CN110438133B

CN110438133B - Application of expression vector containing mung bean flowering gene VrFT2a

Info

Publication number: CN110438133B
Application number: CN201910757986.XA
Authority: CN
Inventors: 叶卫军; 周斌; 杨勇; 田东丰; 张丽亚; 张磊
Original assignee: Institute Of Crops Anhui Academy Of Agricultural Sciences
Current assignee: Institute Of Crops Anhui Academy Of Agricultural Sciences
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2021-06-04
Anticipated expiration: 2039-08-16
Also published as: CN110438133A

Abstract

The invention discloses an application of an expression vector of a mung bean flowering gene VrFT2 a. The expression vector is an over-expression vector obtained by inserting the mung bean flowering gene VrFT2a into a plant expression vector, and a promoter for starting the expression of a corresponding target gene is a strong promoter 35S. The nucleotide sequence of the mung bean flowering gene VrFT2a is shown in a sequence table Seq ID No. 1. The mung bean flowering gene VrFT2a is expressed in plants to regulate the flowering time of the plants. The invention can promote the transgenic plant to bloom in advance, shorten the growth period and has the physiological function of regulating and controlling the flowering period of the plant.

Description

Application of expression vector containing mung bean flowering gene VrFT2a

Technical Field

The invention relates to a mung bean flowering gene in the technical field of plant genetic engineering, in particular to application of an expression vector of a mung bean flowering gene VrFT2 a.

Background

The mung bean is rich in nutrition, the content of the protein in grains is as high as 19.5% -33.1%, the content of the protein in the grains is higher than that in cereal crops such as rice, wheat, corn and the like, and the mung bean belongs to high-protein, medium-starch and low-fat food. Moreover, the mung beans are rich in various mineral elements, vitamins and active substances, have the effects of detoxifying, resisting bacteria, resisting allergy, reducing blood fat, reducing blood pressure, resisting tumors, preventing cancers and the like, and belong to medical and edible crops. The mung beans can be processed into various forms of foods such as powder, soup, porridge, noodles and the like, are popular with the public, and the straws can also be processed into feed. The root system of the mung bean has nitrogen fixation capacity with rhizobia symbiotic with the root system of the mung bean, and the soil fertility and structure can be improved by planting the mung bean, so that the requirements of self growth are met, and the mung bean can be used by succeeding crops. In addition, the mung bean has the characteristics of preference for warmth, short growth period, high seeding elasticity, barren resistance, shade resistance, high economic benefit and the like, so that the mung bean is deeply favored by broad farmers.

At present, the main production areas of mung beans are in Asia, Africa and Europe, and the distribution areas are wide. However, for a single mung bean variety or germplasm resource, due to the limitation of regional adaptability, the planting range of good varieties is severely limited. Previous studies have shown that crop region adaptability is closely related to photoperiod and fertility control genes. Therefore, the genetic analysis and molecular mechanism research on the mung bean photoperiod regulation is beneficial to improving the mung bean molecular breeding level and accelerating the breeding of the wide-range variety. In the prior art, the soybean gene has been studied to regulate the flowering time of plants, but the expression pattern is weak to be regulated by light, and the change of the flowering time of plants by the expression of mung bean gene has not been reported.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the application of the expression vector of the mung bean flowering gene VrFT2a, which can promote the transgenic plant to bloom in advance, shorten the growth period and regulate the flowering period of the plant.

The invention is realized by adopting the following technical scheme: the application of an expression vector containing a mung bean flowering gene VrFT2a is characterized in that the expression vector is an over-expression vector obtained by inserting a mung bean flowering gene VrFT2a into a plant expression vector, and a promoter for promoting the expression of a corresponding target gene is a strong promoter 35S; wherein the nucleotide sequence of the mung bean flowering gene VrFT2a is the nucleotide sequence shown in a sequence table Seq ID No. 1; the mung bean flowering gene VrFT2a is expressed in plants to regulate the flowering time of the plants.

As a further improvement of the scheme, the host cell of the expression vector is obtained by transferring the expression vector into agrobacterium tumefaciens.

As a further improvement of the above scheme, the construction method of the expression vector comprises the following steps:

(1) taking the young leaves of the mung beans containing the mung bean flowering gene VrFT2a, extracting RNA and carrying out reverse transcription on the RNA to obtain full-length cDNA;

(2) carrying out PCR amplification by using the full-length cDNA as a template through a forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCTGGAGGAAGTAGG-3' in a sequence table Seq ID NO.7 and a reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTATTAATATAATCTCCTTCC-3' in a sequence table Seq ID NO.8 to obtain a PCR product;

(3) firstly, performing gel electrophoresis on the PCR product and recovering, and then exchanging the recovered product into an entry vector to obtain a recombinant entry vector;

(4) extracting a plasmid of the recombinant entry vector, and exchanging a VrFT2a gene fragment in the plasmid into the plant expression vector to obtain the over-expression vector.

The expression vector of the mung bean flowering gene VrFT2a and the application thereof have the following beneficial effects:

1. the expression vector of the mung bean flowering gene VrFT2a can promote transgenic plants to bloom in advance and shorten the growth period, so that the expression vector has the physiological function of regulating the flowering period of the plants and can be used for regulating the flowering period of the plants in the field of plant genetic engineering.

Drawings

FIG. 1 is a comparison graph of relative expression levels of mung bean flowering gene VrFT2a in different organs of mung bean according to example 4 of the present invention;

FIG. 2 is a comparison graph of the relative expression of VrFT2a in the transgenic line of the mung bean flowering gene VrFT2a and wild plants in example 9 of the present invention, wherein WT is the wild plant on the left side and the positive transgenic line on the right side;

FIG. 3 is a comparison of flowering-time of mung bean when the mung bean flowering gene VrFT2a is overexpressed in example 10 of the present invention;

FIG. 4 shows a comparison of the phenotype of the transgenic line and the wild type plant of the mung bean flowering gene VrFT2a according to example 10 of the present invention, wherein the wild type plant is shown on the left and the transgenic line is shown on the right.

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

The invention provides a mung bean flowering gene VrFT2a, wherein the mung bean flowering gene VrFT2a comprises a nucleotide sequence selected from the following group:

(a) a nucleotide sequence shown in a sequence table Seq ID No. 1;

(b) a nucleotide sequence complementary to the nucleotide sequence of (a);

(c) a nucleotide sequence having at least 50% homology to the nucleotide sequence of (a); in other embodiments, the nucleotide sequence of (c) may have a nucleotide sequence which is at least 60%, at least 65%, at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, especially at least 95% or 98% or 99% homologous to the nucleotide sequence of (a);

(d) a nucleotide sequence which encodes a protein of the same amino acid sequence as the nucleotide sequence of (a), but which differs in sequence;

(e) a nucleotide sequence encoding one of the following amino acid sequences: an amino acid sequence shown in sequence table Seq ID No.2, or an amino acid sequence which differs from the amino acid sequence shown in sequence table Seq ID No.2 by substitution, deletion and/or insertion of at least one (e.g.1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3) amino acid residue, or an amino acid sequence which has at least 50% (of course, in other embodiments, at least 60%, at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, especially at least 95% or 98% or 99%) homology with the amino acid sequence shown in sequence table Seq ID No. 2;

(f) an active fragment of the nucleotide sequence of any one of (a) - (e); or

(g) A nucleotide sequence complementary to the nucleotide sequence of any one of (a) to (e).

In this example, the sequencing results showed that the CDS sequence of the mung bean flowering gene VrFT2a is composed of 531 bases as shown in Table Seq ID N0.1. In addition, in the present example, the sequence of the encoded protein determined by the CDS sequence of the mung bean flowering gene VrFT2a is shown in Seq ID No.2, and is composed of 176 amino acid residues, and both the protein and GmFT2a contain a PEBP (phospho-binding protein) conserved domain, so it is presumed that VrFT2a may have similar biological functions to the homologous gene in soybean. In the embodiment, related researches show that the mung bean flowering gene VrFT2a can be used for regulating the flowering time of plants by expressing in the plants to regulate the flowering time of the plants. Therefore, the mung bean flowering gene VrFT2a can promote the transgenic plant to bloom in advance and shorten the growth period, so that the mung bean flowering gene VrFT2a has the physiological function of regulating the flowering period of the plant, and can be used for regulating the flowering period of the plant in the field of plant genetic engineering.

Example 2

This example provides a protein encoded by the green bean flowering gene VrFT2a of example 1, having an amino acid sequence selected from the group consisting of:

(1) an amino acid sequence shown in a sequence table Seq ID No. 2;

(2) and (2) an amino acid sequence having an equivalent function, which is obtained by substituting, deleting, adding and/or inserting at least one amino acid in the amino acid sequence of (1).

Example 3

The embodiment provides a method for detecting a coding sequence of a mung bean flowering gene VrFT2a, wherein the gene is the mung bean flowering gene VrFT2a in embodiment 1, and the method for detecting the coding sequence comprises the following steps.

(1) Taking young leaves of mung beans containing a mung bean flowering gene VrFT2a to grind in liquid nitrogen, and extracting total RNA through a kit. In this example, young leaves of medium green 5 can be taken, ground thoroughly in liquid nitrogen, and then total RNA is extracted using the plant total RNA extraction kit TRIzol (Invitrigen).

(2) Total RNA was extracted and reverse transcribed to full-length cDNA. In this example, first strand cDNA was synthesized using TaKaRa PrimeScript^TMII 1st strand cDNA Synthesis Kit, concrete operationThis is done as described.

(3) The full-length cDNA is used as a template, and PCR reaction amplification is carried out through a forward primer 5'-CGAAAAGGCGTGGCTTGA-3' in a sequence table Seq ID NO.3 and a reverse primer 5'-AACAGAGCAAGTGATTTA-3' in a sequence table Seq ID NO. 4. Wherein the total volume of the PCR reaction system is 20. mu.L, and the PCR reaction system comprises cDNA (50 ng. mu.L)^-1) mu.L of each of 2. mu.L of forward and reverse primers (10. mu.M), 2.0. mu.L of 10 XPCR buffer, 2. mu.L of dNTP mix (2 mM), 0.2. mu.L of LATaq enzyme (5U/. mu.L), and water to 20. mu.L. Moreover, the PCR amplification procedure was 94 ℃ pre-denaturation for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; finally, the cells were extended at 72 ℃ for 10min and stored at 4 ℃.

(4) After the PCR reaction is finished, the amplified product is subjected to agarose gel electrophoresis, and then is recovered by a gel recovery kit (AxyPrep can be adopted)^TMDNA Gel Extraction Kit, Axygen) were excised, recovered and sequenced. The sequencing results of this example show that the CDS sequence of the mung bean flowering gene VrFT2a is shown in Table Seq ID No.1 and consists of 531 bases.

Example 4

This example provides a method for detecting the expression level of the mung bean flowering gene VrFT2a in different organs of mung bean in example 1, which uses real-time fluorescent quantitative PCR (RT-PCR) to detect the expression level of the mung bean flowering gene VrFT2a in each organ of mung bean. Wherein, real-time fluorescent quantitative PCR is carried out on a LightCycler 96 platform, and SYBR Green I is used as a fluorescent dye. The detection method of the embodiment includes the following steps.

(1) Taking root, stem, leaf, flower and pod tissues of mung bean containing a mung bean flowering gene VrFT2a, respectively extracting total RNA and carrying out reverse transcription to obtain cDNA. In this example, medium green No.5 root, stem, leaf, flower, and immature pod tissues were taken, total RNA was extracted and reverse transcribed into cDNA.

(2) Carrying out fluorescent quantitative PCR reaction on the cDNA of (1) by using a forward primer 5'-GATTGGGGATGTGTTGGACC-3' in a sequence table Seq ID No.5 and a reverse primer 5'-CGGGATCAACTGCGATCAAG-3' in a sequence table Seq ID No.6 to obtain the mung bean pasteExpression level of flower gene VrFT2a in root, stem, leaf, flower and pod tissues of mung bean. Wherein, the mung bean beta-tubulin gene is used as the fluorescent quantitative PCR internal reference, and the reaction system is 20 mu L: 2.0. mu.L of cDNA, 0.5. mu.L of each of forward and reverse primers (10. mu.M), 10. mu.L of 2 XSSYBR Premix EX-Taq Mix, prepared with RNase free ddH₂Make up to 20. mu.L of O. The PCR reaction program was 94 ℃ hot start 30S, 94 ℃ 5S, 60 ℃ 30S, 40 cycles. Through experiments, the mung bean flowering gene VrFT2a is expressed in roots, stems, leaves, flowers and young pods, but is expressed in lower amounts in the roots and the young pods, and is expressed in higher amounts in the stems and the leaves, as shown in FIG. 1.

Example 5

This example provides an expression vector containing the mung bean flowering gene VrFT2a, wherein the expression vector is an overexpression vector obtained by inserting the mung bean flowering gene VrFT2a of example 1 into a plant expression vector, and the promoter for promoting the expression of the corresponding target gene is strong promoter 35S.

Example 6

This example provides a host cell containing the overexpression vector of the mung bean flowering gene VrFT2a, wherein the host cell is obtained by transferring the expression vector of example 5 into Agrobacterium tumefaciens GV 3101.

Example 7

This example provides a method for constructing an expression vector containing the mung bean flowering gene VrFT2a in example 5, wherein the method comprises the following steps of constructing primers for an over-expression vector, amplifying a CDS sequence of VrFT2a, designing specific primers according to a VrFT2a gene sequence, and adding attB joints at the 5' ends of the primers respectively.

(1) Taking the young leaves of mung bean containing mung bean flowering gene VrFT2a, extracting RNA and reversely transcribing the RNA into full-length cDNA. Wherein, the mung bean is Zhonglv No. 5.

(2) The full-length cDNA is taken as a template through a forward primer 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCTGGAGGAAGTAGG-3' in a sequence table Seq ID NO.7 and a reverse primer 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTATTAATATAATCTCCTTCC-3' in a sequence table Seq ID NO.8And carrying out PCR amplification to obtain a PCR product. In this example, high fidelity enzyme PrimeSTAR HS DNA Polymerase (TaKaRa Code: DR 010A) was used, and the reaction system: 5 mu L of template cDNA; 5 × PrimeSTAR Buffer (Mg 2+ plus) 10 μ L; dNTP mix (2 mM each) 5. mu.L, forward and reverse primers (10. mu.M) 2. mu.L each; PrimeSTAR HS DNA Polymerase 0.5 μ L, ddH₂O make up to 50. mu.L. The PCR amplification procedure was: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 deg.C for 10s, annealing at 57 deg.C for 30s, extension at 72 deg.C for 1.5min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃.

(3) The PCR product is first gel electrophoresed and recovered, and the recovered product is then connected to the entry vector. In this example, the PCR product was gel-electrophoresed and recovered, and the recovered product was exchanged to the Gateway system entry vector pDONR221 by the Gateway BP clone reaction, as follows: 50-150ng of PCR Product (PCR Product), 150ng of destination vector (pDONR 221), 2. mu.L of 5 × close Reaction Buffer, 2. mu.L of BP close enzyme mix, ddH₂And supplementing O to 10 mu L, mixing the mixture by briefly swirling twice, slightly centrifuging the mixture, carrying out water bath at 25 ℃ for 8h, then adding 1 mu L of protease K solution, mixing the mixture evenly, and standing the mixture at 37 ℃ for 10min to finish the reaction.

Wherein, the ligation product is transformed into escherichia coli competence DH5 alpha by a heat shock method, positive clones are selected after bacterial liquid PCR and are sequenced to obtain recombinant clones with the inserted sequence being completely the same as VrFT2a in the vector, and the positive recombinant clones pDONR221-VrFT2a are obtained.

(4) Extracting pDONR221-VrFT2a plasmid recombined into a portal vector, and exchanging VrFT2a gene fragment in the plasmid into the plant expression vector to obtain the over-expression vector. In this example, the pDONR221-VrFT2a plasmid was extracted, and VrFT2a was exchanged into the overexpression vector pK2GW7 using the Gateway LR clone TM Enzyme Mix (Invitrogen, Cat. number 11791-019) system to construct a recombinant plasmid pK2GW7-VrFT2 a. The LR reaction system is as follows:

reaction conditions are as follows: pDONR221-VrFT2a plasmid 50-150ng, destination vector (pK 2GW 7) 150ng, 5 XL Clonase Reaction Buffer 2. mu.L, 2. mu.L LR Clonase enzyme mix, ddH₂O to 10 μL, vortex for two times and mix, centrifugate slightly, after water bath 8h at 25 duC, add 1 uL proteinase K solution and mix, stand 10min at 37 duC and finish the reaction.

And 5 mul of reaction product is taken to transform escherichia coli DH5 alpha, after bacterial liquid PCR, positive clone is picked for sequencing identification, and the obtained sequence is the positive recombinant plasmid pK2GW7-VrFT2a with the same sequence as VrFT2 a.

Example 8

This example provides a method for genetic transformation of Arabidopsis thaliana, which comprises the following steps.

(1) The plasmid containing the expression vector of the mung bean flowering gene VrFT2a in example 7 was mixed with Agrobacterium GV3101 in an ice bath and transformed by the electroporation method.

(2) The transformants were plated on resistant YEP medium and cultured upside down until single colonies grew. In this example, the transformed product was plated on resistant YEP medium containing Rif (rifampicin) 50mg/L and Spec (spectinomycin hydrochloride) 100mg/L, and cultured at 28 ℃ for 2 nights in an inverted state until single colonies were grown. PCR identification of positive clone in bacterial liquid.

(3) Single colonies on resistant YEP medium were picked into YEP liquid medium and shake cultured. And (3) sucking a proper amount of bacterial liquid, adding the bacterial liquid into a YEP liquid culture medium (containing corresponding antibiotics), and culturing until the OD600 is more than 1. In this example, grown Agrobacterium was picked up as a single clone into 5mL YEP liquid medium (Rif 50mg/L for the corresponding antibiotic, spec 100 mg/L) and cultured with shaking at 28 ℃ and 150rpm for 48 hours. In this example, the cultured strain was added to YEP broth (containing the corresponding antibiotic) at a volume ratio of 1:10 and cultured at 28 ℃ until OD600> 1.

(4) Removing the pod of the arabidopsis thaliana fruit in the full-bloom stage, and placing the whole arabidopsis thaliana plant in a bacterial liquid for infection. In this example, arabidopsis thaliana pods were removed cleanly during the full-bloom period of arabidopsis thaliana, and the whole arabidopsis thaliana plant with only inflorescence left was inverted together with the plug and covered in the bacterial solution containing transformed agrobacterium, and seedlings were soaked for 5min while the bacterial solution was continuously shaken.

(5) And taking out the infected arabidopsis thaliana plant for shading growth. In this example, after infection, the plants were removed, placed on their sides in trays, covered with dark black plastic cloth, placed in a growth chamber, and uncovered after 24 h.

(6) Culturing the arabidopsis thaliana plant after shading growth under the illumination, watering 1-2 times per week, and harvesting T0 generation seeds after the seeds are mature.

(7) The harvested T0 generation seeds are sterilized and disinfected, planted in a solid culture medium, vernalized in the dark, cultured in artificial climate and observed for growth. In the embodiment, harvested seeds of T0 generation are sterilized and disinfected, planted in a solid culture medium of MS +25 mg/L Basta, vernalized in the dark at 4 ℃ for 3 days, the culture dish is placed in an artificial climate box for culture, and the growth condition of Arabidopsis plants is observed. Among these, transgenic seeds with BASTA resistance will grow in the selection medium, whereas non-transgenic seeds yellow and die shortly after germination. And harvesting the single plant, planting seeds of T1 generation, continuously screening seedlings by using Basta to obtain positive plants, and harvesting the single plant of T2 generation. And planting T2 generation seeds on the single plant, continuously screening seedlings by using Basta, and if the T3 generation plants all show resistance, indicating that the corresponding T2 generation single plant is a homozygous positive plant.

Example 9

This example provides a mung bean flowering gene VrFT2a, and a comparative experiment was performed based on example 8. In this example, wild-type and homozygous positive Arabidopsis plants were planted, and total RNA was extracted from leaves of wild-type and Arabidopsis T3 generation plants for 3-4 weeks and reverse-transcribed into cDNA. The transgenic Arabidopsis plants 35S:: VrFT2a obtained in example 8 were subjected to fluorescent quantitative PCR analysis using primers (SEQ ID NO.5 and SEQ ID NO. 6) (with Arabidopsis UBQ10 gene as reference gene). The quantitative results are shown in FIG. 2, the expression level of VrFT2a in Arabidopsis thaliana transgenic line 35S:: VrFT2a is much higher than that of wild type, which demonstrates that the VrFT2a gene in the transgenic line 35S:: VrFT2a is over-expressed.

Example 10

This example provides a mung bean flowering gene VrFT2a, and a comparative experiment was performed based on example 8. In this example, Arabidopsis transformed plants obtained in example 8 were grown simultaneously with wild-type control, the same cultivation measures were taken for management, and the flowering time of the transgenic plants and wild-type plants was investigated. The result shows that the mung bean flowering gene VrFT2a can improve the sensitivity of plants to photoperiod and promote flowering in advance when Arabidopsis is transformed. As shown in FIG. 3, the time from sowing to flowering of 14 wild-type controls was 28-31 d, and the average was 29.4 d. The flowering time of the transgenic line is 22-27 days, and the average flowering time is 24.6 days, so that the mung beans VrFT2a can obviously promote flowering of plants. As shown in fig. 4, wild type controls are on the left and transgenic plants are on the right.

Example 11

This example provides a plant transformant comprising the mung bean flowering gene VrFT2a driven by the promoter 35S of example 6 on the genome.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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<120> mung bean flowering gene VrFT2a and application thereof

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Claims

1. The application of an expression vector containing a mung bean flowering gene VrFT2a is characterized in that the expression vector is an over-expression vector obtained by inserting a mung bean flowering gene VrFT2a into a plant expression vector, and a promoter for promoting the expression of a corresponding target gene is a strong promoter 35S; wherein the nucleotide sequence of the mung bean flowering gene VrFT2a is the nucleotide sequence shown in a sequence table Seq ID No. 1; the mung bean flowering gene VrFT2a is expressed in plants to regulate the flowering time of the plants.

2. The use of an expression vector containing a mung bean flowering gene VrFT2a as claimed in claim 1, wherein the host cell of the expression vector is obtained by transferring the expression vector into Agrobacterium tumefaciens.

3. The use of an expression vector containing a mung bean flowering gene VrFT2a as claimed in claim 1, wherein the construction method of the expression vector comprises the following steps: