CN106632635A - Thioredoxin-like gene for preventing and treating Aphelenchoides besseyi, primers for thioredoxin-like gene and application of thioredoxin-like gene - Google Patents

Thioredoxin-like gene for preventing and treating Aphelenchoides besseyi, primers for thioredoxin-like gene and application of thioredoxin-like gene Download PDF

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CN106632635A
CN106632635A CN201611190937.5A CN201611190937A CN106632635A CN 106632635 A CN106632635 A CN 106632635A CN 201611190937 A CN201611190937 A CN 201611190937A CN 106632635 A CN106632635 A CN 106632635A
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thioredoxin
trxl
aphelenchoides besseyi
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CN106632635B (en
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王峰
陈俏丽
李丹蕾
零雅茗
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Northeast Forestry University
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Abstract

Thioredoxin-like gene for preventing and treating Aphelenchoides besseyi has a sequence shown as in sequence table SEQ No. 1. The sequence of the thioredoxin-like gene is shown as in the sequence table SEQ No. 1. A primer for constructing the thioredoxin-like gene for Aphelenchoides besseyi is provided, upstream primer Ab-Trxl-F is shown as in sequence table SEQ No. 2, and downstream primer Ab-Trxl-R is shown as in sequence table SEQ No. 3. The thioredoxin-like gene is applied to prevent and treat Aphelenchoides besseyi. The thioredoxin-like gene experiences up-regulated expression after Aphelenchoides besseyi is stressed by high osmotic pressure, it is indicated that the thioredoxin-like gene plays a role in the process of Aphelenchoides besseyi resisting the stress of high osmotic pressure. Studies on the thioredoxin-like gene show that the thioredoxin-like gene has important biological significance and potential applicable value in preventing and treating Aphelenchoides besseyi.

Description

For preventing and treating similar thioredoxin gene and primer and the application of aphelenchoides besseyi
Technical field
The present invention relates to a kind of similar thioredoxin gene and primer and application for preventing and treating aphelenchoides besseyi.
Background technology
Paddy rice (Oryza sativa L.) is one of world's staple food crop, is the master of 1/3rd mankind in the world Food.The aphelenchoides besseyi disease that aphelenchoides besseyi (Aphelenchoides besseyi Christie) causes, makes paddy rice exist The annual underproduction 10%～70%, causes huge economic loss in world wide.At present, the preventing and treating to aphelenchoides besseyi is arranged Apply, mainly strengthen quarantine, and sow front lukewarm water or Medicament soaked seed, but paddy rice germinating energy often has reduction trend after soaking seed. Research finds, during rice cropping, the work such as fertilising dispenser causes material impact to soil environment osmotic pressure.Major part is raw Thing cannot survive under high osmotic pressure, and aphelenchoides besseyi can be by oozing cryptobiosis, under hyperosmosis environment into change Survival, when environment is suitable, recovers eubolism again, seed soaking preventing and treating is reduced with pesticide control effect, and increase prevents and treats LIPIDS OF DRY RICE EMBRYO The difficulty of sharp nematode.Aphelenchoides besseyi should be studied physiological mechanism during osmotic pressure stress, can be preventing and treating paddy rice Nematode aphelenchoide provides new approaches, it is to avoid because being excessively used for chemical agent causes environmental pollution.
Thioredoxin (thioredoxin, Trx) has the ability such as anti-oxidant, drought resisting, heat-resisting, rises in biology is degeneration-resistant Important effect.Trx is widely present in various biologies in vivo, there is the function of making sulfydryl and disulphide mutually change, and is one Serial physiological reaction provides redox couple, maintains the reducing condition of intracellular protein so as to work orderly, to keeping Biological redox state stable in vivo has important effect.Osmotic pressure stress process is resisted in aphelenchoides besseyi In, Trx plays important booster action.At present, the research both at home and abroad to plant nematode Trx is less.In view of Trx is to biological opposing The important function of abiotic stress, intends the similar thioredoxin gene (thioredoxin of cloning rice nematode aphelenchoide Like protein, Trxl), by gene silent technology, aphelenchoides besseyi is probed into after Trxl gene silencings in hyperosmosis Under the conditions of survival condition, for prevent and treat aphelenchoides besseyi new approaches are provided.
The content of the invention
Based on above weak point, the present invention provides a kind of similar thioredoxin gene for preventing and treating aphelenchoides besseyi And primer and application.
The technology used in the present invention is as follows：A kind of similar thioredoxin gene for preventing and treating aphelenchoides besseyi, its Sequence is 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 similar thioredoxin gene of aphelenchoides besseyi, as follows：
Upstream primer Ab-Trxl-F：As shown in sequence table SEQ No.2,
Downstream primer Ab-Trxl-R：As shown in sequence table SEQ No.3.
2nd, a kind of primer sets for building the similar thioredoxin gene dsRNA of aphelenchoides besseyi, as follows：
Synthesis positive-sense strand RNA upstream primer Ab-Trxl-TTF：As shown in sequence table SEQ No.4,
Synthesis positive-sense strand RNA downstream primer Ab-Trxl-iR：As shown in sequence table SEQ No.5,
Synthesis antisense strand RNA upstream primer Ab-Trxl-iF：As shown in sequence table SEQ No.6,
Synthesis antisense strand RNA downstream primer Ab-Trxl-T7R：As shown in sequence table SEQ No.7.
3rd, the primer sets that a kind of similar thioredoxin enzyme gene Q-PCR for aphelenchoides besseyi is detected, as follows：
Upstream primer Ab-Trxl-qF：As shown in sequence table SEQ No.8,
Downstream primer Ab-Trxl-qR：As shown in sequence table SEQ No.9.
4th, a kind of similar thioredoxin gene of aphelenchoides besseyi as above is in preventing and treating aphelenchoides besseyi Using.
The similar thioredoxin gene of present invention up-regulated after aphelenchoides besseyi receives osmotic pressure stress, shows this Gene works during the anti-osmotic pressure stress of aphelenchoides besseyi.It is that Study On Rice is done by the research to the gene The physiological mechanism of sharp nematode reply osmotic pressure stress sets up basis, can provide new approaches to prevent and treat aphelenchoides besseyi, it is to avoid Because being excessively used for chemical agent causes environmental pollution.There is important biological significance in preventing and treating aphelenchoides besseyi and dive Using value.
Description of the drawings
Fig. 1 is Ab-Trxl relative expression quantity variation diagrams after Q-PCR technical checking osmotic pressure stress in embodiment 1；
Fig. 2 is Q-PCR technical checking Ab-Trxl Gene silencing efficacy figures in embodiment 1；
Fig. 3 is in embodiment 1 aphelenchoides besseyi to be carried out respectively after the process of Ab-Trxl gene silencings and control treatment, Carry out aphelenchoides besseyi survival number comparison diagram during osmotic pressure stress.
Specific embodiment
Provided by the present invention for preventing and treating the similar thioredoxin gene source of aphelenchoides besseyi in aphelenchoides besseyi, Such thioredoxin gene is named as Ab-Trxl, and length is 1,148bp, its gene order as shown in SEQ No.1, with allusion quotation The Trxl domains of type.
Embodiment 1：
The acquisition and its functional verification of the similar thioredoxin gene of preventing and treating aphelenchoides besseyi.
(1) RNA extractings and cDNA synthesis：
DEPC processes water cleaning nematode (female adult：Male worm：Larva=4:2:1), centrifugation is gone after supernatant, is ground under liquid nitrogen process Mill.Abrasive flour is taken, using TRIzol methods (Invitrogen, cat.No.15596-026) aphelenchoides besseyi total serum IgE is extracted, DEPC processes water dissolves.Using AMV reverse transcription systems (Promega, cat.No.A3500), with Oligo (dT)18Carry out for primer First chain cDNA reverse transcriptions, random priming synthesizes the second chain cDNA.Course of reaction is carried out according to test handbook.
(2) aphelenchoides besseyi similar thioredoxin gene entire reading frame clone
Aphelenchoides besseyi similar thioredoxin gene primer group is synthesized according to transcript profile sequencing result,
Ab-Trxl-F:5`-TTC GAC AAA CTC TAT CCA GC-3`,
Ab-Trxl-R：5`-TGA AAT TCC ATT TGA TGG CG-3`.
PCR amplification (the TaKaRa r- of entire reading frame sequence are carried out as template with aphelenchoides besseyi the second chain cDNA The μ L reaction systems of Taq enzyme 25：94 DEG C of 30s, 58 DEG C of 1min, 72 DEG C of 1min, carry out 35 circulations), amplified production send biotech firm Sequence verification, gained sequence designations are Ab-Trxl.
(3) fluorescent quantitation Q-PCR checkings
Choose hyperosmosis (saturation MgSO4·7H2O solution) process LIPIDS OF DRY RICE EMBRYO point line after 4h, 12h, 24h, 48h and 96h Worm, pulverized under liquid nitrogen simultaneously extracts total serum IgE using TRIzol methods.Using GoTaq 2-Step RT-qPCR System (Promega A6010) kit carries out the first chain cDNA reverse transcriptions.
Aphelenchoides besseyi similar thioredoxin gene Q-PCR checking primer sets are synthesized according to sequencing result,
Ab-Trxl-qF：5`-TGA CCG ATG AAG CAA ATA CCA-3`,
Ab-Trxl-qR：5`-TCG ACG AGC TCA ACA CTT AC-3`.
Aphelenchoides besseyi 28s RNA as reference gene,
Ab-28s-qF：5`-TAC GAT CGG TGT TCG TTG C-3`,
Ab-28s-qR：5`-CTC ACA TCG TCG ACA TCC AA-3`.
Using Stratagene Mx3000P qPCR system (Agilent, USA) and GoTaq 2-Step RT-qPCR System kits carry out Q-PCR amplifications.Using 2 footworks PCR, the first step：95 DEG C of 3min of denaturation.Second step：95 DEG C of 30s, 58 DEG C of 1min, 72 DEG C of 30s totally 40 circulations.Melt curve analysis are determined from 55 DEG C to 95 DEG C.Relative quantification method is adopted to Q-PCR amplifications Calculate time repetition test original template amount ratio, two paired-sample t test p<0.01, significant difference.As a result as shown in figure 1, Ab- Trxl has significantly rise in 4h, 12h, 24h, 48h, 72h and 96h after osmotic pressure stress, it was demonstrated that Ab-Trxl is in Thief zone Play a role in pressure stress.
(4) Ab-Trxl gene silencings
With aphelenchoides besseyi cDNA as template, using primer sets：
Ab-Trxl-T7F：5`-TAA TAC GAC TCA CTA TAG GGA ACA AGT GCA TTT TGG AGC G- 3`,
Ab-Trxl-iR：5`-ACA AAC GAT TTG ATG TCC GC-3`
Enter performing PCR amplification production positive-sense strand template.
Using primer sets：
Ab-Trxl-iF：5`-AAC AAG TGC ATT TTG GAG CG-3`,
Ab-Trxl-T7R：5`-TAA TAC GAC TCA CTA TAG GG ACA AAC GAT TTG ATG TCC GC- 3`
Enter performing PCR amplification production antisense strand template.UsingRNAi kits carry out in-vitro transcription reaction, Synthesis positive-sense strand and antisense strand RNA, annealed production dsRNA.
Distilled water diluting dsRNA concentration processes nematode 12h to 3mg/mL using dsRNA feeding methods, is right with distilled water According to.Processing rear portion nematode is used to extract RNA, and Q- is carried out using primer Ab-Trxl-qF and Ab-Trxl-qR after reverse transcription PCR is expanded, and using aphelenchoides besseyi 28s rRNA gene primer Ab-28s-qF and Ab-28s-qR Q-PCR amplification conducts are carried out Control, verifies RNAi effects.As a result as shown in Fig. 2 Ab-Trxl expressions are remarkably decreased after RNAi.Nematode is used for another part Hyperosmosis processes nematode survival number after 1d～6d and determines, and each process randomly selects 100 cestodes and observed, in triplicate. As a result as shown in figure 3, aphelenchoides besseyi survival number is remarkably decreased.Illustrate that aphelenchoides besseyi similar thioredoxin gene sinks It is silent, the anti-hyperosmosis ability of the nematode is played and is significantly affected, can be used for the nematodiasis preventing and treating.
<110>Northeast Forestry University
<120>For preventing and treating similar thioredoxin gene and primer and the application of aphelenchoides besseyi
<160> 9
<210> 1
<211> 1148
<212> DNA
<213> Ab-Trxl
<400> 1
gtaaaagtaa cgatcagtgt gaagatgtct gaatcgcgtt caaatcacga atcagatctc 60
aaaagtttgc gttttcaaac acgttctaaa gtggttgtga aagcggccga agttccagaa 120
gatatgataa gacgagctgt gggattggca gatgaagcaa tcgcaaataa tttgaatgaa 180
gcagacatcg cccaatttct taagtccaat ttcgacaaac tctatccagc accgtggcac 240
tgtttcgttg gcaagcgatt cagctcgttt gtgactcacg aaagcggtag cttcatttac 300
ttttacatcg tcaatttggc cgtcgttttg ttccgctgca aagattgatt tctcttgttg 360
tttttgtacc caaaagattt tgattcgaat ccattggttt ttgtttttgt tggcatcgtg 420
ttcgtgcatt tcattttagc ggccgttcat cgaataaaca agtgcatttt ggagcgttta 480
aagcgaaatg atttcagttt ctcgtctctt caatttcagt cgaactttca atctacgtgg 540
atattgtagc agaatgaccg atgaagcaaa taccatctac tcgttcaaag ccaaggactc 600
cgatggcgcc gaagtctctt tggacaaata tcgcggcaag gtgttgattg tcgtcaacac 660
ggcatctcaa tgcggtaagt gttacagcaa ttacactcaa ttgaaagagc tgctcgacaa 720
gtacaaatcc cagggactag aagttgccgc ttttccgtgc aaccaattcg gaaaccaaga 780
acctggttgt gatgcggaca tcaaatcgtt tgtgtccgac aagttcaaat tcgatcccga 840
cttgtattcc aaggttaagg ttaacggaga cgacgctcat ccgttgttta aattcctcaa 900
gaaagagcaa ggtggtacgt tgttcgacgc catcaaatgg aatttcacca agttcttggt 960
gaaccgtcaa ggaaaagttg ttgctcgtta tgcgccgacc accgagccaa agtcgatggt 1020
gaaagacatc gagaaagcat tgaacggcga gttgtgagaa tcgcgatagt aacgcaaaga 1080
ctgtaatatt ttattgtacg attaagttaa atagagcaag aataaactaa acggttatta 1140
aaaaaaaa 1148
<210> 2
<211> 20
<212> DNA
<213> Ab-Trxl-F
<400> 2
ttcgacaaac tctatccagc 20
<210> 3
<211> 20
<212> DNA
<213> Ab-Trxl-R
<400> 3
tgaaattcca tttgatggcg 20
<210> 4
<211> 40
<212> DNA
<213> Ab-Trxl-T7F
<400> 4
taatacgact cactataggg aacaagtgca ttttggagcg 40
<210> 5
<211> 20
<212> DNA
<213> Ab-Trxl-iR
<400> 5
acaaacgatt tgatgtccgc 20
<210> 6
<211> 20
<212> DNA
<213> Ab-Trxl-iF
<400> 6
aacaagtgca ttttggagcg 20
<210> 7
<211> 40
<212> DNA
<213> Ab-Trxl-T7R
<400> 7
taatacgact cactataggg acaaacgatt tgatgtccgc 40
<210> 8
<211> 21
<212> DNA
<213> Ab-Trxl-qF
<400> 8
tgaccgatga agcaaatacc a 21
<210> 9
<211> 20
<212> DNA
<213> Ab-Trxl-qR
<400> 9
tcgacgagct caacacttac 20

Claims (5)

1. a kind of similar thioredoxin gene 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 similar thioredoxin gene of aphelenchoides besseyi, it is characterised in that as follows：
Upstream primer Ab-Trxl-F：As shown in sequence table SEQ No.2,
Downstream primer Ab-Trxl-R：As shown in sequence table SEQ No.3.
3. a kind of primer sets for building the similar thioredoxin gene dsRNA of aphelenchoides besseyi, it is characterised in that as follows It is shown：
Synthesis positive-sense strand RNA upstream primer Ab-Trxl-TTF：As shown in sequence table SEQ No.4,
Synthesis positive-sense strand RNA downstream primer Ab-Trxl-iR：As shown in sequence table SEQ No.5,
Synthesis antisense strand RNA upstream primer Ab-Trxl-iF：As shown in sequence table SEQ No.6,
Synthesis antisense strand RNA downstream primer Ab-Trxl-T7R：As shown in sequence table SEQ No.7.
4. the primer sets that a kind of similar thioredoxin gene Q-PCR for aphelenchoides besseyi is detected, it is characterised in that as follows It is shown：
Upstream primer Ab-Trxl-qF：As shown in sequence table SEQ No.8,
Downstream primer Ab-Trxl-qR：As shown in sequence table SEQ No.9.
5. a kind of application for preventing and treating the similar thioredoxin gene of aphelenchoides besseyi as claimed in claim 1, it is special Levy and be, application of the gene in preventing and treating aphelenchoides besseyi.
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CN114480447A (en) * 2022-02-25 2022-05-13 广西大学 Application of kenaf thioredoxin analog protein gene HcTrx and recombinant vector thereof in VIGS silencing system

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* Cited by examiner, † Cited by third party
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CN110862973A (en) * 2019-12-16 2020-03-06 武汉大学 Rice thioredoxin gene OsNDU, protein, vector, host cell, molecular marking method and application
CN114480447A (en) * 2022-02-25 2022-05-13 广西大学 Application of kenaf thioredoxin analog protein gene HcTrx and recombinant vector thereof in VIGS silencing system
CN114480447B (en) * 2022-02-25 2023-07-18 广西大学 Kenaf thioredoxin similar protein gene HcTrx and application of recombinant vector thereof in VIGS silencing system

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