CN102718849A - Protein related to chlorophyll synthesis and coding gene and application thereof - Google Patents

Abstract

The invention discloses protein related to chlorophyll synthesis and a coding gene and an application thereof. The protein of the invention is the protein of the following a) or b): a), protein composed of an amino acid sequence shown by SEQ ID NO: 1; b), chlorophyll synthesis-related protein which is derived from a) and is obtained by substitution and/or deletion and/ or addition of one or more than one of amino acid residues in the amino acid sequence shown by SEQ ID NO: 1. The experiment proves that the gene CBR1 of the invention can adjust the chlorophyll synthesis process, and can improve the chlorophyll synthesis capability of a plant; the coding protein of the gene CBR1 can adjust the expression of gene PORA in the chlorophyll synthesis process, and thereby the chlorophyll synthesis is influenced. The gene of the invention can be used for artificial reconstruction or modification of the chlorophyll synthesis.

Description

Proteins related to chlorophyll synthesis and their coding genes and applications

technical field

The invention relates to a protein related to chlorophyll synthesis, its coding gene and application.

Background technique

Photosynthesis is the process of using solar energy on the largest scale on earth, which provides organic matter, energy and oxygen for almost all life activities. 90%-95% of plant dry matter comes from the products of photosynthesis, and photosynthesis is the material basis for crop yield formation. Photosynthesis is mainly carried out in the chloroplast of plant leaves, which requires the participation of a series of pigments and protein complexes. Chlorophyll is the main pigment that absorbs light energy. The reduction or absence of chlorophyll will make the color of leaves lighter and directly affect the efficiency and function of photosynthesis. With the development of plant molecular biology and biochemistry, various enzymes and their genes that catalyze chlorophyll synthesis have been isolated and cloned. However, the synthesis of chlorophyll is regulated by internal and environmental factors, and this regulation is controlled by nuclear genes and (or) plastid genes. Therefore, there is a complex regulatory network for the synthesis and degradation of chlorophyll. The isolation and cloning of key or important regulatory factors is of great significance for revealing the metabolism of chlorophyll and improving the efficiency of light energy utilization through artificial modification.

Contents of the invention

One object of the present invention is to provide a protein synthesized with chlorophyll and its coding gene.

The protein provided by the present invention is derived from Arabidopsis thaliana, and is the protein of the following a) or b):

a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

b) A protein derived from a) wherein the amino acid sequence shown in SEQ ID NO: 1 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and is related to chlorophyll synthesis.

The coding gene is shown in 1) or 2) or 3) as follows:

1) its nucleotide sequence is a DNA molecule shown in SEQ ID NO: 2;

2) a DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the protein;

3) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1) and encodes the protein.

In order to facilitate the purification of the protein in (a) above, the amino-terminus or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 can be linked with the tags shown in Table 1.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK

c-myc 10 EQKLISEEDL

The protein in (a) or (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The coding gene of the protein in (b) above can be deleted by the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carry out the missense of one or several base pairs mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The stringent conditions may be hybridization at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, and the solution is used to wash the membrane.

A pair of primers for amplifying the full length of any of the above-mentioned coding genes or any fragment thereof also falls within the protection scope of the present invention.

The primer pair is specifically as follows: one primer sequence in the primer pair is shown in SEQ ID NO: 3, and the other primer sequence in the primer pair is shown in SEQ ID NO: 4.

Recombinant vectors, recombinant bacteria, transgenic cell lines, recombinant viruses or expression cassettes containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.

In any of the above-mentioned recombinant vectors, the recombinant vector is a recombinant expression vector obtained by inserting the coding gene of the protein described in claim 1 or the coding gene described in claim 2 or 3 at the multiple cloning site of the expression vector pJIM19-9Myc .

Another object of the present invention is to provide a method for growing plants with improved chlorophyll synthesis ability.

The method for cultivating plants with improved chlorophyll synthesis ability provided by the present invention comprises the following steps: introducing a gene encoding any of the above proteins into the starting plant to obtain a transgenic plant with improved chlorophyll synthesis ability compared with the starting plant.

In the above method, the gene encoding the protein is introduced by any one of the above recombinant vectors.

In the above method, the starting plant is a monocot or a dicot; the dicot is Arabidopsis.

Experiments have proved that the gene CBR1 of the present invention can regulate the chlorophyll synthesis process and improve the chlorophyll synthesis ability of plants, and its encoded protein can regulate the expression of PORA gene in the chlorophyll synthesis pathway, thereby affecting the synthesis of chlorophyll. The gene of the invention can be used for artificially transforming or modifying the synthesis of chlorophyll.

Description of drawings

Figure 1 shows the results of yeast screening.

Figure 2 is a comparison of PORA gene expression levels.

Detailed ways

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation of gene

The discovery of genes

Yeast hybridization screening library: DNA fragments (5'-Aattacattaaaatatctataaaatatctataaaatatctactgac-3' and 5'-ctaggtcagtagatattttatagatattttatagatattttaatgt-3') were placed at 95°C for 5 minutes, and then gradually cooled naturally to combine into double-stranded DNA to obtain double-stranded DNA fragments; The pHISi vector (purchased from Clontech Company) was digested with EcoRI and XbaI, recovered and purified, and ligated with double-stranded DNA to obtain the yeast vector pHIS-EE; the pHIS-EE was transformed into yeast Ym4271 cells, and then combined with the cDNA library (purchased from Clontech Company) hybridization, and then screened positive clones on the yeast culture medium lacking His and Leu, and sequenced and analyzed the clones.

One of the clones was obtained from a yeast hybridization screen (Fig. 1, 1, vector control; 2, CBR1). The nucleotide sequence of the gene contained in the clone is shown in SEQ ID NO: 2, and its open reading frame is 1164bp long, which is denoted as gene CBR1; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, It is 387 amino acids.

2. Differences in expression of PORA gene in wild-type Arabidopsis and mutant Arabidopsis

Seeds of cbr1 mutants were purchased from the International Arabidopsis Biological Resource Center.

The cbr1 mutant is a mutant as follows: Compared with the Col wild-type genome, the cbr1 mutant only has a mutation in the cbr1 gene in its genome, except for the mutation site, other sequences in the mutant genome are similar to the Col Wild-type Arabidopsis is completely identical.

PORA is a very important enzyme in the chlorophyll synthesis pathway, and the expression difference of gene PORA in cbr1 mutant and Col wild-type Arabidopsis was detected.

The seeds of the cbr1 mutant and Col wild-type Arabidopsis were grown in the incubator for 6 days to obtain seedlings; the total RNA of the leaves of the seedlings was extracted respectively, and the total RNA of the leaves of the cbr1 mutant and Col wild-type Arabidopsis were respectively extracted. Total RNA of mustard leaves was used as a template for quantitative RT-PCR analysis. The primers for amplifying PORA gene were as follows: (5'-CCTTCAAGCTGCTTCTTTGG-3') and (5'-CCTTGAGGAAGTCTCTGCAC-3').

The results are shown in Figure 2. It was found that compared with the Col wild type, the expression of the PORA gene in the cbr1 mutant was greatly inhibited, indicating that the mutation of the CBR1 gene affects the expression of the PORA gene, that is, the CBR1 gene regulates the expression of the PORA gene.

3. Gene overexpression

The pJIM19-9Myc vector has been disclosed in the literature (Yang et al., Plant Cell, 2005, 17: 804-821), and the public can obtain it from the Institute of Botany, Chinese Academy of Sciences. The Agrobacterium strain GV3101 has been disclosed in literature (Steven J Clough and Andrew F Bent, Plant Journal, 1998, 16: 735-743), and the public can obtain it from the Institute of Botany, Chinese Academy of Sciences.

The total RNA of leaves of Col wild-type Arabidopsis thaliana was used as a template, and the primer pair p1/p2 was used for RT-PCR amplification, and the PCR amplification product obtained was the target gene.

p1: 5'-GTCGACCTATGGCGTCGTCTCCGTTGAC-3' (SEQ ID NO: 3)

p2: 5'-ACTAGTTAAGTGGAGATGAATCTCATGC-3' (SEQ ID NO: 4).

Digest the PCR amplification product with restriction endonuclease XhoI and SpeI, and recover the target gene fragment; digest the pJIM19-9Myc vector with restriction endonuclease XhoI and SpeI, and recover the large vector fragment; combine the target gene fragment with the large vector fragment Ligation; the ligation product was transformed into Escherichia coli DH5α, screened with LB+Kan medium, positive clones were picked, the plasmids of positive clones were extracted, and the plasmids were sequenced. The results were displayed between the XhoI and SpeI restriction sites of the pJIM19-9Myc vector The inserted gene sequence is shown in SEQ ID ID: 2, and the insertion direction is from XhoI to SpeI. The correct recombinant expression vector was designated as pJIM-CBR1.

The plant expression vector pJIM-CBR1 was transformed into Agrobacterium strain GV3101 by electric shock method, positive clones were screened on LB+Spec+Gent resistant medium, and the positive recombinant Agrobacterium was designated as GV3101-pJIM-CBR1.

The seeds of the cbr1 mutant were cultured at 16 hours of light/8 hours of darkness at 22° C. for 4 weeks to obtain cbr1 mutant plants.

Arabidopsis cbr1 mutant plants were transformed by flower soaking method.

Screening and identification of transgenic plants: T1 generation seeds were obtained after Agrobacterium transformation, grown on MS+Kanamycin 50mg/L medium for 7 to 10 days, screened for resistant plants, and transferred to culture soil for growth. After selfing and fruiting, the T2 generation seeds were harvested, the seeds germinated on the medium of kanamycin 50 mg/L, and then the seeds of the T3 generation were obtained through selfing and fruiting, and 100% anti- Sexual plants, indicating that the obtained T3 generation plants are homozygous transgenic plants.

At the same time, the Arabidopsis cbr1 mutant transformed into the empty vector pJIM19-9Myc was used as the empty vector control.

Gene identification of homozygous transgenic plants: grow the seeds of the transgenic plants, the seeds of the Arabidopsis cbr1 mutant, the seeds of the Col wild-type Arabidopsis, and the seeds of the empty vector control in the incubator for 6 days to obtain seedlings respectively; Extract the total RNA of the leaves of the seedlings, and use primers to analyze p1/p2 by RT-PCR. The target product shown in SEQ ID NO: 2 can be obtained in the homozygous transgenic plants and the Col wild type, while the cbr1 mutant and the empty vector None in the control, indicating that the mutants were complemented.

The seeds of the T3 generation transgenic plants, the seeds of the Arabidopsis cbr1 mutant, the seeds of the Col wild-type Arabidopsis, and the seeds of the empty vector control were cultured under the same conditions to obtain complete plants. The chlorophyll content in the leaves of homozygous transgenic plants, Arabidopsis cbr1 mutant plants, Col wild-type Arabidopsis plants, and empty vector control seeds were detected respectively.

The detection method of chlorophyll content: extract leaf chlorophyll with 80% acetone extraction method, measure the absorption at wavelengths of 663nm and 645nm, and calculate the chlorophyll content.

The calculation formula of chlorophyll a content is 12.7×A663-2.69×A645.

The calculation formula of chlorophyll b content is 22.9×A645-4.48×A663.

The experiment was repeated three times, and the results were average ± standard deviation.

The results are as follows:

Chlorophyll a content was as follows: homozygous transgenic plants: 517.2±39.4 (ng/mg FW); Arabidopsis cbr1 mutant plants: 332.8±35.2 (ng/mg FW); Col wild-type Arabidopsis plants: 499.1±40.6 ( ng/mg FW); the results of the empty vector control group were consistent with those of the cbr1 mutant plants.

Chlorophyll b content was as follows: homozygous transgenic plants: 251.8±24.3 (ng/mg FW); Arabidopsis cbr1 mutant plants: 130.6±18.6 (ng/mg FW); Col wild-type Arabidopsis plants: 227.8±24.4 ( ng/mg FW); the results of the empty vector control group were consistent with those of the cbr1 mutant plants.

By comparing the chlorophyll content of the cbr1 mutant and Col wild-type seedlings, it was found that the chlorophyll in the cbr1 mutant was lower than that of the wild type, indicating that the mutation of the CBR1 gene affected the synthesis of chlorophyll, and the exogenous CBR1 transgene could restore the chlorophyll content of the mutant, indicating that CBR1 functions in the regulation of chlorophyll synthesis.

Claims (10)

1. A protein, which is a protein according to a) or b) as follows: a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; b) A protein derived from a) wherein the amino acid sequence shown in SEQ ID NO: 1 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and is related to chlorophyll synthesis. 2. the coding gene of protein described in claim 1. 3. The coding gene according to claim 2, characterized in that: the coding gene is shown in 1) or 2) or 3) as follows: 1) its nucleotide sequence is a DNA molecule shown in SEQ ID NO: 2; 2) a DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the protein; 3) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1) and encodes the protein. 4. A pair of primers for amplifying the full-length gene or any fragment thereof according to claim 2 or 3. 5. The primer pair according to claim 4, characterized in that: a primer sequence in the primer pair is as shown in SEQ ID NO: 3, and another primer sequence in the primer pair is as SEQ ID NO: 4 shown. 6. A recombinant vector, a recombinant bacterium, a transgenic cell line, a recombinant virus or an expression cassette containing the coding gene of claim 2 or 3. 7. The recombinant vector according to claim 6, characterized in that: the recombinant vector is inserted into the multi-cloning site of the expression vector pJIM19-9Myc into the coding gene of the protein described in claim 1 or the protein described in claim 2 or 3 The recombinant expression vector obtained from the coding gene. 8. A method for cultivating plants with improved chlorophyll synthesis ability, comprising the steps of: introducing the coding gene of the protein according to claim 1 into the starting plant to obtain a transgenic plant with improved chlorophyll synthesis ability compared with the starting plant. 9. The method according to claim 8, characterized in that: the gene encoding the protein according to claim 1 is introduced through the recombinant vector according to claim 6 or 7. 10. The method according to claim 8 or 9, characterized in that: the starting plant is a monocot or a dicot; and the dicot is Arabidopsis.

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