CN101210250A - Tea geometrid acetaldehyde dehydrogenase gene adh and application thereof - Google Patents
Tea geometrid acetaldehyde dehydrogenase gene adh and application thereof Download PDFInfo
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- CN101210250A CN101210250A CNA200710164812XA CN200710164812A CN101210250A CN 101210250 A CN101210250 A CN 101210250A CN A200710164812X A CNA200710164812X A CN A200710164812XA CN 200710164812 A CN200710164812 A CN 200710164812A CN 101210250 A CN101210250 A CN 101210250A
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- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
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- 239000003550 marker Substances 0.000 description 1
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- 108010073969 valyllysine Proteins 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an aldehyde dehydrogenase gene (adh) derived from Ectropis oblique, which has the nucleotide sequence shown in SEQ ID NO: 1. The invention also discloses a protein encoded by the gene adh, which has the amino acid sequence shown in SEQ ID NO: 2. The invention further discloses the use of the gene adh in evaluating stress resistance and monitoring feeding environment of insects.
Description
Technical field
The invention belongs to the insect biological technical field.Specifically, the present invention relates to a kind of acetaldehyde dehydrogenase gene clone; Also relate to and utilize this gene thing to carry out evaluation of insect anti-adversity ability and insect growth environmental monitorings such as tea geometrid.
Background technology
In the process of characteristics such as research insect biology, need a large amount of insect materials.Directly catch from nature, waste time and energy, work efficiency is relatively poor.Adopt artificial feeding's method, insect population is enlarged rapidly, but often occur the situations such as death of unknown cause in the insect feeding process, raise to insect and bring loss.
Aldehydes is the important component of organism endoperoxides reaction product, as mda, acetaldehyde etc., organism is had stronger toxic action.(aldehyde dehydrogenase adh) is the important enzyme that is used for degrade acetaldehyde in the organism to acetaldehyde dehydrogenase, and it can be decomposed into acetaldehyde carbonic acid gas and water, thereby eliminates the toxic action of aldehydes to biomass cells.To the clone and the expression study of insect adh gene, help verifying of unusual death reason in the insect feeding process, can utilize the analysis of this expression conditions that feeding environment is assessed simultaneously, thereby make the artificial breeding technical study of insect ripe more.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of tea geometrid acetaldehyde dehydrogenase gene adh and coded protein thereof, and this adh can be used for estimating anti-adversity ability of insect and the feeding environment of monitoring insect.
In order to solve the problems of the technologies described above, the invention provides a kind of tea geometrid acetaldehyde dehydrogenase gene adh, it has the nucleotide sequence shown in the SEQ ID No:1.
The present invention also provides said gene adh encoded protein matter, and it has the aminoacid sequence shown in the SEQ ID NO:2, and this is a kind of protein sequence of tea geometrid adh core area.
The present invention also provides the purposes of said gene adh, can be used for estimating anti-adversity ability of insect and the feeding environment of monitoring insect.
Improvement as the purposes of gene adh of the present invention: insect is a tea geometrid.
The present invention mainly is the unknown cause problem of death that occurs in the insect artificial breeding process, by insect gene expression difference under the different adverse environmental factors of cDNA-AFLP technical study, thereby obtain and raise the relevant important gene of insect unusual death, promptly obtained to react relevant gene with insect adverse circumstance such as tea geometrid; And utilize these expression conditions to carry out the evaluation of insect feeding environment.
Realize that concrete technological step of the present invention is as follows:
One, tea geometrid adh gene isolation and analysis
Ammonia and alcohol can be formed by fermentations such as ight soil and remaining foodstuffs in insect feeding process such as tea geometrid, are that modal feeding environment is coerced the factor, therefore adopt the ammonia and the alcohol blend simulated environment of artificial preparation to coerce.It is some to get the tea geometrid larva, and they are divided into 4 groups, carries out the stifling processing of different ammonia and alcohol blend respectively, cannot not comprise stiflingly that contrast, low dosage are fumigated, middle dosage is stifling and stifling 4 processing of high dosage.Get the stifling some heads of handling after 0.5-12.0 hour of tea geometrid and be used for the cDNA-AFLP analysis.Above-mentioned processing larva places different mortar liquid nitrogen grinding respectively, extracts total RNA with TRIZOL (Invitrogen company) reagent, and obtains 4 kinds of processing larva mRNA with UNIQ-10 pillar mRNA extracting and purifying test kit (worker's biotechnology company limited is given birth in Shanghai).Getting 1-5 μ gmRNA respectively, to carry out cDNA synthetic, and the synthetic TakaRa M-MLV RTase cDNA Synthesis Kit (precious biotechnology (Dalian) company limited) that adopts of double-stranded cDNA carries out.Double-stranded cDNA cuts digestion with TakaRa BamHI/Hha I (precious biotechnology [Dalian] company limited) combination enzyme behind the isopropanol precipitating purifying.2 joint SEQ IDNo:3 of postdigestive fragment and design in advance and SEQ ID No:4 connect with T4 dna ligase (worker's biotechnology company limited is given birth in Shanghai).The cDNA fragment of finishing with connection is a template, carrying out PCR with pre-amplification combination of primers SEQ ID No:5 and SEQID No:6 increases in advance, the PCR program sees Table 1 the 1st row, pre-expansion is increased production thing suitably after the dilution, with selective amplification combination of primers 1. SEQ ID No:7 and SEQ ID No:8, and make up 2. SEQ IDNo:9 and SEQ ID No:10, and carry out the PCR selective amplification respectively, the PCR program sees Table 1 the 2nd row.Amplified production carries out electrophoretic analysis with 6% denaturing polyacrylamide gel, shows band with nucleic acid silver staining test kit (worker's biotechnology company limited is given birth in Shanghai).
Table 1
| PCR increases in advance | The PCR selective amplification | The conventional amplification of PCR |
| 94 ℃ of sex change 30 seconds; Annealed 60 seconds for 56 ℃; 72 ℃ were extended 60 seconds; Totally 30 circulations. | 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 65 ℃; 72 ℃ were extended 60 seconds; 1 circulation.94 ℃ of sex change 30 seconds; Annealed 30 seconds for 65 ℃, and descend 0.7 ℃ with every circulation; 72 ℃ were extended 60 seconds; 12 circulations.94 ℃ of sex change 30 seconds; Annealed 30 seconds for 56 ℃; 72 ℃ were extended 60 seconds; 23 circulations. | 94 ℃ of sex change 4 minutes; A circulation.94 ℃ of sex change 30 seconds; Annealed 30 seconds for 55 ℃; 72 ℃ were extended 60 seconds; 35 circulations.72 ℃ were extended 5 minutes. |
Annotate: PCR or the pcr amplification of not doing special instruction among the present invention all refer to the conventional amplification of PCR
Extract and coerce the difference band that rule matches from gel, after spending the night with the pure water lixiviate, get an amount of vat liquor as template, carry out PCR with the selectivity combination of primers, product carries out electrophoresis.Electrophoresis extracts target stripe after finishing, reclaim test kit (worker's biotechnology company limited is given birth in Shanghai) with UNIQ-10 pillar DNA glue and carry out the fragment recovery, and inserting pUCm-T carrier (worker's biotechnology company limited is given birth in Shanghai), positive colony entrusts Invitrogen company to carry out sequential analysis.Warp and U.S.'s biomolecule information database
Http:// www.ncbi.nlm.nih.govComparison, the significant difference sequence of having determined one of them 524bp is a tea geometrid adh gene.The adh gene order that obtains is seen SEQ ID No:1.Deduce through Editseq software (DNAStar company), obtained 174 amino acid of this gene order coding, see SEQ ID No:2.Biologies such as sequence alignment demonstration tea geometrid adh nucleotide sequence and silkworm, sea anemone have the similarity more than 72%, and aminoacid sequence and silkworm and yellow-fever mosquito etc. have the homology of 79%-92%.
Two, adverse circumstance is to the endogenous adh genetic expression influence of tea geometrid
The tea geometrid larva is divided into two groups, feeds under the normal humidity condition for one group, another group is fed under high humidity.Handle after 6-96 hour, in normal humidity and high humidity are fed group, choose the some heads of larva respectively.Extract total RNA with TRIZOL (Invitrogen company), with the synthetic cDNA of the MMLV first chain cDNA synthetic agent box (worker's biotechnology company limited is given birth in Shanghai).Adopt combination of primers SEQ ID No:11 and SEQ ID No:12 to carry out PCR.The result shows, the tea geometrid larval mortality of high humidity environment growth is higher than normal humidity and handles 30-60%, tea geometrid in the high humidity environment is at the initial stage of coercing simultaneously, and larva adh genetic expression is significantly higher than normal humidity and handles, and handles later stage adh expression decline but coerce.Explanation is coerced the murder by poisoning factor accumulation in vivo that causes coercing the expression that early stage insect is improved some anti contravariance related gene in the body in order to resist extraneous poor environment with minimizing; But when environment-stress continued the long period, insect descended owing to the accumulation murder by poisoning factor too much makes anti-adversity ability, and adversity gene is expressed also and descended.As seen adh genetic expression is a kind of important indicator of weighing feeding environment quality such as tea geometrid, helps to improve experimental insect by the detection to this index and raises surviving rate.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 after the amplified production among the embodiment 1 carries out electrophoretic analysis, shows the figure that is with gained with the nucleic acid silver staining test kit;
Annotate: M is a dna molecular amount mark, and 1 be normal control, and 2 handle for low dosage is stifling, and dosage is stifling in 3 handles, and 4 is that high dosage is fumigated processing.The black arrow indication of entity is a molecular weight marker, and the white arrow indication is for revealing the target stripe of the rule of falling after rising with coercing table of degree.
Fig. 2 is the genetic expression figure among the embodiment 2;
Annotate: M is a dna molecular amount mark, 1 for normal humidity fed 12 hours after the tea geometrid larva, 2 coerce tea geometrid larva after 12 hours for high humidity, 3 is normal humidity 72 hours, 4 coerce processing 72 hours for high humidity.
Embodiment
Embodiment 1
Ammoniacal liquor, industrial spirit and water are blent into three kinds of environment-stress fumigation liquids by different ratios: the low dosage fumigation liquid is the aqueous solution of 0.01-0.5% ammoniacal liquor and 0.1-0.5% alcohol for the inclusion body volume concentrations, the median dose fumigation liquid is the aqueous solution that comprises 0.6-1.5% ammoniacal liquor and 0.5-1% alcohol, and the high dosage fumigation liquid is the aqueous solution that comprises 1.5-6% ammoniacal liquor and 1.1-5% alcohol.Fix a rayon balls with adhesive tape respectively in the culture dish central authorities that 4 diameters are 21cm, the cotton consumption is being as the criterion the complete sticking of fumigation liquid, at the fumigant that drips three kinds of various dose on the cotton balls respectively, every kind of consumption 2-10ml, with the pure water that drips equal volume in contrast.With raise 40 3 age the tea geometrid larva be divided into 4 groups, place 4 culture dish respectively, add the bright leaf foodstuff of an amount of tea after, cover the culture dish lid.Stifling tea geometrid of handling after 6 hours is used for cDNA-AFLP and analyzes.Each processing is got the 2-3 larva and is placed different mortar liquid nitrogen grinding respectively, extract total RNA with TRIZOL (Invitrogen company) reagent, and obtain 4 kinds of different treatment mRNA with UNIQ-10 pillar mRNA extracting and purifying test kit (worker's biotechnology company limited is given birth in Shanghai).Getting 1 μ gmRNA respectively, to carry out cDNA synthetic, and the synthetic TakaRa M-MLV RTase cDNA SynthesisKit (precious biotechnology (Dalian) company limited) that adopts of double-stranded cDNA carries out.Double-stranded cDNA cuts digestion with TakaRa BamHI/HhaI (precious biotechnology [Dalian] company limited) combination enzyme, specifically with reference to BamHI and HhaI restriction endonuclease operation instruction behind the isopropanol precipitating purifying.Enzyme cuts the back enzyme that goes out under 70 ℃ of conditions, and obtains digestion product through isopropanol precipitating.2 the joint sequence SEQ ID No:3 and the SEQ ID No:4 that connect digestion product and design in advance with T4 dna ligase (Shanghai give birth to worker biotechnology company limited).The cDNA fragment of finishing with connection is a template, carrying out PCR with pre-amplification primer SEQ ID No:5 and SEQ ID No:6 increases in advance, after pre-expansion increased production 20 times of thing dilutions, carry out the PCR selective amplification with selective amplification combination of primers SEQ ID No:7 and SEQ ID No:8.Amplified production carries out electrophoretic analysis with 6% denaturing polyacrylamide gel, shows band with nucleic acid silver staining test kit (worker's biotechnology company limited is given birth in Shanghai), sees Fig. 1.
The choice criteria of target stripe is: the band of rule of falling after rising occurring with the coercive intensity increase, is to meet with biological adverse circumstance reacting phase because have the band of above-mentioned rule.After the target stripe that extracts from gel is spent the night with the pure water lixiviate, get 2-4 μ L vat liquor as template, carry out PCR with combination of primers SEQ ID No:7, SEQ ID No:8, product carries out electrophoresis on 1.5% sepharose.Electrophoresis extracts target stripe after finishing, reclaim test kit (worker's biotechnology company limited is given birth in Shanghai) with UNIQ-10 pillar DNA glue and carry out the fragment recovery, and inserting pUCm-T carrier (worker's biotechnology company limited is given birth in Shanghai), positive colony entrusts Invitrogen company to carry out sequential analysis.Warp and U.S.'s biomolecule information database
Http:// www.ncbi.nlm.nih.govComparison, the adh gene of the biologies such as the product of a 524bp of acquisition and silkworm, sea anemone that increased by selective amplification primer SEQ ID No:7 and SEQ IDNo:8 has the homology more than 72%.Deduce through Editseq software (DNAStar company), obtained 174 amino acid of this gene order coding, sequence alignment shows that deducing the aminoacid sequence of the present invention and silkworm and the yellow-fever mosquito etc. that obtain has the homology of 79%-92%.According to the nucleotide sequence that obtains gene with deduce the aminoacid sequence that obtains, the gene fragment relevant with environment-stress that the present invention will obtain named the gene into tea geometrid adh, its concrete nucleotide sequence is seen SEQ ID No:1, and aminoacid sequence is seen SEQ ID No:2.
Embodiment 2
Fix a rayon balls with adhesive tape respectively in the culture dish central authorities that 4 diameters are 21cm, coerce in the water simulation humidity of dripping 2ml or 20ml on the cotton balls respectively, wherein 2ml is treated to the normal humidity group, and 20ml is that high humidity is coerced group.With 200 2 age the tea geometrid larva be divided into two groups, under the normal humidity condition, feed for one group, another group is fed under high humidity.Every group of each 2 ware, a ware are used for mortality ratio to be observed, and another ware is used for gene expression research.Cultivate after 12 and 72 hours, in normal humidity and high humidity are fed group, choose the larva 2-4 head of survival respectively.Extract total RNA with TRIZOL (Invitrogen company) method, with the synthetic cDNA of the MMLV first chain cDNA synthetic agent box (worker's biotechnology company limited is given birth in Shanghai).Adopt combination of primers SEQ ID No:11 and SEQ ID No:12 to carry out PCR and electrophoresis.The result shows, the tea geometrid larval mortality of high humidity environment growth is handled high by 60% than normal humidity, simultaneously the tea geometrid larva is coerced initial stage adh genetic expression at high humidity and handles high more than 2 times than normal humidity, but coerce time lengthening to 72 hour, it only is the 30-40% of normal humidity that high humidity is coerced group adh genetic expression; And the normal humidity group is in medium level all the time in treating processes, sees Fig. 2.Illustrate that adh genetic expression is a kind of important indicator of weighing insect feeding environment quality such as tea geometrid, help to improve experimental insect by detection and raise surviving rate this index.
Therefore, we can draw as drawing a conclusion: at the environment-stress initial stage, the adh genetic expression of tea geometrid significantly strengthens, in order to the formation corresponding proteins, and the unfavorable factor of elimination environment, but when the lasting overtime of environment-stress, tea geometrid adh expression amount descends, the ability drop of synthetic corresponding protein and elimination environment unfavorable factor, and finally cause corresponding resisting to coerce Disability, cause individual death.As seen, adverse environment causes the adh fluctuation, and home adh expresses steadily.Can infer whether the insect culture environment is suitable from the fluctuation situation that adh expresses; Simultaneously can express the resistance of inferring insect by coercing later stage adh, promptly still can keep the adh high expression level coercing the later stage, then be degeneration-resistant strong individuality, vice versa.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ ID No：1
ggatccacag aggtgggtcg catcatcatg agtgctgcct ctgccgtgaa cttgaagaga 60
gttactcttg agctcggtgg caagagccct cttgtcatct tcaacgacgc cgatgtcgaa 120
aaagctgctc tcatcgctca tagagctgct ttcgctaacg ccggccaatg ctgtgtggct 180
ggtaccagga ccttcgtgca atctggcatt tacgacaaat tcgtagcaaa atctgccgaa 240
atcgcccaaa agagatccgt aggcaaccct tacgaagatg ttgaacaagg acctcagatc 300
gacaaagaga tgtttgacaa agtcatggga ttcatcgatg ctggaaaaac gcaaggagct 360
aggtgcattg ctggcggagg tcgtcaaggc aatgttggat ttttcgtcca gcccaccgta 420
ttcgcagatg tcaaggatga catgaaaata gccagagaag agatcttcgg gccagttcag 480
agtattctaa agttcgagac cttcgaagag gtggtagacc gcgc 524
SEQ ID No：2
Gly Ser Thr Glu Val Gly Arg Ile Ile Met Ser Ala Ala Ser Ala
1 5 10 15
Val Asn Leu Lys Arg Val Thr Leu Glu Leu Gly Gly Lys Ser Pro
20 25 30
Leu Val Ile Phe Asn Asp Ala Asp Val Glu Lys Ala Ala Leu Ile
35 40 45
Ala His Arg Ala Ala Phe Ala Asn Ala Gly Gln Cys Cys Val Ala
50 55 60
Gly Thr Arg Thr Phe Val Gln Ser Gly Ile Tyr Asp Lys Phe Val
65 70 75
Ala Lys Ser Ala Glu Ile Ala Gln Lys Arg Ser Val Gly Asn Pro
80 85 90
Tyr Glu Asp Val Glu Gln Gly Pro Gln Ile Asp Lys Glu Met Phe
95 100 105
Asp Lys Val Met Gly Phe Ile Asp Ala Gly Lys Thr Gln Gly Ala
110 115 120
Arg Cys Ile Ala Gly Gly Gly Arg Gln Gly Asn Val Gly Phe Phe
125 130 135
Val Gln Pro Thr Val Phe Ala Asp Val Lys Asp Asp Met Lys Ile
140 145 150
Ala Arg Glu Glu Ile Phe Gly Pro Val Gln Ser Ile Leu Lys Phe
155 160 165
Glu Thr Phe Glu Glu Val Val Asp Arg
170
5’-CTCGTAGACTGCGTACC -3’
SEQ ID No:3 (BamHI joint): 3 '-CATCTGACGCATGGCATG-5 '
SEQ ID No:4 (Hha I joint):
SEQ ID No:5 (upstream primer in advance increases): 5 '-GTAGACTGCGTACCGATCC-3 '
SEQ ID No:6 (downstream primer in advance increases): 5 '-GATGAGTCCTGAGCGC-3 '
SEQ ID No:7 (selective amplification upstream primer): 5 '-GACTGCGTACCGATCCAC-3 '
SEQ ID No:8 (selective amplification downstream primer): 5 '-GATGAGTCCTGACGCGCGG-3 '
SEQ ID No:9 (selective amplification upstream primer): 5 '-GACTGCGTACCGATCCAT-3 '
SEQ ID No:10 (selective amplification downstream primer): 5 '-GATGAGTCCTGACGCGCGT-3 '
SEQ ID No:11 (gene specific upstream primer): 5 '-GGATCCACAGAGGTGGGTCG-3 '
SEQ ID No:12 (gene specific downstream primer): 5 '-GCGCGGTCTACCACCTCTTCG-3 '
Claims (4)
1. a tea geometrid acetaldehyde dehydrogenase gene adh is characterized in that: have the nucleotide sequence shown in the SEQ ID No:1.
2. gene adh encoded protein matter as claimed in claim 1 is characterized in that: have the aminoacid sequence shown in the SEQ ID NO:2.
3. the purposes of gene adh as claimed in claim 1 is characterized in that: be used to estimate anti-adversity ability of insect and the feeding environment of monitoring insect.
4. the purposes of gene adh as claimed in claim 2, it is characterized in that: described insect is a tea geometrid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816297A (en) * | 2010-04-30 | 2010-09-01 | 中国计量学院 | Method for trapping, preventing and controlling adults of ectropis obliqua |
| CN117264976A (en) * | 2023-09-19 | 2023-12-22 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Cotton cell wall intensity regulation gene and application thereof |
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| CN100415870C (en) * | 2005-11-11 | 2008-09-03 | 武汉化工学院 | Nie's brevibacterium new strain and process for preparing enzyme by it |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816297A (en) * | 2010-04-30 | 2010-09-01 | 中国计量学院 | Method for trapping, preventing and controlling adults of ectropis obliqua |
| CN117264976A (en) * | 2023-09-19 | 2023-12-22 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Cotton cell wall intensity regulation gene and application thereof |
| CN117264976B (en) * | 2023-09-19 | 2024-03-19 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Cotton cell wall intensity regulation gene and application thereof |
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