CN106866802B

CN106866802B - PSF protein related to plant photosynthesis as well as encoding gene and application thereof

Info

Publication number: CN106866802B
Application number: CN201510919574.3A
Authority: CN
Inventors: 张立新; 蒋京京; 马今方; 迟伟
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2015-12-11
Filing date: 2015-12-11
Publication date: 2020-08-04
Anticipated expiration: 2035-12-11
Also published as: CN106866802A

Abstract

The invention discloses a PSF protein related to plant photosynthesis as well as a coding gene and application thereof. The PSF protein of the invention is the protein of a) or b) or c) as follows: a) the amino acid sequence is protein shown as a sequence 2 in a sequence table; b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2 in the sequence table; c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table. The PSF protein is proved to have the function of regulating and controlling the photosynthesis of plants by the separation of the PSF gene and the identification and analysis of the gene function.

Description

PSF protein related to plant photosynthesis as well as encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PSF protein related to plant photosynthesis as well as a coding gene and application thereof.

Background

Photosynthesis is the most important chemical reaction on the earth, and research on molecular mechanism of photosynthesis provides theoretical basis for improving light energy conversion efficiency of crops and opening up a new way for solar energy utilization, thereby promoting and promoting development of agriculture and energy science and technology and formation of new industries.

Chloroplasts are very important organelles for higher plants to perform photosynthesis. The chloroplast genome encodes proteins necessary for photosynthesis, gene expression, other essential organelle functions, and the like. The chloroplast symbiotically evolved from the blue algae as an ancestor combines the common characteristics of eukaryotic cells and prokaryotic cells. The expression regulation of chloroplast genes includes transcription, post-transcriptional processing, translation, and post-translational modification, which are controlled by multiple levels of nuclear genes. Chloroplast gene expression is closely related to the development process of chloroplast, and affected individuals of the chloroplast gene expression cause chloroplast development defect, so that the impaired photosynthetic function is shown.

Disclosure of Invention

It is an object of the present invention to provide a protein.

The protein provided by the invention is the protein of a) or b) or c) as follows:

a) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2 in the sequence table;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No.1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

It is another object of the present invention to provide a biomaterial related to the above protein.

The biological material related to the protein provided by the invention is any one of the following A1) to A12):

A1) nucleic acid molecules encoding the above proteins;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

In the above-mentioned related biological material, the nucleic acid molecule according to A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence is cDNA molecule or DNA molecule of sequence 1 in the sequence table;

2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes the protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above-mentioned biological material, the stringent conditions are hybridization and washing of the membrane at 68 ℃ for 2 times, 5min each, in a solution of 2 × SSC, 0.1% SDS, and hybridization and washing of the membrane at 68 ℃ for 2 times, 15min each, in a solution of 0.5 × SSC, 0.1% SDS, or hybridization and washing of the membrane at 65 ℃ in a solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above biological material, the transgenic plant cell line does not comprise propagation material.

It is also an object of the present invention to provide a novel use of the above protein or the above-mentioned related biological material.

The invention provides the application of the protein or the related biological material in regulating and controlling the photosynthesis activity of plants.

In the above application, the regulation of the plant photosynthesis activity is embodied by regulating the plant photosystem activity.

In the above application, the regulation is an improvement.

The invention also provides the application of the protein or the related biological material in culturing transgenic plants with improved photosynthesis activity.

It is a final object of the present invention to provide a method for breeding transgenic plants with increased photosynthetic activity.

The method for cultivating a transgenic plant with improved photosynthetic activity provided by the invention comprises the steps of introducing the coding gene of the protein into a receptor plant to obtain a transgenic plant; the transgenic plant has a photosynthetic activity greater than that of the recipient plant.

In the method, the nucleotide sequence of the coding gene of the protein is a DNA molecule shown as a sequence 1 in a sequence table.

In the above method, the gene encoding the protein is introduced into the recipient plant via a recombinant vector; the recombinant vector is obtained by inserting a DNA molecule shown as a sequence 1 in a sequence table between BamHI and KpnI enzyme cutting sites of a pSN1301 vector and keeping other sequences of the pSN1301 vector unchanged.

In the above method, the transgenic plant has higher photosynthetic activity than the recipient plant in that the transgenic plant has a higher Fv/Fm value than the recipient plant.

In the above method, the recipient plant is a monocot or a dicot, and the dicot is specifically arabidopsis thaliana.

The invention discovers a PSF gene related to plant photosynthetic function from arabidopsis thaliana, the gene encodes a soluble protein positioned in chloroplast, and deletion mutants of the PSF gene show the characteristics of growth retardation, yellow-green leaf color, obviously reduced photosynthetic efficiency, incapability of realizing autotrophic survival in soil and the like. The PSF protein is proved to have the function of regulating and controlling the photosynthesis of plants by the separation of the PSF gene and the identification and analysis of the gene function.

Drawings

FIG. 1 shows the growth phenotype of mutant psf and wild-type Arabidopsis media for 10 days of germination.

FIG. 2 is chlorophyll-excited fluorescence imaging of mutant psf and wild-type Arabidopsis thaliana.

FIG. 3 is a plot of the slow chlorophyll induction of mutant psf and wild-type Arabidopsis thaliana. Wherein, FIG. 3A is a chlorophyll slow induction curve of mutant psf; FIG. 3B is a slow chlorophyll induction curve of wild-type Arabidopsis thaliana.

FIG. 4 shows the localization of PSF proteins.

FIG. 5 shows phenotype and chlorophyll-excited fluorescence imaging of complementary plants.

FIG. 6 is a slow chlorophyll induction curve for complementary, mutant and wild type Arabidopsis plants.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Both wild type and mutant materials in the examples below were Arabidopsis Columbia ecology (Col-0).

Agrobacterium C58 in the examples described below is described in the documents "Jose '-Antonio Daro's and RicardoFlores (2004), Arabidopsis thaliana has the enzymic knowledge of the genetic expressing recombinant viral species of the family Pospiviroidae, PNASVol.101No. 17; 6792-.

Example 1 obtaining of PSF Gene

Acquisition and phenotype of mutants

1. Obtaining of mutants

Wild type Arabidopsis thaliana and mutant seeds (T-DNA insertion mutants ordered from NASC germplasm resource library, the seeds are named as salk _037487 and salk _072637) are treated by low-temperature vernalization (the seeds are treated by shading at 4 ℃ after being soaked in water) and are disinfected by 10% sodium hypochlorite for 10min after 72h treatment, washed by sterile water for 4 times, sown on an MS culture medium containing 2% sucrose and 0.8% agar, and cultured under the culture conditions of 22 ℃ temperature, 100umolm-2s-1 light intensity and 12h/12h light/dark light cycle to obtain wild type Arabidopsis thaliana seedlings (WT), mutant plants (psf-1) and mutant plants (psf-2).

As a result, it was found that: the mutant plants (psf-1) and (psf-2) had yellow leaf color and were slow to grow and failed to grow autotrophically in soil, compared to wild-type plants (FIG. 1).

2. Plant fluorescence imaging observation

After the plants absorb the light energy, part of the energy is emitted in a fluorescence form, the fluorescence emission quantity of the plants growing normally is very low, but when the structure or the function of the plant thylakoid membrane is abnormal, the electron transfer is blocked, the light energy absorbed by chlorophyll cannot be transferred in time and is converted into chemical energy, and the fluorescence emission quantity of the plants is increased at the moment. Wild type Arabidopsis plants (WT) and mutant plants (psf-1) were observed using plant fluorescence imaging techniques.

The results are shown in FIG. 2: the fluorescence emission of the mutant plant (psf-1) was increased compared to the wild type seedlings, indicating that the photosynthetic electron transport chain in the mutant plant (psf-1) was blocked.

3. Chlorophyll fluorescence kinetic analysis

Chlorophyll fluorescence kinetic analysis was performed on wild type arabidopsis plants (WT) and mutant plants (psf-1) using PAM 2000. During the chlorophyll fluorescence slow induction determination, after dark adaptation of leaves for 20min, a saturation pulse (3000 umolm) is applied^-2s^-1) When PSII is temporarily saturated, the PSII electron acceptor QA is completely reduced, and the chlorophyll fluorescence yield rises from the ground state (F0) to a maximum value (Fm). The maximum photochemical efficiency Fv/Fm of PSII (Fm-F0)/Fm can be calculated.

The slow chlorophyll induction curves of wild type Arabidopsis plants (WT) and mutant plants (psf-1) are shown in FIG. 3: the Fv/Fm value of the wild type Arabidopsis plant (WT) was 0.786, while the Fv/Fm value of the mutant plant (psf-1) was 0.347, and the maximum photochemical efficiency of photosystem II was about half that of wild type, indicating that fluorescence in the mutant plant was stabilized at a very high level, that chemical quenching was not activated, indicating that the electronic pathway after photosystem II was not opened, that chlorophyll fluorescence could not fall back to a stable level (Ft), and that the mutant plant (psf-1) was a photosynthetic function defective mutant.

4. Obtaining of homozygous mutant plants (psf-1)

The wild type Arabidopsis thaliana plant (WT) and the mutant plant (psf-1) are verified by PCR amplification through the Bar gene carried in the T-DNA element, and the mutant plant (psf-1) is confirmed to have T-DNA insertion and is screened to be a homozygous mutant plant (psf-1). The PCR verified primer sequences were as follows:

SALK_037487-LP:ATCCCCAAGTTTGACTTGAGC；

SALK_037487-RP:GTGTTGTGGTTTCGGGTAATG；

LBb1.3：ATTTTGCCGATTTCGGAAC。

if primers SA L K _ 037487-L P and SA L K _037487-RP amplify a band with the size of 1042bp, and SA L K _037487-RP and L Bb1.3 amplify a band with the size of 848bp, the hybrid mutant plant is obtained;

if primers SA L K _ 037487-L P and SA L K _037487-RP amplify a band with the size of 1042bp, and SA L K _037487-RP and L Bb1.3 amplify a homozygous mutant plant without bands.

II, obtaining PSF Gene

Extracting genome DNA of arabidopsis (Col-0 ecotype purchased from the center of arabidopsis biological resources), performing PCR amplification by using the obtained genome DNA as a template and adopting primers PSF-pSN1301-BamHI-s and PSF-pSN1301-KpnI-a to obtain a PCR product, cutting and purifying the PCR product, connecting the PCR product with a T-easy vector, transferring the T-easy vector into escherichia coli DH5 α, screening positive clones, and performing PCR enzyme digestion and sequencing.

PSF-pSN1301-BamHI-s:5'–GCCGGATCCATGCAAACGCTTCTCTGTCA-3'；

PSF-pSN1301-KpnI-a:5'-GGCGGTACCTCACTGACTCTCGAACTTGA-3'。

The sequencing result shows that: the nucleotide sequence of the PCR product is shown as sequence 1 in the sequence table, the gene shown as sequence 1 is named as PSF gene, and the amino acid sequence of the protein coded by the PSF gene is shown as sequence 2 in the sequence table.

Third, localization analysis of PSF protein

1. Construction of pPUC18-GFP intermediate vector

(1) Plasmid pCmGFP (source NCBI Gene ID:7011691) was used as template with primers: 5'-GCCTCTAGAATGAGTAAAGGAGAAGAAC-3' and 5'-AAGCTTCTCGAGTTGTATAGTTCATCC-3', and PCR products, namely GFP gene.

(2) The PCR product and pPUC18 vector (purchased from Taraka, cat # D3218) were double-digested with Xba I and HindIII. The intermediate vector pPUC18-GFP is obtained through connection, transformation and identification.

2. Taking a DNA molecule shown in a sequence 1 as a template, and adopting a primer: 5'-GCCGTCGACATGCAAACGCTTCTCTGTCA-3' and 5'-GGCCCATGGGCTGACTCTCGAACTTGAAC-3', and PCR products, namely PSF genes, are obtained.

Carrying out double digestion on the PCR product and the pPUC18-GFP intermediate vector obtained in the step 1 by SalI and NcoI, and carrying out connection, transformation and identification to obtain pPUC 18-PSF-GFP.

3. pPUC18-PSF-GFP obtained in step 2 was digested with SalI and NcoI, ligated, transformed, and a positive clone was identified. And pPUC18-PSF-GFP was transformed into plant protoplasts.

4. The expression of GFP in mesophyll cells is observed by a laser scanning confocal microscope (L SM510META, Zeiss), the magnification of an objective lens is 40 times during observation, the wavelength of excitation light is 488nm, the band pass BP 505-530 nm is adopted, and the band pass L P560 nm is adopted.

The results are shown in FIG. 4: the PSF protein is localized to chloroplasts.

Example 2 acquisition of complementary plants and analysis of their photosynthetic Activity

Obtaining of first, complementary plants

1. The DNA molecule shown in sequence 1 in the sequence table was inserted between BamHI and KpnI cleavage sites of pSN1301 vector (purchased from Biovector plasmid vector strain cell gene Collection center, product No. Biovector105802), and the other sequences of pSN1301 vector were kept unchanged to obtain a recombinant vector.

2. And (3) transforming the recombinant vector obtained in the step (1) into C58 agrobacterium to obtain a recombinant bacterium.

3. And (3) infecting the homozygous mutant plant (psf-1) obtained in the step (4) in the step one of the example 1 with the recombinant bacterium obtained in the step (2) by using a pollen tube channel method.

4. After receiving seeds, screening positive seedlings on MS culture medium plates added with hygromycin, carrying out PCR verification (the primers verified by the PCR are SA L K _ 037487-L P: ATCCCCAAGTTTGACTTGAGC; SA L0K _037487-RP: GTGTTGTGGTTTCGGGTAATG; L1 Bb 1.3: ATTTTGCCGATTTCGGAAC) and specifically carrying out the following steps that the SA L2K _ 037487-L3P and SA L K _037487-RP amplify strips with the size of 1042bp, the SA L K _037487-RP and L Bb1.3 amplify strips with the size of 848bp to form a heterozygous mutant, the SA L K _ 037487-L P and SA L K _037487-RP amplify strips with the size of 1042bp, and the SA L K _037487-RP and L Bb1.3 amplify non-strip to form a homozygous mutant.

5. And (4) verifying the heterozygous mutant, and harvesting seeds of the single plant. About 100 colonies per plant were sown on MS medium plates supplemented with hygromycin. If all positive seedlings are grown, the heterozygous mutant of the previous generation is proved to be homozygote obtained after the agrobacterium infection.

6. And (3) carrying out PCR verification on the homozygote infected by the agrobacterium obtained in the last step, wherein the primers are the same as the primers verified by the PCR in the step (4), SA L K _ 037487-L P and SA L K _037487-RP have no amplified bands, and SA L K _037487-RP and L Bb1.3 amplify bands with the size of 848bp and are positive complementary seedlings, and the positive complementary seedlings are named as complementary plants (Psf-complementary seedlings).

And replacing the recombinant vector in the steps with a pSN1301 vector, and obtaining an empty vector Arabidopsis plant without changing other steps.

Second, the acquisition of complementary plants and the analysis of their photosynthesis activities

1. And (3) observing the complementary plant (Psf-complementary seedling), the wild type arabidopsis plant and the empty vector arabidopsis plant obtained in the first step by using a plant fluorescence imaging technology.

The results are shown in FIG. 5: as can be seen in FIG. 5, the fluorescence of the complementation plants (Psf-complementation seedlings) was not significantly different from that of the wild type Arabidopsis plants, whereas the transformed empty vector plants could not restore normal fluorescence.

2. Chlorophyll fluorescence kinetic analysis was performed on wild type arabidopsis plants, Psf mutants (Psf-1) and complementary plants (Psf-complementation seedlings) using PAM 2000. The analytical method was referred to 2 in step one of example 1.

The results are shown in FIG. 6, from which it can be seen that: the fluorescence of the complementary plant (Psf-complementary seedling) has no significant difference with that of the wild arabidopsis plant, and the Fv/Fm value of the complementary plant falls to a steady-state level of about 0.8 after the fluorescence is excited, which indicates that the complementary plant recovers the normal photosynthetic function.

The above results indicate that the PSF gene has a function of regulating photosynthesis in plants.

Claims

1. A method for cultivating a transgenic plant with improved photosynthesis activity comprises the step of introducing a coding gene of a protein shown in a sequence 2 in a sequence table into a recipient plant to obtain a transgenic plant; the photosynthetic activity of the transgenic plant is higher than that of the recipient plant; the nucleotide sequence of the coding gene of the protein is a DNA molecule shown as a sequence 1 in a sequence table; the transgenic plant has higher photosynthesis activity than the acceptor plant, and the transgenic plant has higher Fv/Fm value than the acceptor plant; the recipient plant is Arabidopsis thaliana.