CN111778265B - Mutant gene, mutant, expression vector and application of zearalenone oxidase - Google Patents
Mutant gene, mutant, expression vector and application of zearalenone oxidase Download PDFInfo
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Abstract
The invention is applicable to the technical field of molecular biology and provides a mutant gene, a mutant, an expression vector and application of zearalenone oxidase, wherein the nucleotide sequence of an original gene of the zearalenone oxidase is shown as a sequence table SEQ ID NO. 1, and the nucleotide sequence of the mutant gene is shown as a sequence table SEQ ID NO. 2 or SEQ ID NO. 3. The mutant gene of zearalanol oxidase provided by the embodiment of the invention can be used as a construction plant expression vector and transformed into crops such as arabidopsis thaliana, and the phenotype, the survival rate and other life states of transgenic arabidopsis thaliana are observed before and after drought stress treatment, and the result shows that the mutant gene of zearalanol oxidase can reduce the plant height of plants such as arabidopsis thaliana and improve the drought tolerance of plants such as arabidopsis thaliana.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a mutant gene, a mutant, an expression vector and application of zearalenone oxidase.
Background
Crops are often subjected to various abiotic stresses, such as drought, salt, low temperature, etc., which severely restrict the growing area of the crop and even lead to yield losses. Corn is an important food and economic crop and, although it has proven to be one of the most environmentally-compatible crops, corn growth and development and yield are still affected by a variety of abiotic stresses. In recent years, China's corn production is more and more seriously affected by regional and stage drought, which causes the production cost to be increased slightly and the yield and quality of the corn to be seriously reduced seriously.
Recent researches find that gibberellin metabolism regulation directly participates in the response and regulation processes of plants for tolerating various abiotic stresses such as drought, low temperature, high salt and weak light, and the tolerance of the plants to the abiotic stresses can be improved through genetic improvement on a few gibberellin metabolism regulation genes. These findings open up a new idea for the study of drought-enduring mechanisms in maize.
ZmGA2ox6 is a GA2ox type negative regulator gene in the synthesis pathway of zearalenone. The expression product (GA 2-oxidases) of the GA2ox gene can regulate and control the content of gibberellin in plants by degrading gibberellin and precursor substances thereof, and further influence various properties of plant types, yield, stress resistance, disease resistance and the like of the plants. Detailed reports of the involvement of GA2 ox-like genes in abiotic stress tolerance responses in plants began with the study of Arabidopsis ddf1 mutants. The AtGA2ox7 gene in the ddf1 mutant is up-regulated and expressed under the condition of salt stress, so that the gibberellin level is obviously reduced, and the survival probability of the plant under the condition of salt stress is finally improved by reducing a series of physiological reactions such as the plant growth rate, the plant leaf area growth rate and the like. Studies in Arabidopsis have also found that AtGA2ox6 and AtGA2ox7 are involved in a similar manner in the response to water stress and cold stress, respectively. The rice OsGA2ox5 gene is over-expressed in Arabidopsis, and the salt tolerance of Arabidopsis is also obviously improved. In addition, AtGA2ox3 and AtGA2ox6 were also involved in the response process of Arabidopsis thaliana to low temperature stress.
In the study on wild emmer ssp dicoccudes, researchers analyzed the change of transcription profiles of wild emmer ssp using a chip technique, and found that the GA2ox gene in roots was down-regulated in drought stress, while the GID1 gene was up-regulated in expression. The result shows that the possible drought tolerance response mechanism in the plant is that the GA2ox gene down-regulated expression leads to the reduction of gibberellin degradation in roots, so that the gibberellin content is increased; meanwhile, the expression of the GID1 gene is up-regulated, so that the GA-GID1 complex in the plant body is increased, the degradation induction effect of the plant body on DELLAs is enhanced, the growth inhibition effect of the DELLAs is weakened, the root elongation is promoted to obtain more water, and the drought tolerance of the plant body is improved. The transcriptional spectrum analysis of different organs under the drought stress of rice also finds a result similar to that of wild emmer. Under drought stress, GA2ox gene expression was significantly down-regulated in rice roots. Although the expression of GID1 gene was not detected up-regulated at the same time, the involvement of GA2ox gene in plant drought-tolerant response was further confirmed. In the research, the GA2ox gene expression in rice leaves under drought stress is obviously up-regulated, contrary to that in roots. Similar results were obtained in other plant leaves or aerial parts studies, i.e., drought induced up-regulation of GA2 ox-like gene expression. For example, in a related study of bread fruits (Artocarpus altis), it was found that all 4 GA2ox genes in the plant are significantly up-regulated under drought stress; for another example, in the process of analyzing the leaf transcription profile of the corn seedling stage, the subject group also finds that the expression of GA2ox genes is obviously up-regulated after drought stress; in addition, the GA2ox gene is overexpressed in the poplar by using a transgenic method, and the drought resistance of transgenic offspring materials is also obviously improved. The results show that the GA2ox gene is not only involved in plant drought tolerance response, but also has difference of expression regulation mechanism in different tissues and organs.
Since the expression change of the GA2ox gene can affect the gibberellin content, most genetic improved materials related to the GA2ox gene have obvious plant morphological change, even the fertility of the plant is affected, and the application of the GA2ox gene is not favorable. However, the overexpression of the rice OsGA2ox6 gene mutant discovered in 2010 not only obtains excellent traits for semi-dwarfing plants, but also eliminates the bad traits of plant sterility and the like caused by the overexpression of OsGA2ox 6. In 2017, in the research on point mutation of different sites of the OsGA2ox6 gene, rice over-expressing wild-type OsGA2ox6 gene is completely dwarf, while rice over-expressing OsGA2ox6 gene containing A141E site mutation shows half dwarf character, and the drought resistance, disease resistance and yield of the rice are all obviously improved. This indicates that there is a GA2ox allele resource available in the natural material or mutant population. At present, no report of improving the drought resistance of plants by using ZmGA2ox genes and alleles thereof is found in maize.
Disclosure of Invention
The present invention provides a mutant gene of zearalenone oxidase, and aims to solve the problems of the background art.
The embodiment of the invention is realized by that the nucleotide sequence of the mutant gene of the zearalenone oxidase is shown as a sequence table SEQ ID NO. 2 or SEQ ID NO. 3. Wherein, the mutant gene with the nucleotide sequence shown as the sequence table SEQ ID NO. 2 is named as ZmGA2ox 6-E144A; the mutant gene with the nucleotide sequence shown in the sequence table SEQ ID NO. 3 is named as ZmGA2ox 6-A145E.
As a preferable scheme of the embodiment of the invention, the nucleotide sequence of the original gene of the zearalenone oxidase is shown as a sequence table SEQ ID NO. 1, the nucleotide sequence is named as ZmGA2ox6, the total length is 1089b p, the initiation codon is ATG, and the termination codon is TAA.
On the basis, a Fast Mutagenesis System is utilized to carry out site-directed Mutagenesis on the ZmGA2ox6 gene, 431 bit A is mutated into C, 434 bit C is mutated into A, and the obtained sequences are named as the mutant genes of ZmGA2ox6-E144A and ZmGA2ox6-A145E respectively, and the sequences are shown as SE Q ID NO 2 and SEQ ID NO 3 respectively.
Another objective of the embodiments of the present invention is to provide a mutant of zearalanol oxidase encoded by the above mutant gene.
As another preferable scheme of the embodiment of the invention, the amino acid sequence of the zearalenone oxidase is shown as SEQ ID NO. 4 in the sequence table, the zearalenone oxidase consists of 362 amino acids, the protein belongs to the family of 2ODDs (2-oxogluterate-dependent dioxygenases), and the 2ODDs in different species such as corn, rice, sorghum, arabidopsis thaliana and the like are subjected to phylogenetic analysis to find that a plurality of branches exist, which indicates that the functions of the two may be different. Whereas the relationship of ZmGA2ox6 to SbGA2ox6 (sorghum) and AtG A2ox8 (Arabidopsis) was recent.
As another preferable scheme of the embodiment of the invention, the amino acid sequence of the mutant is shown in a sequence table S EQ ID NO. 5 or SEQ ID NO. 6.
It is another object of the embodiments of the present invention to provide an expression vector containing the above-mentioned mutant gene.
In the embodiment of the invention, three recombinant plant expression vectors are provided, which respectively contain the Z mGA2ox6 and the mutant genes ZmGA2ox6-E144A and ZmGA2ox 6-A145E.
Specifically, according to the ORF sequences of ZmGA2ox6 and its mutant genes ZmGA2ox6-E144A and ZmGA2ox6-A145E and the related information of the multiple cloning site of the intermediate vector pCHF3300, corresponding enzyme cutting sites are added to amplify ZmGA2ox6 and its mutant genes ZmGA2ox6-E144A and ZmGA2ox6-A145E during the design of primers. The accurate ZmGA2ox6 and its mutant gene ZmGA2ox6-E144A and ZmGA2ox6-A145E which are verified by sequencing are cut by corresponding restriction enzymes, and the small fragment (gene) is recovered. Meanwhile, the expression vector pART-CAM is cut by the same restriction enzyme, and a large fragment (vector) is recovered. The In-fusion kit connects the recovered large and small fragments and recombines the fragments into plant expression vectors of ZmGA2ox6 gene, ZmGA2o x6-E144A gene and ZmGA2ox6-A145E gene respectively. And then transforming the recombinant vector into escherichia coli, extracting plasmids, and performing PCR and enzyme digestion identification.
Another object of the embodiments of the present invention is to provide an application of the above mutant gene in improving plant type of plants.
As another preferred embodiment of the present invention, the plant is Arabidopsis thaliana.
Another objective of the embodiments of the invention is to provide an application of the mutant gene in improving drought tolerance of plants.
As another preferred embodiment of the present invention, the plant is Arabidopsis thaliana.
Specifically, the above-mentioned plant expression vectors containing the genes ZmGA2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E can be introduced into Arabidopsis thaliana by the dipping method, and T can be obtained by several generations of kanamycin screening and molecular identification3Transgenic arabidopsis plants. For homozygous T3The transgenic arabidopsis thaliana plants are subjected to plant type identification and drought tolerance analysis, and the result shows that the over-expression plants are obviously shortened, and the drought tolerance is obviously higher than that of wild plants.
The mutant gene of zearalanol oxidase provided by the embodiment of the invention can be used as a construction plant expression vector and transformed into crops such as arabidopsis thaliana, and the phenotype, the survival rate and other life states of transgenic arabidopsis thaliana are observed before and after drought stress treatment, and the result shows that the mutant gene of zearalanol oxidase can reduce the plant height of plants such as arabidopsis thaliana and improve the drought tolerance of plants such as arabidopsis thaliana.
Drawings
FIG. 1 is a multiple sequence comparison of maize ZmGA2ox6 with other plant GA2ox amino acids. Wherein: SbGA2ox6 is sorghum; AtGA2ox8 was Arabidopsis thaliana.
FIG. 2 is a schematic diagram of phylogenetic tree analysis of ZmGA2ox type genes in maize. Wherein: OsGA2ox is rice; the AtGA2ox family is arabidopsis thaliana.
FIG. 3 shows rosette leaf development of Arabidopsis thaliana material overexpressing ZmGA2ox6 and its mutant genes ZmGA2ox6-E144A and ZmGA2ox 6-A145E.
FIG. 4 shows different T's overexpressing the ZmGA2ox6-A145E gene3Comparison of the height of the generation material and the wild-type material strain.
FIG. 5 shows the overexpression of ZmGA2ox6-E144A gene T3Comparative plots of the generation material after drought stress versus wild type material.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
This example provides a cloning method of the original gene ZmGA2ox6 of zearalenone oxidase and a mutant gene thereof.
The cloning method of the original gene ZmGA2ox6 of the zearalenone oxidase comprises the following steps:
s1, RNA extraction: the maize inbred line B73 was cultured to the trilobate stage, and the total RNA of the leaves was extracted using the Kangshiji ultra pure R NA extraction kit (CW 0581). The concrete steps are as follows:
s11, taking fresh corn leaves, fully grinding in liquid nitrogen, adding 1mL of TRIzo reagent into each 40mg of tissue, and fully and uniformly mixing.
S12, the mixed sample is gently inverted up and down for several times, and the mixture is placed at room temperature for 5min to ensure that the sample is fully cracked.
S13, adding 200 mu L chloroform, covering the centrifuge tube cover, violently shaking for 15S, and standing for 2min at room temperature.
S14, the mixture was centrifuged at 12000rpm at 4 ℃ for 10min, 550. mu.L of the upper aqueous phase was aspirated, and the upper aqueous phase was transferred to a new RNase-Free centrifuge tube.
S15, 550. mu.L of 70% ethanol (prepared without RNase water) is added to the aqueous solution, and the mixture is inverted and mixed.
S16, adding all the solution obtained in the previous step into an adsorption column (Spin columns RM) filled with a collection tube. If the solution can not be added at one time, the solution can be transferred for many times. Centrifuging at 12000rpm for 20s, pouring the waste liquid out of the collecting tube, and replacing the adsorption column into the collecting tube again.
S17, adding 700 mu L of Buffer RW1 into the adsorption column, centrifuging at 12000rpm for 20S, pouring the waste liquid in the collection tube, and replacing the adsorption column in the collection tube again.
S18, adding 500 μ L Buffer RW2 (checking whether absolute ethyl alcohol is added before use) into the adsorption column, centrifuging at 12000rpm for 20S, pouring off the waste liquid in the collection tube, and replacing the adsorption column in the collection tube again.
And S19, repeating the step S18.
S110, centrifuging at 12000rpm for 2min, and pouring waste liquid in the collecting pipe. The column was left at room temperature for several minutes and thoroughly dried.
S111, placing the adsorption column in a new RNase-Free centrifuge tube, adding 40 mu L of RNase-Free Water into the middle part of the adsorption column, placing for 1min at room temperature, centrifuging at 12000rpm for 1min, collecting RN A solution, and storing RNA at-80 ℃ to prevent degradation.
S2, reverse transcription: PrimeScript from Bao Bio IncTMThe extracted RNA was reverse transcribed with RT reagent Kit with gDNA Eraser (RR 047A).
Wherein, the reaction system for removing gDNA is shown in the following table 1:
TABLE 1
| Reagent | Volume of |
| 5×gDNA Eraser Buffer | 2μL |
| gDNA Eraser | 1μL |
| Total RNA | 1μg |
| RNase Free Water | up to 10μl |
The premix, which was mixed as in Table 1, was incubated at 42 ℃ for 2min and then at 4 ℃ for reverse transcription for 5 min. The reverse transcription reaction adopts SYBR Green qPCR method, and the specific reverse transcription reaction system is as follows 2:
TABLE 2
| Reagent | Volume of |
| Premix of Table 1 | 10μL |
| rime Soript RT Enzyme MixⅠ | 1μL |
| RT Primer Mix | 1μL |
| 5× |
4μL |
| RNase Free Water | 4μL |
The whole reverse transcription reaction system in the table 2 is put into a PCR instrument, and the program is set to 37 ℃ for 15 min; 85 ℃ for 5 s.
Amplification of the full length of ORF of S3 and ZmGA2ox6 genes:
according to the ORF gene sequence of corn ZmGA2ox6 published by NCBI, a bioinformatics software Primer 5.0 is used to design a specific cloning Primer of the gene according to the Primer design principle, as follows:
FP: 5'-ATGCGTTACGTAGCTGCCACTCC-3' (shown in SEQ ID NO:7 of the sequence Listing);
RP: 5'-TTAGGCGGGCCGTGATTGAGGGG-3' (shown in SEQ ID NO:8 of the sequence list).
The cDNA obtained by the reverse transcription is taken as a template, and ZmGA2ox6 is cloned by using high-fidelity heat-resistant DNA polymerase PrimeST AR GXL DNA polymerase, the reaction system is shown in Table 3, and the reaction program is shown in Table 4. Note: if the cDNA concentration is too low, the amount of cDNA used may be increased appropriately. If the target band of the first PCR is too shallow, the recovered product can be used to continue the PCR in the same procedure, the reaction system is shown in Table 3, and the reaction procedure is shown in Table 4:
TABLE 3
TABLE 4
Recovery of DNA fragments of S4, ZmGA2ox6 and their point mutations:
the recovery of ZmGA2ox6 target fragment is carried out by using a raw SanPrep column type DNA glue recovery kit, which comprises the following steps:
s41, cutting a gel block containing ZmGA2ox6 mesh fragments after electrophoresis, weighing, and placing in a 1.5mL centrifuge tube. According to the weight of the gel block, 300 mu L of Buffer B2 is added for each 100mg of gel.
And S42, putting the centrifuge tube into a metal bath at 50 ℃ for 10min, and turning the centrifuge tube upside down for several times during the process to uniformly mix the melted liquid and the unmelted glue blocks and accelerate the melting.
S43, placing the obtained solution in an adsorption column and centrifuging the solution at 8000rpm for 30S. If the total volume of the solution is more than 750. mu.L, 750. mu.L of the solution is added each time, and the operation is repeated for a plurality of times.
S44, 300. mu.L of Buffer B2 was added to the adsorption column, the rotation speed was 9000rpm, the mixture was centrifuged for 30 seconds, and then the waste liquid was poured out.
S45, 500. mu.L of Buffer B2 was added to the adsorption column, and the rotation speed was 9000rpm and the centrifugation time was 30S. The waste liquid is decanted. And repeated again.
S46, putting the empty adsorption column and the collection tube into a centrifuge, and centrifuging at 9000rpm for 60S. A new 1.5mL centrifuge tube was placed in the adsorption column and allowed to air for 10 min.
S47, adding 30 mu L of TE buffer or ddH in the center of the adsorption film2O, standing for 2min at room temperature, and centrifuging for 60s at 9000 rpm. The DNA solution obtained in this step was stored in a refrigerator at-20 ℃ or used for subsequent tests.
S48, site-directed Mutagenesis was performed using the Fast Mutagenesis System kit of all-Kaiki GmbH using the recovered DNA as a template. Mutation of 431-bit A to C and 434-bit C to A, respectively. The obtained sequences are named as ZmGA2ox6-E144A and ZmGA2ox6-A145E respectively, the nucleotide sequences are shown as sequence tables SEQ ID NO:2 and SEQ ID NO:3, and the manufactured SanPrep column type DNA gel recovery kit is used for recovering ZmGA2ox6-E144A and ZmGA2ox6-A145E target fragments.
S5, ZmGA2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E are respectively connected into pMD18-T Vector:
the target fragment was ligated with pMD18-T Vector using the DNA A-labeling Kit of TaKaRa and pMD18-T Vector Cloning Kit to obtain a recombinant Vector for gene sequencing.
S51, carrying out an A addition reaction on the 3' end of the DNA fragments recovered from the three target fragment gels.
(1) The following ligation reaction systems were prepared in 200 μ L centrifuge tubes as in table 5 below:
TABLE 5
(2) The ligation reaction system in Table 5 was mixed well and reacted at 72 ℃ for 20 min.
(3) After the reaction is finished, the mixture is placed in ice and stands for 2min to obtain the A-Tailing DNA fragment.
S52, the A-labeling DNA fragment was ligated with pMD18-T vector. Wherein, the p MD18-T connection reaction system is shown in the following table 6:
TABLE 6
| Components | Volume of |
| pMD18-T Vector | 1μL |
| A-Tailing DNA | 4μL |
| Solution I | 5μL |
The reaction procedure is as follows: reacting at 16 ℃ for 1 h.
S6, Top10 Escherichia coli competence transformation and PCR detection:
s61, 50. mu.L of TOP10 E.coli competent cells were thawed on ice.
S62, pipette 5. mu.L of the ligation product obtained in step S5 into 50. mu.L of TOP10 E.coli competent cells.
S63, ice-cooling for 30min, heat-shocking for 90S at 42 ℃ and ice-cooling for 5 min. Subsequently, 800. mu.L of LB liquid medium was added.
S64, shake culturing at 37 ℃ for 1h, and centrifuging at 8000rpm for 5 min. The supernatant was discarded, leaving approximately 50. mu.L of medium in the centrifuge tube. And (3) uniformly blowing and mixing by using a pipette, coating the mixture on an LB solid culture medium containing corresponding antibiotics, keeping the temperature at 37 ℃, and performing inverted culture for 14 hours.
S65, picking up single colony in 800 microliter LB liquid culture medium containing corresponding antibiotic, placing the centrifuge tube into a shaker, culturing at 37 deg.C and 180rpm for about 10 h.
S66, using Ex Taq enzyme to perform PCR molecular detection on the bacterial liquid. And (3) amplifying and shaking the bacterial liquid with the positive PCR result, adding 15% of glycerol into the bacterial liquid, sending the bacterial liquid to Huada Gen company for sequencing, and storing the original bacterial liquid in a refrigerator at the temperature of-80 ℃.
Example 2
This example provides two zearalenone oxidase mutants, which are encoded by the above-mentioned mutant genes ZmGA2ox6-E144A and ZmGA2ox6-A145E, respectively, and the corresponding amino acid sequences are shown in the sequence tables SEQ ID NO. 5 and SEQ ID NO. 6.
In addition, ZmGA2ox6 gene is used to code zearalenone oxidase, the open reading frame of which has 1089bp nucleotides and codes 362 amino acids, and the amino acid sequence of the obtained zearalenone oxidase is shown in the sequence table SEQ ID NO. 4.
The TMHMM Server v.2.0 online website is used for preliminarily predicting the transmembrane domain of the protein, and the result shows that the transmembrane domain does not appear in the protein, so that ZmGA2ox6 is preliminarily presumed to be a non-transmembrane protein.
As shown in FIGS. 1 and 2, the zearalenone oxidase ZmGA2ox6 protein belongs to the family of 2ODDs (2-ox-dependent dioxigens), and by carrying out phylogenetic analysis on the 2ODDs proteins in different species such as corn, rice, sorghum, Arabidopsis thaliana and the like, a plurality of branches are found, which indicates that the functions of the two may be different. Whereas the affinity of ZmGA2ox6 to SbGA2ox6 (sorghum) and AtGA2ox8 (Arabidopsis) was recent. Relevant studies have demonstrated that L-S-W-S-E-A motif is an important domain affecting the binding of C20-GA2ox genes to the substrate GA12/GA 53. Thus, point mutations were made to potential functional sites in ZmGA2ox6 (E144A and a 145E).
Example 3
This example provides three recombinant plant expression vectors, which contain the aforementioned ZmGA2ox6 and its mutant gene ZmGA2ox6-E144A and ZmGA2ox6-A145E, respectively. The construction method of the recombinant plant expression vector comprises the following steps:
s1, adding corresponding enzyme cutting sites during primer design according to corresponding sequences at the multiple cloning sites of the intermediate vector.
S2, determining the correctness of the expressed gene through a series of steps of recovering, adding A, connecting with T vector, transforming, PCR enzyme digestion identification, sequencing and the like, wherein the gene amplified by the high fidelity enzyme has a complete open reading frame, no mismatch and no frame shift.
S3, the gene with correct sequence is cut by enzyme with the designed enzyme cutting site, and the small fragment (gene) is recovered.
Specifically, restriction endonucleases Psp XI and Xba I were selected, and the correctly sequenced target fragments were excised from the intermediate vector, and the digestion system was shown in Table 7 and Table 8. Because the two enzymes have different enzyme cutting temperatures, one enzyme is used for enzyme cutting, and then the enzyme cutting product is recovered and then the other enzyme is used for enzyme cutting. The Psp XI cleavage system is shown in the following table 7, and the reaction procedure is as follows: water bath at 30 ℃ for 1 h.
TABLE 7
| Components | Volume of |
| Psp XI | 2.5μL |
| 10×T Buffer | 5μL |
| 0.1％BSA | 5μL |
| DNA or vector | 20μL |
| Sterilization water | 17.5μL |
Xba I was digested as follows in Table 8, and the reaction sequence was as follows: water bath at 37 ℃ for 1 h.
TABLE 8
S4, the expression vector pCAMBIA-3301 is cut by the two same restriction enzymes, and the large fragment (vector) is recovered.
S5, connecting the recovered large fragment with the small fragment, then transforming the competence of the escherichia coli, extracting the plasmid, and carrying out PCR and enzyme digestion identification. Specifically, the small fragments (ZmG A2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E) digested from the intermediate vector and the large fragment of pCAMBIA-3301 vector are connected for 3h at 16 ℃ to obtain the corresponding recombinant plant expression vector, wherein the connection system is shown in Table 9.
TABLE 9
| Components | Volume of |
| Small fragment of interest | 5.5μL |
| Vector large fragment | 2.5μL |
| 10×T4 ligase buffer | 1μL |
| T4 DNA ligase | 1μL |
Example 4
This example provides the acquisition and molecular testing of transgenic Arabidopsis thaliana genes ZmGA2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E, which is carried out by transformation of the genes with the dipping method, as follows:
s1, inverting flowering Arabidopsis to make flower buds face downwards, and soaking in Agrobacterium liquid for 2 min.
S2, the transformed Arabidopsis plants are flatly placed, covered with a preservative film, grown for 24 hours under low light intensity, placed under normal illumination conditions for culture and growth, and infected once again after one week.
S3, the transformed plants can normally blossom and grow, and seeds can be harvested when the hornberries are completely withered and yellow and are about to crack.
S4, harvested fraction T0The generation seed is screened by kanamycin and identified by PCR to obtain T1Generating transgenic plants, and obtaining T through twice generation addition3Can be used for subsequent phenotype screening for Arabidopsis thaliana plants.
Example 5
This embodiment provides T3Drought stress treatment and growth vigor determination of ZmGA2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E transgenic Arabidopsis thaliana:
the wild Arabidopsis thaliana and the transgenic Arabidopsis thaliana seeds obtained in example 4 were randomly sown, and then cultured under the illumination condition of 22 ℃. And observing the germination condition, counting the germination rate, and repeating the experiment for three times. The experimental results are shown in the attached figures 3-5.
As can be seen from FIGS. 3 and 4, the plant morphology of the Arabidopsis progeny material overexpressing the ZmGA2ox6 gene is severely affected, the rosette leaves become smaller and severely dwarfed; the phenotype of two materials of over-expression ZmGA2ox6-E144A and ZmGA2ox6-A145E is not seriously influenced, the rosette leaves are slightly smaller than wild type, can normally develop and fruit, and the plant height has a half-dwarf phenotype; it is shown that overexpression of the ZmGA2ox6 gene significantly affects GA biosynthesis, and further affects the phenotype of Arabidopsis; on the other hand, the ZmGA2ox6-E144A and ZmGA2ox6-A145E have reduced catalytic effect of the products due to the modification of key sites, so that the decomposition effect of the products on GA synthesis precursor substances is reduced, the biosynthesis inhibition effect on GA is reduced, and the semi-dwarf trait is generated.
In addition, as can be seen from FIG. 5, the survival rate of rehydration of the material over-expressing ZmGA2ox6-E144A after 25 days of drought stress reached about 90%; the survival rate of the material which over-expresses ZmGA2ox6-A145E after being rehydrated reaches about 75 percent, which is obviously higher than that of the control material after being rehydrated by about 25 percent. The drought tolerance of the transgenic arabidopsis plant is relatively high, the drought tolerance of the arabidopsis can be improved by over-expressing the ZmGA2ox6-E144A gene and the ZmGA2ox6-A145E gene, and the drought tolerance of the transgenic arabidopsis plant is obviously higher than that of a non-transgenic arabidopsis material.
In conclusion, the invention obtains a gibberellin oxidase gene ZmGA2ox6 from corn, and carries out mutation on specific sites on a key structural domain of the gibberellin oxidase gene ZmGA2ox6 according to the protein structure of the gibberellin oxidase gene, so as to obtain point mutant genes Zm GA2ox6-E144A and ZmGA2ox 6-A145E. Through agrobacterium-mediated transformation method, plant expression vectors respectively containing ZmGA2ox6, ZmGA2ox6-E144A and ZmGA2ox6-A145E are successfully transformed into Arabidopsis thaliana to obtain homozygous T3Transgenic Arabidopsis plants were generated. The results show that the plant morphology of several transgenic Arabidopsis thaliana is obviously changed. Indicating that the ZmGA2ox6 and the point mutation gene thereof can change the plant type of Arabidopsis. Among them, overexpression of two point mutation genes ZmGA2ox6-E144A and ZmGA2ox6-A145E can cause the appearance of a semi-dwarf trait. Transgenic Arabidopsis thaliana, particularly transgenic ZmG A2ox6-E144A and ZmGA2ox6-A145E gene material, showed higher survival rates under drought stress. The over-expression of two point mutation genes ZmGA2ox6-E144A and ZmGA2ox6-A145E of the zeatin oxidase ZmGA2ox6 gene can obviously improve the drought tolerance of arabidopsis.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Jilin province academy of agricultural sciences
<120> mutant gene, mutant, expression vector and application of zearalenone oxidase
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1089
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcgttacg tagctgccac tccgaccatg ccgtcccttg tcgcggagag cgccgccgaa 60
ccgcctctgg tggacagcta cctggagctg ctccggcgcg gcggcggcgg cggcggcatt 120
gcggcggcga ccgagggctg cgtgcaggag cgcgagctgc ccttgatcga cctgacgtgc 180
ctgcagggca gcgcgggcga ggcggcgagg acgacgtgcg cggacgccat ggcgagggcg 240
gcctcggagt ggggcttttt ccaggtgacc gggcacggcg tgagccgggc gctgttggag 300
cggctgcggg cggagcaggc gcggctgttc cggctgccgt tcgaaaccaa ggccaaggcc 360
gggcttctca acggctccta ccgctggggc gcccccacgg ccacgtcgct ccgccacctc 420
tcgtggtcgg aggcgttcca cgtcccgctc gccagcatct ccggcactgc ctgcgacttc 480
ggagagctca gctccttgag ggacgtggtg caggaggtgg cggacgcgat gtcgcgggtg 540
gccaagaccg tggcggtggc gctggcgggg agccttctgg gccacgacga ggcggcggcg 600
ttcccggcgg ggtgcggcga gaccacctgc tacctgcggc tcaatcggta cccggcgtgc 660
ccgttcgcgg cgaacacctt cgggctggtg ccccacacgg acagcgactt cctgacggtg 720
ctgtcccagg accaggtcgg gggcctgcag ctcatgacgg acgccggctg ggtggccgtc 780
aagccccgcc ccgacgcgct catcgtcaac atcggcgatc tgtttcaggc ctggagcaac 840
aacctgtaca agagcgtgga gcacaaggtg gtggccaacg ccgcggcgga gcgcttctcg 900
gcggcctact tcctgtgccc gtcctacgac tcgctcgtcg gcacgtgcgg cgagccgtca 960
ccgtacagag acttcacctt cggggagtac aggaggaagg tgcaggagga cgtcaagagg 1020
accggaagaa agattgggct ccccaacttt ctcaaacacc ggccaccccc tcaatcacgg 1080
cccgcctaa 1089
<210> 2
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atgcgttacg tagctgccac tccgaccatg ccgtcccttg tcgcggagag cgccgccgaa 60
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gcggcggcga ccgagggctg cgtgcaggag cgcgagctgc ccttgatcga cctgacgtgc 180
ctgcagggca gcgcgggcga ggcggcgagg acgacgtgcg cggacgccat ggcgagggcg 240
gcctcggagt ggggcttttt ccaggtgacc gggcacggcg tgagccgggc gctgttggag 300
cggctgcggg cggagcaggc gcggctgttc cggctgccgt tcgaaaccaa ggccaaggcc 360
gggcttctca acggctccta ccgctggggc gcccccacgg ccacgtcgct ccgccacctc 420
tcgtggtcgg cggcgttcca cgtcccgctc gccagcatct ccggcactgc ctgcgacttc 480
ggagagctca gctccttgag ggacgtggtg caggaggtgg cggacgcgat gtcgcgggtg 540
gccaagaccg tggcggtggc gctggcgggg agccttctgg gccacgacga ggcggcggcg 600
ttcccggcgg ggtgcggcga gaccacctgc tacctgcggc tcaatcggta cccggcgtgc 660
ccgttcgcgg cgaacacctt cgggctggtg ccccacacgg acagcgactt cctgacggtg 720
ctgtcccagg accaggtcgg gggcctgcag ctcatgacgg acgccggctg ggtggccgtc 780
aagccccgcc ccgacgcgct catcgtcaac atcggcgatc tgtttcaggc ctggagcaac 840
aacctgtaca agagcgtgga gcacaaggtg gtggccaacg ccgcggcgga gcgcttctcg 900
gcggcctact tcctgtgccc gtcctacgac tcgctcgtcg gcacgtgcgg cgagccgtca 960
ccgtacagag acttcacctt cggggagtac aggaggaagg tgcaggagga cgtcaagagg 1020
accggaagaa agattgggct ccccaacttt ctcaaacacc ggccaccccc tcaatcacgg 1080
cccgcctaa 1089
<210> 3
<211> 1089
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcgttacg tagctgccac tccgaccatg ccgtcccttg tcgcggagag cgccgccgaa 60
ccgcctctgg tggacagcta cctggagctg ctccggcgcg gcggcggcgg cggcggcatt 120
gcggcggcga ccgagggctg cgtgcaggag cgcgagctgc ccttgatcga cctgacgtgc 180
ctgcagggca gcgcgggcga ggcggcgagg acgacgtgcg cggacgccat ggcgagggcg 240
gcctcggagt ggggcttttt ccaggtgacc gggcacggcg tgagccgggc gctgttggag 300
cggctgcggg cggagcaggc gcggctgttc cggctgccgt tcgaaaccaa ggccaaggcc 360
gggcttctca acggctccta ccgctggggc gcccccacgg ccacgtcgct ccgccacctc 420
tcgtggtcgg aggagttcca cgtcccgctc gccagcatct ccggcactgc ctgcgacttc 480
ggagagctca gctccttgag ggacgtggtg caggaggtgg cggacgcgat gtcgcgggtg 540
gccaagaccg tggcggtggc gctggcgggg agccttctgg gccacgacga ggcggcggcg 600
ttcccggcgg ggtgcggcga gaccacctgc tacctgcggc tcaatcggta cccggcgtgc 660
ccgttcgcgg cgaacacctt cgggctggtg ccccacacgg acagcgactt cctgacggtg 720
ctgtcccagg accaggtcgg gggcctgcag ctcatgacgg acgccggctg ggtggccgtc 780
aagccccgcc ccgacgcgct catcgtcaac atcggcgatc tgtttcaggc ctggagcaac 840
aacctgtaca agagcgtgga gcacaaggtg gtggccaacg ccgcggcgga gcgcttctcg 900
gcggcctact tcctgtgccc gtcctacgac tcgctcgtcg gcacgtgcgg cgagccgtca 960
ccgtacagag acttcacctt cggggagtac aggaggaagg tgcaggagga cgtcaagagg 1020
accggaagaa agattgggct ccccaacttt ctcaaacacc ggccaccccc tcaatcacgg 1080
cccgcctaa 1089
<210> 4
<211> 362
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Arg Tyr Val Ala Ala Thr Pro Thr Met Pro Ser Leu Val Ala Glu
1 5 10 15
Ser Ala Ala Glu Pro Pro Leu Val Asp Ser Tyr Leu Glu Leu Leu Arg
20 25 30
Arg Gly Gly Gly Gly Gly Gly Ile Ala Ala Ala Thr Glu Gly Cys Val
35 40 45
Gln Glu Arg Glu Leu Pro Leu Ile Asp Leu Thr Cys Leu Gln Gly Ser
50 55 60
Ala Gly Glu Ala Ala Arg Thr Thr Cys Ala Asp Ala Met Ala Arg Ala
65 70 75 80
Ala Ser Glu Trp Gly Phe Phe Gln Val Thr Gly His Gly Val Ser Arg
85 90 95
Ala Leu Leu Glu Arg Leu Arg Ala Glu Gln Ala Arg Leu Phe Arg Leu
100 105 110
Pro Phe Glu Thr Lys Ala Lys Ala Gly Leu Leu Asn Gly Ser Tyr Arg
115 120 125
Trp Gly Ala Pro Thr Ala Thr Ser Leu Arg His Leu Ser Trp Ser Glu
130 135 140
Ala Phe His Val Pro Leu Ala Ser Ile Ser Gly Thr Ala Cys Asp Phe
145 150 155 160
Gly Glu Leu Ser Ser Leu Arg Asp Val Val Gln Glu Val Ala Asp Ala
165 170 175
Met Ser Arg Val Ala Lys Thr Val Ala Val Ala Leu Ala Gly Ser Leu
180 185 190
Leu Gly His Asp Glu Ala Ala Ala Phe Pro Ala Gly Cys Gly Glu Thr
195 200 205
Thr Cys Tyr Leu Arg Leu Asn Arg Tyr Pro Ala Cys Pro Phe Ala Ala
210 215 220
Asn Thr Phe Gly Leu Val Pro His Thr Asp Ser Asp Phe Leu Thr Val
225 230 235 240
Leu Ser Gln Asp Gln Val Gly Gly Leu Gln Leu Met Thr Asp Ala Gly
245 250 255
Trp Val Ala Val Lys Pro Arg Pro Asp Ala Leu Ile Val Asn Ile Gly
260 265 270
Asp Leu Phe Gln Ala Trp Ser Asn Asn Leu Tyr Lys Ser Val Glu His
275 280 285
Lys Val Val Ala Asn Ala Ala Ala Glu Arg Phe Ser Ala Ala Tyr Phe
290 295 300
Leu Cys Pro Ser Tyr Asp Ser Leu Val Gly Thr Cys Gly Glu Pro Ser
305 310 315 320
Pro Tyr Arg Asp Phe Thr Phe Gly Glu Tyr Arg Arg Lys Val Gln Glu
325 330 335
Asp Val Lys Arg Thr Gly Arg Lys Ile Gly Leu Pro Asn Phe Leu Lys
340 345 350
His Arg Pro Pro Pro Gln Ser Arg Pro Ala
355 360
<210> 5
<211> 362
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Arg Tyr Val Ala Ala Thr Pro Thr Met Pro Ser Leu Val Ala Glu
1 5 10 15
Ser Ala Ala Glu Pro Pro Leu Val Asp Ser Tyr Leu Glu Leu Leu Arg
20 25 30
Arg Gly Gly Gly Gly Gly Gly Ile Ala Ala Ala Thr Glu Gly Cys Val
35 40 45
Gln Glu Arg Glu Leu Pro Leu Ile Asp Leu Thr Cys Leu Gln Gly Ser
50 55 60
Ala Gly Glu Ala Ala Arg Thr Thr Cys Ala Asp Ala Met Ala Arg Ala
65 70 75 80
Ala Ser Glu Trp Gly Phe Phe Gln Val Thr Gly His Gly Val Ser Arg
85 90 95
Ala Leu Leu Glu Arg Leu Arg Ala Glu Gln Ala Arg Leu Phe Arg Leu
100 105 110
Pro Phe Glu Thr Lys Ala Lys Ala Gly Leu Leu Asn Gly Ser Tyr Arg
115 120 125
Trp Gly Ala Pro Thr Ala Thr Ser Leu Arg His Leu Ser Trp Ser Ala
130 135 140
Ala Phe His Val Pro Leu Ala Ser Ile Ser Gly Thr Ala Cys Asp Phe
145 150 155 160
Gly Glu Leu Ser Ser Leu Arg Asp Val Val Gln Glu Val Ala Asp Ala
165 170 175
Met Ser Arg Val Ala Lys Thr Val Ala Val Ala Leu Ala Gly Ser Leu
180 185 190
Leu Gly His Asp Glu Ala Ala Ala Phe Pro Ala Gly Cys Gly Glu Thr
195 200 205
Thr Cys Tyr Leu Arg Leu Asn Arg Tyr Pro Ala Cys Pro Phe Ala Ala
210 215 220
Asn Thr Phe Gly Leu Val Pro His Thr Asp Ser Asp Phe Leu Thr Val
225 230 235 240
Leu Ser Gln Asp Gln Val Gly Gly Leu Gln Leu Met Thr Asp Ala Gly
245 250 255
Trp Val Ala Val Lys Pro Arg Pro Asp Ala Leu Ile Val Asn Ile Gly
260 265 270
Asp Leu Phe Gln Ala Trp Ser Asn Asn Leu Tyr Lys Ser Val Glu His
275 280 285
Lys Val Val Ala Asn Ala Ala Ala Glu Arg Phe Ser Ala Ala Tyr Phe
290 295 300
Leu Cys Pro Ser Tyr Asp Ser Leu Val Gly Thr Cys Gly Glu Pro Ser
305 310 315 320
Pro Tyr Arg Asp Phe Thr Phe Gly Glu Tyr Arg Arg Lys Val Gln Glu
325 330 335
Asp Val Lys Arg Thr Gly Arg Lys Ile Gly Leu Pro Asn Phe Leu Lys
340 345 350
His Arg Pro Pro Pro Gln Ser Arg Pro Ala
355 360
<210> 6
<211> 362
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Arg Tyr Val Ala Ala Thr Pro Thr Met Pro Ser Leu Val Ala Glu
1 5 10 15
Ser Ala Ala Glu Pro Pro Leu Val Asp Ser Tyr Leu Glu Leu Leu Arg
20 25 30
Arg Gly Gly Gly Gly Gly Gly Ile Ala Ala Ala Thr Glu Gly Cys Val
35 40 45
Gln Glu Arg Glu Leu Pro Leu Ile Asp Leu Thr Cys Leu Gln Gly Ser
50 55 60
Ala Gly Glu Ala Ala Arg Thr Thr Cys Ala Asp Ala Met Ala Arg Ala
65 70 75 80
Ala Ser Glu Trp Gly Phe Phe Gln Val Thr Gly His Gly Val Ser Arg
85 90 95
Ala Leu Leu Glu Arg Leu Arg Ala Glu Gln Ala Arg Leu Phe Arg Leu
100 105 110
Pro Phe Glu Thr Lys Ala Lys Ala Gly Leu Leu Asn Gly Ser Tyr Arg
115 120 125
Trp Gly Ala Pro Thr Ala Thr Ser Leu Arg His Leu Ser Trp Ser Glu
130 135 140
Glu Phe His Val Pro Leu Ala Ser Ile Ser Gly Thr Ala Cys Asp Phe
145 150 155 160
Gly Glu Leu Ser Ser Leu Arg Asp Val Val Gln Glu Val Ala Asp Ala
165 170 175
Met Ser Arg Val Ala Lys Thr Val Ala Val Ala Leu Ala Gly Ser Leu
180 185 190
Leu Gly His Asp Glu Ala Ala Ala Phe Pro Ala Gly Cys Gly Glu Thr
195 200 205
Thr Cys Tyr Leu Arg Leu Asn Arg Tyr Pro Ala Cys Pro Phe Ala Ala
210 215 220
Asn Thr Phe Gly Leu Val Pro His Thr Asp Ser Asp Phe Leu Thr Val
225 230 235 240
Leu Ser Gln Asp Gln Val Gly Gly Leu Gln Leu Met Thr Asp Ala Gly
245 250 255
Trp Val Ala Val Lys Pro Arg Pro Asp Ala Leu Ile Val Asn Ile Gly
260 265 270
Asp Leu Phe Gln Ala Trp Ser Asn Asn Leu Tyr Lys Ser Val Glu His
275 280 285
Lys Val Val Ala Asn Ala Ala Ala Glu Arg Phe Ser Ala Ala Tyr Phe
290 295 300
Leu Cys Pro Ser Tyr Asp Ser Leu Val Gly Thr Cys Gly Glu Pro Ser
305 310 315 320
Pro Tyr Arg Asp Phe Thr Phe Gly Glu Tyr Arg Arg Lys Val Gln Glu
325 330 335
Asp Val Lys Arg Thr Gly Arg Lys Ile Gly Leu Pro Asn Phe Leu Lys
340 345 350
His Arg Pro Pro Pro Gln Ser Arg Pro Ala
355 360
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgcgttacg tagctgccac tcc 23
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttaggcgggc cgtgattgag ggg 23
Claims (5)
1. A mutant gene of zearalenone oxidase is characterized in that the nucleotide sequence of the mutant gene is shown in a sequence table SEQ ID NO. 2 or SEQ ID NO. 3.
2. A mutant zearalanol oxidase encoded by the mutant gene of claim 1.
3. An expression vector comprising the mutant gene of claim 1.
4. Use of the mutant gene of claim 1 to improve drought tolerance in a plant.
5. Use according to claim 4, wherein the plant is Arabidopsis thaliana.
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| CN112626113B (en) * | 2020-12-22 | 2022-06-24 | 吉林省农业科学院 | Method for creating maize dwarfing material by using gene editing technology |
| CN113817756A (en) * | 2021-10-12 | 2021-12-21 | 河南大学三亚研究院 | Application of corn Zm00001d045563 gene in regulation of establishment of corn plant type |
| CN114540529B (en) * | 2022-02-16 | 2023-07-14 | 吉林省农业科学院 | Corn genotype and functional molecular marker InDel-K2 for increasing yield of semi-dwarf and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607989A (en) * | 2008-06-20 | 2009-12-23 | 中国科学院遗传与发育生物学研究所 | A kind of rice dwarf-related protein and encoding gene thereof and application |
| CN107365795A (en) * | 2017-08-11 | 2017-11-21 | 吉林省农业科学院 | The method of application and cultivation resistance plant of the AtGA2ox1 genes in stress resistance of plant is adjusted |
| CN108728446A (en) * | 2017-04-25 | 2018-11-02 | 吉林省农业科学院 | Corn resistant gene of salt ZmHKT2;1 and its application |
| CN109844107A (en) * | 2016-08-17 | 2019-06-04 | 孟山都技术公司 | By manipulation gibberellin metabolism increase can crop the method and composition for short plant type plant |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060253933A1 (en) * | 1998-02-10 | 2006-11-09 | Brown Sherri M | Nucleic acid molecules and other molecules associated with the gibberellin pathway |
| US8344211B2 (en) * | 2008-08-13 | 2013-01-01 | Ceres, Inc. | Plant nucleotide sequences and corresponding polypeptides |
| TWI595092B (en) * | 2012-11-28 | 2017-08-11 | 中央研究院 | Mutant gibberellin 2-oxidase genes and uses thereof |
| CN110818785B (en) * | 2019-11-26 | 2022-04-19 | 吉林省农业科学院 | Corn sucrose transporter ZmSUT3J and coding gene and application thereof |
-
2020
- 2020-07-14 CN CN202010675989.1A patent/CN111778265B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607989A (en) * | 2008-06-20 | 2009-12-23 | 中国科学院遗传与发育生物学研究所 | A kind of rice dwarf-related protein and encoding gene thereof and application |
| CN109844107A (en) * | 2016-08-17 | 2019-06-04 | 孟山都技术公司 | By manipulation gibberellin metabolism increase can crop the method and composition for short plant type plant |
| JP2019524147A (en) * | 2016-08-17 | 2019-09-05 | モンサント テクノロジー エルエルシー | Method and composition for obtaining low plant height plants through gibberellin metabolic manipulation to increase harvestable yield |
| CN108728446A (en) * | 2017-04-25 | 2018-11-02 | 吉林省农业科学院 | Corn resistant gene of salt ZmHKT2;1 and its application |
| CN107365795A (en) * | 2017-08-11 | 2017-11-21 | 吉林省农业科学院 | The method of application and cultivation resistance plant of the AtGA2ox1 genes in stress resistance of plant is adjusted |
Non-Patent Citations (10)
| Title |
|---|
| Cloning, expression and subcellular localization of Gibberellin 2-oxidase gene NtGA2ox1 from Nicotiana tobacum;Jiang Shan等;《Genomics and Applied Biology》;20211006;第39卷(第1期);第132-137页 * |
| Comparisons within the Rice GA 2-Oxidase Gene Family Revealed Three Dominant Paralogs and a Functional Attenuated Gene that Led to the Identification of Four Amino Acid Variants Associated with GA Deactivation Capability;Hsieh KT等;《RICE》;20210808;第14卷(第1期);全文 * |
| Ectopic expression of GA 2-oxidase 6 from rapeseed (Brassica napus L.) causes dwarfism, late flowering and enhanced chlorophyll accumulation in Arabidopsis thaliana;Yan, JD等;《PLANT PHYSIOLOGY AND BIOCHEMISTRY》;20170201;第111卷;第10-19页 * |
| Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice;Shuen-Fang Lo等;《Plant Biotechnol J》;20170323;第15卷(第7期);第850-864页 * |
| gibberellin 2-beta-dioxygenase 7 [Zea mays];Alexandrov NN等;《NCBI GenBank database》;20081210;Accession No：ACG30311 * |
| The role of gibberellins in improving the resistance of tebuconazole-coated maize seeds to chilling stress by microencapsulation;Lijuan Yang等;《Sci Rep》;20161107;第6卷;全文 * |
| 利用CRISPR/Cas9基因编辑技术定点编辑水稻赤霉素2氧化酶基因OsGA2ox10;惠索祯等;《分子植物育种》;20190924;第18卷(第18期);第6016-6024页 * |
| 玉米GA2ox类基因的发掘及其在苗期干旱胁迫后的表达分析;姜志磊等;《玉米科学》;20181113;全文 * |
| 赤霉素在非生物胁迫中的作用;蒋超等;《生物技术通报》;20160526(第05期);全文 * |
| 赤霉素生物合成及其分子机理研究进展;石琰等;《西北植物学报》;20060728(第07期);全文 * |
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