CN108315334B

CN108315334B - Transcription factor for improving chlorophyll content of plant in high-temperature environment and application of transcription factor in regulating and controlling synthesis of chlorophyll in plant petals

Info

Publication number: CN108315334B
Application number: CN201810070207.4A
Authority: CN
Inventors: 宁国贵; 申玉晓; 王桢; 杜星; 刘芳; 包满珠
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-01-24
Filing date: 2018-01-24
Publication date: 2021-08-20
Anticipated expiration: 2038-01-24
Also published as: CN108315334A

Abstract

The invention discloses a transcription factor for improving chlorophyll content of a plant in a high-temperature environment and application thereof in regulating and controlling chlorophyll synthesis in plant petals. The transcription factor comprises Fbp21 and Fbp21, and the nucleotide sequence of the transcription factor is shown in a sequence table SEQ ID NO: 1 and SEQ ID NO: 3, and the sequence of the corresponding protein is shown as SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The two gene segments can change the flower color of the plant in a high-temperature environment, the two gene segments are sequentially transformed into the plant to obtain a transgenic plant simultaneously containing the two genes, the flower color of the transgenic plant is changed in the high-temperature environment, the chlorophyll content is increased, and a reference is provided for improving the chlorophyll content in the plant in the future plant heat-resistant research.

Description

Transcription factor for improving chlorophyll content of plant in high-temperature environment and application of transcription factor in regulating and controlling synthesis of chlorophyll in plant petals

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a transcription factor for improving the chlorophyll content of a plant in a high-temperature environment and application thereof in regulating and controlling synthesis of chlorophyll in plant petals.

Background

With the global warming, the greenhouse effect is increasingly remarkable, and extreme climates occur occasionally, and various stress conditions, such as high temperature, drought, freeze injury, salt and alkali, are suffered in the plant growth, and the stress conditions seriously affect the plant growth and development process. Important economic and ornamental plants cultivated in the Yangtze river, the middle and lower river regions and other areas of China are often attacked by abnormal high temperature in summer, and the high temperature is accompanied by drought and various diseases, so that the resistance of the plants is reduced, the growth vigor is weakened, and the yield and the quality are reduced. Under the condition of high temperature, the plant cells lose a large amount of water, so that photosynthesis and respiration are inhibited, plant leaves are curled, leaf color turns yellow, the water utilization rate is reduced, electrolyte is leaked out, the permeability of cell membranes is increased, and the microstructure of cells is damaged. The function of photosynthetic organs is hindered or completely lost due to high-temperature stress, which is one of the main causes of the final crop yield reduction (Yexing Yuan, et al, 2004; Chaves et al, 2009; Huiqujun, et al, 2017; Lijiajia, et al, 2017).

A large number of researches show that when plants are stressed by high temperature, structures of chloroplasts and mitochondria are directly and irreversibly damaged, photosynthetic pigments are degraded, and then photosynthesis is inhibited. High temperature stress causes damage to chloroplast outer membrane and thylakoid membrane structures, resulting in H⁺Increased permeability results in decreased ATP production and a concomitant decrease in chlorophyll content (Yang Hui bin, 2012; Huo Jun et al, 2017). How to improve the heat resistance of plants, slow down the inhibition of high temperature on photosynthesis and relieve the damage caused by high temperature stress is a research hotspot all the time. The moshaw 37574f (2017) summarizes that the technology for relieving the high-temperature stress injury of the vegetables mainly has 3 aspects of physical approaches, chemical approaches, biological approaches and the like. The physical way is mainly through the use of cooling equipment to increase the air humidity and CO₂Enrichment, etc.; the chemical approach is mainly characterized in that substances such as plant hormones, saccharides, spermidine, melatonin and the like are applied by external sources; for example, bear blinding et al (2006) increased chlorophyll and soluble sugar content of perennial ryegrass by spraying paclobutrazol, enhanced activities of superoxide dismutase (SOD) and Peroxidase (POD), to alleviate high temperature stress and improve heat resistance of ryegrass. Muyu et al (2017) reasonably apply nitrogen fertilizers with different proportions to the flowering wheat under a high-temperature environment to reduce the reduction range of chlorophyll, so that the damage of high-temperature stress to spring wheat is reduced. The biological approaches mainly comprise stress response related gene overexpression or RNA interference and the like. Genes such as the stress response genes DREB, NAC, HSFs, HsP, WRKY and MYB are reported to be overexpressed in crops and ornamental plants such as corn, tomato, cucumber, ground cover chrysanthemum and the like to improve the heat resistance of the plants (Mishra and the like, 2002; Weqian and the like, 2011; rejuvenation and the like, 2016).

Therefore, the method improves the content of chlorophyll by a transgenic method, relieves the damage of high temperature to the photosynthesis of plants, and is feasible to improve the high-temperature adaptability of the plants.

Disclosure of Invention

The invention aims to provide transcription factors Fbp21 and Fbp22 for improving the chlorophyll content of plants in a high-temperature environment and application of the transcription factors Fbp21 and Fbp22 in regulation and control of synthesis of chlorophyll in plant petals. Firstly, the complete translation region of the transcription factor Fbp21 is combined with a cauliflower mosaic virus promoter and then transferred into a general plant body, and then the complete translation region of the transcription factor Fbp22 is combined with the cauliflower mosaic virus promoter and then transferred into a transformed Fbp21 transgenic plant, so that a transgenic plant Fbp21 x 22 jointly transformed by Fbp21 and Fbp22 is obtained. The color of the transgenic plant is changed under the high-temperature environment (40 ℃,16h/28 ℃,8h), and the chlorophyll content in the plant tissue is obviously improved.

The transcription factor for improving the chlorophyll content of the plants in the high-temperature environment comprises a transcription factor Fbp21 and a transcription factor Fbp22, and the nucleotide sequences of the transcription factors are respectively shown as SEQ ID No.1 and SEQ ID No. 3.

The invention also provides a protein coded by the transcription factor, which comprises a protein Fbp21 and a protein Fbp22, and the amino acid sequences of the proteins are respectively shown as SEQ ID No.2 and SEQ ID No. 4.

The invention also provides a primer pair for obtaining the transcription factor,

the transcription factor Fbp21 primer pair is as follows:

fbp21 forward primer: 5'-ATGGTAAGAGGGAAAACTCAGAT-3'

Fbp21 reverse primer: 5'-TCAGTTTTGTGAGGGACGCCTTATTC-3', respectively;

the transcription factor Fbp22 primer pair is as follows:

fbp22 forward primer: 5'-ATGCAGGAAATGGTGAGAGG-3' the flow of the air in the air conditioner,

fbp22 reverse primer: 5'-TCAGGATCGAAGGCCAATA-3' are provided.

The invention also provides a method for obtaining the transcription factor for improving the chlorophyll content of the plants in the high-temperature environment, which comprises the following steps:

1) designing primer pairs according to the transcription factor Fbp21 sequence and the transcription factor Fbp22 sequence shown in SEQ ID No.1 and SEQ ID No.3 respectively:

the transcription factor Fbp21 primer pair is as follows:

fbp21 forward primer: 5'-ATGGTAAGAGGGAAAACTCAGAT-3'

Fbp21 reverse primer: 5'-TCAGTTTTGTGAGGGACGCCTTATTC-3', respectively;

the transcription factor Fbp22 primer pair is as follows:

fbp22 reverse primer: 5'-TCAGGATCGAAGGCCAATA-3', respectively;

2) carrying out PCR by taking the sequence of the transcription factor Fbp21 as a template and the primer pair of the transcription factor Fbp21, and purifying to obtain a transcription factor Fbp 21;

3) carrying out PCR by taking the sequence of the transcription factor Fbp22 as a template and the primer pair of the transcription factor Fbp22, and purifying to obtain a transcription factor Fbp 22;

preferably, in the step 2) and the step 3),

and (3) PCR system:

10×Buffer	2μl
		10mM dNTP	0.4μl
forward primer	1μl
		Reverse primer	1μl
Taq DNA polymerase	0.2μl
		Form panel	1μl
ddH₂O	14.4μl
		Total up to	20

Conditions of PCR:

the invention also provides a recombinant expression vector which contains the plant expression vector of the transcription factor Fbp21 and the transcription factor Fbp22, wherein the plant expression vector is pCAMBIA2300s and pMOG 22.

The invention also provides a construction method of the recombinant expression vector, which comprises the following steps:

1) the fragment of the transcription factor Fbp21 is introduced into a cloning vector

18T, then carrying out the cloning vector Fbp21-

Carrying out enzyme digestion on 18T and pCAMBIA2300s by Kpn I and BamH I, and connecting the obtained target fragment to an enzyme-digested pCAMBIA2300s vector to obtain a recombinant expression vector pCAMBIA2300s-Fbp 21;

2) the cloning vector containing the target gene Fbp22-

18T and pMOG22 are cut by EcoR I and BamH I, and the obtained target fragment is connected to the cut pMOG22 vector to obtain the recombinant expression vector pMOG22-Fbp 22.

The invention also provides a host cell containing the recombinant expression vector, and the host cell is agrobacterium EHA 105.

The application of one of the following items in improving the chlorophyll content of plants in high-temperature environment,

(1) the transcription factors Fbp21 and Fbp22 described above;

(2) the above recombinant expression vector;

(3) the above Agrobacterium EHA 105.

Preferably, the plant is tobacco.

The invention introduces the coding genes of Fbp21 and Fbp22 into plant cells or tissues together by an agrobacterium-mediated conventional biotechnology method, and cultivates the transformed plant tissues into plants. When the gene fragment of the present invention is used to construct a plant expression vector, any one of an enhancer promoter and an inducible promoter may be added in front of the transcription initiation nucleotide. To facilitate the identification and selection of transgenic plant cells or plants, the vectors used may be engineered, for example by the addition of antibiotic markers which confer resistance (e.g.kanamycin or hygromycin, etc.). The host to be transformed is tobacco. Firstly, a transgenic plant Fbp21 is obtained by independently transforming the Fbp21 gene, then the Fbp22 gene is transferred into a transgenic plant Fbp21 carrying the Fbp21, and finally, a transgenic plant Fbp21 x 22 containing the Fbp21 and Fbp22 genes is obtained. Then, the transgenic plant Fbp21 x 22 is transformed in a high-temperature environment (40 ℃,16h/28 ℃,8h), the chlorophyll content in petals and leaves of the transgenic plant is obviously increased, and the transgenic petals are subjected to transmission electron microscopic observation of subcellular organelles to find that organelles with chloroplast structures appear in the transgenic petals. The result of all experiments is combined to show that the transgenic plant Fbp21 x 22 can increase the chlorophyll content in the plant body under the high-temperature environment.

The invention has the beneficial effects that:

the transcription factors of the invention are named Fbp21 and Fbp22 respectively. Firstly, an overexpression vector pCAMBIA2300s-Fbp21 is constructed and introduced into wild tobacco, then pMOG22-Fbp22 is introduced into a transgenic plant Fbp21 through secondary transformation, and finally two transcription factors Fbp21 and Fbp22 are integrated into the same plant to obtain a transgenic plant Fbp21 x 22. Under the stress of a high-temperature environment (40 ℃,16h/28 ℃,8h), the transgenic tobacco leaves are changed into dark green, the flower color is changed from pink into green, and the chlorophyll content is obviously increased. The transcription factors Fbp21 and Fbp22 provide a molecular biological basis for regulating and controlling the chlorophyll content in petals and leaves of horticultural flowers by means of plant genetic engineering, increasing the high-temperature adaptability of plants, improving the quality of the plants and cultivating new varieties.

Drawings

FIG. 1: the structure of the original excessive plant expression vector pCAMBIA2300s is shown schematically (the size of the vector is 11 Kbp);

FIG. 2: the structural schematic diagram of an original excess plant expression vector pMOG22 (the vector size is 11 Kbp);

FIG. 3: wild type tobacco, Fbp21 × 22 transgenic tobacco flower color comparison map;

in the figure: FIG. 3A is flower color at normal temperature of wild-type tobacco petals; FIG. 3B flower color of wild type tobacco petals at high temperature; FIG. 3C shows the flower color of Fbp21 x 22 transgenic tobacco petals of the present invention at normal temperature; FIG. 3D shows the flower color of Fbp21 x 22 transgenic tobacco petals of the present invention under high temperature conditions.

FIG. 4: positive detection of the transgenic tobacco Fbp21 × 22 resistant seedlings;

FIG. 5: detecting the chlorophyll content in Fbp 21-22 transgenic tobacco petals;

in the figure: FIG. 5A shows the chlorophyll content of Fbp21 × 22 transgenic tobacco petals detected at normal temperature; FIG. 5B shows the chlorophyll content detection of the Fbp21 × 22 transgenic tobacco petals in the high-temperature environment.

FIG. 6: transmission electron microscope observation of chloroplast in transgenic tobacco Fbp 21X 22 petals

In the figure: FIG. 6A is an observation and detail view of ultrastructure at normal temperature of a wild-type tobacco petal; FIG. 6B is an observation and detail view of an ultrastructure of wild type tobacco petals at a high temperature; FIG. 6C is an observation and detail view of the ultrastructure of Fbp21 x 22 transgenic tobacco petals at normal temperature; FIG. 6D is the ultrastructural observation and detail drawing of Fbp21 x 22 transgenic tobacco petals of the present invention at high temperature.

FIG. 7: the expression vector pCAMBIA2300s-Fbp21 constructs picture (the size of the insert is 657 bp).

FIG. 8: expression vector pMOG22-Fbp22 construction map (insert size 657 bp).

Detailed Description

In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.

Example 1: isolation of transcription factors Fbp21 and Fbp22 (abbreviated Fbp21 and Fbp22)

Fbp21 and Fbp22 (petunia transcription factors) are respectively obtained by separating petunia, the sequences of which are shown as SEQ ID No.1-4, plasmids containing the genes are taken as templates to amplify required fragments, and the specific steps are as follows:

1) designing Fbp21 primer pair according to the Fbp21 sequence shown in SEQ ID No. 1:

fbp21 forward primer:

5’-ATGGTAAGAGGGAAAACTCAGAT-3’

fbp21 reverse primer:

5’–TCAGTTTTGTGAGGGACGCCTTATTC-3’；

and using Fbp21 sequence as a template and the Fbp21 primer pair: the PCR is carried out, and the PCR is carried out,

and (3) PCR system:

10×Buffer	2μl
		10mM dNTP	0.4μl
forward primer	1μl
		Reverse primer	1μl
Taq DNA polymerase	0.2μl
		Form panel	1μl
ddH₂O	14.4μl
		Total up to	20μl

Conditions of PCR:

purifying to obtain an amplification product Fbp 21;

2) designing Fbp22 primer pair according to the Fbp22 sequence shown in SEQ ID No. 3:

fbp22 forward primer:

5’-ATGCAGGAAATGGTGAGAGG-3’，

fbp22 reverse primer:

5’-TCAGGATCGAAGGCCAATA-3’；

and using Fbp22 sequence as a template and the Fbp22 primer pair: PCR was performed, and the amplification product Fbp22 was obtained by purification. The PCR system and reaction conditions were the same as those of 1).

The transcription factors Fbp21 and Fbp22 obtained by amplification are respectively connected into

18-T vector (purchased from Takara Bio-engineering, Inc.), screening positive clone and sequencing to obtain the required full-length gene. We named this clone

18-Fbp21 and

18-Fbp22 plasmid.

Example 2: construction and transformation of Fbp21 and Fbp22 overexpression vectors

In order to better elucidate the function of the gene, the cloned gene is overexpressed in tobacco, and the function of the cloned gene is verified from the phenotype of a transgenic plant in a high-temperature environment. The method comprises the following specific steps:

the positive clones obtained in example 1 were first cloned

Carrying out double enzyme digestion on the 18-Fbp21 plasmid by using Kpn I and BamH I to recover a target fragment; meanwhile, the genetic transformation vector pCAMBIA2300S (constructed and donated by the national emphasis laboratory of the university of agriculture crop genetic improvement, Huazhong) carrying the double tobacco mosaic virus promoter 35S was digested in the same manner. After the enzyme digestion, the enzyme digestion fragment containing the Fbp21 gene and the digested pCAMBIA2300s (figure 1) vector are used for connection reaction to transform the Escherichia coli DH5 alpha (the Escherichia coli strain is purchased from Takara Bio-engineering, Inc.). Positive clones were screened by restriction enzyme digestion to obtain a transformation vector, which was named pCAMBIA2300s-Fbp 21.

Will be provided with

The 18-Fbp22 plasmid is subjected to double enzyme digestion by EcoRI and BamHI to recover a target fragment; meanwhile, the genetic transformation vector pMOG22 carrying the double tobacco mosaic virus promoter 35S was digested by the same method. After the completion of the digestion, the digested fragment containing Fbp22 was ligated with the digested pMOG22 (FIG. 2) vector to transform E.coli DH 5. alpha. (E.coli strain was purchased from Takara Bio engineering Co., Ltd.). Positive clones were screened by restriction enzyme digestion to obtain a transformation vector, which was named pMOG22-Fbp 22.

By an agrobacterium-mediated tobacco genetic transformation method, firstly, pCAMBIA2300s-Fbp21 is introduced into wild tobacco, a transformed seedling with resistance is obtained through infection, co-culture and screening, and then a transgenic plant Fbp21 is obtained through conventional steps of rooting, seedling hardening, transplanting and the like. Then, pMOG22-Fbp22 is introduced into the obtained transgenic tobacco Fbp21 again through an agrobacterium-mediated tobacco genetic transformation method, and finally, a transgenic plant Fbp21 x 22 containing two target genes is obtained.

The main steps of genetic transformation and reagents used are as follows:

(1) reagent and solution abbreviations

The abbreviations for the phytohormones used in the culture media are as follows: 6-BA (6-BenzylaminoPurine ); NAA (Naphthalene acetic acid); kan (Kanamycin ); cef (Cefotaxime, cephamycin); hyg (Hygromycin ).

(2) Culture medium formula for tobacco transformation

Table 1 shows the composition and the amount of each of the media of the present invention.

TABLE 1 relevant Medium design for transgenic tobacco

Note: MS medium formulation see: murashige T.and F.Skoog.Physiol.plant,1962, 15: 473-.

Kan (Kanamycin), Cef (cefetaxime, cefamycin), and Hyg (Hygromycin ) in Table 1 were sterilized by filtration through a 0.45 μm filter, and the culture medium was sterilized by the conventional method except for the components Kan, Hyg, and Cef: sterilizing with high pressure steam at 121 deg.C for 20min, cooling the culture medium to 50-60 deg.C, and adding the above components Kan, Hyg, and Cef on a clean bench.

(3) Agrobacterium-mediated genetic transformation procedure

1) Cultivation of Agrobacterium

First, solid LB medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, Kan100 mg/L; agar) with selection for corresponding resistanceLipid 1.5g/L) for 48 hours at 28 ℃; selecting single colony of pre-cultured Agrobacterium, inoculating into liquid LB culture medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, Kan100mg/L) corresponding to resistance selection, shake culturing at 28 deg.C and 200rpm overnight until bacterial liquid concentration OD₆₀₀The value is about 0.6.

2) Agrobacterium transformed plant tissue and resistant seedling acquisition

Tobacco-leaf disc conversion method

a. Cutting young and tender leaves completely unfolded at the upper part of the transgenic early flowering tobacco aseptic seedling, cutting the leaves into small blocks of 0.8cm multiplied by 0.8cm, and putting the small blocks into an aseptic beaker;

b. the prepared bacterial solution was poured into a beaker, and the beaker was gently shaken. Soaking the leaves in the bacterial liquid for 10 min;

c. taking out the leaves in the step b, transferring the leaves to sterilized filter paper, and sucking the leaves to be dry; then placing the mixture on the co-culture medium for dark culture for three days, wherein the culture temperature is 28 ℃;

d. after three days, the leaves are transferred to the budding selection culture medium as shown in the table 1, and are alternately cultured by adopting illumination and dark culture (the illumination intensity is 1000-1500lx, the illumination time is 16h/d, and the dark time is 8h/d), Kan and Hyg resistant buds are screened and differentiated, and the culture temperature is 28 ℃;

e. after the resistant buds are formed, cutting the resistant buds, transferring the cut resistant buds to a strong seedling selection culture medium as shown in the table 1, alternately culturing by adopting illumination and dark culture (the illumination intensity is 1000-1500lx, the illumination time is 16h/d, and the dark time is 8h/d), and screening Kan and Hyg resistant seedlings at the culture temperature of 28 ℃;

f. transferring the screened resistant seedlings to a rooting selection culture medium described in the table 1 to root the resistant seedlings, and alternately culturing the resistant seedlings by adopting illumination and dark culture (the illumination intensity is 1000-.

3) Transplanting

The residual medium on the roots of the transgenic plants was washed off and seedlings with good root systems were transferred to the greenhouse while keeping the moisture moist for the first week.

The T of the transgenic strain obtained in this example, which was positive by PCR detection and simultaneously transferred into plasmids pCAMBIA2300s-Fbp21 and pMOG22-Fbp22₀Transgenic tobacco Fbp21 × 22 was generated.

Example 3: phenotypic observation of Fbp 21-22 transgenic progeny in the field and after high temperature treatment

After Fbp 21-22 transgenic tobacco T3 generation sowing, comparing the flower color of the transgenic plant with the flower color of an untransformed plant (wild type 'WT'), and finding that the flower color of the transgenic Fbp 21-22 tobacco has no obvious difference with that of the wild type tobacco at normal temperature; the colors of the transgenic and non-transgenic tobacco are pink. The wild tobacco and the Fbp21 x 22 transgenic tobacco have obvious difference of the flower color phenotype in the environment with higher temperature. Therefore, wild tobacco and transgenic tobacco are subjected to temperature treatment, the temperature treatment is carried out in two identical illumination incubators, and one incubator is set to be under normal growth conditions, namely 26 ℃,16h/22 ℃,8 h; the other incubator is set to high temperature conditions, namely 40 ℃,16h/28 ℃,8h, and the temperature treatment strategy is as follows: at the early stage, transgenic and wild tobacco plants (more than T3 generation, 3 lines) are grown under normal conditions, and when the tobacco plants have visible small flower buds, the plants are transferred to a high-temperature incubator to grow, and flowers and fruits are produced. After being treated in a high-temperature environment at 40 ℃, the petals of Fbp21 and Fbp22 tobacco are obviously green.

Example 4 extraction and determination of transgenic plants Fbp21 × 22 chlorophyll

The chlorophyll content was measured by an ultraviolet spectrophotometer.

(1) Grinding 0.2g of sample in liquid nitrogen, adding 2ml of 95% absolute ethyl alcohol, standing for 4-5h in a dark place, uniformly mixing the mixture by reversing every 2h, centrifuging the mixture at 12000rpm for 5min, carefully sucking the supernatant into a new centrifugal tube to be measured;

(2) and respectively reading OD values under A665 and A649, and calculating the corresponding content of Chla and Chlb through a formula.

Ca 13.95a665-6.88a 649; cb ═ 24.96a649-7.32a 665. See the literature Arnon 1949.

The measurement results show that under the high-temperature environment, the chlorophyll content of the Fbp21 × 22 transgenic tobacco and the chlorophyll content of the normal tobacco show a significant difference, and the chlorophyll content of the Fbp21 × 22 is more than 4 times that of the wild tobacco under the same conditions (shown in figure 5).

Example 5 transgenic tobacco Fbp21 x 22 petals Transmission Electron microscopy

Collecting petals of wild type and Fbp21 x 22 transgenic tobacco plants under normal growth condition and high temperature growth condition respectively, and carrying out transmission electron microscopic observation of subcellular organelles.

The specific operation steps are as follows:

(1) cutting the corolla part of the petal into small strips, quickly putting into 2% glutaraldehyde stationary liquid, vacuumizing to prevent the material from floating upward, and storing at 4 ℃;

(2) osmium tetroxide (OsO) 1% at 4 deg.C₄) Fixing for 3 h;

(3) gradient dehydration treatment of 30-100% ethanol;

(4) embedding the sample with epoxy resin 812 embedding medium;

(5) slicing with ultrathin slicer, and placing in uranyl acetate [ UO ]₂(CH₃COO)₂·2H₂0]Lead citrate (C)₆H₅O₇Dyeing in Pb);

(6) observed under a transmission electron microscope (H-7650, Hitachi, Japan). Reference may be made in particular to the document Bonora et al 2000.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

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210 215

Claims

1. Use of one of the following for increasing the chlorophyll content of tobacco in a high temperature environment, characterized in that:

(1) transcription factorFbp21AndFbp22(ii) a The nucleotide sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 3;

(2) a recombinant expression vector; the recombinant expression vector contains the transcription factorFbp21And transcription factorsFbp22The plant expression vector of (1), wherein the plant expression vector is pCAMBIA2300s and pMOG 22; transcription factorFbp21AndFbp22(ii) a The nucleotide sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 3;

(3) agrobacterium EHA105 containing a recombinant expression vector; wherein the recombinant expression vector contains the transcription factorFbp21And transcription factorsFbp22The plant expression vector of (1), wherein the plant expression vector is pCAMBIA2300s and pMOG 22; transcription factorFbp21AndFbp22(ii) a The nucleotide sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 3.