CN106591336A - ATP synthetase gene for prevention and control of aphelenchoides besseyi christie, and primers and application thereof - Google Patents
ATP synthetase gene for prevention and control of aphelenchoides besseyi christie, and primers and application thereof Download PDFInfo
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- CN106591336A CN106591336A CN201611190938.XA CN201611190938A CN106591336A CN 106591336 A CN106591336 A CN 106591336A CN 201611190938 A CN201611190938 A CN 201611190938A CN 106591336 A CN106591336 A CN 106591336A
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Abstract
The invention relates to an ATP synthetase gene for prevention and control of aphelenchoides besseyi christie, and primers and an application thereof. The gene has the sequence shown in a sequence table SEQ No.1. A primer group for construction of the aphelenchoides besseyi christie ATP synthase gene comprises an upstream primer Ab-ATPs-F shown in the sequence table SEQ No.2 and a downstream primer Ab-ATPs-R shown in the sequence table SEQ No.3. The application of the gene in prevention and control of the aphelenchoides besseyi christie is provided. The ATP synthetase gene has the expression up-regulated after the aphelenchoides besseyi christie is subjected to hypoxia stress, so the gene is indicated to play a role in the hypoxia stress resisting process of the aphelenchoides besseyi christie. Through the study of the gene, the gene has important biological significance and potential application value in the prevention and control of the aphelenchoides besseyi christie.
Description
Technical field
The present invention relates to a kind of ATP synthase genes and primer and application for preventing and treating aphelenchoides besseyi.
Background technology
Paddy rice (Oryza sativa L.) is China or even one of most important cereal crops in the world.Paddy rice receives paddy rice
Nematode aphelenchoide is caused harm (Aphelenchoides besseyi Christie), causes huge economic loss, every in world wide
The year underproduction 10～20%, the underproduction is more than 30% under serious conditions.At present, to the prophylactico-therapeutic measures of aphelenchoides besseyi, predominantly add
Seed soaking before strong quarantine and sowing, but prevention effect of soaking seed is limited, and affect paddy rice germinating energy.Aphelenchoides besseyi is with adult and children
Worm hides and is survived the winter between the clever shell and the grain of rice of grain, and when presoaking and germinating, nematode comes into play in seed.Seeding belt disease
Afterwards, it is free on nematode in water and soil, most of gradually dead, small part runs into young shoot, seedling, from bud scale, leaf sheath gap more
Place invades.Due to lacking respiratory circulatory system, the oxygen content in nematode body depends primarily on external environment and its body size.Research
It was found that, during Rice Cropping, the oxygen content in soil is affected by many factors.Under anoxic conditions, most of dynamic plant
Thing cannot survive.But aphelenchoides besseyi can be by into anoxic cryptobiosis, surviving a couple of days under anoxic conditions, when environment is fitted
When preferably, eubolism is recovered again, be that preventing and treating aphelenchoides besseyi increased difficulty.
ATP (atriphos) is the direct energy source material of intracellular most vital movements.In biologic artifact,
Organism needs to be continuously available energy and carrys out maintenance metabolism activity.ATP synzyme is the core enzyme in the mitochondria conservation of energy, extensively
It is distributed on the plasma membrane of mitochondrial inner membrane, chloroplast thylakoids, heterotroph and photosynthetic bacteria, it utilizes the matter that electron stream cross-film is produced
Sub- motive power synthesizes ATP by ADP (adenosine diphosphate (ADP)) and inorganic phosphate, and for organism energy is provided, while it is true to exercise hydrolysis
The dual-use function of ATP in nucleus and bacterium.Numerous studies show, biological close with energy metabolism to the stress response of adverse circumstance
Cut is closed, and stress from outside can cause consuming excessively for ATP in organism, produces substantial amounts of active oxygen (ROS, reactive
Oxygen species), so as to damage to biology.ATP synzyme as a kind of important coenzyme and response enzyme,
Play an important role in biological degeneration-resistant reaction.At present, the research both at home and abroad to plant ATP synzyme is relatively more, and ATP is synthesized
Enzyme gene regulation and control nematode anti anoxia stress functional study is less.Based on the importance of ATP synzyme, by gene silent technology,
After research ATP synthase gene silences, aphelenchoides besseyi survival condition under anoxic conditions probes into ATP synthase genes
Potentiality in biological control, are to prevent and treat the nematode to open up new road.
The content of the invention
Based on above weak point, the present invention provide a kind of ATP synthase genes for preventing and treating aphelenchoides besseyi and
Primer and application.
The technology used in the present invention is as follows：A kind of ATP synthase genes for preventing and treating aphelenchoides besseyi, its sequence
As shown in sequence table SEQ No.1.
The present invention also has following technical characteristic：
1st, a kind of primer sets for building the ATP synthase genes of aphelenchoides besseyi, as follows：
Upstream primer Ab-ATPs-F：As shown in sequence table SEQ No.2,
Downstream primer Ab-ATPs-R：As shown in sequence table SEQ No.3.
2nd, a kind of primer sets for building the ATP synthase gene dsRNA of aphelenchoides besseyi, as follows：
Synthesis positive-sense strand RNA upstream primer Ab-ATPs-TTF：As shown in sequence table SEQ No.4,
Synthesis positive-sense strand RNA downstream primer Ab-ATPs-iR：As shown in sequence table SEQ No.5,
Synthesis antisense strand RNA upstream primer Ab-ATPs-iF：As shown in sequence table SEQ No.6,
Synthesis antisense strand RNA downstream primer Ab-ATPs-T7R：As shown in sequence table SEQ No.7.
3rd, the primer sets that a kind of ATP synthase genes Q-PCR for aphelenchoides besseyi is detected, as follows：
Upstream primer Ab-ATPs-qF：As shown in sequence table SEQ No.8,
Downstream primer Ab-ATPs-qR：As shown in sequence table SEQ No.9.
4th, the ATP synthase genes of a kind of aphelenchoides besseyi as above answering in preventing and treating aphelenchoides besseyi
With.
The ATP synthase genes of present invention up-regulated after aphelenchoides besseyi is coerced by anoxic, shows that the gene exists
Work in aphelenchoides besseyi anti anoxia stress procedure.By the research to the gene, ATP synthase genes are probed in biology
Potentiality in preventing and treating, are that the nematode sets up basis using anoxic stress preventing and treating, there is provided prevent and treat new approaches, are preventing and treating LIPIDS OF DRY RICE EMBRYO point line
There is important biological significance and potential using value in worm.
Description of the drawings
Fig. 1 is Q-PCR technical checking Ab-ATPs Gene silencing efficacy figures in embodiment 1；
Fig. 2 is in embodiment 1 aphelenchoides besseyi to be carried out respectively after the process of Ab-ATPs gene silencings and control treatment,
Carry out aphelenchoides besseyi survival number comparison diagram when anoxic is coerced.
Specific embodiment
ATP synthase genes provided by the present invention for preventing and treating aphelenchoides besseyi derive from aphelenchoides besseyi, should
Unnamed gene is Ab-ATPs, its length be Isosorbide-5-Nitrae 45bp, its gene order as shown in SEQ No.1, with typical ATPs structures
Domain.
Embodiment 1：
The acquisition and its functional verification of the ATP synthase genes of preventing and treating aphelenchoides besseyi.
(1) RNA extractings and cDNA synthesis：
DEPC processes water cleaning aphelenchoides besseyi (female adult：Male worm：Larva=4:2:1), centrifugation is gone after water, is ground under liquid nitrogen
Mill.Abrasive flour is taken, using TRIzol methods (Invitrogen, cat.No.15596-026) total serum IgE is extracted.DEPC process is water-soluble
After solution RNA, using AMV reverse transcription systems (Promega, cat.No.A3500), with Oligo (dT)18For primer, synthesize the first chain
cDNA.According to test handbook, using random priming the second chain cDNA is synthesized.
(2) aphelenchoides besseyi ATP synthase genes entire reading frame clone
Aphelenchoides besseyi ATP synthase gene primer sets are synthesized according to transcript profile sequencing result：
Ab-ATPs-F:5`-TGT GAT GAT GCC GTC GAT TC-3`,
Ab-ATPs-R:5`-CCT TAG CGT TCT TGG CTG TT-3`。
PCR amplification (the μ L reaction systems of TaKaRar-Taq enzymes 50 of entire reading frame sequence are carried out as template with cDNA：94
DEG C 30s, 58 DEG C of 1min, 72 DEG C of 1min, carry out 35 circulations), amplification products therefrom send biotech firm to be sequenced, and is named as after checking
Ab-ATPs。
(3) Ab-ATPs gene silencings
With aphelenchoides besseyi cDNA as template, using primer sets：
Ab-ATPs-T7F：5`-TAA TAC GAC TCA CTA TAG GGA GAA GGC AAA TCG GTT GGT G-
3`,
Ab-ATPs-iR：5`-ATT TCA GCG ACT CCT TCC TTC-3`
Enter performing PCR amplification production positive-sense strand template.
Using primer sets：
Ab-ATPs-iF：5`-AGA AGG CAA ATC GGT TGG TG-3`,
Ab-ATPs-T7R：5`-TAA TAC GAC TCA CTA TAG GGC ATT TCA GCG ACT CCT TCC
TTC-3`
Enter performing PCR amplification production antisense strand template.UsingRNAi kits synthesize positive-sense strand and antisense
Chain RNA, annealed production dsRNA.Distilled water diluting dsRNA concentration processes nematode 12h to 3mg/mL using dsRNA feeding methods,
With distilled water as control.
Processing rear portion nematode is used to extract RNA, and Stratagene Mx3000P qPCR are applied after reverse transcription
System (Agilent, USA) and GoTaq 2-Step RT-qPCR System kits carry out Q-PCR amplifications.
Using primer sets：
Ab-ATPs-qF：5`-CAG TTG TTG GCA AAG CTG AG-3`,
Ab-ATPs-qR：5`-GGA GTC GGC CAACTT TCT C-3`
Q-PCR amplifications are carried out, using 2 footworks PCR, the first step：95 DEG C of 3min of denaturation.Second step：95 DEG C of 30s, 58 DEG C
1min, 72 DEG C of 30s totally 40 circulations.Melt curve analysis are determined from 55 DEG C to 95 DEG C.
Using aphelenchoides besseyi 28s rRNA gene primer groups：
Ab-28s-qF：5`-TAC GAT CGG TGT TCG TTG C-3`,
Ab-28s-qR：5`-CTC ACA TCG TCG ACATCC AA-3`
Q-PCR amplifications are carried out as control, RNAi effects are verified.Time weight is calculated using relative quantification method to Q-PCR amplifications
Original template amount ratio, two paired-sample t test p are tested in retrial<0.01, significant difference.As a result as shown in figure 1, Ab- after RNAi
ATPs expressions are remarkably decreased.
Another part nematode is used for anaerobic water immersion carries out anoxic treatment 1～6 day, and each process randomly selects 100 lines
Worm determines survival number, in triplicate.As a result as shown in Fig. 2 aphelenchoides besseyi survival number is remarkably decreased.Illustrate LIPIDS OF DRY RICE EMBRYO point line
Worm ATP synthase gene silences, play on the nematode anti-anoxia ability and significantly affect, and can be used for the preventing and treating of the nematodiasis.
<110>Northeast Forestry University
<120>For preventing and treating ATP synthase genes and primer and the application of aphelenchoides besseyi
<160> 9
<210> 1
<211> 1445
<212> DNA
<213> Ab-ATPs
<400> 1
gaaggggttt attaacccaa gtttgagaaa ttccaaacat gaagttttct gcaatctttt 60
tcttaacgat catcgcattc acgggattgt tgtacgttca ctgtgatgat gccgtcgatt 120
cggcagctga caaagtgaag gaaggagccg attatactcg ggacaaagtt catgagggtg 180
cgagcaaagt gaatgaaaag gccgaagacg tgaaggatgg agccagtgcg tatctgcaca 240
aaacgttcga caaggacaaa tgcactggtg aagacggttt cttacccgaa tcgtgtcaac 300
aagtggagga aggaattgag aagattcact cgaagatcga cgattcaatt aacggtaatt 360
gcgtgcactt ttgcttatca cacgtctaac tccgctgggt ttcaagatga tggtaccgta 420
aaccattggg ttcaatcggc caaggaaaaa attcgtgccg ccgccaactt tgtgatgggt 480
catgccgaag aggcgaccga acatttgccg aattcggttg atgaagcgaa acagaagtcc 540
aaggatttgg ctgaagatat tagcgaacat gctcgtgagg gatataacaa agcggaagaa 600
cgagcgaagg accgtgccga acaactccgc gaagaaggca aatcggttgg tgaaactatt 660
gccgacaaag tttctggagc tgctgaatcc gtaaaaaatg ttgctgtcgg agccaaagac 720
aaagttgtcg atgctgctgt tggagccaaa gaagcagttg ttggcaaagc tgaggaagtt 780
caagaggccg ccgaagagaa gaaggatcgt ttgaaacgtg attccgagac tgtcaaagac 840
aaattgggac aagctgctga tgatgtttcg caaaaggctg gtgaggcaaa ggacgcagtc 900
aaagagaaag ttggccgact ccgtcgtgat tccgagactg tcaaagacaa attgggacaa 960
gctgctgatg atgtttcgca aaaggctggt gaggcaaagg acgcagtcaa agagaaagtt 1020
ggcaaagtta aacgagatat tggatcggga gccgaaaaag tgaaggaagg agtcgctgaa 1080
attggctcgg gagttgccga caaagcttcg gatgctgcgg ctggtgcacg cgacgccgcc 1140
gtgaacgccg ccgaaacggc caaagagagt gcagcggccg caggtcaagc cgcagctgaa 1200
acagccaaga acgctaagga tggagtacaa aacgctgccg ccaacgtccg cgatgccact 1260
ggagaaaaga tcgaagatgt tggaaaatcg atccaaggaa aagactccta aacatctgtc 1320
aatgttgcca taaacaagct ttattgttct acaattgttt ctctctttaa cttcattgtg 1380
tatgtaattt gtgttttgtc catatgattt tctgtttata aacaatgtac gagattaaaa 1440
aaaaa 1445
<210> 2
<211> 20
<212> DNA
<213> Ab-ATPs-F
<400> 2
tgtgatgatg ccgtcgattc 20
<210> 3
<211> 20
<212> DNA
<213> Ab-ATPs-R
<400> 3
ccttagcgtt cttggctgtt 20
<210> 4
<211> 40
<212> DNA
<213> Ab-ATPs-TTF
<400> 4
taatacgact cactataggg agaaggcaaa tcggttggtg 40
<210> 5
<211> 21
<212> DNA
<213> Ab-ATPs-iR
<400> 5
atttcagcga ctccttcctt c 21
<210> 6
<211> 20
<212> DNA
<213> Ab-ATPs-iF
<400> 6
agaaggcaaa tcggttggtg 20
<210> 7
<211> 42
<212> DNA
<213> Ab-ATPs-T7R
<400> 7
taatacgact cactataggg catttcagcg actccttcct tc 42
<210> 8
<211> 20
<212> DNA
<213> Ab-ATPs-qF
<400> 8
cagttgttgg caaagctgag 20
<210> 9
<211> 19
<212> DNA
<213> Ab-ATPs-qR
<400> 9
ggagtcggcc aactttctc 19
Claims (5)
1. a kind of ATP synthase genes for preventing and treating aphelenchoides besseyi, it is characterised in that its sequence such as sequence table SEQ
Shown in No.1.
2. a kind of primer sets for building the ATP synthase genes of aphelenchoides besseyi, it is characterised in that as follows：
Upstream primer Ab-ATPs-F：As shown in sequence table SEQ No.2,
Downstream primer Ab-ATPs-R：As shown in sequence table SEQ No.3.
3. a kind of primer sets for building the ATP synthase gene dsRNA of aphelenchoides besseyi, it is characterised in that following institute
Show：
Synthesis positive-sense strand RNA upstream primer Ab-ATPs-TTF：As shown in sequence table SEQ No.4,
Synthesis positive-sense strand RNA downstream primer Ab-ATPs-iR：As shown in sequence table SEQ No.5,
Synthesis antisense strand RNA upstream primer Ab-ATPs-iF：As shown in sequence table SEQ No.6,
Synthesis antisense strand RNA downstream primer Ab-ATPs-T7R：As shown in sequence table SEQ No.7.
4. the primer sets that a kind of ATP synthase genes Q-PCR for aphelenchoides besseyi paddy is detected, it is characterised in that as follows
It is shown：
Upstream primer Ab-ATPs-qF：As shown in sequence table SEQ No.8,
Downstream primer Ab-ATPs-qR：As shown in sequence table SEQ No.9.
5. a kind of application for preventing and treating the ATP synthase genes of aphelenchoides besseyi as claimed in claim 1, its feature exists
In application of the gene in preventing and treating aphelenchoides besseyi.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899291A (en) * | 2021-01-20 | 2021-06-04 | 华南农业大学 | ATP synthetase of rice tip nematode and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098282A2 (en) * | 2003-04-15 | 2004-11-18 | Divergence, Inc. | Nematode atp synthase subunit e-like sequences |
| CN102732554A (en) * | 2011-03-31 | 2012-10-17 | 中国科学院上海生命科学研究院 | Method for raising insect resistance of plants |
| CN103687952A (en) * | 2011-07-18 | 2014-03-26 | 德福根有限公司 | Down regulating gene expression in insect pests |
| CN104404042A (en) * | 2014-11-14 | 2015-03-11 | 重庆大学 | Application of Locusta migratoria ATP synthase beta subunit gene and its dsRNA in pest control |
-
2016
- 2016-12-21 CN CN201611190938.XA patent/CN106591336B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098282A2 (en) * | 2003-04-15 | 2004-11-18 | Divergence, Inc. | Nematode atp synthase subunit e-like sequences |
| CN102732554A (en) * | 2011-03-31 | 2012-10-17 | 中国科学院上海生命科学研究院 | Method for raising insect resistance of plants |
| CN103687952A (en) * | 2011-07-18 | 2014-03-26 | 德福根有限公司 | Down regulating gene expression in insect pests |
| CN104404042A (en) * | 2014-11-14 | 2015-03-11 | 重庆大学 | Application of Locusta migratoria ATP synthase beta subunit gene and its dsRNA in pest control |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899291A (en) * | 2021-01-20 | 2021-06-04 | 华南农业大学 | ATP synthetase of rice tip nematode and application thereof |
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