CN101139610A - Cultivated silkworm glutathione-S-transferase BmGSTe5 gene - Google Patents
Cultivated silkworm glutathione-S-transferase BmGSTe5 gene Download PDFInfo
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- CN101139610A CN101139610A CNA200710092538XA CN200710092538A CN101139610A CN 101139610 A CN101139610 A CN 101139610A CN A200710092538X A CNA200710092538X A CN A200710092538XA CN 200710092538 A CN200710092538 A CN 200710092538A CN 101139610 A CN101139610 A CN 101139610A
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Abstract
The invention provides a glutathione-S-transferase BmGSTe5 gene for silkworms, which is obtained by getting Epsilon homogenetic gene through retrieving in silkworm gene group by taking use of the known Epsilon family members of other species' GST and by cloning. The activity of the expression product over the mode background CDNB (1-Cl-2,4-dinitrobenzene) is 12 micro mol/min/mg protein. With this gene target, it is hopeful to design a medicine that can prevent and treat Lepidoptera destructive insects but is no harm to those useful insects. The invention is of high value for developing medicine and preventing and treating destructive insects.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of cultivated silkworm glutathione-S-transferase BmGSTe 5 gene that comes from anti-agricultural chemicals of having of silkworm or anti-oxidant activity.
Background technology
Agricultural chemicals plays important effect in pest control, but use along with a large amount of agricultural chemicals, insect resistance increasingly sharpens, force people to develop the consumption of new Pesticidal products or increasing medicament, this has not only restricted expanding economy, also brought serious burden, had influence on economic and human sustainable development to environment.In addition, anti-agricultural chemicals of insect and the anti-oxidant of insect often link together, for example: insect is in metabolic detoxification organophosphorus pesticide process, may produce materials such as some active oxygens, thereby induce the response to oxidative stress of insect, therefore insect is when improving pesticide resistance, and oxidation resistant ability is also increasing thereupon.
Glutathione-S-transferase (Glutathione S-transferase, GSTs, EC 2.5.1.18) is the important isozyme superfamily of a class, extensively is distributed in the organisms such as animal, plant, yeast, bacterium to have the function of eliminating endogenous and exogenous toxic substance.It is not only exercised outside the metabolism to multiple synthetic organic pesticide in insect body, and has unnecessary free radical in the insect body of elimination, keeps intravital oxidation and oxidation resistant running balance.Silkworm is cloned the silkworm gst gene as the model animals of lepidopterous insects, and its function is studied, and for illustrating the relation of GSTs and resistance to insecticides, and important theory and practice significance is arranged for the control of agriculture and forestry injurious insect.Up to the present, the research report that does not still have the gene of anti-agricultural chemicals of having of relevant silkworm or anti-oxidant activity.
Summary of the invention
The object of the present invention is to provide a kind of cultivated silkworm glutathione-S-transferase BmGSTe 5 gene that comes from anti-agricultural chemicals of having of silkworm or anti-oxidant activity, by this gene is further studied, can be development control lepidoptera pest medicine and provide fundamental basis.
The GST that participates in metabolic detoxification in the insect mainly is special Delta of insect and Epsilon family member, and they play important effect (Li etc., 2007) in the anti-organophosphorus of insect, organochlorine and pyrethroid pesticide.The present patent application people utilizes the Epsilon family member of known other species GST among the NCBI, retrieves in the silkworm genome, has obtained the homologous gene of Epsilon.We clone one of them Epsilon member BmGSTe5 gene.Concrete grammar is as follows:
(1) coding region sequence (Coding sequence at first to predict in the domestic silkworm gene group, CDS), (Expressed sequence tag EST) seeks the EST evidence for the BmGSTe5 gene in the database, with CDS and est sequence design Auele Specific Primer in the silkworm expressed sequence tag; The forward primer of BmGSTe5: 5 ' CATATGGTGTTCATCCTGTACAAAA, 3 ' reverse primer: 5 ' GGATCCTGTAATATACTTAGTTGCCGA 3 '; Be added with NdeI and BamHI restriction enzyme site in forward and reverse primer respectively;
(2) Auele Specific Primer with design is that template increases at the midgut cDNA in 5 ages 3 of silkworm, obtained the purpose band, the PCR product reclaims the back and is connected with the pMD18-T carrier, transform DH5 α competent cell, obtain to be sent to Shanghai Sangon order-checking behind the positive colony, sequencing result shows that the sequence of amplification is consistent with expected result;
(3) will contain the pulsating T clone of purpose and carry out double digestion with NdeI and BamHI respectively with the pET28a empty plasmid, reclaim the purpose segment respectively, connect and transform B121 (DE3) competent cell with the T4 ligase enzyme, screening obtains to contain the clone of pET28a-BmGSTe5, extract plasmid and be sent to Shanghai Sangon order-checking, obtain correct clone; The mono-clonal that will contain the pET28a-BmGSTe5 recombinant plasmid is 37 ℃ of shaking culture in containing that LB substratum of card, to OD600=0.6, it is 1mmol/L that adding IPTG makes its final concentration, continued inducing culture 5 hours, get 50ml bacterium liquid 10000g and collected bacterial sediment in centrifugal 5 minutes, with His label purifying target protein matter, purification process carries out according to the pET treasured book; Target protein is 12 μ mol/min/mgprotein to the activity of pattern substrate CDNB (1-chloro-2,4-dinitrobenzene).
(4) sequence homology analysis: the similarity comparison is finished with BLAST on the NCBI website, the BLAST analysis revealed, and the special Epsilon family member similarity of BmGSTe5 and insect is higher.
The conserved domain of predicting in NCBI is:
BmGSTe5 and pattern substrate CDNB's is active higher, and the ability that stronger metabolism agricultural chemicals is arranged in vivo is described, so BmGSTe5 is the relevant important gene of resistance.
The CDS of the BmGSTe5 gene complete that aforesaid method obtains and the aminoacid sequence of deduction thereof are as follows:
atggtgttcatcctgtacaaaaaggacacaagtcctccttgcagatcagttcagatggta
M V F I L Y K K D T S P P C R S V Q M V
ctccacgagctcggaatttatgacgtggaactcattgaagtgaatctgccagaaagggac
L H E L G I Y D V E L I E V N L P E R D
catctgaaagaagaatttctgaggatgaatccgcaacacactgttcccacgttaatagac
H L K E E F L R M N P Q H T V P T L I D
ggagatttcataatatgggacagccacgccatagtcacatatttggtgaacagatatgct
G D F I I W D S H A I V T Y L V N R Y A
aaaaatgacacattatatcctaaagagcctaaacaaagagccattgttgatcagaggtta
K N D T L Y P K E P K Q R A I V D Q R L
cattttgataccggagtgctcttcgccatattacgagccactgctgaaccggtattatac
H F D T G V L F A I L R A T A E P V L Y
aacaatgaaaagagctttaaacaagaaaatctagagaaaatggaggcagcctacgaattc
N N E K S F K Q E N L E K M E A A Y E F
gttgaaaaattcctgacttccgattggctagccggagaccaagttacattagccgatatc
V E K F L T S D W L A G D Q V T L A D I
tgctgtgtatctacaatcagttccatgaacgtaattgtccctattgacaaaaagaagtac
C C V S T I S S M N V I V P I D K K K Y
ccaaaaataatttcatggctacagcgctgttccgaacaagaattttacaagaaagccaac
P K I I S W L Q R C S E Q E F Y K K A N
gaaccaggcttgaagaaattcatagaaatgttcaaaaataaaatcggcaactaa
E P G L K K F I E M F K N K I G N -
Advantage of the present invention is:
Known Epsilon member retrieves in the silkworm genome by other species, a plurality of Epsilon homologous genes in the silkworm genome, have been obtained, we clone the BmGSTe5 gene, its expression product is 12 μ mol/min/mg protein to the activity of pattern substrate CDNB (1-chloro-2,4-dinitrobenzene).By this gene target spot, be expected to design control lepidoptera pest medicine, and nontoxic to the useful insect of other purposes.Therefore in drug development and pest control, significant values is arranged.
Embodiment:
Embodiment:
Coding region sequence (Coding sequence with the BmGSTe5 gene, CDS), (Expressed sequence tag EST) retrieves in the database in the silkworm expressed sequence tag, for the BmGSTe5 gene is sought the EST evidence, then with CDS and est sequence splicing back design gene-specific primer; The forward primer of BmGSTe5: 5 ' TTCCGACTATGGTGTTCATCCTGTA 3 ', reverse primer: 5 ' TTGTATAATACCGGTTCAGCAGTGG 3 '; Auele Specific Primer with design is that template increases at the middle cDNA in 5 ages 3 of silkworm, obtained the purpose band, the PCR product reclaims the back and is connected with the pMD18-T carrier, transform DH5 α competent cell, obtain to be sent to Shanghai Sangon order-checking behind the positive colony, sequencing result shows that the sequence of amplification is consistent with expected result; To contain purpose segment and pET28a, connect and conversion B121 (DE3) competent cell with the T4 ligase enzyme, screening obtains to contain the clone of pET28a-BmGSTe7, and the mono-clonal that will contain the pET28a-BmGSTe7 recombinant plasmid is 37 ℃ of shaking culture in containing that LB substratum of card, to OD
600About=0.6, it is 1mmol/L that adding IPTG makes its final concentration, continues inducing culture 5 hours, gets 50ml bacterium liquid 10000g and collects bacterial sediment in centrifugal 5 minutes, and with His label purifying target protein matter, purification process carries out according to the pET treasured book; Target protein is 12 μ mol/min/mgprotein to the activity of pattern substrate CDNB (1-chloro-2,4-dinitrobenzene); On the NCBI website, finish sequence homology analysis, show that the BmGSTe5 gene is a new gene with BLAST.
Claims (2)
1. cultivated silkworm glutathione-S-transferase BmGSTe 5 gene is characterized in that its sequence is as follows:
ATGGTGTTCA?TCCTGTACAA?AAAGGACACA?AGTCCTCCTT?GCAGATCAGT?TCAGATGGTA 60
CTCCACGAGC?TCGGAATTTA?TGACGTGGAA?CTCATTGAAG?TGAATCTGCC?AGAAAGGGAC?120
CATCTGAAAG?AAGAATTTCT?GAGGATGAAT?CCGCAACACA?CTGTTCCCAC?GTTAATAGAC?180
GGAGATTTCA?TAATATGGGA?CAGCCACGCC?ATAGTCACAT?ATTTGGTGAA?CAGATATGCT?240
AAAAATGACA?CATTATATCC?TAAAGAGCCT?AAACAAAGAG?CCATTGTTGA?TCAGAGGTTA?300
CATTTTGATA?CCGGAGTGCT?CTTCGCCATA?TTACGAGCCA?CTGCTGAACC?GGTATTATAC?360
AACAATGAAA?AGAGCTTTAA?ACAAGAAAAT?CTAGAGAAAA?TGGAGGCAGC?CTACGAATTC?420
GTTGAAAAAT?TCCTGACTTC?CGATTGGCTA?GCCGGAGACC?AAGTTACATT?AGCCGATATC?480
TGCTGTGTAT?CTACAATCAG?TTCCATGAAC?GTAATTGTCC?CTATTGACAA?AAAGAAGTAC?540
CCAAAAATAA?TTTCATGGCT?ACAGCGCTGT?TCCGAACAAG?AATTTTACAA?GAAAGCCAAC?600
GAACCAGGCT?TGAAGAAATT?CATAGAAATG?TTCAAAAATA?AAATCGGCAA?CTAA 654
2. cultivated silkworm glutathione-S-transferase BmGSTe 5 gene encoded protein matter is characterized in that its sequence is as follows:
1 Met?Val?Phe?Ile?Leu?Tyr?Lys?Lys?Asp?Thr?Ser?Pro?Pro?Cys?Arg 15
16 Ser?Val?Gln?Met?Val?Leu?His?Glu?Leu?Gly?Ile?Tyr?Asp?Val?Glu 30
31 Leu?Ile?Glu?Val?Asn?Leu?Pro?Glu?Arg?Asp?His?Leu?Lys?Glu?Glu 45
46 Phe?Leu?Arg?Met?Asn?Pro?Gln?His?Thr?Val?Pro?Thr?Leu?Ile?Asp 60
61 Gly?Asp?Phe?Ile?Ile?Trp?Asp?Ser?His?Ala?Ile?Val?Thr?Tyr?Leu 75
76 Val?Asn?Arg?Tyr?Ala?Lys?Asn?Asp?Thr?Leu?Tyr?Pro?Lys?Glu?Pro 90
91 Lys?Gln?Arg?Ala?Ile?Val?Asp?Gln?Arg?Leu?His?Phe?Asp?Thr?Gly 105
106 Val?Leu?Phe?Ala?Ile?Leu?Arg?Ala?Thr?Ala?Glu?Pro?Val?Leu?Tyr 120
121 Asn?Asn?Glu?Lys?Ser?Phe?Lys?Gln?Glu?Asn?Leu?Glu?Lys?Met?Glu 135
136 Ala?Ala?Tyr?Glu?Phe?Val?Glu?Lys?Phe?Leu?Thr?Ser?Asp?Trp?Leu 150
151 Ala?Gly?Asp?Gln?Val?Thr?Leu?Ala?Asp?Ile?Cys?Cys?Val?Ser?Thr 165
166 Ile?Ser?Ser?Met?Asn?Val?Ile?Val?Pro?Ile?Asp?Lys?Lys?Lys?Tyr 180
181 Pro?Lys?Ile?Ile?Ser?Trp?Leu?Gln?Arg?Cys?Ser?Glu?Gln?Glu?Phe 195
196 Tyr?Lys?Lys?Ala?Asn?Glu?Pro?Gly?Leu?Lys?Lys?Phe?Ile?Glu?Met 210
211 Phe?Lys?Asn?Lys?Ile?Gly?Asn
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| Application Number | Priority Date | Filing Date | Title |
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| CNB200710092538XA CN100523195C (en) | 2007-08-07 | 2007-08-07 | Cultivated silkworm glutathione-S-transferase BmGSTe5 gene |
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| CNB200710092538XA CN100523195C (en) | 2007-08-07 | 2007-08-07 | Cultivated silkworm glutathione-S-transferase BmGSTe5 gene |
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| CN101139610A true CN101139610A (en) | 2008-03-12 |
| CN100523195C CN100523195C (en) | 2009-08-05 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604906A (en) * | 2012-03-27 | 2012-07-25 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTD4 and genes thereof |
| CN103160524A (en) * | 2013-03-27 | 2013-06-19 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTe4 gene |
| CN103170089A (en) * | 2012-06-13 | 2013-06-26 | 中国矿业大学(北京) | Application of glutathione-S transferase (GST) protein to degradation of multiple pesticides |
-
2007
- 2007-08-07 CN CNB200710092538XA patent/CN100523195C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604906A (en) * | 2012-03-27 | 2012-07-25 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTD4 and genes thereof |
| CN102604906B (en) * | 2012-03-27 | 2013-06-19 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTD4 and genes thereof |
| CN103170089A (en) * | 2012-06-13 | 2013-06-26 | 中国矿业大学(北京) | Application of glutathione-S transferase (GST) protein to degradation of multiple pesticides |
| CN103170089B (en) * | 2012-06-13 | 2015-05-27 | 中国矿业大学(北京) | Application of glutathione-S transferase (GST) protein to degradation of multiple pesticides |
| CN103160524A (en) * | 2013-03-27 | 2013-06-19 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTe4 gene |
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| Publication number | Publication date |
|---|---|
| CN100523195C (en) | 2009-08-05 |
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