CN112266904B - Gene BnaC08-CYP705a12 involved in cabbage type rape chlorophyll synthesis and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene BnaC08-CYP705a12 related to chlorophyll synthesis and application thereof. The gene BnaC08-CYP705a12 is obtained by cloning and separating a cabbage type rape yellowing seedling mutant material, and the gene has 1 base substitution with a wild type. The invention constructs a PBI121-BnaC08-CYP705a12 over-expression vector and converts the vector into a wild type to obtain a PBI121-BnaC08-CYP705a12 over-expression transgenic plant, and the leaves have a yellowing phenotype; meanwhile, an RNAi-BnaC08-CYP705a12 vector is constructed and transformed into the yellowing seedling, and an RNAi-BnaC08-CYP705a12 transgenic plant is obtained, and the transgenic plant restores the green leaf phenotype. The cloned gene mutation can lead to the reduction of the chlorophyll content of the brassica napus, thereby leading to the yellowing character of the rape leaves.

Description

Gene BnaC08-CYP705a12 involved in cabbage type rape chlorophyll synthesis and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to separation cloning, functional verification and application of DNA fragments (genes) related to chlorophyll synthesis of brassica napus.

Background

The leaf is a main place for photosynthesis of higher plants, the leaf color change of the plants can cause the change of the photosynthesis efficiency of the leaf, and the research on leaf color mutants of the plants can clarify the regulation and control mechanism of the leaf color on the photosynthesis, thereby providing good theoretical basis and research materials for the research on the high light efficiency of the plants.

Chlorophyll synthesis and heme biosynthesis are two important branches of tetrapyrrole anabolism. Biosynthesis of higher plant chlorophyll ALA is synthesized from glutamic acid and a-ketoglutaric acid. 2 molecules of ALA are catalyzed by ALA dehydratase ALAD to produce 1 molecule of porphobilinogen, which has a contiguous ring. Deamination catalysis is carried out on 4 molecules of porphyrinogen to form uroporphyrinogen III, and 4 acetic acid side chains of uroporphyrinogen III generate coproporphyrinogen III with 4 methyl groups through uroporphyrinogen III decarboxylase. Coproporphyrinogen III is shuttled, dehydrogenated, oxidized to form protoporphyrin IV in coproporphyrinogen III oxidase and protoporphyrinogen oxidase, and protoporphyrin IX is a watershed for chlorophyll or heme formation. If combined with magnesium, mg-protoporphyrin IV is formed, and protochlorophyllin is formed by Mg-protoporphyrin IV methyltransferase, mg-protoporphyrin IV monomethyl ester cyclase and divinyl reductase. The protochlorophyllin is reduced by light to generate chlorophyllin a, then chlorophyll a is formed by catalysis of chlorophyll synthase, and chlorophyll b is converted from chlorophyll a. The process of chlorophyll synthesis involves the participation of multiple enzymes. The mutation of any gene in the chlorophyll synthesis process can cause the inhibition of chlorophyll synthesis, so that the content of various pigments in chloroplasts is changed, and the leaf color variation of plants is caused. If protoporphyrin IV is combined with iron to form Fe ²⁺ Chelate, then to produce heme b, in the plant body heme b is formed into photosensitive pigment chromophore through a series of oxidation reduction. If the gene related to heme metabolic reaction is mutated, the balance of tetrapyrrole metabolism is likely to be broken, so that the content of heme in cells is increased, accumulation of heme influences the acid value contents of ALA and protochlorophyll through feedback inhibition of Glu-tRNA reductase activity, synthesis of chlorophyll is inhibited, and the mutant causes variation of leaf color due to lack of chlorophyll. Heme feedback inhibition is a critical regulatory step in the chlorophyll synthesis process. In tomato aurea and yenow-green-2 mutants, genetic variation encoding heme oxidase and photopigment chromophore synthase resulted in excessive intracellular heme accumulation, feedback of which inhibited synthesis of the chlorophyll precursor aminoketovalerate, rendering the mutants appear as leaf yellowing.

Cytochrome P450 monooxygenases (P450 s) are a conserved superfamily of heme-sulfur genes and play an important role in a wide variety of stromal metabolism, including the metabolism of endogenous and exogenous compounds. The three-dimensional structure of cytochrome P450 is very conserved, and a conserved characteristic heme binding domain FxxGxRxCxG is positioned in the catalytic center of CYP450 with all known structures, and the sequence is very conserved, and the region is also a key basis for identifying the cytochrome P450. Cysteine (C) in this domain site is fully conserved in all P450s and is the 5 th axis ligand of the heme binding site. The cytochrome P450 catalyzes a reaction in which electrons are transferred from NADPH/NADP to flavoproteins and iron-sulfur proteins and then to cytochrome P450 oxidase. Since most of P450 has low and unstable content in plants, it is very difficult to directly separate and purify P450 enzyme, so cloning P450 gene, researching P450 gene expression and regulation, and researching P450 function by heterologous expression, deletion mutation and mutant complementation becomes an important way.

Disclosure of Invention

The invention aims to provide a gene related to chlorophyll synthesis of brassica napus, and the BnaC08-CYP705a12 gene can participate in chlorophyll synthesis so as to control the properties of rape leaves.

The gene related to the chlorophyll synthesis of the brassica napus provided by the invention is named as BnaC08-CYP705a12 (Brassica napus CYTOCHROME P, FAMILY 705,SUBFAMILY A,POLYPEPTIDE 12), and a gene fragment cloned from a brassica napus yellowing mutant material cde1 is replaced by 1 base from a cloned sequence in a wild type ZS 11.

A gene BnaC08-CYP705a12 related to cabbage type rape chlorophyll synthesis is one of the following amino acid residue sequences, has 1 amino acid residue substitution with wild type ZS11, and is positioned in aa-320:

(1) SEQ ID NO:1, a step of;

(2) SEQ ID NO:1 through substitution and/or deletion and/or addition of one to ten amino acid residues and has the function of participating in the chlorophyll synthesis of brassica napus.

SEQ ID NO:1 consists of 492 amino acid residues, and 15 th to 491 th amino acid residues from the amino terminal (N terminal) are conserved sequences.

One to ten amino acid residues of the substitutions and/or deletions and/or additions are amino acid residues in the non-domain, the alteration of which does not affect the function of the protein.

The cDNA of the gene which codes for the gene BnaC08-CYP705a12 (Brassica napus CYTOCHROME P450, FAMILY 705,SUBFAMILY A,POLYPEPTIDE 12) involved in the chlorophyll synthesis of the brassica napus is one of the following nucleotide sequences, has 1 base substitution with the cloned sequence in the wild ZS11, and is positioned at 959 base from the 5' end:

(1) SEQ ID NO:2, a DNA sequence of seq id no;

(2) Encoding SEQ ID NO:1, a DNA sequence of seq id no;

(3) And SEQ ID NO:2 and has nucleotide sequence which has more than 90 percent of homology and participates in the chlorophyll synthesis of the brassica napus;

(4) Can be matched with SEQ ID NO in a sequence table under high-stringency conditions: 2, and a nucleotide sequence to which the defined DNA sequence hybridizes.

The high stringency conditions are hybridization and washing of the membranes in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

SEQ ID NO:2 consists of 1479 bases, the coding sequence of which is the 1 st to 1479 th bases of the 5' end, and the coding sequence of which has the sequence shown in SEQ ID NO:1, and the base at the 43-1473 position from the 5' end codes a conserved sequence.

The genome gene is one of the following nucleotide sequences:

(1) SEQ ID NO:3, a DNA sequence of 3;

(2) And SEQ ID NO:3 has more than 90 percent of homology and has a nucleotide sequence which participates in the chlorophyll synthesis of the brassica napus;

(3) Can be matched with SEQ ID NO in a sequence table under high-stringency conditions: 3, and a nucleotide sequence to which the defined DNA sequence hybridizes.

The high stringency conditions are hybridization and washing of the membranes in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

SEQ ID NO:3 consists of 1896 bases, the 1 st to 672 nd bases from the 5' end are the first exon of the genome gene, the 673 th to 699 th bases from the 5' end are the first intron of the genome gene, the 700 th to 907 th bases from the 5' end are the second exon of the genome gene, the 908 th to 1297 th bases from the 5' end are the second intron of the genome gene, and the 1298 th to 1896 th bases from the 5' end are the third exon of the genome gene.

The BnaC08-CYP705a12 gene is related to the chlorophyll synthesis of the brassica napus. After the complete coding sequence of the gene is fused with an over-expression vector PBI121, a PBI121-BnaC08-CYP705a12 vector is constructed and is transformed into double 11 (ZS 11) in a conventional good variety of brassica napus, and leaves of a transgenic plant are obviously yellowing compared with those of a control plant; the constructed gene RNAi-BnaC08-CYP705a12 expression vector is transformed into a homozygous brassica napus yellowing mutant cde1, and leaves of a transgenic plant can be restored to a green leaf phenotype. The BnaC08-CYP705a12 gene can participate in the synthesis of chlorophyll, so that the properties of rape leaves can be controlled.

The BnaC08-CYP705a12 protein is positioned in a cell membrane by subcellular. The coding sequence of the gene without the terminator is directly transferred into tobacco after being combined with subcellular localization carrier pA7-GFP, and fluorescence is observed under a laser confocal microscope, and the result shows that BnaC08-CYP705a12 protein is localized in cell membranes and plays a role in the cell membranes.

Expression vectors, transgenic cell lines, host bacteria and encoded proteins containing the genes of the invention are all within the scope of the invention.

Primers and primer pairs for amplifying any fragment of the BnaC08-CYP705a12 gene are also within the scope of the invention.

The cloned gene BnaC08-CYP705a12 can provide good theoretical basis and research materials for research on plant high photosynthetic efficiency.

The specific operation steps are as follows:

(1) Introducing the gene BnaC08-CYP705a12 into a brassica napus receptor by using an agrobacterium-mediated transgenic method to obtain a transformed plant;

(2) Analyzing and identifying positive transgenic plants by means of a PCR method;

(3) Planting the transgenic plant in the step (2) and observing the characters of the transgenic plant;

(4) Analysis of expression of the gene BnaC08-CYP705a12 involved in chlorophyll synthesis of brassica napus in transgenic plants and wild plants by means of qRT-PCR.

The beneficial effects are that:

the invention finds a gene BnaC08-CYP705a12 which is derived from rape and participates in the chlorophyll synthesis of the brassica napus, the gene can reduce the chlorophyll synthesis in the leaves of plants after being over-expressed, thereby reducing the chlorophyll content in the leaves and finally causing the yellowing of the leaves of the plants, and when the gene is interfered in expression, the plants can recover the chlorophyll synthesis of the brassica napus which participates in the gene, thereby recovering the chlorophyll content in the leaves of the plants and enabling the leaves of the yellowing materials to recover the phenotype of green leaves.

The gene BnaC08-CYP705a12 is related to the chlorophyll synthesis of the brassica napus. After the complete coding sequence of the gene is fused with an over-expression vector PBI121, a PBI121-BnaC08-CYP705a12 vector is constructed and is transformed into double 11 (ZS 11) in a conventional good variety of brassica napus, and leaves of a transgenic plant are obviously yellowing compared with those of a control plant; the constructed gene RNAi-BnaC08-CYP705a12 expression vector is transformed into a homozygous brassica napus yellowing mutant cde1, and leaves of a transgenic plant can be restored to a green leaf phenotype. The BnaC08-CYP705a12 gene can participate in the synthesis of chlorophyll, so that the properties of rape leaves can be controlled.

Drawings

FIG. 1 is a framework structure of the BnaC08-CYP705a12 genomic gene;

FIG. 2 is a protein sequence analysis of BnaC08-CYP705a 12;

FIG. 3 is a schematic diagram of plant over-expression vector PBI121 construction;

FIG. 4 is a schematic diagram of plant PFGC5941 vector construction;

FIG. 5 is a BnaC08-CYP705a12 gene overexpression and RNAi expression vector transgenic plant phenotype; wherein, the left 1 is a wild ZS11 single plant, the left 2 is a phenotype of a PBI121-BnaC08-CYP705a12 transgenic plant, the left 3 is a cde1 plant, and the left 4 is a phenotype of an RNAi-BnaC08-CYP705a12 transgenic plant;

FIG. 6 shows subcellular localization of BnaC08-CYP705a12 protein in tobacco leaf cells.

Detailed Description

The methods used in the examples described below are conventional methods unless otherwise specified.

Example 1

Cloning of gene BnaC08-CYP705a12 involved in cabbage type rape chlorophyll synthesis

The present study used the modified CTAB method of the laboratory to extract total DNA. Total RNA from fresh leaves of Brassica napus (Brassica napus) was extracted with TRIZAL reagent and with reference to the kit instructions, then cDNA was synthesized by reverse rotation with Reverse Transcription kit from Takara and according to the kit instructions, and primers were designed on primer P1 (upstream primer) using the synthesized cDNA as a template and the published genome of Brassica napus as a reference: 5'-ATGGCAGCAATGATAGTTGA-3' and P2 (downstream primer): and (3) amplifying genes related to chlorophyll synthesis in rape by PCR under a primer of 5'-TTAAAGGCTGAGTCGAGAAA-3', detecting a PCR amplified product by 1% agarose gel electrophoresis after the reaction is finished, recovering a target band according to the instruction by adopting an AXYGEN centrifugal column type gel recovery kit, purifying the target band, connecting the recovered product into a carrier Easy Blunt Simple, and converting escherichia coli (E.coli) DH5 alpha competent cells by a heat shock method. Screening positive clones from blue and white spots, inoculating the positive clones into a liquid culture medium containing kanamycin LB, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and displaying that amplified fragments have SEQ ID NO in a sequence table: 2, consisting of 1479 bases, wherein the coding sequence has the position point from the 1 st to 1479 th bases of the 5' end, and the coding sequence has the sequence shown in SEQ ID NO:1, wherein bases 43-1473 from the 5' end encode a conserved sequence. The genome gene thereof has the sequence shown in SEQ ID NO:3, which consists of 1896 bases, the 1 st to 672 nd bases from the 5' end are the first exon of the genome gene, the 673 th to 699 th bases from the 5' end are the first intron of the genome gene, the 700 th to 907 th bases from the 5' end are the second exon of the genome gene, the 908 th to 1297 th bases from the 5' end are the second intron of the genome gene, and the 1298 th to 1479 th bases from the 5' end are the third exon of the genome gene. The frame structure of the gene is shown in figure 1.

Example 2

Acquisition of BnaC08-CYP705a12 overexpressing transgenic plants

1. Construction of plant overexpression vector containing BnaC08-CYP705a12 Gene

An Xba I restriction enzyme site is added to the CDS sequence upstream of the BnaC08-CYP705a12 gene, and an upstream primer P3:5'-GCTCTAGAATGGCAGCAATGATAG-3' Sma I restriction endonuclease site downstream primer P4 is added to the CDS sequence downstream of BnaC08-CYP705a12 gene: 5'-TCCCCCGGGTTAAAGGCTGAGTCGA-3'. The sequence listing cloned in example 1 was subjected to the sequence listing of SEQ ID NO:2, after the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplified product, adopting an AXYGEN centrifugal column type gel recovery kit, recovering target bands according to the instruction, purifying the target bands, connecting the recovered product into a carrier Easy Blunt Simple, and converting E.coli (E.coli) DH5 alpha competent cells by a heat shock method. Screening positive clones from blue and white spots, inoculating the positive clones into a liquid culture medium containing kanamycin LB, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and displaying that amplified fragments have SEQ ID NO in a sequence table: 2 adding nucleotide sequences of restriction enzymes Xba I and Sma I sites, using restriction enzymes Xba I and Sma I to carry out enzyme digestion on the constructed plasmid containing BnaC08-CYP705a12 gene, carrying out 1% agarose gel electrophoresis detection on the enzyme digestion product, recovering BnaC08-CYP705a12 gene fragment with the length of about 1496bp (adding enzyme digestion site), purifying the fragment, connecting the recovered fragment with a vector PBI121 subjected to the same enzyme digestion by using T4 DNA ligase (Takara), transforming the E.coli (E.coli) DH5 alpha competent cells by using a heat shock method, screening positive clones, inoculating the positive clones into LB liquid medium containing kanamycin, culturing at 37 ℃ and 200rpm, extracting plasmids, carrying out enzyme digestion identification on the recombinant plasmids by using the restriction enzymes Xba I and Sma I, carrying out further identification by using primers P3 and P4, obtaining DNA fragments with the length of 1496bp by PCR, and obtaining the DNA fragments with the expected result, and indicating that the DNA fragments with the length of 1496bp are identical to the expected result and the DNA fragments have the right position of the BnaC 705a and have the right position of BnaC 12-75 a, and have the right position of the vector BnaC 12-75 a.

2. Obtaining of PBI121-BnaC08-CYP705a12 transgenic rape

The plant expression vector PBI121-BnaC08-CYP705a12 constructed in the first step is transformed into agrobacterium EHA105 competent cells by a heat shock method, the agrobacterium is coated on LB resistant plates containing kanamycin and rifampicin, the agrobacterium is cultured at 28 ℃ and 150rpm, single colonies of the agrobacterium which grow out are picked up and inoculated into 20ml LB liquid culture medium containing kanamycin and rifampicin, the agrobacterium is cultured for 2 days at 28 ℃ and 150rpm, and then bacterial solution is inoculated into 300ml LB liquid culture medium containing kanamycin and rifampicin according to 2% inoculum size, and the agrobacterium is cultured for 16-18 hours at 28 ℃ and 150 rpm. After completion of the culture, the cells were collected by centrifugation at 5000rpm for 20 minutes, and then suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. Finally, transferring the bacterial liquid into a 250ml beaker, removing the cabbage type rape flowers after pollination, and inverting the plants into the beaker to enable inflorescences of the plants to completely invade the bacterial liquid, and repeating the steps once again after one week in order to improve the conversion efficiency. And (3) conventionally culturing the transformed plant, harvesting seeds, and screening the obtained seeds by kanamycin and PCR identification to obtain the BnaC08-CYP705a12 transgenic plant.

3. Phenotypic observation of PBI121-BnaC08-CYP705a12 transgenic plant

And (3) collecting seeds of the positive transgenic plant over-expressing BnaC08-CYP705a12 obtained in the step (III) and planting the seeds under the same condition with the wild type, wherein the wild type of the brassica napus is used as a control. Observation of growth of BnaC08-CYP705a12 over-expressed plants and wild type plants the phenotype of BnaC08-CYP705a12 over-expressed plants grown under natural conditions is shown in FIG. 5, and leaves of BnaC08-CYP705a12 over-expressed plants yellow.

Example 3

RNAi-BnaC08-CYP705a12 transgenic plant acquisition

1. Construction of plant PFGC5941 vector containing BnaC08-CYP705a12 Gene

According to the conserved sequence of BnaC08-CYP705a12 gene, selecting an interference fragment 170bp (SEQ ID NO: 4), and performing the following steps: 4, and an Asc I restriction enzyme site, an upstream primer P5:5'-TTACAATTACCATGGGGCGCGCCATCCCTCATCACACCATA-3', a nucleotide sequence shown in SEQ ID NO:4, downstream of the sequence, a Swa I restriction enzyme site was added, downstream primer P6:5'-TTAAATCATCGATTGGGCGCGCATCTACCAAACCTCCCTT-3'. The sequence listing cloned in example 1 was subjected to the sequence listing of SEQ ID NO:2, and the amplified product is the sense strand of the BnaC08-CYP705a12 gene. In SEQ ID NO:4, and an Sma I restriction enzyme site, an upstream primer P7:5'-GGACTCTAGAGGATCCCCGGGATCCCTCATCACACCATA-3', a sequence set forth in SEQ ID NO:4, a BamH I restriction enzyme site was added downstream of the sequence, downstream primer P8:5'-ATAAGGGACTGACCACCCGGGATCTACCAAACCTCCCTT-3'. The sequence listing cloned in example 1 was subjected to the sequence listing of SEQ ID NO:2, and the amplified product is the antisense strand of the BnaC08-CYP705a12 gene. After the PCR reaction is finished, the PCR amplified products are respectively subjected to 1% agarose gel electrophoresis detection, the target bands are recovered and purified by adopting an AXYGEN centrifugal column type gel recovery kit according to the description, the recovered products are connected into a carrier Easy Blunt Simple, and E.coli (E.coli) DH5 alpha competent cells are transformed by a heat shock method. Screening positive clones from blue and white spots, inoculating the positive clones into a liquid culture medium containing kanamycin LB, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids respectively, and sequencing results show that the P5 nuclear P6 amplified fragment has the sequence shown by SEQ ID NO:4 plus restriction enzymes Asc I and Swa I sites, the fragments amplified by P7 and P8 have the sequence of SEQ ID NO:4 plus the antisense strand sequence of the restriction enzymes SmaI and BamHI sites. To construct the gene silencing vector, the interfering sense strand was first ligated to the PFGC5941 expression vector, and the restriction endonucleases Asc I and Swa I were used to encode a gene sequence comprising SEQ ID NO:4, performing enzyme digestion on the sense strand plasmid of the sequence, performing 1% agarose gel electrophoresis detection on the enzyme digestion product, and recovering the sequence with the length of about 170bp and containing SEQ ID NO:4 and purifying the sense strand fragment, and ligating the recovered fragment with the same digested vector PFGC5941 using T4 DNA ligase (Takara). The restriction enzymes Sma I and BamH I are used for the DNA sequence containing SEQ ID NO:4, performing enzyme digestion on the antisense strand plasmid of the sequence, performing 1% agarose gel electrophoresis detection on the enzyme digestion product, and recovering the sequence with the length of about 170bp and containing SEQ ID NO:4 and purifying it, and ligating the recovered fragment with T4 DNA ligase (Takara) to the vector PFGC5941 which had been digested with the same enzymes and to which the sense strand had been ligated. Then, the ligation product is transformed into E.coli DH5 alpha competent cells by a heat shock method, positive clones are screened, the positive clones are inoculated into LB liquid medium containing kanamycin, the culture is carried out at 37 ℃ and 200rpm, plasmids are extracted, PCR identification is carried out by using primers P5/P6 and P7/P8, and the sequences of inserted sense strand and antisense strand are consistent with the expected results, and the sequence and the position of the inserted sense strand and the antisense strand containing SEQ ID NO:4, designated RNAi-BnaC08-CYP705a12.

2. RNAi-BnaC08-CYP705a12 transgenic rape acquisition

The plant RNAi-BnaC08-CYP705a12 vector constructed in the step one is transformed into agrobacterium EHA105 competent cells by a heat shock method, the agrobacterium is coated on LB resistant plates containing kanamycin and rifampicin, the agrobacterium is cultured at 28 ℃ and 150rpm, single colonies of the agrobacterium which grow out are picked up and inoculated into 20ml LB liquid medium containing kanamycin and rifampicin, the agrobacterium is cultured at 28 ℃ and 150rpm for 2 days, and then bacterial liquid is inoculated into 300ml LB liquid medium containing kanamycin and rifampicin according to 2% inoculum size, and the agrobacterium is cultured at 28 ℃ and 150rpm for 16-18 hours. After completion of the culture, the cells were collected by centrifugation at 5000rpm for 20 minutes, and then suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. Finally, transferring the bacterial liquid into a 250ml beaker, removing cabbage type rape flowers with the cde1 material being pollinated, and then inverting the plants into the beaker to enable inflorescences of the plants to completely invade the bacterial liquid, so as to improve the conversion efficiency, and repeating the steps once a week. And (3) conventionally culturing the transformed plant, harvesting seeds, and screening the obtained seeds by kanamycin and PCR identification to obtain the RNAi-BnaC08-CYP705a12 transgenic plant.

3. Phenotype observation of RNAi-BnaC08-CYP705a12 transgenic plant

And (3) collecting seeds of the RNAi-BnaC08-CYP705a12 vector positive transgenic plant obtained in the second step and planting the seeds and the yellow mutant under the same condition, wherein the yellow mutant of the brassica napus is used as a control. The growth of RNAi-BnaC08-CYP705a12 transgenic plants and a control group was observed, and the phenotype of the RNAi-BnaC08-CYP705a12 transgenic plants grown under natural conditions is shown in FIG. 5, and the yellowing mutant cde1 plants restored the phenotype of leaf green leaves.

Example 4

BnaC08-CYP705a12 subcellular localization

1. Construction of fusion expression vector containing BnaC08-CYP705a12 Gene

The fusion vector for subcellular localization in the laboratory is pA7-GFP, and two enzyme cutting sites of Xho I and Sal I are selected for vector enzyme cutting, and a vector enzyme cutting system (50 μl system) is adopted: 1. Mu.l each of the two enzymes, 1 XH buffer, 16. Mu.l of vector, ddH ₂ O is added to 50 μl system, the mixture is digested in a water bath at 37 ℃ for 3h, immediately after digestion, the mixture is inactivated in a water bath at 60 ℃ for 15 minutes, and then the mixture is preserved at-20 ℃ or 4 ℃. The CDS obtained by cloning is used as a template, a pair of primers added with corresponding restriction sites (Xho I and Sal I) are utilized, an upstream primer is a vector sequence of 15 bp+restriction site+19 bp gene sequence (19 bp from the front of the ORF), the sequence is CATTTACGAACGATA +CTCGAG+ ATGGCAGCAATGATAG, a downstream primer is a 19bp gene sequence (19 bp from the rear of the gene) +restriction site+15 bp vector sequence, and the sequence is +GTCGAC+ TTCTCGACTCAGCCTT, and then the reverse complementation is carried out. PCR amplification was performed using the above primers, followed by gel recovery. And (3) connecting the target fragment with an enzyme digestion carrier. Ligation system (10 μl): mu.l homologous recombinase+2. Mu.l buffer+3. Mu.l digestion vector+4. Mu.l target fragment, were ligated in a water bath at 37℃for 30 min, and then placed on ice 5The reaction was terminated in minutes. The ligation product was transformed into E.coli DH 5. Alpha. Competent cells by heat shock, positive clones were selected, inoculated into LB liquid medium of ampicillin, cultured at 37℃and 200rpm, plasmids were extracted, and the recombinant plasmids were identified by restriction enzymes Xho I and Sal I, which were consistent with the expected results, indicating that the subcellular fusion vector containing BnaC08-CYP705a12 with correct insert sequence and position was obtained, designated BnaC08-CYP705a12-GFP.

2. Agrobacterium-mediated transient transformation of tobacco

The fusion expression vector BnaC08-CYP705a12-GFP constructed in the first step is transformed into agrobacterium EHA105 competent cells by a heat shock method, the competent cells are coated on LB resistant plates containing ampicillin and rifampicin, and activated agrobacterium is inoculated in 50ml of culture solution containing corresponding antibiotics in a single colony at 28 ℃ and 200rpm overnight. When the bacterial liquid OD ₆₀₀ When the value is between 0.6 and 1.0, the agrobacterium is collected by centrifugation at 5000rpm for 5 min. By heavy suspension (MgCL) ₂ ·6H ₂ O,10mM; MES,10 μm, ph=0.7; AS, 100. Mu.M) was washed twice with 10ml each. Dilution of the heavy suspension to OD ₆₀₀ The value is between 0.6 and 0.8. Placing at 25deg.C for 3 hr, injecting, dark culturing for 12-16 hr, transferring to light culturing, and observing after 3 days. Fluorescence was observed under a confocal laser microscope, and the result showed that the fluorescence of the GFP protein expressed by BnaC08-CYP705a12-GFP had a clear signal on the cell membrane (FIG. 6), indicating that the BnaC08-CYP705a12 protein was co-localized in the cell membrane and functioned in the cell membrane.

Sequence listing

<110> Nanjing agricultural university

<120> a gene BnaC08-CYP705a12 involved in cabbage type rape chlorophyll synthesis and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 492

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Met Ala Ala Met Ile Val Asp Phe Gln Asn Cys Ser Ile Phe Ile Leu

1 5 10 15

Leu Cys Phe Phe Ser Phe Leu Cys Tyr Ser Val Phe Phe Phe Phe Lys

20 25 30

Lys Thr Asn Asp Leu Gly Pro Ser Pro Pro Ser Leu Pro Ile Ile Gly

35 40 45

His Leu His His Phe Leu Ser Val Leu Pro His Lys Ala Phe Gln Lys

50 55 60

Ile Ser Thr Lys Tyr Gly Pro Leu Leu His Leu His Ile Phe Ser Phe

65 70 75 80

Pro Ile Val Leu Val Ser Ser Pro Thr Met Ala His Glu Ile Phe Thr

85 90 95

Thr His Asp Leu Asn Ile Ser Ser Arg Asn Thr Pro Ala Ile Asp Glu

100 105 110

Ser Leu Leu Phe Gly Pro Ser Gly Phe Thr Val Ala Pro Tyr Gly Asp

115 120 125

Tyr Val Lys Phe Ile Lys Lys Leu Leu Ala Thr Lys Leu Leu Arg Pro

130 135 140

Arg Ala Ile Glu Lys Ser Arg Gly Val Arg Ala Glu Glu Leu Lys Gln

145 150 155 160

Phe Tyr Leu Lys Leu His Asp Lys Ala Leu Lys Lys Glu Ser Ile Glu

165 170 175

Ile Gly Lys Glu Thr Met Lys Phe Thr Asn Asn Met Ile Cys Arg Met

180 185 190

Ser Ile Gly Arg Ser Phe Ser Glu Glu Asn Gly Glu Val Glu Thr Leu

195 200 205

Arg Glu Leu Ile Ile Lys Ser Phe Ala Leu Ser Lys Gln Ile Leu Phe

210 215 220

Met Leu Gly Leu Met Ser Leu Phe Lys Lys Asp Ile Met Asp Val Ser

225 230 235 240

Arg Gly Phe Asp Glu Leu Leu Glu Arg Val Leu Ala Glu His Glu Glu

245 250 255

Lys Arg Glu Glu Asp Gln Asp Met Asp Met Met Asp Leu Leu Leu Glu

260 265 270

Ala Cys Thr Asp Glu Asn Ala Glu Tyr Lys Ile Thr Arg Asn Gln Ile

275 280 285

Lys Ser Leu Phe Val Glu Ile Phe Leu Gly Gly Thr Asp Thr Ser Ala

290 295 300

His Thr Thr Gln Trp Thr Met Ala Glu Leu Val Asn Asn Leu Asn Thr

305 310 315 320

Leu Gly Arg Leu Arg Asp Glu Ile Asp Leu Val Val Gly Lys Glu Arg

325 330 335

Leu Ile Gln Glu Thr Asp Leu Pro Asn Leu Pro Tyr Leu Gln Ala Val

340 345 350

Val Lys Glu Gly Leu Arg Leu His Pro Pro Ala Pro Leu Leu Val Arg

355 360 365

Met Phe Asp Lys Lys Cys Val Ile Lys Asp Phe Phe Lys Val Pro Glu

370 375 380

Lys Thr Thr Leu Val Val Asn Val Tyr Gly Val Met Arg Asp Pro Asp

385 390 395 400

Ser Trp Glu Asp Pro Asn Glu Phe Lys Pro Glu Arg Phe Leu Thr Ser

405 410 415

Lys Gln Glu Glu Glu Lys Val Leu Lys Tyr Leu Pro Phe Ala Ala Gly

420 425 430

Arg Arg Gly Cys Pro Ala Thr Asn Val Gly Tyr Ile Phe Val Gly Ile

435 440 445

Ser Ile Gly Met Met Val Gln Cys Phe Asp Trp Ser Ile Lys Asp Lys

450 455 460

Val Ser Met Lys Glu Val Tyr Ala Gly Met Ser Leu Ser Met Ala His

465 470 475 480

Pro Pro Lys Cys Thr Pro Val Ser Arg Leu Ser Leu

485 490

<210> 2

<211> 1479

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggcagcaa tgatagttga ctttcaaaac tgctccatct tcatcctcct ctgcttcttc 60

tcatttctct gttactccgt cttcttcttc ttcaagaaga caaatgactt gggtccgagc 120

cctccctctt tgccgatcat cggccatctt caccattttc tctcagttct accccacaag 180

gcttttcaga aaatctcgac caagtatgga cctctcctcc atctccacat tttcagtttt 240

cccatagtcc ttgtttcttc tcccacaatg gcccacgaga tattcacgac acacgactta 300

aacatctcgt ctcgcaacac acctgccatc gacgagtctc tcctgtttgg accttccggc 360

ttcacggtag ctccttatgg agactacgtt aagttcataa agaagcttct tgcgacgaag 420

cttcttcgac cgcgggcaat cgagaagtca cgaggtgtcc gtgcagagga gctaaagcaa 480

ttttatctta aacttcacga taaggcgttg aagaaagaaa gcattgagat tggtaaggaa 540

acgatgaagt tcactaacaa catgatctgc aggatgagca ttgggaggag tttttcagag 600

gagaacggtg aggtagagac tctgagggaa ttgattatca aatcgtttgc cttatcgaag 660

cagattctgt ttatgcttgg actaatgtca ctgtttaaga aagatataat ggatgtttca 720

agagggtttg atgagttgtt ggagagggtt cttgcggagc atgaagagaa acgggaggag 780

gatcaagata tggacatgat ggatttgctg ttggaagctt gtacagacga aaacgcagag 840

tataaaatca ctaggaacca gatcaaatca ttgttcgtgg aaattttttt gggaggcaca 900

gacacctcgg cacacacaac gcagtggaca atggcggagc tcgttaacaa cctaaacact 960

cttgggagat taagagacga aattgatctc gttgtaggga aagaaagatt gattcaagaa 1020

acagatctac caaacctccc ttatttgcaa gcagtggtta aggaagggct acgcttgcac 1080

ccaccggcac ctttactggt tagaatgttc gacaaaaaat gtgtgatcaa agatttcttc 1140

aaagtaccgg aaaaaacaac acttgttgtt aatgtttatg gtgtgatgag ggatccagat 1200

tcttgggaag atcctaatga gttcaagcca gagaggtttc taacttcaaa gcaagaagaa 1260

gagaaagtat taaagtacct tccttttgca gctggaagaa ggggatgtcc tgcaacaaat 1320

gtaggctata tctttgtagg aatctcaatt ggaatgatgg tgcagtgctt tgactggagt 1380

atcaaagata aggttagtat gaaagaggtc tatgcaggaa tgagtctttc catggctcat 1440

cccccaaagt gcactccagt ttctcgactc agcctttaa 1479

<210> 3

<211> 1896

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggcagcaa tgatagttga ctttcaaaac tgctccatct tcatcctcct ctgcttcttc 60

tcatttctct gttactccgt cttcttcttc ttcaagaaga caaatgactt gggtccgagc 120

cctccctctt tgccgatcat cggccatctt caccattttc tctcagttct accccacaag 180

gcttttcaga aaatctcgac caagtatgga cctctcctcc atctccacat tttcagtttt 240

cccatagtcc ttgtttcttc tcccacaatg gcccacgaga tattcacgac acacgactta 300

aacatctcgt ctcgcaacac acctgccatc gacgagtctc tcctgtttgg accttccggc 360

ttcacggtag ctccttatgg agactacgtt aagttcataa agaagcttct tgcgacgaag 420

cttcttcgac cgcgggcaat cgagaagtca cgaggtgtcc gtgcagagga gctaaagcaa 480

ttttatctta aacttcacga taaggcgttg aagaaagaaa gcattgagat tggtaaggaa 540

acgatgaagt tcactaacaa catgatctgc aggatgagca ttgggaggag tttttcagag 600

gagaacggtg aggtagagac tctgagggaa ttgattatca aatcgtttgc cttatcgaag 660

cagattctgt ttgtaaacgt attacgtagg ccactggaga tgcttggact aatgtcactg 720

tttaagaaag atataatgga tgtttcaaga gggtttgatg agttgttgga gagggttctt 780

gcggagcatg aagagaaacg ggaggaggat caagatatgg acatgatgga tttgctgttg 840

gaagcttgta cagacgaaaa cgcagagtat aaaatcacta ggaaccagat caaatcattg 900

ttcgtggtaa aactaatatc atcagaaact ataaccgttt tttatatact aaaaaaatag 960

aaatttctta ggatagtctt tttagtttat tttcacaaaa aatagttttc aaaaaaaaaa 1020

ataccaaaat ttttttatta aaagataaat atacatttat actctaaagt taattaatat 1080

atacttacga tttagagtta agagcttagg ttttgaggtg gattttcaaa ttaaaaagaa 1140

ataaaagtta aaaatttcaa aataaaaaaa ggctattttg gtaaatgttt tttttttaga 1200

actattttga tcacaaaatt ttaaacaaga ctatttgaaa gaattgccct tacaaaaaat 1260

gtgtaatgtg ttaaatgcta atacttataa ctgcaggaaa tttttttggg aggcacagac 1320

acctcggcac acacaacgca gtggacaatg gcggagctcg ttaacaacct aaacactctt 1380

gggagattaa gagacgaaat tgatctcgtt gtagggaaag aaagattgat tcaagaaaca 1440

gatctaccaa acctccctta tttgcaagca gtggttaagg aagggctacg cttgcaccca 1500

ccggcacctt tactggttag aatgttcgac aaaaaatgtg tgatcaaaga tttcttcaaa 1560

gtaccggaaa aaacaacact tgttgttaat gtttatggtg tgatgaggga tccagattct 1620

tgggaagatc ctaatgagtt caagccagag aggtttctaa cttcaaagca agaagaagag 1680

aaagtattaa agtaccttcc ttttgcagct ggaagaaggg gatgtcctgc aacaaatgta 1740

ggctatatct ttgtaggaat ctcaattgga atgatggtgc agtgctttga ctggagtatc 1800

aaagataagg ttagtatgaa agaggtctat gcaggaatga gtctttccat ggctcatccc 1860

ccaaagtgca ctccagtttc tcgactcagc ctttaa 1896

<210> 4

<211> 170

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atccctcatc acaccataaa cattaacaac aagtgttgtt ttttccggta ctttgaagaa 60

atctttgatc acacattttt tgtcgaacat tctaaccagc aaaggtgccg gtgggtgcaa 120

gcgtagccct tccttaacca ctgcttgcaa ataagggagg tttggtagat 170

Claims (1)

1. A method for regulating and controlling chlorophyll synthesis of cabbage type rape is characterized in that agrobacterium-mediated transgenic method is used for regulating and controlling chlorophyll synthesis of cabbage type rapeBnaC08-CYP705a12The gene is introduced into a cabbage type rape receptor to obtain a transformed plant;BnaC08-CYP705a12the sequence of the gene is shown in SEQ ID NO:2, the coded amino acid sequence is shown as SEQ ID NO:1 is shown in the specification;

extracting total RNA of fresh leaves of brassica napus, synthesizing cDNA in a reverse way, taking the synthesized cDNA as a template, and carrying out primer P1: 5'-ATGGCAGCAATGATAGTTGA-3' and P2: PCR amplification under 5'-TTAAAGGCTGAGTCGAGAAA-3'; at the position ofBnaC08- CYP705a12Addition of the CDS sequence of the Gene upstreamXba IRestriction enzyme site, upstream primer P3:5'-GCTCTAGAATGGCAGCAATGATAG-3', atBnaC08-CYP705a12Downstream of the CDS sequence of the GeneSma IRestriction endonuclease site downstream primer P4:5'-TCCCCCGGGTTAAAGGCTGAGTCGA-3'; obtaining the product containingBnaC08- CYP705a12Plant expression vector, namedPBI121-BnaC08-CYP705a12The method comprises the steps of carrying out a first treatment on the surface of the After transformation, yellowing of the leaves is obtainedBnaC08-CYP705a12And (5) over-expressing plants.