CN114908069A - Application of rice kinase gene OsMKKK70 and homologous gene thereof in negative regulation of cold resistance of rice in booting stage - Google Patents
Application of rice kinase gene OsMKKK70 and homologous gene thereof in negative regulation of cold resistance of rice in booting stage Download PDFInfo
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Abstract
Application of a rice kinase gene OsMKKK70 and a homologous gene thereof in negative regulation of cold tolerance of rice at a booting stage relates to the field of rice genetic engineering, in particular to new application of the rice kinase gene OsMKKK70 and the homologous gene thereof. Provides an important theoretical basis for improving the yield of rice under low-temperature stress. The invention discovers that the rice kinase gene OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof can negatively regulate and control cold tolerance of rice in the booting stage. The OsMKKK70 gene, the OsMKKK62 gene and the OsMKKK55 gene are jointly involved in regulating and controlling cold tolerance of rice in the booting stage. OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof influence the GA content in anthers by negatively regulating GA biosynthesis under low-temperature stress, and finally negatively regulate cold resistance of rice at the booting stage. The invention is applied to improving the yield of rice under low-temperature stress.
Description
Technical Field
The invention relates to the field of rice genetic engineering, in particular to a new application of a rice kinase gene OsMKKK70 and a homologous gene thereof.
Background
Rice is an important food crop, and more than half of the world population uses rice as staple food. Heilongjiang province is an important commodity grain area of rice in China, but in recent years, cold injury in booting period becomes a local main natural disaster due to abnormal climate change, and the cold injury can occur once every 3-4 years on average. Therefore, the method solves the problem of cold injury during the booting stage, excavates cold-resistant genes and analyzes the functions of the cold-resistant genes, and has very important practical significance for improving the yield of rice and ensuring the grain safety. GA, gibberellin, is a common plant hormone, positively regulates cold tolerance of rice at booting stage, and low temperature treatment results in bioactive GA mainly in rice anthers 4 、GA 7 The content is reduced, the function of a flower organ is influenced, the fertility is reduced, the rice is seriously reduced, the phenotype of reduced setting rate can be recovered to a certain degree by applying GA from an external source, the setting rate of GA signal mutants slr1-d1, slr1-d3 and gid1-8 after low-temperature treatment is obviously reduced compared with that of a control material, and the direct relation between GA and the cold resistance regulation of the rice in the booting stage is fully demonstrated.
If the rice meets low-temperature cold damage in the booting stage, the maturing rate is greatly reduced, and the yield of the rice is easily reduced. Therefore, the research on the influence of the low-temperature stress in the booting stage on the rice yield is of great significance for cultivating low-temperature resistant rice varieties to guarantee the high yield of grains in China.
Disclosure of Invention
The invention aims to provide a new application of a rice kinase gene OsMKKK70 and a homologous gene thereof in negative regulation of cold resistance of rice at the booting stage, provides an important theoretical basis for increasing yield of rice under low-temperature stress, and has a wide application prospect.
The invention provides application of a rice kinase gene OsMKKK70 and a homologous gene thereof in negative regulation of cold tolerance of rice in a booting stage.
Furthermore, pollen fertility of the OsMKKK70 gene overexpression transgenic rice is poor, and maturing rate is reduced.
Furthermore, pollen fertility and seed setting rate of the OsMKKK70 and the double mutant of the homologous gene OsMKKK62 after low-temperature treatment are both obviously higher than those of the wild type.
Furthermore, pollen fertility and seed setting rate of OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 after low-temperature treatment are both obviously higher than those of wild type.
Furthermore, the GA content in the double mutant anther of OsMKKK70 and the homologous gene OsMKKK62 is obviously higher than that of the wild type.
Furthermore, GA content in the anthers of OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 of the three mutants is obviously higher than that of the wild type.
The invention has the beneficial effects that:
the invention discovers for the first time that the rice kinase gene OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 can negatively regulate and control the cold tolerance of rice at the booting stage. The OsMKKK70 gene, the OsMKKK62 gene and the OsMKKK55 gene are jointly involved in regulating and controlling cold tolerance of rice in the booting stage.
The invention discovers that the pollen fertility of the OsMKKK70 gene overexpression transgenic rice is poor, the maturing rate is reduced, and the GA-deficient phenotype is shown;
the pollen fertility and the seed setting rate of the OsMKKK70 and the double mutant of the homologous gene OsMKKK62, the OsMKKK70 and the homologous gene OsMKKK62 and the OsMKKK55 after the three mutants are treated at low temperature are obviously higher than those of wild type; the GA content determination finds that the GA content in the anthers of OsMKKK70 and homologous genes thereof OsMKKK62, OsMKKK70 and homologous genes thereof OsMKKK62 and OsMKKK55 after low-temperature treatment is obviously higher than that of the wild type, qRT-PCR experiments show that the expression levels of GA-related synthetic genes KA0, GA20ox1, GA20ox3 and GA3ox1 in OsMKKK70 and homologous genes thereof OsMKKK 4642 and OsMKKK55 after low-temperature treatment and three mutants thereof are obviously higher than that of the wild type, the OsMKKK70 and homologous genes thereof OsKKK 62 and OsMKKK55 influence the ear content in the anthers by negatively regulating GA biosynthesis under low-temperature stress, and finally the cold tolerance of rice in the negative direction is regulated.
The discovery that the rice kinase gene OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof are used as negative control factors of cold tolerance of rice at the booting stage excavates new functions of OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof, provides an important theoretical basis for improving yield of rice under low-temperature stress, and has wide application prospect.
Drawings
FIG. 1 is a diagram of the induction of OsMKKK70 by low temperature;
FIG. 2 is a diagram of panicle type and anther form of Longjing 11 and OsMKKK70 overexpression transgenic rice;
FIG. 3 shows the statistical results of the maturing rate of Longjing 11 and OsMKKK70 overexpression transgenic rice;
FIG. 4 shows the results of sequencing and identification of osmkk 70/62 double mutant plants;
FIG. 5 shows the results of sequencing and identification of the osmkk 70/62/55 triple mutant plants;
FIG. 6 is a spike pattern of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice under normal conditions and after low temperature treatment at 15 ℃;
FIG. 7 shows the statistics of the setting percentage of Longjing 11, osmkk 70/62 double mutants, osmkk 70/62/55 triple mutant rice under normal conditions and after 15 ℃ low-temperature treatment;
FIG. 8 is an anther activity diagram of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice after being treated at 15 ℃ at low temperature;
FIG. 9 shows the statistics of anther activity of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice after low temperature treatment at 15 ℃;
FIG. 10 is the anther shape chart of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice after low temperature treatment at 15 ℃;
FIG. 11 is a graph showing the anther length of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice after being treated at 15 ℃ at low temperature;
FIG. 12 shows the statistics of anther length of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice after low temperature treatment at 15 ℃;
FIG. 13 shows GA in anther treated at 18 ℃ under normal conditions of Longjing 11 and osmkkk70/62 double mutants 1 、GA 3 、GA 4 、GA 7 The detection result of the content;
FIG. 14 shows GA in anther after Longjing 11 and osmkkk70/62 double mutants treated under normal conditions and at 18 ℃ at low temperature 9 、GA 12 、GA 24 The detection result of the content;
FIG. 15 shows GA in anther treated at 18 ℃ under normal conditions of Longjing 11 and osmkkk70/62 double mutants 5 、GA 19 、GA 53 The detection result of the content;
FIG. 16 shows GA in anther treated at 18 ℃ under normal conditions of Longjing 11 and osmkkk70/62 double mutants 8 、GA 34 、GA 51 The detection result of the content;
FIG. 17 shows the results of the expression level detection of GA biosynthetic gene KAO in young ears of Longjing 11, osmkk 70/62 double mutants, and osmkk 70/62/55 triple mutant rice after low temperature treatment at 15 ℃ under normal conditions;
FIG. 18 shows the results of the expression levels of GA biosynthetic genes GA20ox1 in young ears of Longjing 11, osmkk 70/62 double mutants and osmkk 70/62/55 triple mutant rice treated at 15 ℃ under normal conditions;
FIG. 19 shows the results of the expression levels of GA biosynthetic genes GA20ox3 in young ears of Longjing 11, osmkk 70/62 double mutants and osmkk 70/62/55 triple mutant rice treated at 15 ℃ under normal conditions;
FIG. 20 shows the results of the expression levels of GA biosynthetic genes GA3ox1 in young ears of Longjing 11, osmkk 70/62 double mutants and osmkk 70/62/55 triple mutant rice treated at 15 ℃ under normal conditions;
FIG. 21 shows the results of detecting the expression level of GAYB gene in young ears of Longjing 11, osmkk 70/62 double mutants and osmkk 70/62/55 triple mutant rice after low temperature treatment at 15 ℃ under normal conditions;
FIG. 22 shows the results of the expression level detection of CYP703A3 gene in young ears of Longjing 11, osmkk 70/62 double mutant and osmkk 70/62/55 triple mutant rice treated at 15 ℃ and normal conditions.
Detailed Description
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1: biological function verification of the rice booting stage negative regulation kinase gene OsMKKK 70:
the OsMKKK70 overexpression transgenic plant is obtained by the following method:
firstly, vector construction: taking cDNA of rice variety Longjing 11 as template, refer to TaKaRa
The HS DNA Polymerase operation instruction uses a primer pair F1 and R1 to amplify the OsMKKK70 gene, and clones the amplified fragment into a plant over-expression vector PC1390U to form an OsMKKK70 gene over-expression vector driven by a Ubiquitin promoter.
Forward primer F1: 5'-GTTACTTCTGCACTAGGTACCATGGCTAAGCAGCTCAGGC-3'
Reverse primer R1: 5'-TCTTAGAATTCCCGGGGATCCTCAGCATGTGGTTGCCAATT-3'
Secondly, transforming agrobacterium tumefaciens EHA105 by the target vector: taking out EHA105 from a refrigerator at-80 deg.C, and thawing on ice; 500 ng-1 mu g of the target plasmid is added into 100 mu l of EHA105 competence, and is placed on ice for 30 min; rapidly placing in liquid nitrogen for 5 min; taking out from liquid nitrogen, and rapidly placing in water pre-pot at 37 deg.C for 5 min; ice for 2 min; adding 800 mul of liquid LB culture medium, placing in a full-temperature oscillator (purchased from MKN company), and incubating at 28 ℃ and 120rpm for 4-5 h; after centrifugation, most of the supernatant was discarded, and the remaining bacterial solution was applied to LB solid medium containing kanamycin (50. mu.g/ml) (obtained from Amresco) and rifampicin (50. mu.g/ml) (obtained from Amresco), and cultured at 28 ℃ for 3 days.
Thirdly, after the colonies grow out, carrying out colony PCR identification to identify positive clones; selecting positive clone to liquid LB culture medium containing corresponding antibiotic and rifampicin, culturing at 28 deg.C and 180rpm for about 16h, storing the bacterial liquid with 30% glycerol at a volume ratio of 1:1, storing in a refrigerator at-80 deg.C, and activating at-80 deg.C when infecting callus.
Fourthly, infecting the rice callus by agrobacterium: removing the target bacteria from the refrigerator at-80 deg.C, adding into liquid LB medium containing kanamycin (50 μ g/ml) and rifampicin (50 μ g/ml) at a ratio of 1:100, culturing at 180rpm at 28 deg.C overnight; culturing the bacterial liquid to a color (OD is about 1.0) which looks like orange juice to naked eyes, and taking out the bacterial liquid from the incubator; taking about 500ul of bacterial liquid into a 1.5ml centrifuge tube, centrifuging for 3min at the temperature of 28 ℃ and 5000rpm, and discarding supernatant to see white bacterial colonies at the bottom of the tube; the basilar cenobium was gently pipetted with 300. mu.l of liquid co-culture medium containing 20. mu.g/ml acetosyringone (purchased from Aldrich) to be suspended uniformly in the liquid medium; selecting the callus with good growth state to a 50ml centrifuge tube, wherein the volume of the callus is about 5ml of the scale of the centrifuge tube; adding 20ml of liquid co-culture medium containing 20 mu g/ml acetosyringone, and then adding the suspended 300 mu l of bacterial liquid into a 50ml centrifuge tube; and (4) continuously and softly mixing for 2-3 min for infection. Pouring the liquid co-culture medium, transferring the infected callus into a culture dish paved with filter paper, and adsorbing the redundant culture medium, wherein the process is about 1 min; spreading a layer of filter paper on the solid co-culture medium to soak the filter paper, and transferring the infected callus to the solid culture medium; dark culture is carried out for 2-3 days at 28 ℃.
Fifthly, recovering and culturing the callus of the infected rice: after the infected callus is cultured in dark for 2-3 days, transferring the callus particles into a 50ml centrifuge tube; washing the callus with sterile water containing 400 mu g/ml carbenicillin (purchased from Amresco) for 4-5 times, each time lasting for about 1min, and sterilizing; cleaning the callus with sterile water for 2-3 times, transferring the callus to a culture dish paved with filter paper, and sucking off excessive water; and transferring the callus onto a recovery culture medium containing 400 mu g/ml carbenicillin, and performing recovery culture in a climatic incubator (24h light culture) at 28 ℃ for 4-5 days.
Sixthly, screening and culturing infected rice callus tissues: after 4-5 days of recovery culture, transferring the callus on the recovery culture medium to a screening culture medium containing 400 μ g/ml carbenicillin and 50 μ g/ml hygromycin (purchased from Roche); the cells were transferred to a 28 ℃ climatic incubator (24h light culture) and cultured for about 30 days.
Seventhly, differential culture of the resistant rice callus: transferring the resistant callus on the screening culture medium to a differentiation culture medium, and transferring each bottle to a cluster of callus; culturing in a 28 deg.C artificial climate incubator (24h light culture) for about 30 days to obtain transgenic seedling.
The expression conditions of OsMKKK70 at the ear part after the rice is processed at 15 ℃ for 1h and 3h at the booting stage are detected, and the OsMKKK70 is transiently induced to express under the low-temperature regulation (as shown in figure 1). Observing OsMKKK70 overexpression transgenic plants under the background of wild rice varieties Longjing 11 and Longjing 11, finding that the fruiting rate of the OsMKKK70 overexpression plants is remarkably reduced compared with that of a control material Longjing 11 (as shown in figures 2 and 3). This suggests that OsMKKK70 is likely to be involved in the regulation of cold tolerance of rice at booting stage.
CDS sequences of OsMKKK70, OsMKKK62 and OsMKKK55 genes are input into CRISPR Primer Designer software, 3 pairs of target site primers (F2 and R2; F3 and R3; F4 and R4) are designed and used for construction of a subsequent knockout vector. Transforming agrobacterium tumefaciens EHA105 by using the target vector; an osmkk 70 osmkk 62 double mutant plant and an osmkk 70 osmkk 62 osmkk 55 triple mutant plant are obtained by using a CRISPR-CAS9 technology under the background of a wild rice variety Longjing 11 by adopting an agrobacterium-mediated genetic transformation method, and sequencing identification results are shown in figures 4 and 5.
Forward primer F2: 5'-GGCACCCGAAATCCGCGATCTTGGCCC-3'
Reverse primer R2: 5'-AAACGGGCCAAGATCGCGGATTTCGGG-3'
Forward primer F3: 5'-GCCGGGCAATGGCTAAGCAGCTC-3'
Reverse primer R3: 5'-AAACGAGCTGCTTAGCCATTGCCT-3'
Forward primer F4: 5'-GTTGCCGACCATGATGTTCCTGGCCTT-3'
Reverse primer R4: 5'-AAACAAGGCCAGGAACATCATGGTCGG-3'
The osmkk 70 osmkk 62 double mutant plants and the osmkk 70 osmkk 62 under the background of wild rice varieties Longjing 11 and Longjing 11The osmkkk55 three-mutant plant is used as an experimental material, when the main tiller of the rice grows until the distance between the flag leaf and the inverted two-leaf pillow of the rice is-5-0 cm (namely, the anther is in the meiosis stage to the mononuclear stage), the rice is subjected to low-temperature treatment at 15 ℃ for 4 days, and is transferred back to the normal growth condition after 5 days, and the total grain number, the solid number and the seed setting rate of the main spike are counted during maturation. As shown in FIGS. 6 and 7, □ in FIG. 7 indicates Longjing 11,
represents osmkk 70 osmkk 62 double mutant plants,
represents osmkkk70 osmkkk62 osmkkk55 triple mutant plants. The fruit set percentage of the osmkk 70 osmkk 62 double mutant plants and the osmkk 70 osmkk 62 osmkk 55 triple mutant plants after low temperature treatment were significantly increased compared to the control material.
Under the same treatment conditions, after 4 days, 5 days of treatment, the plants were transferred back to normal growth conditions, and a sample was taken the day before flowering (i.e. the anthers were in trinuclear stage), fixed in FAA fixative, and treated with 1% I 2 KI staining and pinching off the anthers to release pollen grains, pollen viability was observed under Olympus BX53 microscope. As shown in FIGS. 8 and 9, □ in FIG. 9 indicates Longjing 11,
represents osmkk 70 osmkk 62 double mutant plants,
represents osmkkk70 osmkkk62 osmkkk55 triple mutant plants. The osmkk 70 osmkk 62 double mutant plants and the osmkk 70 osmkk 62 osmkk 55 triple mutant plants had significantly better pollen viability after cold treatment than their control materials.
After sampling under the same low-temperature treatment conditions, the anthers in the day before flowering (i.e., the anthers were in the trinuclear stage) were observed under an Olympus SZX16 microscope and the length of the anthers was counted. The result is shown in figures 10, 11 and 12, □ in figure 12 represents Longjing 11,
represents osmkk 70 osmkk 62 double mutant plants,
represents osmkkk70 osmkkk62 osmkkk55 triple mutant plants. The osmkk 70 osmkk 62 double mutant plants and the osmkk 70 osmkk 62 osmkk 55 triple mutant plants had longer, more upright and more yellow anthers after the cold treatment, while the control material had significantly shorter anther length, bent, deteriorated texture and white color after the cold treatment for 4 days and 5 days.
The results show that the OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof can negatively regulate cold tolerance of rice at the booting stage.
Example 2: detection of GA content in OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 negative regulation rice anther:
wild rice variety Longjing 11 and osmkk 70 osmkk 62 double mutant plants under the genetic background thereof are used as experimental materials, when main tillers of rice grow to have the distance between flag leaves and inverted two-leaf pillows of the rice of-8 to-6 cm (namely, the anthers are in the meiosis stage), the rice is subjected to low-temperature treatment at 18 ℃ for 15 days (the anthers are in the trinuclear stage), and about 30 anthers of the main tillers of the rice are sampled to detect the GA content in the anthers through Thermo Scientific Ultimate 3000 UHPLC and TSQ Quantiva in Wuhan Jiang corporation (http:// www.greenswordcreation.com).
The results are shown in FIGS. 13-16, in which
Represents that wild Longjing 11 is at normal temperature,
shows osmkkk70 osmkkk62 double mutant plants at normal temperature,
shows that the wild Longjing tea 11 is processed at low temperature,
represents double mutant plants after the low temperature treatment of osmkk 70 osmkk 62. Low temperature stress resulted in a decrease in GA content in rice anthers, but the decrease was not significant in osmkk 70 osmkk 62 double mutant plants, resulting in a significantly higher GA content in the anthers of osmkk 70 osmkk 62 double mutant plants than in control material.
Example 3: OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof can be used for detecting GA biosynthesis in rice in negative regulation mode:
firstly, using wild rice variety Longjing 11 and osmkk 70 osmkk 62 double mutant plants and osmkk 70 osmkk 62 osmkk 55 triple mutant plants under the genetic background thereof as experimental materials, carrying out low-temperature treatment on rice for 4 days at 15 ℃ when main tillers of the rice grow until the distance between flag leaves and inverted two-leaf pillows of the rice is-5-0 cm (namely, anthers are in a meiosis stage to a single nucleus stage), sampling young ears of the main tillers and young ears of main tillers of the rice growing under normal conditions with the same leaf pillow distance, and extracting the total RNA of the young ears by referring to an operation manual of a TRIzol kit purchased from Invitrogen company;
secondly, treating the total RNA extracted in the step one by adopting DNase I;
third, 1. mu.g of the total RNA treated in the second step was used for cDNA synthesis according to BD SMART from BD Biosciences Clontech TM The manual of RACE cDNA Amplification Kit;
fourthly, the obtained cDNA is taken as a template to pass through 4 GA biosynthesis genes and OsGAYB gene specifically expressed in anther and a target gene OsCYP703A3 primer: OsKAO gene (forward primer F5 and reverse primer R5), OsGA20ox1 gene (forward primer F6 and reverse primer R6), OsGA20ox3 gene (forward primer F7 and reverse primer R7), OsGA3ox1 gene (forward primer F8 and reverse primer R8), OsGAYB gene (forward primer F9 and reverse primer R9), OsCYP703A3 gene (forward primer F10 and reverse primer R10) and rice internal reference actin (forward primer F11 and reverse primer R11), and performing Quantitative real-time PCR by SYBR Green PCR master mix (TransStart); data were obtained from a Bio-Rad chromo 4real-time PCR detector; by 2 -△△CT Methods fold changes were analyzed.
Forward primer F5: 5'-CATCTCCGGCAAATCCT-3'
Reverse primer R5: 5'-CCAGCCTCGACATCGC-3'
Forward primer F6: 5'-ATCGGCTGGAGATGAAGAGG-3'
Reverse primer R6: 5'-CGGCTCATCTCGTGGCAGTA-3'
Forward primer F7: 5'-AATACCGCCACATGGGGGAGGT-3'
Reverse primer R7: 5'-GTAGTGGTTCAGCCGCATCACC-3'
Forward primer F8: 5'-GACGATTCACCTCAACATGTTCCCT-3'
Reverse primer R8: 5'-GGCTCTGCAGGATGAAGGTGAA-3'
Forward primer F9: 5'-GGAGGACGGACAACGAGAT-3'
Reverse primer R9: 5'-GAGTGCGGGAACTGGAAGA-3'
Forward primer F10: 5'-AGACTTCGCTCGATTCTGCAC-3'
Reverse primer R10: 5'-GCTTTAGCCCACACAAACTGA-3'
Forward primer F11: 5'-AGACCTTCAACACCCCTGCTATG-3'
Reverse primer R11: 5'-TCACGCCCAGCAAGGTCG-3'
As shown in fig. 17, 18, 19, 20, 21 and 22, wherein
The representation indicates that the shape of Longjing 11,
representing the osmkk 70 osmkk 62 double mutant plants,
represents osmkkk70 osmkkk62 osmkkk55 triple mutant plants. Is the expression characteristics of 4 GA biosynthetic genes KAO, GA20ox1, GA20ox3, GA3ox1, OsGAMYB genes and target genes thereof, namely OsCYP703A3, in osmkk 70 osmkk 62 double mutant plants and osmkk 70 osmkk 62 osmkk 55 triple mutant plants, rice and control materials thereof under normal and low-temperature treatment conditions. The results show that low temperature treatment can cause the expression of GA related genes to be reduced, and the biosynthesis of 4 GAs in the knocked-out double-mutant and triple-mutant riceThe expression level of the formed gene, the GAYB gene and the target gene OsCYP703A3 under the low-temperature treatment condition is obviously higher than that of a control material, which shows that OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 thereof can negatively regulate GA biosynthesis in rice.
The experimental results show that the OsMKKK70 and the homologous genes OsMKKK62 and OsMKKK55 thereof can negatively regulate the GA content in the rice anther, so that the cold tolerance of the rice at the booting stage can be negatively regulated.
Sequence listing
<110> institute of geography and agroecology of northeast China academy of sciences
<120> application of rice kinase gene OsMKKK70 and homologous gene thereof in negative regulation of cold tolerance of rice at booting stage
<141> 2022-05-31
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gttacttctg cactaggtac catggctaag cagctcaggc 40
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcttagaatt cccggggatc ctcagcatgt ggttgccaat t 41
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggcacccgaa atccgcgatc ttggccc 27
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aaacgggcca agatcgcgga tttcggg 27
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<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gccgggcaat ggctaagcag ctc 23
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaacgagctg cttagccatt gcct 24
<210> 7
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttgccgacc atgatgttcc tggcctt 27
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aaacaaggcc aggaacatca tggtcgg 27
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
catctccggc aaatcct 17
<210> 10
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccagcctcga catcgc 16
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aataccgcca catgggggag gt 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtagtggttc agccgcatca cc 22
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gacgattcac ctcaacatgt tccct 25
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggctctgcag gatgaaggtg aa 22
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggaggacgga caacgagat 19
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gagtgcggga actggaaga 19
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agacttcgct cgattctgca c 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gctttagccc acacaaactg a 21
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
agaccttcaa cacccctgct atg 23
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tcacgcccag caaggtcg 18
Claims (7)
1. The application of the rice kinase gene OsMKKK70 and the homologous gene thereof in negative regulation of cold tolerance of rice at the booting stage is characterized in that the pollen fertility of transgenic rice over-expressed by the OsMKKK70 gene is poor and the maturing rate is reduced.
2. The application of the rice kinase gene OsMKKK70 and homologous genes thereof in negative regulation of cold resistance of rice at the booting stage is characterized in that pollen fertility and setting percentage are higher than those of a wild type after gene knockout double mutants of OsMKKK70 and homologous genes OsMKKK62 of the rice are subjected to low-temperature treatment.
3. The use according to claim 2, characterized in that OsMKKK70 and its homologous gene OsMKKK62 are knocked out to obtain transgenic plants with improved cold tolerance in rice booting stage.
4. The rice kinase gene OsMKKK70 and the homologous gene thereof are applied to negative regulation of cold tolerance of rice at the booting stage, and are characterized in that pollen fertility and setting percentage of gene knockout triple mutants of OsMKKK70 and the homologous gene OsMKKK62 and OsMKKK55 are higher than those of wild type after low-temperature treatment.
5. The use according to claim 4, characterized in that OsMKKK70 and its homologous genes OsMKKK62 and OsMKKK55 are knocked out to obtain transgenic plants with improved cold tolerance in rice booting stage.
6. The application of the rice kinase gene OsMKKK70 and the homologous gene thereof in negative regulation of cold tolerance of rice at the booting stage is characterized in that the GA content in a gene knockout double-mutant anther of the OsMKKK70 and the homologous gene OsMKKK62 is higher than that of a wild type.
7. The application of the rice kinase gene OsMKKK70 and homologous genes thereof in negative regulation of cold tolerance of rice at the booting stage is characterized in that GA content in gene knockout triple-mutant anthers of OsMKKK70 and homologous genes OsMKKK62 and OsMKKK55 of the OsMKKK70 is higher than that of wild type.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006137574A1 (en) * | 2005-06-22 | 2006-12-28 | Incorporated Administrative Agency National Agriculture And Food Research Organization | Cold-hardy plant and method for development of the same |
| CN112592394A (en) * | 2021-01-07 | 2021-04-02 | 中国科学院东北地理与农业生态研究所 | Application of rice transcription factor OsWRKY53 in negative regulation of cold tolerance of rice in booting stage |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006137574A1 (en) * | 2005-06-22 | 2006-12-28 | Incorporated Administrative Agency National Agriculture And Food Research Organization | Cold-hardy plant and method for development of the same |
| CN112592394A (en) * | 2021-01-07 | 2021-04-02 | 中国科学院东北地理与农业生态研究所 | Application of rice transcription factor OsWRKY53 in negative regulation of cold tolerance of rice in booting stage |
Non-Patent Citations (3)
| Title |
|---|
| ENYANG MEI ET AL.: "OsMKKK70 Negatively Regulates Cold Tolerance at Booting Stage in Rice" * |
| MARKUS TEIGE ET AL.: "The MKK2 Pathway Mediates Cold and Salt Stress Signaling in Arabidopsis" * |
| ZHIQI LIU ET AL.: "OsMKKK70 regulates grain size and leaf angle in rice through the OsMKK4‐OsMAPK6‐OsWRKY53 signaling pathway" * |
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