CN112048488A - OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, protein and application thereof - Google Patents
OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, protein and application thereof Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses an OsPEAMT2 gene for improving the heading stage maturing rate of paddy rice under high-temperature stress, and a protein and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The invention provides a new rice panicle stage high temperature related gene, and provides a new gene resource for improving the rice yield character and breeding and utilizing the rice temperature-sensitive sterile line.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an OsPEAMT2 gene for improving the heading stage maturing rate of paddy rice under high temperature stress, and a protein and application thereof.
Background
In recent years, global warming trends are exacerbated, and extreme high temperatures are frequent, which poses serious challenges to world food safety (pittel kow C M et al, 2015). As a high-temperature short-day crop, rice is more vulnerable to high-temperature stress in the growth process. However, the studies on high temperature in rice are still relatively delayed due to the difficulty of high temperature studies and environmental limitations. Although a large number of high-temperature related genes are reported in rice at present, a large blank still exists on how a plant senses high temperature and conducts downstream signal conduction, so that the function of the high-temperature related genes is researched, a molecular mechanism in the high-temperature related genes is disclosed, and the high-temperature related genes are mined and utilized, so that the high-temperature related genes and the low-temperature related genes have important practical significance on guaranteeing high and stable yield of the rice under high-temperature stress.
Temperature is one of the important regulatory factors affecting rice growth. The growth and development of rice at various stages need to be in an appropriate temperature range. Data show that the heading and flowering stage is the most temperature sensitive stage of rice, later in meiosis, next to the filling stage, and minimal in vegetative growth (Jagadish S V K et al, 2007). Currently, a large number of heat-resistant related genes have been cloned by using mutants in rice, and the genes are mainly divided into four types in function: the heat shock protein family, transcription factors, various enzyme proteins and other proteins (Ai-Li Qu et al, 2013).
Phosphoethanolamine N-methyltransferase (pemto) is an important class of enzyme proteins that catalyze phosphoethanolamine to ultimately produce choline and phosphatidic acid. The genes in arabidopsis are reported to be involved in root system and pollen development, choline synthesis of mutant xipotl is inhibited, and early maturity and temperature-sensitive male sterility are shown (Zhang, k.p. et al, 2004). Surprisingly, promoter sequence analysis of the PEAMT1 (zmpoamt 1) gene in maize showed that the gene was subject to external salt stress and high temperature repression (Eastmond, p.j. et al, 2010). Rice contains a gene encoding pemat in chromosome 1 and 5 segments, respectively, in which ospemit 1 on chromosome 1 is reported to be associated with salt stress (Yu j.d. et al, 2014), and no segment 5 is reported.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the OsPEAMT2 gene for improving the maturing rate of the rice in the heading stage under high-temperature stress, and the protein obtained by encoding and the application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
an OsPEAMT2 gene for improving the maturing rate of paddy rice in the heading stage under high-temperature stress, wherein the nucleotide sequence of the OsPEAMT2 gene is shown as SEQ ID No. 1.
The amino acid sequence of the protein obtained by the gene coding is shown in SEQ ID NO. 2.
The gene is applied to improvement of rice germplasm resources and improvement of the seed setting rate of paddy rice in the heading stage under high-temperature stress.
The application of the gene in culturing thermosensitive sterile rice.
An expression vector containing the gene.
A preparation for improving the fructification rate of paddy rice in the heading stage under high-temperature stress comprises the protein.
A gene chip comprises a specific primer for detecting an OsPEAMT2 gene, and the nucleotide sequence of the specific primer is shown in SEQ ID No. 3-4.
A kit for screening rice with high maturing rate in heading stage comprises specific primers shown as SEQ ID No. 3-4.
The invention has the beneficial effects that:
1. compared with the traditional mutant research mode, the research provides a new research thought of spike-stage heat-resistant genes, and provides a good research basis for the molecular mechanism of rice fructification.
2. The gene belongs to a new ear period high temperature related gene in rice, and provides a new gene resource for improving the yield and the character of the rice.
3. The gene of the invention shows high temperature-sensitive sterility after being knocked out, and provides a new genetic resource for breeding and utilization of a rice temperature-sensitive sterile line.
Drawings
FIG. 1 shows a homology alignment of two PEAMT genes in rice of the first embodiment of the present invention;
FIG. 2 is the expression profile of OsPEAMT2 gene in the first embodiment of the present invention;
FIG. 3 is a schematic diagram of the OsPEAMT2 gene editing vector in the second embodiment of the present invention;
FIG. 4 is a schematic diagram showing the editing of the knockout target site of OsPEAMT2 gene in the second embodiment of the present invention;
FIG. 5 shows the high temperature treatment conditions of the OsPEAMT2 gene knockout strain in the third embodiment of the present invention;
FIG. 6 shows the result of high temperature treatment of the OsPEAMT2 gene knockout strain in the third embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 PEAMT family protein sequence homology alignment analysis in Rice
1. Homologous Gene sequence acquisition
Protein sequence Rice was derived from Rice Genome Annotation Project (http:// Rice. plant biology. msu. edu). The code numbers of the obtained protein sequences are as follows: OsPEAMT1(LOC _ Os01g50030), OsPEAMT2(LOC _ Os05g 47540).
2. Evolution tree making
The ospermt 1 and ospermt 2 protein sequences were loaded into different lanes using DNAMAN7, respectively, and then protein homology analysis was performed using a two-sequence alignment. The sequence analysis results are shown in fig. 1, the total number of the pemto genes in rice is 2, the genes are named as ospemit 1 and ospemit 2, and the data show that the homology of the two genes is as high as 86.46%. The application develops research on OsPEAMT2, and explores the biological function of the gene in rice through a design knockout experiment.
3. Expression profiling
Before verifying the function of ospemit 2 by using transgenes, qPCR primers were first designed, and the specific sequences thereof are as follows:
U565:TCGAGGCCATGATGCTCGAC；(SEQ ID NO.3)
U566:CCAGTTCCCCAGTAAAGCGTCC(SEQ ID NO.4)
and PCR detection is carried out on the spatiotemporal expression pattern of OsPEAMT2 at different parts of rice. The detection result is shown in FIG. 2, the gene is highly expressed at different stages of ear development, thereby suggesting that the gene is likely to have a certain relation with the resistance of the ear stage adversity stress.
Example 2 OsPEAMT2 Gene editing vector construction
1. Gene editing site design
According to the existing CRISPR/Cas9 related experimental method, ACTAATGTACCTTTCAGATGAGG containing AGG as a recognition site is selected on the 5 th exon of OsPEAMT2 to serve as a knockout target site of OsPEAMT2 (fig. 3 and fig. 4), and a loss-of-function transgenic plant is expected to be obtained to determine the biological function of the OsPEAMT2 gene.
CRISPR/Cas9 vector construction
Mixing 5 mul of primers before and after 10 mul of target site, annealing at 99 ℃ for 5min, naturally cooling to obtain a double-stranded sequence containing a knockout target site, connecting the target site sequence to an intermediate vector pYLsgRNA-OsU6a by using Bas1(NEB company) while enzyme cutting (37 ℃), then combining universal amplification primers CTCCGTTTTACCTGTGGAATCG(UF) and CGGAGGAAAATTCCATCCAC (gR) with a target site rear primer and a front primer respectively, obtaining one large fragment and one small fragment containing the target site from the intermediate vector pYLsgRNA-OsU6a by PCR amplification, then fusing the two fragments by once overlapping, and finally connecting the fused fragment to a final vector pyLCrispr/Cas9P35S-N (15011bp) by using Bas1(NEB company) while enzyme cutting (37 ℃) for 3 hours, wherein the successfully constructed final vector is shown in figure 3.
3. Transformation of E.coli
Taking out Escherichia coli T1 competent (whole gold) from-80 ℃, then placing on ice for thawing, quickly adding the ligation product in the previous step after the cell is dissolved, gently mixing by using a pipette, standing on ice for 30 minutes, then thermally shocking at 42 ℃ for 30 seconds, standing on ice again for 2 minutes, then adding 10 times of LB non-resistant culture solution into the transformation product, culturing at 37 ℃ for 50 minutes at 200rpm, taking out, centrifuging at 4000rpm, removing most of supernatant, re-suspending the thallus by using residual liquid (about 100 mu L), plating (LB + kanamycin resistance), standing and culturing at 37 ℃ overnight, picking out a single colony for expanding and culturing for 3mL (LB + kanamycin resistance), and sequencing by using a universal primer (SP-L1: GCGGTGTCATCTATGTTACTA) to verify a target site sequence to obtain a positive clone.
4. Agrobacterium transformation
Taking out agrobacterium EHA105 cell competence (prepared by a chemical transformation method) from-80 ℃, then placing the cell competence on ice for unfreezing, adding 2 mu L of prepared positive clone plasmid, placing the cell competence on ice for 30 minutes after being gently mixed, then freezing the cell competence in liquid nitrogen for 2 minutes, quickly taking out the cell competence, placing the cell lysis product in a metal bath at 37 ℃ for 2 minutes, then adding 10 times of LB nonreactive culture solution into a transformation product, culturing the cell lysis product at 28 ℃ for 2-3 hours at 250rpm, taking out the cell lysate, centrifuging the cell lysate at 5000rpm, removing most of supernatant, using residual liquid (about 100 mu L) to re-suspend the cell, plating the cell on a plate (LB + rifampicin + kanamycin), picking out a monoclonal colony after overnight standing and culturing at 28 ℃, detecting by using G418 primer to obtain positive clone, and expanding and culturing 3mL (.
5. Genetic transformation of rice
Taking about 500 Japanese nitrile seeds, respectively cleaning sterile water and 50% sodium hypochlorite, then sucking water by filter paper, and inducing callus generation by using an NMB culture medium; then, carrying out enlarged culture on the standby agrobacterium tumefaciens bacterial liquid by 50mL, collecting the thallus at 5000rpm, then, resuspending the thallus by using an AAM liquid culture medium added with AS (acetosyringone), then, infiltrating the selected callus in the environment of the resuspended thallus for about 30 minutes, then, sucking out the thallus, and continuously culturing the callus on the culture medium for 2 days; respectively cleaning the cultured callus sterile water and the sterile water containing the cefamycin, and then placing the cleaned callus sterile water and the sterile water on a selective culture medium for about three weeks; and finally, continuously inducing the callus to take root by using a differentiation culture medium respectively, and hardening the seedling indoors when the height of the seedling is about 10cm to prepare subsequent detection and transplantation.
Example 3 OsPEAMT2 Gene editing in Nippon background
1. Transgenic seedling detection
Agrobacterium infection transformation to obtain 10 seedlings, detecting the transformation condition of a vector by using G418, amplifying a sequence in a positive transformation single plant by using a cross-target-site amplification primer U1028(TTCTGTAGAATGAGAACAT) + U1029(ACTAGCTTCTCGACCTACAGTA), and then sending the amplified sequence to a sequencing company of the department of Onychidae for sequencing, wherein the result is shown in figure 4, and the obtained sequence shows that a transgenic plant ospeamt2 with protein translation termination in advance is obtained by comparison and analysis.
2. Phenotypic analysis
In the T1 generation planting, different strains are planted in 3 times of field experiments repeatedly, each time 2 rows are planted repeatedly, and the stable mutation of the target site is confirmed by sequencing detection. When the agronomic characters are inspected, the boundary lines and the mixed plants are repeatedly removed, 5-10 stable lines are selected, the agronomic characters of the single plants are inspected, and statistical analysis is carried out. The results show that the knockout progeny of the stable line has no obvious change in other agronomic traits except for the setting rate, wherein the setting rate of the knockout line is significantly reduced (fig. 6C).
3. Design of temperature treatment experiment
In combination with the fructification phenotypic changes of knockout lines and the biological function analysis of the PEAMT gene in other species, the present study guesses that OSPEAMT2 is likely to participate in the spike-stage high-temperature stress process. To verify the hypothesis of the experiment, the experiment utilizes an intelligent greenhouse to set a strict temperature gradient, and the mutant and the wild type in the flowering period are subjected to a heading period high-temperature treatment experiment, wherein the specific treatment conditions are shown in fig. 5. Then, about 10 wild type and knockout mutants in the field at the heading stage are prepared, seedlings are dug out and placed in small barrels (2 seedlings per barrel) with the diameter of 37 centimeters and the depth of 40 centimeters 3 days before most of main ears bloom, and sufficient water is supplied to prevent the seedlings from losing water when the seedlings are placed in a cool place and recovered for about 2 days after the seedlings are dug out. And then, marking the blossoming ear and sword leaves on the next day of all the single plants by using a branch binding machine (purchased from Taobao) one day and one night before blossoming, placing the marked seedlings in a set intelligent greenhouse for high-temperature treatment (except for adding water in the morning and the evening, cutting without opening a door), taking out the seedlings after 48 hours of treatment, carefully planting the seedlings back to the field again, and cutting the marked single ears after the seedlings grow to be mature to check the seed setting rate.
4. Ear stage high temperature treatment analysis
After 2 days of high temperature treatment of the wild type and ospeamt2 at flowering stage, the single ear setting rate was counted and compared. The results show that the knockout mutant ospeamt2 shows complete sterility after high temperature treatment. Therefore, we hypothesize that OSPEAMT2 is probably a new temperature-sensitive fertility-related gene in rice.
Sequence listing
<110> Sichuan university of agriculture
<120> OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, and protein and application thereof
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<170> SIPOSequenceListing 1.0
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<213> Rice (Oryza sativa)
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tgggaggagc actccaagga cctcaccgtc gaggccatga tgctcgactc ccgcgccgcc 120
gacctcgaca aggaggagcg ccccgaggtc ctgtctgtac tcccttctta caaagggaaa 180
tcagtactgg agcttggtgc tggaatagga cgctttactg gggaactggc aaaagaagct 240
ggccatgttt tagccctaga cttcattgaa agtgtgatta agaagaatga gaacataaat 300
gggcatcaca agaacataac ctttatgtgc gctgatgtca cgtctccgga cctgacgatc 360
gaagataact ctattgatct catattctca aactggctac taatgtacct ttcagatgag 420
gaggtcgaga agctagtagg aagaatggtg aaatggctga aggtaggtgg ccatatattc 480
tttagggagt catgctttca ccaatctgga gattccaaaa ggaaggtgaa tccaacacat 540
taccgggagc caaggttcta tacaaagata tttaaagaat gccattccta tgataaagat 600
gggggttctt atgaactttc tctagaaaca tgcaagtgca ttggggctta tgtgaaaagc 660
aagaaaaatc aaaatcagtt atgttggcta tgggaaaagg ttaagtcaac agaagacaga 720
ggattccaaa gattcctgga caatgtgcag tacaaaacca ctggaatctt acgctatgag 780
cgtgtcttcg gagagggtta tgtcagcact ggtggaattg aaaccacaaa ggaatttgtg 840
gataagctgg atcttaaacc tggacagaaa gtgcttgatg ttgggtgcgg aattggaggc 900
ggcgacttct atatggctga aaactacgat gcccatgttc ttggtattga tctttcaatc 960
aacatggttt catttgcaat cgaacgtgcc attggacgca agtgttcggt tgagtttgaa 1020
gtagctgatt gcaccacaaa gacctacgca ccaaatacat ttgatgtgat ctacagccgt 1080
gacaccattc ttcacataca tgataaacct gctttgttca gaagtttctt caagtggctg 1140
aaacctgggg gcaaagtcct catcagtgat tactgtagga atcctgggaa accatcagaa 1200
gaatttgctg cttacattaa gcagagaggc tatgacctcc acgatgtgaa gacttacgga 1260
aagatgcttg aggatgctgg tttccatcat gtcattgctg aagaccgcac ggaccagttc 1320
ctgcgtgttc ttcaaaggga gcttgctgaa gttgagaaga acaaagaagc cttcatggca 1380
gacttcaccc aggaggacta cgatgacatt gtgaacggct ggaacgcgaa gctgaagcgg 1440
agctctgccg gtgagcagag gtgggggctg ttcattgcaa ccaaatga 1488
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Glu Asn Ile Asn Gly His His Lys Asn Ile Thr Phe Met Cys Ala Asp
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Phe Arg Glu Ser Cys Phe His Gln Ser Gly Asp Ser Lys Arg Lys Val
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Asn Pro Thr His Tyr Arg Glu Pro Arg Phe Tyr Thr Lys Ile Phe Lys
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Glu Cys His Ser Tyr Asp Lys Asp Gly Gly Ser Tyr Glu Leu Ser Leu
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Glu Thr Cys Lys Cys Ile Gly Ala Tyr Val Lys Ser Lys Lys Asn Gln
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Asn Gln Leu Cys Trp Leu Trp Glu Lys Val Lys Ser Thr Glu Asp Arg
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Gly Phe Gln Arg Phe Leu Asp Asn Val Gln Tyr Lys Thr Thr Gly Ile
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Leu Arg Tyr Glu Arg Val Phe Gly Glu Gly Tyr Val Ser Thr Gly Gly
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Ile Glu Thr Thr Lys Glu Phe Val Asp Lys Leu Asp Leu Lys Pro Gly
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Gln Lys Val Leu Asp Val Gly Cys Gly Ile Gly Gly Gly Asp Phe Tyr
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Met Ala Glu Asn Tyr Asp Ala His Val Leu Gly Ile Asp Leu Ser Ile
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Asn Met Val Ser Phe Ala Ile Glu Arg Ala Ile Gly Arg Lys Cys Ser
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Val Glu Phe Glu Val Ala Asp Cys Thr Thr Lys Thr Tyr Ala Pro Asn
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Thr Phe Asp Val Ile Tyr Ser Arg Asp Thr Ile Leu His Ile His Asp
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Lys Pro Ala Leu Phe Arg Ser Phe Phe Lys Trp Leu Lys Pro Gly Gly
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tcgaggccat gatgctcgac 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ccagttcccc agtaaagcgt cc 22
Claims (8)
1. An OsPEAMT2 gene for improving the heading stage maturing rate of paddy rice under high-temperature stress is characterized in that the nucleotide sequence of the OsPEAMT2 gene is shown as SEQ ID No. 1.
2. The protein encoded by the gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.
3. The application of the gene of claim 1 in rice germplasm resource improvement.
4. The application of the gene of claim 1 in cultivating temperature-sensitive sterile rice.
5. An expression vector comprising the gene of claim 1.
6. An agent for increasing the heading-stage fructification rate of paddy rice under high-temperature stress, which comprises the protein according to claim 2.
7. A gene chip is characterized by comprising a specific primer for detecting an OsPEAMT2 gene, wherein the nucleotide sequence of the specific primer is shown in SEQ ID NO. 3-4.
8. A kit for screening rice with high maturing rate in heading stage under high temperature stress is characterized by comprising specific primers shown as SEQ ID No. 3-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010952455.9A CN112048488B (en) | 2020-09-11 | 2020-09-11 | OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, protein and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010952455.9A CN112048488B (en) | 2020-09-11 | 2020-09-11 | OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, protein and application thereof |
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| CN111909912A (en) * | 2020-09-11 | 2020-11-10 | 四川农业大学 | MAP3K-19 gene for improving high-temperature tolerance of rice in heading stage, protein obtained by encoding same and application thereof |
| CN114751969A (en) * | 2022-04-25 | 2022-07-15 | 江西省超级水稻研究发展中心(江西省农科院海南水稻育种中心) | High-temperature-resistant protein and nucleic acid molecule of rice and method for cultivating high-temperature-resistant rice strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111909912A (en) * | 2020-09-11 | 2020-11-10 | 四川农业大学 | MAP3K-19 gene for improving high-temperature tolerance of rice in heading stage, protein obtained by encoding same and application thereof |
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| CN114751969A (en) * | 2022-04-25 | 2022-07-15 | 江西省超级水稻研究发展中心(江西省农科院海南水稻育种中心) | High-temperature-resistant protein and nucleic acid molecule of rice and method for cultivating high-temperature-resistant rice strain |
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| CN112048488B (en) | 2022-05-10 |
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