacmid. Other systems, ET-recombination and phage lambda red system were used to modify the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) bacmid (Pijlman et al., 2002) and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) bacmid (Hou et al., 2002). However, all the above studies only resulted in the knockout of certain baculovirus genes, and offered a repaired or substituted gene at the polh gene locus using Bac-to-Bac system. In this paper, we describe the use and modification of a technique called RecA-mediated homologous recombination which was reported to carry out easily gene substitution in those baculovirus that had been cloned as a bacmid (Deng et al., 2000; Luckow et al., 1993; Pijlman et al., 2002; Zhu et al., 1997). This technique was described originally by Yang et al. (1997) and improved by Lalioti and Heath (2001).
2. Materials and methods 2.1. Viruses, plasmids, and primers AcMNPV strain accompanying the Bac-to-Bac® system from Invitrogen was used in this study. The SpltMNPV G2 strain was isolated from suburban of Guangzhou and kept in our laboratory, and the viral genome was sequenced recently (Pang et al., 2001). The AcMNPV bacmid-polh containing gentamicin (Gm)-resistant gene was constructed by introducing polyhedrin gene from pFastBac1 into the commercial AcMNPV bacmid of the Invitrogen Bac-to-Bac® system using the method described by Luckow et al. (1993). The plasmid pDF25 (Lalioti and Heath, 2001) contains a temperature-sensitive replicon RepA, a recA gene and a tetracycline (Tet)-resistant gene. The temperature-sensitive replicon RepA makes pDF25 replicate at the permissive temperature (30 ◦ C) and to stop replication at the non-permissive temperature (43 ◦ C). pfu DNA polymerase (Bioasia Biotech, Shanghai, PR China) was used to amplify DNA fragments from the virus genomic DNA. The AcMNPV p74 gene upstream fragment from genome position of 121661–121073 was amplified using the primers ACP74R1 and ACP74R2 and then digested with BamH1. SpltMNPV p74 gene fragment from genome position of 19508–21679 was amplified using the primers SLP741 and SLP742 and then digested with Sac1. The AcMNPV p74 gene downstream fragment from genome position of 118729–119268 was amplified using the primers ACP74L1 and ACP74L2 and then digested
with Sac1 and Not1. The resulted fragments were then ligated into the plasmid pKOV-Kan (Lalioti and Heath, 2001) between the BamH1 and Not1 sites, producing plasmid pKOV-S74I which contains the temperature-sensitive replicon RepA, a chloramphenicol (Cm)-resistant gene, and a counter selection marker sacB gene. The cloned fragments were confirmed by sequencing using an ABI PRISM® 377 automated DNA sequencer (PE Biosystems). Two pairs of PCR primers were designed to distinguish the p74 gene of AcMNPV and SpltMNPV: primer AT1 and AT2 amplify the AcMNPV p74 gene fragment from position 119713–120335, primer ST1 and ST2 amplify the SpltMNPV p74 gene fragment from position 20365–21224. The position of primers used and the structure of plasmid pKOV-S74I are shown in Fig. 1. Sequences of the primers are shown in Table 1. 2.2. Substitution of the p74 gene The pKOV-S74I DNA (0.5 g) and pDF25 DNA (0.5 g) were co-transformed into competent E. coli strain DH10B (100 l) harboring the AcMNPV bacmid-polh. The transformed bacteria were incubated at 30 ◦ C overnight on an LB plate containing 7 g/ml Gm, 20 g/ml Cm and 10 g/ml Tet. Three to five colonies were then picked and washed into 1 ml LB media and 100 l of this were spread onto a LB plate containing 7 g/ml Gm. After incubation at 43 ◦ C overnight, colonies were again washed and spread onto a LB plate containing 20 g/ml Cm and incubated at 43 ◦ C overnight. Survived co-integrant colonies should appear on the Cm plate and three to five colonies were picked and cultured in the LB media containing 20 g/ml Cm at 43 ◦ C for 2 h. The bacteria were harvested and made competent with 100 mM CaCl2 , and then transformed with plasmid pDF25 (0.5 g). The transformed cells were plated on LB plates containing 7 g/ml Gm and 10 g/ml Tet and left to resolve at 30 ◦ C overnight. Three to five colonies were picked and washed into 1 ml LB media and then diluted 100-fold to obtain single colonies after aliquots of 100 l of the diluted cells were spread on an LB plate containing 20 g/ml Gm and 7% sucrose and grew at 43 ◦ C overnight. Ten to 20 larger colonies were then selected and streaked on Gm and Cm simultaneously and left at 30 ◦ C overnight. Those colonies that only grew on Gm were selected for further analysis. The procedure for p74 gene substitution is shown in Fig. 2. 2.3. PCR and RT-PCR confirmation of substitution and transcription of p74 gene Bacmid DNA was isolated from each selected colony. For the recombinant bacmid-polh-slp74, PCR amplification with primer pair ST1 and ST2 would expect an 860 bp product whereas primer pair AT1 and AT2 would produce nothing.
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
97
Fig. 1. The primers position and structure of plasmid pKOV-S74I. (A) Three pairs of primers were designed to amplify AcMNPV p74 gene upand downstream fragments and the SpltMNPV p74 gene fragment. Two pairs of primers, AT1, AT2, ST1, and ST2, were designed for p74 gene confirmation. (B) Structure of plasmid pKOV-S74I. Showing the SpltMNPV p74 gene flanked by AcMNPV p74 gene up- and downstream fragments. Cm: chloramphenicol-resistant gene; RepA: a temperature-sensitive replicon; and SacB: a counter selection marker that blocks the growth of colony on the medium containing sucrose.
DNA of the recombinant bacmid was transfected into the insect cell line Tn-5B1-4 (Hi5) using lipofectin (Gibco BRL). The supernatant was collected at 72 h posttransfection and used to infect the cells at 5 MOI (multiplic-
ity of infection). Total RNA was extracted from about 106 cells at 20 h post-infection using TriPure isolation reagent (Boehringer Mannheim). One unit of RQ1 RNase-free DNase (Promega) was added to 5 g total RNA to digest
Table 1 Primers used in this study Primer
Sequencea
Virus
Positionb
ACP74L1 ACP74L2 ACP74R1 ACP74R2 AT1 AT2 SLP741 SLP742 ST1 ST2
5 -AGGCGGCCGCGACCTTTAATTCAACCC-3 5 -CGAGCTCTGGCGTTTACAGCATTTGTT-3 5 -GTTATATAGGACTTAAAATAAAC-3 5 -TCGGATCCAGGGAAACATACAC-3 5 -AAACCAAATCTGCCAGCGTCAAT-3 5 -TTCCAGCATACTACCGCCACGAC-3 5 -CAGAGCTCGATTGTCCGGGTGGCG-3 5 -ATGAGCGCGCACGTAAATTCTCCGA-3 5 -CTGAATTGCCATCCGAGTCAGTG-3 5 -TCTAAATTCCCCGTACCTACGCA-3
AcMNPV AcMNPV AcMNPV AcMNPV AcMNPV AcMNPV SpltMNPV SpltMNPV SpltMNPV SpltMNPV
118729 119268 121073 121661 119713 120335 19508 21679 20365 21224
a b
The restriction sites are underlined and the real viral sequences are shown in bold letters. Genome position of the 5 nucleotide of real viral sequence of the corresponding primer.
98
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
Fig. 2. Substituting the p74 gene of AcMNPV bacmid-polh. The substitution begins with the integration of the plasmid pKOV-S741 containing SpltMNPV p74 gene into the AcMNPV bacmid-polh through homologous recombination. The co-integrant may then resolve into either the original or the recombinant bacmid-polh. The diagram shows only the recombinant one.
for 5 min. The DNase was inactivated by heating at 80 ◦ C and the resulting RNA was used for reversed transcription (RT) using OligdT and 1 l of the RT product was used as the PCR template using primers ST1 and ST2. A control PCR was also undertaken with DNase treated RNA as the template.
3. Results
cloned fragment in the plasmid pKOV-S74I was sequenced and confirmed to be correct. Partial results are shown in Fig. 3, and we can see the ATG codon of SpltMNPV p74 gene follows immediately the upstream sequence of AcMNPV p74 gene. A recombinant bacmid AcMNPV bacmid-polhSL74 was produced by integration of plasmid pKOV-S74I into AcMNPV bacmid-polh forming the co-integrant at the first step and resolution of the co-integrant into either the original or the recombinant bacmid at the second step.
3.1. p74 gene substitution 3.2. Analysis of the recombinant bacmid SpltMNPV and AcMNPV p74 gene up- and downstream fragments were PCR amplified, ligated, and inserted into the plasmid pKOV-Kan (Lalioti and Heath, 2001) between the BamH1 and Not1 sites to construct plasmid pKOV-S74I. The
Recombination was confirmed by PCR test. In PCR experiments, an expected 860 bp band was observed when amplified with the SpltMNPV p74 gene-specific primers ST1
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
99
Fig. 3. Partial sequence results of plasmid pKOV-S74I. Showing the sequence upstreaming ATG codon is exactly the upstream sequence of AcMNPV p74 gene and there was no nucleotide insertion or deletion found in the sequence.
and ST2 while no band appeared when using the AcMNPV p74 gene-specific primers AT1 And AT2, showing the successful replacement of the AcMNPV p74 gene by SpltMNPV p74 gene. The PCR result is shown in Fig. 4. The SpltMNPV p74 gene-specific primers ST1 and ST2 were also used to perform RT-PCR to determine expression of SpltMNPV p74 gene in the recombinant AcMNPV bacmid. The RT-PCR result was shown in Fig. 4, showing that the substitute gene was able to express in the recombinant bacmid. 3.3. Methodology improvement
Fig. 4. PCR and RT-PCR test of the recombinant bacmid-polhSL74. (A) PCR identification of SpltMNPV p74 gene. Only the SpltMNPV p74 gene-specific primers ST1 and ST2 produced an 860 bp PCR product, whereas with the AcMNPV p74 gene-specific primer AT1 and AT2, no product was obtained. Lane M: marker; lane 1: with primers ST1 and ST2; and lane 2, with primers AT1 and AT2. (B) RT-PCR identification of the transcription of the SpltMNPV p74 gene in the Tn-5B1-4 cell infected by AcMNPV bacmid-polhSL74. Using the SpltMNPV p74 gene-specific primers ST1 and ST2, an expected 860 bp RT-PCR product was obtained in lane 1. Lane M: marker; lane 1: cDNA template; and lane 2: control with RNA template.
The method described by Lalioti and Heath (2001) was modified to facilitate the selection of the co-integrant colonies from un-integrated pKOV-S74I colonies. Since temperature selection accompanied with Cm selection pressure was not able to efficiently suppress the growth of the un-integrated colonies, a step with temperature selection but without Cm selection pressure was added to eliminate pKOV-S74I colonies before selection of the co-integrant colonies. This modification resulted in big co-integrant colonies at clear background. The result was shown in Fig. 5.
Fig. 5. Comparison of the co-integrant selection plate obtained by Lalioti method (B) and the modified method (A). Co-integrant colonies are indicated by black arrows. The co-integrant colonies obtained by the modified method are big and clear compared to the relative smaller ones at the background of tiny un-integrant colonies obtained by Lalioti’s original method.
100
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101
4. Discussions p74 gene is an essential gene for ODV infectivity of baculovirus, and therefore gene function should be studied with a polh-positive baculovirus. The commercial AcMNPV bacmid is a polh-negative protein expression tool with an F-factor-based plasmid backbone at the polyhedrin locus (Luckow et al., 1993). Therefore, we began the study by constructing AcMNPV bacmid-polh, a polyhedrin-positive bacmid, by transposing the polyhedrin gene, which had been cloned into the plasmid pFastBac1 (Invitrogen), into the commercial AcMNPV bacmid using Bac-to-Bac® protein expression system (data not shown). Many other methods had been used for gene modification of bacmid (Bideshi and Federici, 2000; Hou et al., 2002; Pijlman et al., 2002), and all use linear DNA as the gene target tool. Comparing to our method of RecA-mediated recombination, those methods are less convenient at in situ gene substitution: When undertaking in situ gene substitution, all those methods, including the E. coli BJ5183 system, ET-recombination and the phage lambda red system, require two steps. One step is to co-insert a selection and anti-selection marker into the target site; and the second step is to replace the marker by the modified gene. Unfortunately, the lack of good counter selection marker makes the second step very inefficient (Nefedov et al., 2000; Zhang et al., 1998). Therefore, it is essential to confirm that there are no mutations in anti-selection marker when the marker is amplified by PCR to produce the linear insertion DNA used in the first co-insert step. However, the RecA-mediated recombination does not require sequencing of the anti-selection marker, and this save a lot of time. Moreover, further improvement of the RecA-mediated recombination method in this study makes the selection of the modified bacmid much easier. In plasmid pKOVSL74I, the translation start codon of SpltMNPV p74 gene follows immediately the upstream sequence of AcMNPV p74 gene, so the AcMNPV p74 promoter directly controls the expression of SpltMNPV p74 gene. Fig. 2 shows the flowchart of p74 gene substitution procedure containing two steps, co-integration and resolution. Each step may have one recombination event taking place between the homologous sequences of AcMNPV bacmid-polh and pKOVSL74I. For example, if the first recombination event took place at the downstream sequence of AcMNPV p74 gene and the second recombination event took place at the upstream sequence of AcMNPV p74 gene, the recombinant AcMNPV bacmid-polh-slp74 was produced. At the step when selecting the first co-integration recombinant, the protocol described by Dr. Lalioti was modified. After obtaining the colonies containing three plasmids (AcMNPV bacmid-polhSL74, pDF25 and pKOV-S74I), the Lalioti method was to select four to six colonies and wash them into 1 ml LB, then spread them onto the plate containing Cm and Gm, and grown at 43 ◦ C overnight. Tens
to hundreds larger colonies will appear on a thick lawn of non-recombinant small colonies, and these larger colonies were selected for testing of the co-integrants by PCR or Southern blots (Lalioti and Heath, 2001). Due to the persisting existence of selection pressure (chloramphenicol), the un-integrated pKOV-S74I will not be lost from the colony even when the colony was grown at 43 ◦ C. Therefore, using the Lalioti method co-integration recombinants must be selected from a thick lawn of non-recombinant small colonies background, thus performing PCR or Southern blots to confirm the co-integrants is necessary. In order to simplify the procedures and decrease the non-recombinant background, we first pool those recombinants from the three antibiotics plate and spread them on the Gm plate and incubate at 43 ◦ C overnight. This treatment will make the recombinants lose the un-integrated pKOV-S74I. As the selection pressure (chloramphenicol) is withdrawn at this step, the un-integrated pKOV-S74I will be lost quickly from the colony. Then, the Gm-resisted colonies were pooled and spread on the Cm plate at 43 ◦ C to select the co-integration colony. After this treatment, the background of the co-integration step decreases remarkably comparing to the former protocol, so that the step of testing the co-integration is no longer required. Using the SpltMNPV p74-specific primers (ST1 and ST2), an expected 860 bp fragment can be amplified from the recombinant AcMNPV while using the AcMNPV p74-specific primers (AT1 And AT2), no PCR product was obtained, thus demonstrating that the new bacmid only has the SpltMNPV p74 gene. RT-PCR result also showed that the SpltMNPV p74 gene in the recombinant AcMNPV bacmid was expressed. It was also found that the SpltMNPV p74 gene of the recombinant bacmid was transcripted at the same time phase as that of the wild-type bacmid (data not shown). This result is consistent with the fact that the 5 UTR and 3 UTR of p74 gene were all un-touched in the recombinant AcMNPV bacmid. The successful substitution of p74 gene using AcMNPV bacmid provides an easy method for further research of gene function of the p74 gene and other genes in AcMNPV genome or even in other types of large viral genome that had been cloned into the BAC or PAC plasmid.
Acknowledgements We thank Dr. Maria Lalioti for providing the plasmids pDF25 and pKOV-Kan, Ms. Xiaoxin Zhang for kindly reviewing the draft. This work was supported by National Natural Science Foundation, No. 30170039.
References Bideshi, D.K., Federici, B.A., 2000. The Trichoplusia ni granulovirus helicase is unable to support replication of Autographa californica
W. Wu et al. / Journal of Virological Methods 113 (2003) 95–101 multicapsid nucleopolyhedrovirus in cells and larvae of T. ni. J. Gen. Virol. 81, 1593–1599. Court, D.L., Sawitzke, J.A., Thomason, L.C., 2002. Genetic engineering using homologous recombination. Annu. Rev. Genet. 36, 361–388. Deng, X., Zhu, Y., Diao, Z., Qi, Y., Zhou, Z., 2000. Construction of a novel Bm NPV Bac to Bac system. Acta Microbiol. Sin. 40, 155–160. Faulkner, P., Kuzio, J., Williams, G.V., Wilson, J.A., 1997. Analysis of p74, a PDV envelope protein of Autographa californica multicapsid nucleopolyhedrovirus required for occlusion body infectivity in vivo. J. Gen. Virol. 78, 3091–3100. Hou, S., Chen, X., Wang, H., Tao, M., Hu, Z., 2002. Efficient method to generate homologous recombinant baculovirus genomes in E. coli. Biotechniques 32 (4), 783–788. Jones, I., Morikawa, Y., 1996. Baculovirus vectors for expression in insect cells. Curr. Opin. Biotechnol. 7 (5), 512–516. Kitts, P.A., Possee, R.D., 1993. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14 (5), 810–817. Lalioti, M., Heath, J., 2001. A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli. Nucleic Acids Res. 29 (3), E14. Lin, G., Blissard, G.W., 2002. Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication. J. Virol. 76 (6), 2770–2779. Lu, A., Miller, L.K., 1996. Generation of recombinant baculoviruses by direct cloning. Biotechniques 21 (1), 63–68. Luckow, V.A., Lee, S.C., Barry, G.F., Olins, P.O., 1993. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579.
101
Nefedov, M., Williamson, R., Ioannou, P.A., 2000. Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli. Nucleic Acids Res. 28 (17) E79. Pang, Y., Yu, J., Wang, L., Hu, X., Bao, W., Li, G., Chen, C., et al., 2001. Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287 (2), 391–404. Patel, G., Nasmyth, K., Jones, N., 1992. A new method for the isolation of recombinant baculovirus. Nucleic Acids Res. 20 (1), 97–104. Peakman, T.C., Harris, R.A., Gewert, D.R., 1992. Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination. Nucleic Acids Res. 20 (3), 495– 500. Pijlman, G.P., Dortmans, J.C., Vermeesch, A.M., Yang, K., Martens, D.E., Goldbach, R.W., Vlak, J.M., 2002. Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J. Virol. 76 (11), 5605–5611. Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., Simon, M., 1992. Cloning and stable maintenance of 300 kilobase pair fragments of human DNA in Escherichia coli using an F factor based vector. Proc. Natl. Acad. Sci. U.S.A. 89, 8794–8797. Yang, X.W., Model, P., Heintz, N., 1997. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865. Zhang, Y., Buchholz, F., Muyrers, J.P., Stewart, A.F., 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20 (2), 123–128. Zhu, Y., Qi, Y.P., Liu, D.L., 1997. Construction of a shuttle vector using HaNPV and expression of GFP gene in insect cell and larvae. J. Wuhan Univ. (Nat. Sci. Ed.) 43 (6), 819–820.