CN106632635B - Thioredoxin-like gene for preventing and treating aphelenchoides besseyi, primer and application - Google Patents
Thioredoxin-like gene for preventing and treating aphelenchoides besseyi, primer and application Download PDFInfo
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Abstract
A thioredoxin-like gene for preventing and treating aphelenchoides besseyi has a sequence shown in a sequence table SEQ No. 1. The sequence of the gene is shown in a sequence table SEQ No. 1. A primer group for constructing thioredoxin-like genes of aphelenchoides besseyi, an upstream primer Ab-Trxl-F: as shown in a sequence table SEQ No.2, a downstream primer Ab-Trxl-R: as shown in a sequence table SEQ No. 3. The gene is applied to the prevention and treatment of aphelenchoides besseyi. The thioredoxin-like gene is up-regulated after the aphelenchoides besseyi is stressed by high osmotic pressure, and shows that the gene plays a role in the process of resisting the high osmotic pressure stress of the aphelenchoides besseyi. Through the research on the gene, the gene has important biological significance and potential application value in preventing and treating aphelenchoides besseyi.
Description
Technical Field
The invention relates to a thioredoxin-like gene for preventing and treating aphelenchoides besseyi, a primer and application thereof.
Background
Rice (Oryza sativa L.) is one of the major food crops in the world and is the staple food for one third of the world's mankind. The Aphelenchoides besseyi Christie disease caused by Aphelenchoides besseyi causes the rice to reduce 10 to 70 percent of yield every year in the world, and causes huge economic loss. At present, the prevention and treatment measures for the aphelenchoides besseyi are mainly to strengthen quarantine and soak seeds with warm soup or medicament before sowing, but the germination potential of the rice after seed soaking is always reduced. Researches find that in the rice cultivation process, work such as fertilization and pesticide application has important influence on the osmotic pressure of the soil environment. Most organisms can not survive under high osmotic pressure, and the aphelenchoides besseyi can survive under the high osmotic pressure environment by entering a variable osmotic cryptobiosis state, and when the environment is proper, the normal metabolism is recovered, so that the seed soaking prevention and control effect and the pesticide prevention and control effect are reduced, and the difficulty of preventing and controlling the aphelenchoides besseyi is increased. The research on the physiological mechanism of the aphelenchoides besseyi under the stress of high osmotic pressure can provide a new idea for preventing and treating the aphelenchoides besseyi and avoid environmental pollution caused by excessive use of chemical agents.
Thioredoxin (Trx) has the capacities of oxidation resistance, drought resistance, heat resistance and the like and plays an important role in biological stress resistance. Trx is widely present in various organisms, has the function of interconverting sulfydryl and disulfide, provides redox pairs for a series of physiological reactions, maintains the reduction state of proteins in cells, enables the proteins to normally play a function, and has an important role in maintaining the stable redox state in the organisms. Trx plays an important auxiliary role in the process of resisting high osmotic pressure stress of aphelenchoides besseyi. At present, few researches on plant parasitic nematodes Trx are carried out at home and abroad. In view of the important function of Trx on biological resistance to abiotic stress, thioredoxin like protein (Trxl) of aphelenchoides besseyi is planned to be cloned, and the survival condition of aphelenchoides besseyi under the condition of high osmotic pressure after the Trxl gene is silenced is explored through a gene silencing technology, so that a new idea is provided for preventing and treating aphelenchoides besseyi.
Disclosure of Invention
Based on the defects, the invention provides a thioredoxin-like gene for preventing and treating aphelenchoides besseyi, a primer and application.
The technology adopted by the invention is as follows: a thioredoxin-like gene for preventing and treating aphelenchoides besseyi has a sequence shown in a sequence table SEQ No. 1.
The invention also has the following technical characteristics:
1. a primer group for constructing thioredoxin-like genes of aphelenchoides besseyi comprises the following components:
the upstream primer Ab-Trxl-F: as shown in a sequence table SEQ No.2,
the downstream primer Ab-Trxl-R: as shown in a sequence table SEQ No. 3.
2. A primer group for constructing thioredoxin like gene dsRNA of aphelenchoides besseyi comprises the following components:
synthesizing a sense strand RNA upstream primer Ab-Trxl-T7F: as shown in a sequence table SEQ No.4,
synthesizing a sense strand RNA downstream primer Ab-Trxl-iR: as shown in a sequence table SEQ No.5,
synthesizing an antisense strand RNA upstream primer Ab-Trxl-iF: as shown in a sequence table SEQ No.6,
synthesizing an antisense strand RNA downstream primer Ab-Trxl-T7R: as shown in SEQ No.7 of the sequence table.
3. A primer group for the thioredoxin analog gene Q-PCR detection of aphelenchoides besseyi comprises the following components:
upstream primer Ab-Trxl-qF: as shown in a sequence table SEQ No.8,
the downstream primer Ab-Trxl-qR: as shown in SEQ No.9 of the sequence table.
4. The thioredoxin-like gene of the aphelenchoides besseyi is applied to the prevention and the treatment of the aphelenchoides besseyi.
The thioredoxin-like gene is up-regulated after the aphelenchoides besseyi is stressed by high osmotic pressure, and shows that the gene plays a role in the process of resisting the high osmotic pressure stress of the aphelenchoides besseyi. Through the research on the gene, a foundation is established for researching the physiological mechanism of the aphelenchoides besseyi to respond to high osmotic pressure stress, a new thought can be provided for preventing and treating the aphelenchoides besseyi, and environmental pollution caused by excessive use of chemical agents is avoided. Has important biological significance and potential application value in preventing and controlling aphelenchoides besseyi.
Drawings
FIG. 1 is a graph showing the relative expression level changes of Ab-Trxl after examination of high osmotic stress by Q-PCR in example 1;
FIG. 2 is a graph showing the effect of Ab-Trxl gene silencing by Q-PCR in example 1;
FIG. 3 is a comparison graph of survival rates of Aphelenchoides besseyi under high osmotic stress after Ab-Trxl gene silencing treatment and control treatment on Aphelenchoides besseyi in example 1, respectively.
Detailed Description
The thioredoxin-like gene for preventing and treating aphelenchoides besseyi provided by the invention is derived from aphelenchoides besseyi, is named Ab-Trxl, has the length of 1,148bp, has a gene sequence shown in SEQ No.1, and has a typical Trxl structural domain.
Example 1:
the acquisition of thioredoxin-like gene for preventing and controlling aphelenchoides besseyi and the functional verification thereof.
RNA extraction and cDNA Synthesis:
the DEPC treated water was used to wash the nematodes (female: male: larva: 4:2:1), centrifuged to remove the supernatant, and ground under liquid nitrogen treatment. Taking the ground powder, extracting the total RNA of the Aphelenchoides besseyi by using a TRIzol method (Invitrogen, cat.No.15596-026), and dissolving the total RNA in DEPC (diethyl phthalate) treated water. Using AMV reverse transcription System (Promega, cat. No. A3500), with oligo (dT)18First strand cDNA reverse transcription is performed for the primers, and second strand cDNA is synthesized by a random primer method. The reaction procedure was carried out according to the experimental manual.
(II) complete reading frame cloning of thioredoxin genes of aphelenchoides besseyi
Synthesizing thioredoxin gene primer group of aphelenchoides besseyi according to the sequencing result of the transcriptome,
Ab-Trxl-F:5`-TTC GAC AAA CTC TAT CCA GC-3`，
Ab-Trxl-R：5`-TGA AAT TCC ATT TGA TGG CG-3`。
the second strand cDNA of the aphelenchoides besseyi is taken as a template to carry out PCR amplification of a complete reading frame sequence (TaKaRa r-Taq enzyme 25 muL reaction system is carried out for 35 cycles at 94 ℃ for 30s, 58 ℃ for 1min and 72 ℃ for 1 min), an amplification product is sent to a biological company for sequencing verification, and the obtained sequence is named Ab-Trxl.
(III) fluorescent quantitative Q-PCR verification
Selecting high osmotic pressure (saturated MgSO)4·7H2O solution), grinding under liquid nitrogen and extracting total RNA by a TRIzol method after 4h, 12h, 24h, 48h and 96h of treatment. First strand cDNA reverse transcription was performed using the GoTaq 2-Step RT-qPCR System (Promega A6010) kit.
Synthesizing a thioredoxin gene Q-PCR verification primer group of the aphelenchoides besseyi according to a sequencing result,
Ab-Trxl-qF：5`-TGA CCG ATG AAG CAA ATA CCA-3`，
Ab-Trxl-qR：5`-TCG ACG AGC TCAACA CTT AC-3`。
the 28s RNA of the aphelenchoides besseyi is used as an internal 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`。
Q-PCR amplification was performed using Stratagene Mx3000P qPCR system (Agilent, USA) and GoTaq 2-Step RT-qPCRSystem kit. PCR using the 2-step method, first step: pre-denaturation at 95 ℃ for 3 min. The second step is that: 40 cycles of 95 ℃ for 30s, 58 ℃ for 1min and 72 ℃ for 30 s. The dissolution curve was measured from 55 ℃ to 95 ℃. And (3) calculating the ratio of the initial template amount of the repeated tests by adopting a relative quantification method for Q-PCR amplification, wherein the t test p of two paired samples is less than 0.01, and the difference is obvious. The results are shown in FIG. 1, Ab-Trxl is significantly up-regulated at 4h, 12h, 24h, 48h, 72h and 96h after high osmotic stress, and Ab-Trxl is proved to play a certain role in high osmotic stress.
(tetra) Ab-Trxl Gene silencing
Taking the Aphelenchoides besseyi cDNA as a template, and applying a primer group:
Ab-Trxl-T7F：5`-TAA TAC GAC TCA CTA TAG GGA ACA AGT GCA TTT TGG AGC G-3`，
Ab-Trxl-iR：5`-ACA AACGAT TTG ATG TCC GC-3`
PCR amplification is carried out to produce a sense strand template.
The application of the primer set:
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`
PCR amplification is performed to produce the antisense strand template. Applications of
RNAi kit carries out in vitro transcription reaction, synthesizes RNA of a sense strand and RNA of an antisense strand, and produces dsRNA through annealing.
The dsRNA concentration is diluted to 3mg/mL by distilled water, and the nematodes are treated by a dsRNA feeding method for 12h, and the distilled water is used as a control. And (3) extracting RNA from the treated part of nematodes, performing Q-PCR amplification by using primers Ab-Trxl-qF and Ab-Trxl-qR after reverse transcription, and performing Q-PCR amplification by using 28s rRNA gene primers Ab-28s-qF and Ab-28s-qR of aphelenchoides besseyi as a control to verify RNAi effect. The results are shown in fig. 2, and the expression level of Ab-Trxl is significantly reduced after RNAi. And the other part of nematodes are used for determining the survival number of the nematodes after the treatment for 1-6 days under high osmotic pressure, and 100 nematodes are randomly selected for observation after each treatment and are repeated for three times. The results are shown in fig. 3, where the survival rate of aphelenchoides besseyi is significantly decreased. Shows that thioredoxin genes of aphelenchoides besseyi have silence, has obvious influence on the high osmotic pressure resistance of the nematode and can be used for preventing and treating diseases of the nematode.
<110> northeast university of forestry
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Claims (5)
1. A thioredoxin-like gene for preventing and treating aphelenchoides besseyi is characterized in that the sequence of the thioredoxin-like gene is shown as SEQ No.1 in a sequence table.
2. A primer group for constructing thioredoxin-like genes of aphelenchoides besseyi is characterized by comprising the following components in parts by weight:
the upstream primer Ab-Trxl-F: as shown in a sequence table SEQ No.2,
the downstream primer Ab-Trxl-R: as shown in a sequence table SEQ No. 3.
3. A primer group for constructing thioredoxin-like gene dsRNA of aphelenchoides besseyi is characterized by comprising the following components:
synthesizing a sense strand RNA upstream primer Ab-Trxl-T7F: as shown in a sequence table SEQ No.4,
synthesizing a sense strand RNA downstream primer Ab-Trxl-iR: as shown in a sequence table SEQ No.5,
synthesizing an antisense strand RNA upstream primer Ab-Trxl-iF: as shown in a sequence table SEQ No.6,
synthesizing an antisense strand RNA downstream primer Ab-Trxl-T7R: as shown in SEQ No.7 of the sequence table.
4. A primer group for the thioredoxin-like gene Q-PCR detection of aphelenchoides besseyi is characterized by comprising the following components in parts by weight:
upstream primer Ab-Trxl-qF: as shown in a sequence table SEQ No.8,
the downstream primer Ab-Trxl-qR: as shown in SEQ No.9 of the sequence table.
5. The use of the thioredoxin-like gene for controlling aphelenchoides besseyi according to claim 1, wherein the gene is used for controlling aphelenchoides besseyi.
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