CN118240050A - Method for marking plant callus and seeds by using red fluorescent protein TdTMT - Google Patents

Abstract

The invention provides a method for marking plant callus and seeds by utilizing red fluorescent protein TdTMT, which is characterized in that a red fluorescent protein TdTMT gene is optimized and synthesized and is constructed at the downstream of a constitutive promoter (such as a 35S promoter of cauliflower mosaic virus) and a seed specific promoter (such as a rice glutelin GluB-4 gene promoter). Transforming rice, screening to obtain transgenic positive callus, and obtaining the selfed seed of the transgenic positive plant. The callus and seed have obvious red fluorescence when observed under a fluorescence microscope. The method can distinguish transgenic callus from non-transgenic callus, and can distinguish transgenic seed from non-transgenic seed, and has good application prospect in transgenic marking and production.

Description

Method for marking plant callus and seeds by using red fluorescent protein TdTMT

Technical Field

The invention relates to the field of plant molecular biology, in particular to a method for marking plant callus and seeds by using red fluorescent protein TdTMT.

Background

The use of transgenic technology enables the introduction of genes of interest with superior traits into the plant genome, thereby allowing the genetic trait of plants to be improved, opening new avenues for modifying plants to have improved characteristics or traits such as plant disease resistance, insect resistance, herbicide resistance, improved yield quality, improved nutritional quality of the edible parts of plants, etc. However, only a few plant cells are able to take up the exogenous DNA and integrate into the plant genome during transformation, most of the cells being untransformed. Thus, the introduction of the gene of interest often requires the provision of specific selectable markers to the transformed cells by means of screening marker genes in order to identify and identify the transgenic plants.

The fluorescent protein is used as a marker molecule and is derived from marine organisms, can emit fluorescence to carry out optical detection, has small molecular weight, is harmless to cells, does not need exogenous chemical substance treatment, and has higher biological safety. Starting from the discovery and isolation of the earliest fluorescent protein green fluorescent protein (Green fluorescent protein, GFP) in jellyfish named Aequorea victoria in villages under 1962, etc., further research and use of fluorescent proteins has never been stopped. In 1999, red fluorescent protein is reported for the first time, and the earliest red fluorescent protein used for research is DsRed, which is a red fluorescent protein separated from coral, and has the advantages of high fluorescence intensity, high stability and the like. However, there are many problems that DsRed oligomeric state is tetramer, and when protein fusion is performed, multimer is easily formed to affect target protein, toxicity is generated in intracellular expression, and scientists have to make structural modification on the protein. Modern red fluorescent proteins have enhanced fluorescent properties and reduced cytotoxicity compared to the first generation conventional red fluorescent proteins, for example mScarlet, studied such as Bindels, is a monomeric red fluorescent protein, and is more suitable as a fusion tag than other red fluorescent proteins. The optimized DsRed derivative has good fluorescence characteristics, including brightness, rapid maturation and high light stability by optimizing the solubility of the protein.

The protein is the second largest storage substance after relay starch in endosperm of rice seeds, and accounts for 8% -10% of dry weight of brown rice. They can be divided into two main classes according to protein function, namely seed storage proteins as seed storage substances and structural proteins that maintain normal metabolism of seed cells. Rice seed proteins are mostly storage proteins, forming grains (aleurone layers) in cotyledons or endosperm. The seed proteins are mainly classified into 4 types according to their solubility: alkali-soluble gluten, alcohol-soluble prolamin, salt-soluble globulin and water-soluble albumin. The seed-specific promoter may drive expression and storage of a foreign protein or a seed self-protein in the seed.

Disclosure of Invention

The invention aims to provide a method for marking plant callus and seeds by using red fluorescent protein TdTMT.

To achieve the object of the present invention, in a first aspect, the present invention provides a red fluorescent protein TdTMT, which comprises or consists of the amino acid sequence as follows:

i) An amino acid sequence shown as SEQ ID NO. 2; or (b)

Ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)

Iii) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).

In a second aspect, the invention provides a nucleic acid molecule (tdTomato) encoding the protein TdTMT, the nucleotide sequence of which is optimized in full sequence is shown in SEQ ID NO. 1.

In a third aspect, the invention provides a biological material comprising a nucleic acid molecule encoding the protein TdTMT or expressing the protein TdTMT.

Such biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cells.

In a fourth aspect, the invention provides an expression cassette comprising a promoter-a nucleic acid molecule encoding said protein TdTMT-a terminator.

Further, the promoter is at least one of a constitutive promoter, a plant seed specific promoter, a Ubi promoter of rice or corn, an action promoter, a Rubisco small subunit promoter or a Cab promoter, and the like; wherein the constitutive promoter is preferably 35S promoter of cauliflower mosaic virus, and the plant seed specific promoter is preferably rice glutelin GluB-4 gene promoter.

The terminator is AtHSP Ter terminator or Ubi terminator.

Preferably, the promoters are the 35S promoter of cauliflower mosaic virus and the glutelin GluB-4 gene promoter, and the terminator is AtHSP Ter terminator.

In a specific embodiment of the invention, the expression cassette comprises the nucleotide sequence shown as SEQ ID NO. 3.

In a fifth aspect, the invention provides for the use of the protein TdTMT, a nucleic acid molecule encoding the protein TdTMT, a nucleic acid molecule comprising a nucleic acid encoding the protein TdTMT or a biological material expressing the protein TdTMT, or any of the following of the expression cassettes:

1) Used for screening positive callus in plant callus by red fluorescence;

2) For transgenic plant seed markers;

3) Used for preparing transgenic plants.

In a sixth aspect, the present invention provides a method for marking plant calli and seeds using red fluorescent protein TdTMT, the method comprising: allowing the plant to contain a nucleic acid molecule encoding said protein TdTMT or express said protein TdTMT.

Such plants include, but are not limited to, rice, maize, wheat, barley, soybean, cotton, canola, sorghum, or millet.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

The invention provides a method for marking plant seeds by utilizing red fluorescent protein, which is characterized in that a red fluorescent protein TdTMT gene is synthesized, the red fluorescent protein TdTMT gene is constructed at the downstream of a plant seed specific promoter, a genetic transformation vector is transferred into embryogenic callus of rice through agrobacterium mediation, positive callus is selected and differentiated into seedlings, the positive callus and the seed are observed under a fluorescent microscope, and the transgenic seeds have obvious red fluorescence. The method can distinguish transgenic seeds from non-transgenic seeds, can be used for screening markers of crops, and can also be used for marking callus as a transformation screening method.

And (II) fluorescent protein TdTMT nm in excitation wavelength, 635nm in emission wavelength, red in color, has stronger tissue penetrating capability and is easier to distinguish from plant autofluorescence.

And thirdly, the red fluorescent protein is expressed in positive callus, and the transgenic seed and the non-transgenic callus can be easily distinguished through fluorescent detection.

And fourthly, the red fluorescent protein is expressed in positive callus and plant seeds, and the transgenic seeds and the non-transgenic seeds can be easily distinguished through fluorescent detection, so that the red fluorescent protein has good application prospects in transgenic marking and production.

Drawings

FIG. 1 is a map of a skeletal support pCDEEnGluB in accordance with preferred embodiment 1 of the present invention.

FIG. 2 is a map of recombinant plasmid pCDEEnGluB.sup.4-TdTMT in preferred embodiment 2 of the present invention.

FIG. 3 is an electrophoresis chart of recombinant plasmids pCDEEnGluB-TdTMT digested with BamHI and HindIII in accordance with the preferred embodiment of the present invention; wherein M is DL15000 Marker, ck is pCDEEnGluB-TdTMT recombinant plasmid which is not digested, 1 and 2 are pCDEEnGluB-TdTMT recombinant plasmids which are digested, and the size of the digested fragments is 3362bp.

FIG. 4 shows the result of PCR detection electrophoresis of transformed Agrobacterium provided in example 2 of the present invention; wherein M is Marker, H ₂ O is blank control, 1 is pCDEEnGluB-TdTMT recombinant plasmid positive control, 2-10 is pCDEEnGluB-TdTMT recombinant plasmid agrobacterium monoclonal bacteria liquid sample.

FIG. 5 is a PCR detection electrophoresis diagram of hygromycin primers for transgenic sample plants in the preferred embodiment 2 of the present invention; wherein M is 2000bp marker, H is water as blank control, ck-is non-transgenic ZH11 genome DNA, ck+ is pCDEEnGluB-TdTMT recombinant plasmid positive control, and 1-20 is pCDEEnGluB-TdTMT genome DNA of the transgenic plant obtained by screening.

FIG. 6 shows the fluorescence detection results of pCDEEnGluB4-TdTMT transgenic rice calli in the preferred embodiment 3 of the present invention. Wherein ZH11 is a blank control, tdTMT is a fluorescent detection result of the transgenic rice callus of pCDEEnGluB-TdTMT, tdTMT is a fluorescent detection result of the transgenic rice callus of pCDEEnGluB-TdTMT.

FIG. 7 is a bar graph showing the resistant and positive calli of transformed TbRFP and TdTMT calli in preferred embodiment 3 of the invention.

FIG. 8 shows the fluorescence detection results of pCDEEnGluB-TdTMT transgenic T0-generation positive line rice seeds in preferred embodiment 4 of the present invention. Wherein ZH11 is blank control, tbRFP is the fluorescence detection result of rice seeds of pCDEEnGluB-TbRFP transgenic T0 generation positive strain, tdTMT is the fluorescence detection result of rice seeds of pCDEEnGluB-TdTMT transgenic T0 generation positive strain.

Detailed Description

The invention provides a method for marking plant callus and seeds by using red fluorescent protein TdTMT.

The invention provides a red fluorescent protein TdTMT gene for marking plant callus and seeds, which is as follows:

A) A nucleotide sequence shown in SEQ ID NO. 1;

B) The nucleotide sequence with equivalent gene function is obtained by substituting, deleting and adding one or more nucleotide sequences in the nucleotide sequence shown in the A).

The amino acid sequence of the red fluorescent protein TdTMT coded by the gene is shown as SEQ ID NO. 2.

The invention further provides a biological material comprising the red fluorescent protein TdTMT, wherein the biological material is an expression cassette, a vector or a transgenic cell.

Further, when the biological material is an expression cassette, the expression cassette further includes a constitutive promoter, a seed-specific promoter, a functional gene, and a terminator.

Further, the functional gene is a marker gene.

Further, the application is that the red fluorescent protein TdTMT gene is constructed into a vector and then is introduced into a plant to prepare a transgenic plant; alternatively, the biological material is introduced into a plant to prepare a transgenic plant.

Further, after the transgenic plants are prepared, they are screened by the marker gene.

As a preferred embodiment, the present invention provides a method for preparing transgenic rice comprising: transforming a vector comprising the red fluorescent protein TdTMT gene into rice callus by an agrobacterium transformation method;

The rice callus is subjected to resistance screening and differentiation to obtain rice seedlings;

And carrying out rooting culture on the rice seedlings to obtain transgenic rice.

Further, the rice callus is prepared by the following method:

after the rice seeds are shelled and disinfected, inoculating the mature embryo into an induction culture medium, inducing embryogenic callus, and culturing in dark at 28-30 ℃ for 30-50 days;

further, after the pCDEEnGluB-TdTMT vector is transformed into the rice callus, co-culture is further included, wherein the co-culture is performed until thalli appear on the surface of the callus after dark culture at 22-24 ℃;

Further, the resistance screening is to inoculate the co-cultured calli into a screening culture medium added with hygromycin, and perform the resistance screening after dark culture for 30-50 days at 28-30 ℃;

Further, the differentiation is to add the calli subjected to the resistance selection to a differentiation medium to which hygromycin is added, and to culture the calli at 28-30 ℃ for 25-40 days under light.

Further, the rooting culture is to inoculate rice seedlings to a rooting culture medium added with hygromycin for rooting, and the rooting culture is carried out for 5-20 days at the temperature of 30-32 ℃.

Further, after rooting culture, PCR detection is included, and plants with positive detection are selected for planting.

The invention further provides the use of said plant expression cassette or said biological material for marking plant calli and seeds, for example for expression in plant calli and/or seeds.

Further, the plants include, but are not limited to, rice, maize, wheat, barley, soybean, cotton, canola, sorghum, or millet.

The invention constructs and obtains an expression cassette which consists of a constitutive promoter (35S promoter of cauliflower mosaic virus (CaMV)) and a seed specific promoter (rice glutelin GluB-4 gene promoter) and a red fluorescent protein TdTMT gene at the downstream thereof, and can be expressed in callus tissues or seeds of rice, corn or wheat.

The invention provides a method for marking plant callus and seeds by utilizing red fluorescent protein TdTMT, which is characterized in that a red fluorescent protein TdTMT gene is optimally synthesized and is constructed at the downstream of a constitutive promoter (35S promoter of cauliflower mosaic virus) and a seed specific promoter (glutelin GluB-4 gene promoter), the method has the characteristics of high brightness and strong observability, and has good application prospect in marking and production of plant tissues.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 construction of recombinant expression vector containing Red fluorescent protein TdTMT

1. Construction of expression vectors

The synthetic fragment DEOsGluB P-MCS-AtHSPT (Kirschner Biotechnology Co., ltd.) was cut with SacI and HindIII and ligated into vector pC1300, and the cut site was retained to construct backbone vector pCDEEnGluB (FIG. 1). The synthetic fragment TdTMT (Jinsri Biotechnology Co., ltd.) was digested with AfeI and MluI, and ligated into intermediate vector pCDEEnGluB (SEQ ID NO: 4), the AfeI cleavage site was retained, and the MluI cleavage site was not retained, to construct recombinant expression vector pCDEEnGluB-TdTMT, the sequence of which is shown in SEQ ID NO: 5.

2. Transformation

Adding 1 μl of the vector pCDEEnGluB-TdTMT plasmid into competent cells of Escherichia coli, slightly mixing, and ice-bathing for half an hour; e.coli competent cells are transformed by 1.8KV electric shock through an electrotransformation instrument; 1ml of SOC medium was added, the culture was performed at 37℃and 220rpm, the mixture was centrifuged at 5000rpm for 30 seconds, 800. Mu.l of the supernatant was discarded, and the remaining cells were mixed with the medium and plated on LB plates containing kanamycin. Culturing at 37deg.C for about 16 hr, picking single colony, performing colony PCR verification with specific primer (sequence shown in SEQ ID NO: 6-7), selecting positive colony, shaking at 37deg.C and 220rpm overnight, extracting plasmid with high purity plasmid small extraction kit (ZHONGKERITAI), enzyme cutting, detecting correctly (FIG. 3), preserving bacteria, and selecting positive clone for sequencing. Designated pCDEEnGluB-TdTMT, and the carrier map is shown in FIG. 2.

3. Verification pCDEEnGluB4-TdTMT vector

PCDEEnGluB4-TdTMT positive calli and seeds appear red under fluorescent microscopy. pCDEEnGluB4-TdTMT vector contains hygromycin gene and is used for PCR detection of fluorescent and non-fluorescent T1 generation plant genotypes (primer sequences are shown in SEQ ID NO: 6-7).

Example 2 Agrobacterium transformation, identification and Rice genetic transformation

In this example, pCDEEnGluB-TdTMT vector plasmids were transformed into plants to prepare corresponding transgenic plants, and the specific procedures are as follows:

1. agrobacterium transformation and identification

Agrobacterium EHA105 was streaked onto a plate containing Kan (50. Mu.g/ml) +Rif (25. Mu.g/ml) +streptomycin (50. Mu.g/ml) and cultured at 28 ℃. Single colonies were picked and inoculated into 50ml of YEB liquid medium and cultured with shaking at 28℃and 220rpm for 12-16h. 2ml of the bacterial liquid was transferred to 100ml of YEP liquid medium (containing antibiotics) and cultured with shaking at 28℃and 220rpm until OD ₆₀₀ =0.5. Pre-cooling on ice for 10min, centrifuging at 5000rpm for 10min (pre-cooling to 4 ℃ C. In a refrigerated centrifuge). 2 washes (10 ml each) with sterile deionized water and 1 wash with 10% glycerol in 3ml of 10% glycerol. Mu.l of competent cells were transformed with 1. Mu.l of pCDEEnGluB-TdTMT plasmid prepared in example 1 by electric shock at 2.5 KV. Positive clones were selected by culturing on YEP plates containing kanamycin and rifampicin at 28℃and PCR-verified with pCDEEnGluB-TdTMT vector-specific primers (SEQ ID NO: 6-7), and the results were shown in FIG. 4, positive clones (engineering Agrobacterium) were selected, shaken for 36-48 hours, and the bacterial solution was preserved for infection.

2. Agrobacterium-mediated genetic transformation

Taking rice as an example, the engineering agrobacterium obtained above is transformed into rice ZH11 callus by agrobacterium-mediated genetic transformation, and links such as screening, differentiation, rooting and the like under hygromycin screening are carried out. And obtaining positive calli, differentiating and rooting the positive calli, and finally obtaining transgenic positive seedlings.

3. Identification of transgenic lines

In order to identify whether the obtained strain is a transgenic strain, the present example performs PCR verification on a part of positive transgenic plants obtained through screening culture, differentiation culture and rooting culture.

Firstly, extracting sample DNA, wherein the DNA extraction steps are as follows: taking rice leaves with the length of about 2cm, and placing the rice leaves into a 2ml centrifuge tube; 800 μl of 1.5 XCTAB was added to the mortar, the leaves were ground to homogenate and poured back into the centrifuge tube; water bath at 65 ℃ for 20-30min, and mixing for 1 time after reversing every 5min; centrifuging at 12000rpm for 10min; sucking 400 μl of supernatant into a new centrifuge tube, adding 2 times of absolute ethanol precooled by ice, and placing in ice at-20deg.C for 20min; centrifuging at 12000rpm for 10min; the supernatant was discarded, 500. Mu.l of 75% ethanol was added, rinsed upside down, and centrifuged at 8000rpm for 5min; the supernatant was discarded, and the mixture was placed on a super clean bench for drying or naturally airing, and 100. Mu.l of ddH ₂ O was added to dissolve DNA.

PCR amplification detection is carried out on a transgenic strain genome DNA sample by using hygromycin primer Hn-F/Hn-R (SEQ ID NO: 8-9), the primer pair takes an endogenous rice genome as a template and cannot be amplified to obtain fragments, and the size of the fragments obtained by amplification of transgenic seedlings is 561bp.

ZH11 genomic DNA was used as a negative control and water was used as a blank control.

The PCR reaction procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 45s and annealing at 55-65℃for 45s; extending at 72 ℃ for 1.5min for 30-35 cycles; extending at 72 ℃ for 10min; ending at 16 ℃.

The PCR reaction system is as follows:

The PCR product is subjected to agarose gel electrophoresis, the result is shown in figure 5, and the result shows that the transgenic sample contains 561bp transgenic strips which have the same size as the vector contrast (11 samples in 20 samples are transgenic positive plants); whereas the blank and negative control ZH11 failed to spread out the band.

EXAMPLE 3 transgenic plant positive callus fluorescent observations

In the practice of transgenesis, one common problem is: the obtained transgenic lines are screened as positive calli and positive after the later-stage PCR verification, but many lines cannot show phenotype, which may be related to the integration or expression of the target genes in the genome, and the like, and the proportion of phenotype is about 20-30%. This results in many strains, although positive transgenes, which subsequently have no value for continued research, resulting in a significant amount of ineffective work. If the phenotype transgenic lines or transgenic calli can be distinguished in the early stage, the workload can be greatly reduced, and the working efficiency can be improved. This experiment was thus performed for observation.

Red fluorescence was observed during the 3-4 week screening period of calli, and the calli transformed with TdTMT fluorescent protein exhibited bright red spots in some areas under yellow excitation, indicating that these calli had been successfully transformed, whereas calli transformed with TbRFP (NCBI number: ADK 12947.1) exhibited a less bright red state with less visible spots, despite the red fluorescence, indicating that TdTMT was better than TbRFP when observed in calli, and the results are shown in FIG. 6. Meanwhile, the proportion of the callus quantity with red fluorescence is counted, so that the resistance callus rate of TdTMT vectors is 63.16 percent, the resistance callus rate of TbRFP vectors is 57.21 percent, and the resistance callus rate of TdTMT vectors is 5.95 percent higher than that of TbRFP vectors; and the fluorescent callus rate of TdTMT carrier was 25.73%, the fluorescent callus rate of TbRFP carrier was only 14.90%, and the fluorescent callus rate of TdTMT carrier was about 1.8 times that of TbRFP carrier, and the results are shown in table 1 and fig. 7.

TABLE 1 fluorescent protein resistant calli and fluorescent calli statistics

Therefore, tdTMT red fluorescent protein widens the application range of the fluorescent protein in screening rice callus and seeds.

Example 4 fluorescent observations of transgenic T0-generation positive lines seed

The strain with positive transgenosis is planted in a greenhouse, the fluorescence of the seeds is observed when the seeds are harvested, the seeds with TdTMT fluorescent proteins are in bright red under the excitation of yellow light, and the brightness is uniform and expressed in the endosperm of the whole seeds; however, the fluorescence intensity of the seed at rotation TbRFP was significantly weaker and was expressed only in a localized region of the endosperm, which significantly affected the efficiency of sorting the seed by fluorescence, as shown in figure 8.

Comparative example

ZH11 callus is wild type control, and callus with TbRFP fluorescent protein only has a less bright red color and no obvious red bright point under the excitation of fluorescent microscope yellow light, and as shown in figure 4, the fluorescence intensity is obviously weaker than TdTMT positive callus. Seeds with TbRFP fluorescent protein exhibited less bright red color in a partial region under excitation of yellow light and the fluorescence intensity was much weaker than TdTMT positive seeds, as shown in fig. 8.

The invention provides a method for marking plant callus and seeds by utilizing red fluorescent protein TdTMT, which is characterized in that a red fluorescent protein TdTMT gene is optimally synthesized and is constructed at the downstream of a constitutive promoter (such as a 35S promoter of cauliflower mosaic virus) and a seed specific promoter (such as a rice glutelin GluB-4 gene promoter). Transforming rice, screening to obtain transgenic positive callus, and obtaining the selfed seed of the transgenic positive plant. The callus and seed have obvious red fluorescence when observed under a fluorescence microscope. The method can distinguish transgenic callus from non-transgenic callus, and can distinguish transgenic seed from non-transgenic seed, and has good application prospect in transgenic marking and production.

Example 5 use of TdTMT Red fluorescent protein in maize

PCDEEnGluB 4A 4-TdTMT vector plasmid was transformed into maize and the result was similar to rice. Observing red fluorescence during the period of the selected callus, wherein the callus transformed by TdTMT fluorescent protein presents bright red spots in partial areas under the excitation of yellow light, so that the callus is successfully transformed; when transgenic positive maize seeds are harvested, seed fluorescence is observed, the seeds with TdTMT fluorescent proteins appear very bright red under yellow light excitation, and the brightness is uniform and expressed in the whole seed endosperm.

Example 6 use of TdTMT Red fluorescent protein in barley

PCDEEnGluB 4A 4-TdTMT vector plasmid was transformed into barley and the result was similar to rice. Observing red fluorescence during the period of the selected callus, wherein the callus transformed by TdTMT fluorescent protein presents bright red spots in partial areas under the excitation of yellow light, so that the callus is successfully transformed; when transgenic positive barley seeds are harvested, fluorescence of the seeds is observed, the seeds with TdTMT fluorescent proteins show very bright red under the excitation of yellow light, and the brightness is uniform and expressed in the whole seed endosperm.

Example 7TdTMT application of Red fluorescent protein in Soybean

PCDEEnGluB 4A 4-TdTMT vector plasmid was transformed into soybean and the result was similar to rice. Observing red fluorescence during the period of the selected callus, wherein the callus transformed by TdTMT fluorescent protein presents bright red spots in partial areas under the excitation of yellow light, so that the callus is successfully transformed; when transgenic positive soybean seeds are harvested, seed fluorescence is observed, the seeds with TdTMT fluorescent proteins show very bright red under yellow light excitation, and the brightness is uniform and expressed in the whole seed endosperm.

The experimental result shows that TdTMT red fluorescent protein can not only distinguish transgenic callus, seeds and non-transgenic callus, seeds in rice, but also can be used as a transgenic marker in corn, barley and soybean, and has wide application prospect in production.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. Red fluorescent protein TdTMT, characterized in that the protein comprises or consists of the amino acid sequence:

i) An amino acid sequence shown as SEQ ID NO. 2; or (b)

Ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)

Iii) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).

2. A nucleic acid molecule encoding the protein of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 1.

3. A biological material comprising the nucleic acid molecule of claim 2 or expressing the protein of claim 1;

the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineering bacteria.

4. An expression cassette comprising a promoter-the nucleic acid molecule of claim 2-a terminator.

5. The expression cassette of claim 4, wherein the promoter is at least one of a constitutive promoter, a plant seed specific promoter, a Ubi promoter of rice or maize, an action promoter, a Rubisco small subunit promoter, or a Cab promoter; wherein the constitutive promoter is preferably a 35S promoter of cauliflower mosaic virus, and the plant seed specific promoter is preferably a rice gluten GluB-4 gene promoter;

the terminator is AtHSP Ter terminator or Ubi terminator.

6. The expression cassette of claim 5, wherein the promoters are the 35S promoter of cauliflower mosaic virus and the rice glutelin GluB-4 gene promoter, and the terminator is AtHSP Ter terminator.

7. The expression cassette of claim 6, wherein the expression cassette comprises the nucleotide sequence set forth in SEQ ID NO. 3.

8. The protein of claim 1, the nucleic acid molecule of claim 2, the biological material of claim 3 or any of the following uses of the expression cassette of any of claims 4-7:

1) Used for screening positive callus in plant callus by red fluorescence;

2) For transgenic plant seed markers;

3) Used for preparing transgenic plants.

9. A method for marking plant calli and seeds using red fluorescent protein TdTMT, comprising: comprising the nucleic acid molecule of claim 2 or expressing the protein of claim 1.

10. The method of claim 9, wherein the plant is selected from the group consisting of rice, maize, wheat, barley, soybean, cotton, canola, sorghum, and millet.