CN116606362A - Mung bean VrAP1 gene and application thereof in regulating plant type and flowering - Google Patents

Mung bean VrAP1 gene and application thereof in regulating plant type and flowering Download PDF

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CN116606362A
CN116606362A CN202310459086.3A CN202310459086A CN116606362A CN 116606362 A CN116606362 A CN 116606362A CN 202310459086 A CN202310459086 A CN 202310459086A CN 116606362 A CN116606362 A CN 116606362A
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李帅
张慧莹
郭旭
侯瑞
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Qingdao Agricultural University
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Abstract

The invention discloses a mung bean VrAP1 gene and application thereof in regulating plant type and flowering, belonging to the technical field of molecular biology. The nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 2. And constructing a plant expression vector by the gene, and transforming an arabidopsis plant to obtain the transgenic arabidopsis. The research on flowering and plant type of transgenic arabidopsis thaliana shows that the mung bean VrAP1 gene can obviously change the plant type of arabidopsis thaliana VrDet1:tfl1 and lead the arabidopsis thaliana VrAP1 to bloom in advance, so that the VrAP1 gene has a certain application prospect in regulating and controlling plant type and flowering period.

Description

Mung bean VrAP1 gene and application thereof in regulating plant type and flowering
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a mung bean VrAP1 gene and application thereof in regulating plant type and flowering.
Background
Mung beans (Vigna radiata L.) have long cultivation history, are traditional export agricultural products, have short growth cycle and strong stress resistance, are rich in nutrition, belong to high-protein and low-fat medicine and food homologous crops, are ideal nutritional health-care food, and are important economic crops. But the mung bean scientific research is relatively late in starting, the basic research is relatively weak, the field production unit yield level is low, the plant type is reasonably improved, the ecological adaptability of crops can be enhanced, and the mung bean ecological cultivation method is one of important ways for improving the crop yield.
The former person finds important genes capable of regulating and controlling plant types in plants such as arabidopsis thaliana, tomatoes, soybeans, beans and the like, and the laboratory earlier-stage finds main genes capable of regulating and controlling mung bean plant types: vrDet1, which is a homologous gene of Arabidopsis AtTFL1, and after the Arabidopsis AtTFL1 mutant tfl1 is subjected to heterologous transformation, the transgenic plant can be transformed from the tfl1 mutant with a limited type plant type to an infinite type plant type, and the transgenic plant is named as VrDet1:tfl1.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a mung bean VrAP1 gene and application thereof in regulating plant type and flowering.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the amino acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 2.
In a specific embodiment, the nucleic acid sequence of the gene is shown in SEQ ID No. 1.
The application of the mung bean VrAP1 gene in plant type and/or flowering regulation is that the plant is mung bean or Arabidopsis thaliana.
A recombinant vector and an expression box for regulating plant type and/or flowering contain coding region sequences of mung bean VrAP1 genes, and the nucleic acid sequences of the mung bean VrAP1 genes are shown as SEQ ID No. 1.
A recombinant bacterium for regulating plant type and/or flowering contains a coding region sequence of a mung bean VrAP1 gene, and the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
Based on the above scheme, the recombinant bacteria are bacteria or fungi, and in a specific embodiment, the recombinant bacteria are escherichia coli, agrobacterium or yeast.
A method for converting plants from infinite type to finite type comprises constructing coding region sequence of mung bean VrAP1 gene into plant expression vector, transforming plant cells, and expressing in plants to convert plants from infinite type to finite type; the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
A method for advancing the flowering of a plant comprises constructing a coding region sequence of a mung bean VrAP1 gene into a plant expression vector, transforming plant cells to express in the plant so as to advance the flowering of the plant; the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
The technical scheme of the invention has the advantages that:
the invention clones and obtains mung bean VrAP1 gene, constructs a plant expression vector by the gene, and transforms an Arabidopsis plant to obtain transgenic Arabidopsis. Comparing the flowering and plant type of the arabidopsis plant transferred with the VrDet1:tfl1 plant, and finding that the bolting time of the transgenic plant is shorter, the number of rosette leaves is smaller, and the flowering time is earlier, namely the transgenic plant has an early flowering phenotype; the arabidopsis plant transformed with the VrAP1 gene is shorter than VrDet1:tfl1, and is changed from an infinite type plant type to a finite type plant type (tfl 1-like mutant), the plant is dwarfed, and the gene can regulate and control the plant type. Therefore, the mung bean VrAP1 gene has a certain application prospect in regulating plant type and flowering period.
Drawings
FIG. 1 is a phylogenetic tree diagram of the VrAP1 gene;
FIG. 2 is an alignment of amino acid sequences of multi-species AP 1;
FIG. 3 is a diagram of the toxicity verification of the VrAP1 protein on yeast cells;
FIG. 4 is a graph showing the transcriptional activation activity of VrAP1 protein;
FIG. 5 is a schematic diagram of the vector construction of the overexpression vector pCAMBIA1300-VrAP 1;
FIG. 6 is a statistical plot of bolting time, rosette number and flowering time of Arabidopsis overexpressing the VrAP1 gene (where OE-1, OE-2, OE-3 are three different overexpressing lines);
FIG. 7 is a statistical plot of plant heights of Arabidopsis thaliana overexpressing the VrAP1 gene (where OE-1, OE-2, OE-3 are three different overexpressing strains);
FIG. 8 is a graph of strain observations of Arabidopsis that overexpresses the VrAP1 gene (where OE-1, OE-2, OE-3 are three different overexpressing strains).
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples of the present invention,
the mung beans are provided by the application genomics of crops in Qingdao agricultural university;
yeast competent Y2HGold was purchased from Shanghai Biotechnology Inc.;
pGBKT7 vectors are provided by Qingdao university crop application genomics;
pGBKT7-53 and pGBKT7-lam vectors are provided by Qingdao university crop application genomics;
coli competent DH 5. Alpha. Was purchased from TSINGKE;
agrobacterium tumefaciens strain competent GV3101 was purchased from Shanghai Biotechnology Inc.;
the transgenic acceptor material is Arabidopsis VrDet1:tfl1, which is provided by the crop application genomics of Qingdao university.
Arabidopsis thaliana tfl1 mutants were purchased from the American Arabidopsis thaliana biological resource center and stored by the present laboratory;
the pCAMBIA1300 vector is provided by the crop application genomics of the Qingdao university of agriculture.
EXAMPLE 1 cloning of the VrAP1 Gene
1. RNA and cDNA acquisition
And (5) fully grinding young leaves of mung beans in liquid nitrogen, and extracting total RNA by using a plant RNA extraction kit of TARAKA. After RNA was obtained, it was reverse transcribed into cDNA, and the first strand of cDNA was synthesized using the Evo M-MLV Plus cDNA synthesis kit of the Ai Kerui organism, the synthesis steps of which were carried out with reference to the kit instructions.
2. Acquisition of the VrAP1 Gene
The upstream and downstream primers designed to amplify the CDS fragment of the VrAP1 gene were designated P1 and P2, respectively.
Using cDNA obtained by reverse transcription of the RNA of the mung bean as a template, and amplifying by using P1 and P2; the reaction system was 50. Mu.L; the reaction condition is 95 ℃ for 5min;95 ℃, 45sec,58 ℃, 30sec,72 ℃, 1min,32 cycles; 72 ℃, 5min,4 ℃ and infinity. The PCR product can be used for subsequent experiments immediately or stored at-20 ℃.
The nucleic acid sequence of the cloned VrAP1 gene is shown as SEQ ID No.1, the coding sequence of the gene comprises 711 nucleotides, and the coded amino acid sequence is shown as SEQ ID No. 2.
SEQ ID No.1(5’→3’)
ATGGGAAGGGGTAGGGTTCAGCTGAAGAGAATAGAGAACAAGATCAATCGCCAGGTAACCTTCTCCAAAAGGAGAGCTGGGTTACTCAAGAAAGCACACGAGATCTCTGTGCTCTGTGACGCTGAGGTCGCTTTGATTGTCTTCTCCCACAAAGGAAAGCTCTTTGAATATGCCACTGATTCTTGCATGGAGAAGATACTGGAACGCTATGAAAGGTATGCCTATGCTGAGAGACAACTTGTTGCCAATGATTCCGAATCACAGGGAAATTGGACCATTGAGTACACCAGACTCAAGGCAAAGATTGACCTTTTGCAGAGAAACCATAGGCACTATATGGGAGAAGATTTAGGTTCAATGAGCCTAAAAGAGCTTCAGAGTCTGGAGCAGCAGTTAGATACTGCTCTCAAACAAATTCGTACCCGCAGAAACCAACTCATGTACGAGTCGATCTCGGAGCTTCAGAAGAAGGAGAAAGTTATACAGGAGCAGAATAACATGCTTGCAAAGAAGATAAAGGAGAAAGAAAAGGTTGCAGCACAGCAGGCACAATGGGAGCATCCAAACCATGGAGTTAATGCATCTTTCTTGCTACCTCAGCCACTTCTGAACATGGGTGGCAATTATCGTGAGGAAGCACCAGAAGTAGGAAGGAATGAACTTGACCTCACTCTGGAACCCTTGTATTCCTGCCACCTTGGATGCTTTTGA
SEQ ID No.2
MGRGRVQLKRIENKINRQVTFSKRRAGLLKKAHEISVLCDAEVALIVFSHKGKLFEYATDSCMEKILERYERYAYAERQLVANDSESQGNWTIEYTRLKAKIDLLQRNHRHYMGEDLGSMSLKELQSLEQQLDTALKQIRTRRNQLMYESISELQKKEKVIQEQNNMLAKKIKEKEKVAAQQAQWEHPNHGVNASFLLPQPLLNMGGNYREEAPEVGRNELDLTLEPLYSCHLGCF
The P1 and P2 primer sequences are as follows:
P1：5'-ATGGGAAGGGGTAGGGTTCA-3'(SEQ ID No.3)；
P2：5'-TCAAAAGCATCCAAGGTGGCA-3'(SEQ ID No.4)。
example 2 phylogenetic analysis and amino acid sequence alignment of VrAP1
The amino acid sequences of the AtAP1 genes are obtained from an Arabidopsis website, the gene numbers of soybean GmAP1, medicago sativa MtPIM and pea PIM are obtained from published documents, the amino acid sequences of the leguminous plant genes are obtained by utilizing an NCBI website, the amino acid sequences of the VrAP1 and the amino acid sequences in other species are arranged into a file in a fasta format, the fasta file is imported into ClustalX software for sequence comparison, then Draw a Tree is clicked to generate a file with ph suffix, and the file is imported into software MEGA7.0 to generate an evolutionary Tree, as shown in figure 1. The fasta files which are arranged and contain the amino acid sequences of the AP1 of different species are imported into DNAMAN software, the multiple sequences are aligned, and the alignment result is output, as shown in figure 2.
Example 3
Construction of VrAP1-pGBKT7 fusion vector
The cDNA of mung bean is used as a template, ecoRI and BamHI enzyme digestion sites are respectively added into an upstream primer and a downstream primer, the upstream primer and the downstream primer are respectively P3 and P4, and the primer sequences are as follows:
P3：5'-GGAATTCATGGGAAGGGGTAGGGTTCA-3'(SEQ ID No.5)；
P4：5'-CGGATCCAAAGCATCCAAGGTGGCAGG-3'(SEQ ID No.6)；
PCR amplification is carried out by using the primer and the high-fidelity enzyme, and the reaction system is 30 mu L; the reaction condition is 95 ℃ for 5min;95 ℃, 45sec,58 ℃, 30sec,72 ℃, 1min,32 cycles; 72 ℃, 5min,4 ℃ and infinity. The CDS fragment of the VrAP1 gene containing EcoRI and BamHI cleavage sites was obtained, the PCR product was recovered by purification, and the PCR purified product and pGBKT7 vector were digested with EcoRI and BamHI restriction enzymes, and 1% agarose gel was prepared to recover the PCR purified product and the vector enzyme fragment. The PCR gene fragment was ligated with the vector by T4 DNA ligase, and the ligation product was transformed into E.coli DH 5. Alpha. By a heat shock method and plated on LB plates containing kanamycin (kan) resistance. After overnight culture at 37 ℃, single colony is selected for activation, and the bacterial liquid is sent to a biological company for sequencing verification through colony PCR verification, so that a fusion vector VrAP1-pGBKT7 containing VrAP1 genes is obtained.
Example 4
Toxicity verification and transcriptional activation activity verification of VrAP1 protein on yeast cells
The AtAP1 gene belongs to a transcription factor, and in order to verify whether VrAP1 is a transcription factor, transcriptional activation activity is verified.
Toxicity detection: vrAP1-pGBKT7, pGBKT7-53 (positive control) and pGBKT7-lam (negative control) prepared in example 3 were transformed into Y2HGold yeast competent cells individually, and coated on a single-defect plate (SD/-Trp), and the yeast cells were grown normally, as shown in FIG. 3, which revealed that VrAP1 protein had no toxic effect on the yeast cells.
Transcriptional activation Activity assay: the well-grown yeast cells on the single-segment plates were streaked onto X-alpha-gal-containing double-segment plates (SD/-Trp/-His), and the yeast containing the VrAP1-pGBKT7 fusion plasmid turned blue as positive controls, indicating that the VrAP1 protein had transcriptional activation activity, as shown in FIG. 4.
Example 5
Construction of VrAP1 Gene plant expression vector
The cDNA of mung bean is used as a template, sacI and BamHI enzyme cutting sites are respectively added into an upstream primer and a downstream primer, the upstream primer and the downstream primer are respectively P5 and P6, and the primer sequences are as follows:
P5：5'-CGAGCTCATGGGAAGGGGTAGGGTTCAGC-3'(SEQ ID No.7)；
P6：5'-CGGATCCTCAAAAGCATCCAAGGTGGCA-3'(SEQ ID No.8)；
PCR amplification is carried out by using the primer and the high-fidelity enzyme, and the reaction system is 30 mu L; the reaction condition is 95 ℃ for 5min;95 ℃, 45sec,58 ℃, 30sec,72 ℃, 1min,32 cycles; 72 ℃, 5min,4 ℃ and infinity. The CDS fragment of the VrAP1 gene containing SacI and BamH I cleavage sites was obtained, and after the PCR product was recovered by purification, the PCR purified product and pCAMBIA1300 vector were digested with SacI and BamH I restriction enzymes to prepare 1% agarose gel to recover the PCR purified product and vector enzyme fragments. The PCR gene fragment was ligated with the vector by T4 DNA ligase, and the ligation product was transformed into E.coli DH 5. Alpha. By a heat shock method and plated on LB plates containing kanamycin (kan) resistance. After overnight culture at 37 ℃, single colony is selected for activation, and the plant expression vector pCAMBIA1300-VrAP1 containing the target fragment is obtained through colony PCR verification and sequencing verification of the bacterial liquid by a biological company, wherein the vector diagram is shown in figure 5.
Example 6
Obtaining recombinant bacteria containing plant expression vector pCAMBIA1300-VrAP1
The plant expression vector pCAMBIA1300-VrAP1 prepared in example 5 was used to transform Agrobacterium strain competent cells GV3101 by electric shock and plated on YEP plates containing rifampicin (rif) and kanamycin (kan) resistance. And (3) after a single colony grows out of the flat plate, picking the single colony for activation, and verifying through colony PCR to obtain the recombinant strain containing the plant expression vector pCAMBIA1300-VrAP1, mixing the strain and glycerol into a 1.5mL centrifuge tube according to a ratio of 1:1, and preserving at-80 ℃.
Example 7
Acquisition of Arabidopsis thaliana overexpressing the VrAP1 Gene
(1) Planting of Arabidopsis thaliana
Washing Arabidopsis seed VrDet1:tfl1 in ultra clean bench, soaking in 70% alcohol for 5min, soaking in 10% NaClO for 10min, and finally using ddH 2 O is washed for 5 times, the mixture is placed on an MS flat plate containing glufosinate, and after the mixture is subjected to dark treatment for 3d at the temperature of 4 ℃, the mixture is cultured for 10d in an illumination incubator, and 4 green leaves are grown and moved into substrate soil.
(2) Preparation of dyeing liquor
20. Mu.L of the recombinant strain containing the plant expression vector pCAMBIA1300-VrAP1 prepared in example 6 and stored in glycerol was pipetted into 20mL of YEP liquid medium, and simultaneously 20. Mu.L of rib and 20. Mu.L of kan were added, and cultured overnight at 28℃and 240 rpm; after the concentration of the bacterial liquid OD600 = 0.6-0.8, centrifuging the bacterial liquid at room temperature and 5000rpm for 10min, and discarding the supernatant to obtain bacterial liquid sediment. Adding 20mL of 5% sucrose solution into the bacterial liquid precipitate, re-suspending the bacterial liquid, centrifuging at 4000rpm at room temperature for 10min to obtain bacterial liquid precipitate; 20mL of 5% sucrose solution was added to the bacterial suspension pellet again for resuspension, and 4. Mu.L of SILWETL-L-77 was added to complete the preparation of the invaded solution.
(3) Genetic transformation of Arabidopsis thaliana
The day before infection, the flowers or flower buds of the arabidopsis with flowers and white exposure are subtracted, and the arabidopsis plants with good growth vigor are selected for experiments the next day. The arabidopsis plants are infected in the following way, gloves are firstly needed to be worn, then a pipetting gun is used for sucking agrobacterium suspension to beat on green buds, and secondary treatment is carried out after 20min, so that 3 times of infection are carried out. Placing the infected plants in a relatively airtight dark place (a paper box or a black plastic bag) for 24 hours, taking out the plants the next day, placing the plants in an illumination culture room, and growing under normal illumination conditions.
(4) PCR detection of transgenic plants
The infected plants mature after 2 months, and the mature seeds, namely T, are collected 0 Seed generation. Will T 0 The seeds of the generation are disinfected according to the cleaning process of the seeds of the arabidopsis thaliana, and are evenly coated on an MS culture medium containing hygromycin, after being subjected to dark treatment at 4 ℃ for 3 days, the seeds are cultivated in an illumination incubator, and after green seedlings with four leaves grow on the culture medium, the seedlings are moved into matrix soil.
The Arabidopsis green leaves are selected, DNA thereof is extracted by TPS method, and amplification primers of target genes are used as primers for detecting transgenic plants, namely P1 (SEQ ID No. 3) and P2 (SEQ ID No. 4). The PCR reaction system is 50 mu L; the reaction condition is 98 ℃ for 3min;98 ℃, 10sec,59 ℃, 30sec,72 ℃, 30sec,32 cycles; 72 ℃, 5min,4 ℃ and infinity. The PCR products were gel-electrophoresed and positive lines were determined.
After the positive strain is mature, collecting its seed as T 1 Seed substitution, T 1 The generation seeds are treated according to the process of cleaning the Arabidopsis seeds, and inoculated on an MS culture medium containing hygromycin, and cultured in an illumination incubator, if the ratio of positive plants to negative plants in the plant lines is about 3:1, the seeds of the positive plant lines are collected by the single plant, and T is obtained 2 Seed generation. Will T 2 After the disinfection treatment of the generation seeds, the arabidopsis strains which can grow on the resistant culture medium are selected for planting, and finally, the seeds of the arabidopsis strains are collected singly, namely the transgenic homozygous T 3 Seed generation.
Example 8
Statistics of bolting time, rosette number and flowering time of arabidopsis through over-expressing VrAP1 gene
Arabidopsis seeds VrDet1:tfl1, tfl1 mutant seeds and the Arabidopsis seeds over-expressing the VrAP1 gene obtained by the method of example 7 are cultured on an MS culture medium, after green four-leaf seedlings grow out, the seedlings are moved into substrate soil and planted in a negative one-layer illumination culture room of Qingdao university, and are cultured under long sunlight (16 hours of illumination time/8 hours of darkness). The day of arabidopsis that receives light in the incubator is regarded as the first day of plant growth, after which the plants are observed when to bolt, and the number of rosette leaves and the bolting date are recorded when the plants are bolting. After bolting from arabidopsis, observing when the plants bloom and counting the flowering time.
The statistical results of the bolting time of the plants are shown in (a) of fig. 6: the bolting time of the transgenic arabidopsis thaliana (OE) is obviously earlier than that of a VrDet1:tfl1 plant, and is relatively close to that of a comparative group tfl1 mutant plant; the rosette leaf number statistics for the above plants are shown in fig. 6 (b): the number of rosette leaves of transgenic arabidopsis is obviously less than that of VrDet1:tfl1 plants, and has no obvious difference with that of the rosette leaves of the tfl1 mutant plants of the control group; flowering-time statistics of the above plants are as shown in fig. 6 (c): the flowering time of the transgenic arabidopsis thaliana is obviously earlier than that of a VrDet1:tfl1 plant, and is not obviously different from that of a control group tfl1 mutant plant. From the above statistics, it can be seen that: the arabidopsis thaliana over-expressing the VrAP1 gene has obvious early flowering phenomenon compared with a VrDet1:tfl1 plant.
Example 9
Plant height statistics and plant type observation of arabidopsis thaliana over-expressing VrAP1 gene
The arabidopsis plants in example 8 were photographed 7 weeks after flowering, and the main stem height of the plants was measured and counted as the plant height of arabidopsis. The statistical result of plant height is shown in figure 7, the over-expression plant is obviously shorter than a VrDet1:tfl1 plant, the phenomenon that the strain type of the VrDet1:tfl1 gene is changed by over-expression in the VrDet1:tfl1 plant can be more intuitively observed through figure 8, the infinite strain type VrDet1:tfl1 is changed into a finite strain type, and the over-expression plant type is similar to the strain type of the tfl1 mutant plant.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (9)

1. The mung bean VrAP1 gene is characterized in that the amino acid sequence coded by the gene is shown as SEQ ID No. 2.
2. The mung bean VrAP1 gene of claim 1, wherein the nucleic acid sequence of the gene is shown in SEQ id No. 1.
3. The use of the mung bean VrAP1 gene according to claim 1 in plant type and/or flowering regulation.
4. A recombinant vector and an expression cassette for regulating plant type and/or flowering are characterized by comprising a coding region sequence of a mung bean VrAP1 gene, wherein the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
5. The recombinant bacterium for regulating plant type and/or flowering is characterized by comprising a coding region sequence of a mung bean VrAP1 gene, wherein the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
6. Recombinant bacterium for use in regulating plant type and/or flowering according to claim 5, wherein the recombinant bacterium is of the bacterial or fungal type.
7. The recombinant bacterium for controlling plant type and/or flowering according to claim 6, wherein the recombinant bacterium is escherichia coli, agrobacterium or yeast.
8. A method for converting a plant from an infinite type plant type to a finite type plant type is characterized in that a coding region sequence of a mung bean VrAP1 gene is constructed into a plant expression vector, and plant cells are transformed to be expressed in the plant so as to convert the plant from the infinite type plant type to the finite type plant type; the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
9. A method for advancing flowering of a plant, which is characterized in that a coding region sequence of a mung bean VrAP1 gene is constructed into a plant expression vector, and plant cells are transformed to be expressed in the plant so as to advance flowering of the plant; the nucleic acid sequence of the mung bean VrAP1 gene is shown as SEQ ID No. 1.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004106531A1 (en) * 2003-05-22 2004-12-09 E.I. Dupont De Nemours And Company Method for manipulating growth, yield, and architecture in plants
CN101845085A (en) * 2009-12-07 2010-09-29 南京农业大学 Wild soybean AP1 type protein and coding gene and application thereof
CN102206262A (en) * 2011-04-11 2011-10-05 南京农业大学 Soybean activating protein-1(AP1) transcription factor and coding gene and use thereof
CN110317249A (en) * 2019-07-02 2019-10-11 西南大学 Albumen and the application of wax plum CpAP1 gene and its coding

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004106531A1 (en) * 2003-05-22 2004-12-09 E.I. Dupont De Nemours And Company Method for manipulating growth, yield, and architecture in plants
CN101845085A (en) * 2009-12-07 2010-09-29 南京农业大学 Wild soybean AP1 type protein and coding gene and application thereof
CN102206262A (en) * 2011-04-11 2011-10-05 南京农业大学 Soybean activating protein-1(AP1) transcription factor and coding gene and use thereof
CN110317249A (en) * 2019-07-02 2019-10-11 西南大学 Albumen and the application of wax plum CpAP1 gene and its coding

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENBANK: "PREDICTED: Vigna radiata var. radiata truncated transcription factor CAULIFLOWER A-like(LOC106767147), mRNA", 《GENBANK》, 12 October 2017 (2017-10-12), pages 014651982 *
LIYU CHEN: "Soybean AP1 homologs control flowering time and plant height", 《JOURNAL OF INTEGRATIVE PLANT BIOLOGY》, vol. 62, no. 12, 20 December 2020 (2020-12-20), pages 1868 - 1879 *
于静静: "绿豆开花相关基因VrFT2a 的克隆及表达分析", 《农业生物技术学报》, vol. 30, no. 6, 31 December 2022 (2022-12-31), pages 1058 *

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