Original Paper |
|
|||
|
Acta Biochim Biophys |
||||
|
doi:10.1111/j.1745-7270.2008.00471.x |
Transgenic breeding of anti-Bombyx mori L. nuclear
polyhedrosis virus silkworm Bombyx mori
Huijuan Yang1, Wei Fan1, Hao Wei1, Jinwei Zhang2, Zhonghua Zhou1, Jianying Li1, Jianrong Lin3, Nong Ding2, and Boxiong Zhong1*
1 College of Animal Sciences, Zhejiang
University, Hangzhou 310029, China
2 Huzhou Academy of Agriculture Sciences,
Huzhou 313000, China
3 College of Animal Sciences, South China
Agricultural University, Guangzhou 510642, China
Received: July 07,
2008
Accepted: August
27, 2008
This work was
supported by grants from the National Basic Research Program of China (No.
2005CB121003), the National Hi-Tech Research and Development Program of China
(No. 2006AA10A118), and Doctoral Fund of the Ministry of Education of China
(No. 20070335148)
*Corresponding
author: Tel/Fax, 86-571-86971302; E-mail, [email protected]
Silkworm strains resistant to Bombyx
mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac
transposon with an A3 promoter were randomly inserted into the silkworm,
driving the enhanced green fluorescent protein (EGFP) reporter gene into
the silkworm genome. Polymerase chain reaction results verified the insertion
of the extraneous EGFP gene, and fluorescence microscopy showed that the
EGFP was expressed in the midgut tissue. The morbidity ratio of the
nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7%
in the transgenic silkworm strain. Compared with the resistance to the Bombyx
mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly
used in the production, there was an increase of 33 centesimal points in the
transgenic silkworms. The antivirotic character in the Chunhua´Qiuyue
strain, which was bred from a different transgenic family, was about 10
centesimal points higher than that in the Qiufeng´Baiyu,
another crossbreed used in production. Our results indicated a good application
value of the transposon-inserted mutation in the breeding of anti-BmNPV
silkworm strain.
Keywords silkworm; transgenic; piggyBac;
BmNPV; antivirotic
Nuclear polyhedrosis is caused by the Bombyx mori L. nuclear
polyhedrosis virus (BmNPV), a member of the subfamily Eubaculovirinae of the
family Baculoviridae [1]. Nuclear polyhedrosis is one of the deadliest diseases
that can strike the mulberry silkworm, Bombyx mori. The most economical
and effective way to prevent the disease is by breeding the antivirotic
silkworm strain. Previous genetic development research has shown that
resistance to BmNPV is controlled by one major gene and several minor genes
belonging to the incomplete patroclinous heredity with the effective gene
number of 2.31 [2,3]. There are significant differences among the various
silkworm strains, such as between the antivirotic strain and the normal strain,
as the latter is three times as susceptible to nuclear polyhedrosis. Our
results showed that the resistance to the virus was positively related to the
silkworm’s cocoon weight, suggesting that the successful breeding of
antivirotic silkworm strains may possibly be the economical merit strains.
Rcently, the application of transgenic technology conducted by the
injection of piggyBac transposon has become widely used [4]. Previous
research has shown that the Nistari strain has high transformation efficiency
when injected with the piggyBac transposon [5]. Also, extraneous target
genes have been successfully inserted and expressed in the silkworm by piggyBac
transformation system [6–8].
Random insertion of a piggyBac transposon into the silkworm genome
changes gene expression. Based on this, we screened transgenic silkworm stranis
with high resistance to BmNPV.
Materials and Methods
Materials
Nistari, a non-diapause, multivoltine silkworm strain, were bred and
raised by Zhejiang University’s Laboratory of Germplasm Innovation and
Molecular Breeding in Silkworm and Bee (Hangzhou, China). The piggyBac
transposon (pPIGA3GFP) with A3 promoter and enhanced green fluorescent protein
(EGFP) reporter gene as well as the helper plasmid (pHA3PIG) were provided
by Dr. Toshiki
Tamura (National Institute of Sericultural and Entomological Science, Tsukuba,
Japan).
Embryo injection and screening of transgenic silkworms
Embryo injection and polymerase chain reaction (PCR) analysis were carried
out [5]. In brief, eggs were harvested at the syncytial preblastoderm stage
1–6 h after being laid in at 25 ºC environment. The mixture of vector pPIGA3GFP
and helper plasmids (15–20 nl; 1:1, 0.4 mg/ml total DNA concentration)
in 0.5 mM phosphate buffer (pH 7.0) containing 5 mM KCl was microinjected into
each egg. The embryos were allowed to develop at 25 ºC and 75% humidity. G0 moths were mated randomly. In G1
generation, we screened the positive individuals by observing EGFP
fluorescence with an Olympus SZX12 stereomicroscope (Tokyo, Japan)
Excitation filter spectral width was set between 460 nm and 490 nm,
and emission filter spectral width between 510 nm and 550 nm was used for
detection. G1 positive transgenic individuals were mated and generated G2 generation.
Total DNA was extracted from the posterior silk glands of G2 fifth instar larvae that had been frozen in liquid nitrogen and
stored at –20 ºC. The obtained DNA (<1 mg) was used for PCR analysis. The target
fragment of 1341 bp nucleotide of pPIGA3GFP was amplified using the primer pair
5¢-ACGACGGCAACTACAAGACC-3¢ and 5¢-GCGGAGAATGGGCGGAACT-3¢. Amplification
was carried out with a 4 min denaturing step at 96 ºC, followed by 35 cycles
of 1 min at 96 ºC, 1 min at 60.9 ºC, 1.5 min at 72 ºC and a final extension at
72 ºC for 10 min. Amplified products were separated on 1% agarose gel and
visualized by ethidium bromide staining.
Comparing the resistance to BmNPV
The single G2 batch was reared, and the
positive individuals mated to generate G3
silkworms. We chose 90 silkworms from each family for resistance testing, and
others were reared for the strain reserve. Each set of 90 silkworms were
divided into three groups at the beginning of the third instar. Three mulberry
leaves (4 cm´4 cm) incubated with 200 ml BmNPV (5´107/ml) were added to each test group. The
silkworms were kept at room temperature (26 ºC –27 ºC). The
morbidity ratio was investigated in the forth instar.
Results
Screening positive transgenic silkworm
In total 25,058 eggs were microinjected, and 708 female moths and
869 male moths were obtained from G0 generation. All of them
were fertile and had a percent of 6.29% in the total microinjected eggs. In G1 larvae, the positive transgenic individuals with EGFP were
found in 214 broods with a percent of 13.57% to the total living moths
(708+869) and 0.854% in the total eggs (25,058). PCR analysis in G2 larvae showed that the EGFP gene had been inserted and
expressed in the genome of transgenic silkworms (data not shown). The
anatomical images observed under fluorescence indicated that the EGFP
driven by the A3 promoter, which mainly promotes gene expression in muscle
tissue, could be expressed in midgut (Fig. 1).
Comparing resistance to BmNPV in transgenic silkworm strains
Ten transgenic families numbered T1–T10 were chosen for
antivirotic testing, and the normal Nistari strain was used as control. The
transgenic families’ resistance to BmNPV was elevated compared with the
control’s. The morbidity ratio decreased from 90% in the control group and to
66.7%, the lowest ratio, in T1 transgenic group, suggesting that resistance to
BmNPV approximately be increased by 23.3 centesimal points (Table 1).
Comparing the results of the T1, T4, T6, T7 and the most commonly used silkworm
strain, Qiufeng, showed that resistance to BmNPV was elevated by approximately
33.4 centesimal points in the T7 silkworm family (Table 2).
Hybridized combinations of anti-BmNPV silkworm strains
The T4 and T6 families were crossbred with the strains Feng1 and
Qiufeng, two strains used in the production. The T7 family was hybridized with
the strains 54A and Baiyu. In the crossbreeding process, the EGFP was
used as a maker and the strains with high resistance to BmNPV were reserved. At
the same time, other economic characters were also analyzed. After five
generations of selection, two hybridized combination strains, Chunhua´Qiuyue and Qiuyue´Chunhua,
from the transgenic silkworm strain were obtained. The hybridized strains’
resistance to BmNPV was compared to that of strains Qiufeng´Baiyu and Baiyu´Qiufeng,
which were used in production. The concentrations of BmNPV used for the test
were 5´106/ml and 5´107/ml. Evidence of high resistance to BmNPV in
the hybridized transgenic silkworm strains became more apparent when treated
with higher concentrations of BmNPV. The Chunhua´Qiuyue reciprocal hybrids had a 10.28 centesimal point resistance
to BmNPV, which was higher than that of the Qiufeng´Baiyu reciprocal hybrids (Table 3).
Discussion
It has been proven that the piggyBac transposon was most
probably inserted into the function genome areas when being transformed [9].
Using RNA interference technology, researchers have found that target silence of
baculoviral immediate early-1 gene as well as other preventative mechanisms
could increase the silkworm resistance to baculovirus [10,11]. The silence of
essential viral gene lef-1 of BmNPV may also induce silkworm resistance
to BmNPV [12]. Our results showed that in the transgenic silkworms, A3 promoter
in the piggyBac transposon could induce extraneous gene expression in
the midgut tissue (Fig. 1), and some transgenic silkworm families showed
high resistance to BmNPV (Tables 1–3). These results excluded the target
gene silence of BmNPV related genes that acquire a rather common resistance to
BmNPV in several transgenic silkworm strains.
There are two possible reasons for this phenomenon. One is that the
transposon may be inserted into a functional locus in the genome that is
related to silkworm resistance to BmNPV, thus resulting in the low morbidity
ratio in transgenic silkworms as the insertion of transposon affected the
physiology of resistance to BmNPV. The mechanism may be related to the
activation of BmNPV receptor in midgut or the reduced binding ability between
them. The other possibility relates to the EGFP protein expressed in the midgut
as a result of the A3 promoter. Interestingly, no matter where the transposon
was inserted into the genome and regardless of the transgenic family, all the
resistances to BmNPV increased, indicating that the expression of EGFP in the
midgut might be protective to the silkworm (Tables 1–3). Previous
research also reported that EGFP, RFP (red fluorescent protein) afford some protection
against the damaging effects of ultraviolet light in baculoviruse [13]. We
assumed that EGFP functioned similarly in this case. Both of the above
hypotheses are possible, while the latter holds greater potential. However,
these mechanisms still need to be verified by further researches.
References
1 Francki RIB, Fauquet CM, Knudson DL, Brown F.
Classification and Nomenclature of Viruses. Vienna & New York:
Springer-Verlag 1991
2 Chen K, Lin C, Yao Q. Studies on the resistance
to NPV and its hereditary regularity in the silkworm (Bombyx mori L.).
Acta Sericologia Sinica 1996, 22: 160–164
3 Zhao Y, Qian H, Chen K, He S. Studies on
resistance of current silkworm races Bombyx mori to fluoride and nuclear
polyhedrosis virus. Acta Sericologia Sinica 1996, 22: 219–223
4 Tamura T, Thibert C, Royer C, Kanda T,
Abraham E, Kamba M, Komoto N et al. Germline transformation of the
silkworm Bombyx mori L. using a piggyBac transposon-derived
vector. Nat Biotechnol 2000, 18: 81–84
5 Zhong B, Li J, Chen J, Ye J, Yu S. Comparison
of transformation efficiency of piggyBac transposon among three different
silkworm Bombyx mori strains. Acta Biochim Biophys Sin 2007, 39: 117–122
6 Tomita M, Munetsuna H, Sato T, Adachi T, Hino
R, Hayashi M, Shimizu K et al. Transgenic silkworms produce recombinant
human type III procollagen in cocoons. Nat Biotechnol 2003, 21: 52–56
7 Royer C, Jalabert A, Da Rocha M, Grenier AM,
Mauchamp B, Couble P, Chavancy G. Biosynthesis and cocoon-export of a
recombinant globular protein in transgenic silkworms. Transgenic Res 2005, 14:
463–467
8 Yang HJ, Zhuang LF, Fan W, Lan TY, Ding N, Lu
CD, Zhong BX. The transgenic research of A3 promoter-trap piggyBac-derived
transposon in silkworm Bombyx mori. Acta Sericologia Sinica 2008, 34: 41–44
9 Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T.
Efficient transposition of the piggyBac (PB) transposon in mammalian
cells and mice. Cell 2005, 122: 473–483
10 Kanginakudru S, Royer C, Edupalli SV, Jalabert
A, Mauchamp B; Chandrashekaraiah, Prasad SV et al. Targeting ie-1
gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in
transgenic silkworms. Insect Mol Biol 2007, 16: 635–644
11 Valdes VJ, Sampieri A, Sepulveda J, Vaca L.
Using double-stranded RNA to prevent in vitro and in vivo viral
infections by recombinant baculovirus. J Biol Chem 2003, 278: 19317–19324
12 Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda
T, Tamura T, Sahara K et al. Use of RNAi technology to confer enhanced
resistance to BmNPV on transgenic silkworms. Arch Virol 2004, 149: 1931–1940
13 McIntosh AH, Grasela JJ, Lua L, Braunagel SC.
Demonstration of the protective effects of fluorescent proteins in
baculoviruses exposed to ultraviolet light inactivation. J Insect Sci 2004, 4:
31