CN112266904A - Gene BnaC08-CYP705a12 related to 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 cabbage type rape etiolating seedling mutation materials, and the gene has 1 base substitution with a wild type. The invention constructs a PBI121-BnaC08-CYP705a12 overexpression vector and converts the vector into a wild type to obtain a PBI121-BnaC08-CYP705a12 overexpression transgenic plant, and the leaves of the plant have a etiolated phenotype; meanwhile, an RNAi-BnaC08-CYP705a12 vector is constructed and transformed into etiolated seedlings, so that RNAi-BnaC08-CYP705a12 transgenic plants are obtained, and the green leaf phenotype of the transgenic plants is restored. The cloned gene mutation can cause the chlorophyll content of the cabbage type rape to be reduced, thereby causing the yellowing character of rape leaves.

Description

Gene BnaC08-CYP705a12 related to 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 and cloning, functional verification and application of a DNA fragment (gene) related to cabbage type rape chlorophyll synthesis.

Background

The leaves are the main places for photosynthesis of higher plants, the change of the leaf color of the plants can cause the change of the photosynthetic efficiency of the leaves, the research on the leaf color mutants of the plants can clarify the regulation and control mechanism of the influence of the leaf color on the photosynthesis, and a good theoretical basis and research materials can be provided for the high-light efficiency research of the plants.

Chlorophyll synthesis and heme biosynthesis are two important branches of tetrapyrrole anabolism. Biosynthesis of higher plant chlorophyll ALA is synthesized by using glutamic acid and alpha-ketoglutaric acid as raw materials. 2 molecules of ALA are catalyzed by ALA dehydratase ALAD to generate 1 molecule of porphobilinogen which has a corroborative ring. 4 molecules of porphobilinogen are catalyzed by deamination to form uroporphyrinogen III, and 4 acetic acid side chains of the uroporphyrinogen III are subjected to uroporphyrinogen III decarboxylase to generate coproporphyrinogen III with 4 methyl groups. Coproporphyrinogen III is recyclized, dehydrogenated and oxidized in coproporphyrinogen III oxidase and protoporphyrinogen oxidase to form protoporphyrin IV, which is the watershed of chlorophyll or heme formation. If bound to magnesium, Mg-protoporphyrin IV is formed, which in turn is converted to the orthochlorophyllin ester by Mg-protoporphyrin IV methyltransferase, Mg-protoporphyrin IV monomethyl ester cyclase and divinyl reductase. The orthochlorophyllin ester is subjected to photoreduction to generate chlorophyllin ester a, and then the chlorophyllin ester a is catalyzed to form chlorophyll a under the action of chlorophyll synthase, and the chlorophyll b is converted from the chlorophyll a. The process of chlorophyll synthesis involves the involvement of multiple enzymes. Any mutation of the gene during chlorophyll synthesis may cause chlorophyllSynthesis is hindered, and the content of various pigments in chloroplast is further changed, so that the leaf color variation of plants is caused. If protoporphyrin IV is combined with iron to form Fe²⁺Chelate, then heme b is generated, and the heme b in the plant body finally forms a phytochrome chromophore through a series of oxidation reduction. If genes related to the heme metabolic reaction are mutated, the balance of tetrapyrrole metabolism is likely to be broken, the content of heme in cells is increased, the accumulation of heme influences the content of ALA and the acid value of protochlorophyll by feedback inhibition of the activity of Glu-tRNA reductase, so that the synthesis of chlorophyll is inhibited, and the mutant has variation of leaf color due to the lack of chlorophyll. Heme feedback inhibition is a key regulatory step in the chlorophyll synthesis process. In tomato aurea and yenow-green-2 mutants, genetic variations encoding heme oxidase and the phytochrome chromophore synthase lead to an excess of intracellular heme accumulation, which feedback inhibits the synthesis of the chlorophyll precursor aminolevulinic acid, causing the mutants to exhibit leaf yellowing.

Cytochrome P450 monooxygenase (P450s) is a conserved superfamily of heme-sulfur protein genes that play important roles in a wide variety of substrate metabolism, including the metabolism of endogenous and exogenous compounds. The three-dimensional structure of cytochrome P450 is well conserved, and CYP450 with all known structures contains a conserved characteristic heme binding domain FxxGxRxCxG at the catalytic central part, and the sequence is well conserved, so that the region is also a key basis for identifying cytochrome P450. Cysteine (C) in this domain site is completely conserved in all P450s and is the 5 th axis ligand of the heme binding site. The reaction process catalyzed by cytochrome P450 is the transfer of electrons from NADPH/NADP to flavoproteins and iron-sulfur proteins, and then to cytochrome P450 oxidase. Because most of P450 in plants has low and unstable content, and it is very difficult to directly separate and purify P450 enzyme, it becomes an important way to clone P450 gene, study the expression and regulation of P450 gene, and study the function of P450 by heterologous expression, deletion mutation, mutant complementation and other methods.

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 leaf traits of rape.

The gene related to the synthesis of the cabbage type rape chlorophyll is named as BnaC08-CYP705a12(Brassica napus CYTOCHROME P450, FAMILY 705, SUBFAMILY A and POLYPEPTIDE 12), and a gene fragment cloned from a cabbage type rape yellowing mutant material "cde 1" has 1 base substitution with a sequence cloned from 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 between aa and 320:

(1) SEQ ID NO: 1;

(2) and (3) mixing the amino acid sequence shown in SEQ ID NO: 1 by substitution and/or deletion and/or addition of one to ten amino acid residues and has a function of participating in the synthesis of cabbage type rape chlorophyll.

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

The one to ten amino acid residues substituted and/or deleted and/or added are amino acid residues in the non-structural domain, and the change thereof does not affect the function of the protein.

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

(1) SEQ ID NO: 2;

(2) encoding the amino acid sequence shown in SEQ ID NO: 1;

(3) and SEQ ID NO: 2 has more than 90 percent of homology with the DNA sequence and has a nucleotide sequence which is involved in the chlorophyll synthesis of the cabbage type rape;

(4) can be combined with the sequence shown in SEQ ID NO: 2 to the DNA sequence defined in the specification.

The high stringency conditions are hybridization and membrane washing 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 the coding sequence is the 1 st to 1479 th bases at the 5' end, and the coding sequence has the nucleotide sequence shown in SEQ ID NO: 1, and a conserved sequence encoded by bases 43 to 1473 from the 5' end.

The genome gene is one of the following nucleotide sequences:

(1) SEQ ID NO: 3 in the sequence listing;

(2) and SEQ ID NO: 3, the DNA sequence has over 90 percent of homology and has a nucleotide sequence participating in the chlorophyll synthesis of the cabbage type rape;

(3) can be combined with the sequence shown in SEQ ID NO: 3 to the DNA sequence defined in the specification.

The high stringency conditions are hybridization and membrane washing 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 th bases from the 5 ' end are the first exon of the genome gene, the 673 th and 699 th bases from the 5 ' end are the first intron of the genome gene, the 700 th and 907 th bases from the 5 ' end are the second exon of the genome gene, the 908 th and 1297 th bases from the 5 ' end are the second intron of the genome gene, and the 1298 th and 1896 th bases from the 5 ' end are the third exon of the genome gene.

The BnaC08-CYP705a12 gene is related to cabbage type rape chlorophyll synthesis. After the complete coding sequence (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(ZS11) in a conventional excellent variety of brassica napus, and the leaves of a transgenic plant are obviously yellowed compared with the leaves of a control plant; the constructed expression vector of the gene RNAi-BnaC08-CYP705a12 is transformed into the homozygous cabbage type rape yellowing mutant cde1, and the leaves of the transgenic plant can be restored to the green leaf phenotype. The BnaC08-CYP705a12 gene can participate in chlorophyll synthesis, so that the leaf traits of rape can be controlled.

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

The expression vector, the transgenic cell line, the host bacterium containing the gene and the protein coded by the host bacterium belong to the protection scope of the invention.

The primer and the primer pair for amplifying any fragment of the BnaC08-CYP705a12 gene are also within the protection scope of the invention.

The gene BnaC08-CYP705a12 cloned by the invention can provide good theoretical basis and research materials for the research of high photosynthetic efficiency of plants.

The specific operation steps are as follows:

(1) introducing the gene BnaC08-CYP705a12 into a cabbage type rape receptor by utilizing an agrobacterium-mediated transgenic method to obtain a transformed plant;

(2) analyzing and identifying the positive transgenic plant by means of a PCR method;

(3) planting the transgenic plant in the step (2) and observing the character of the transgenic plant;

(4) and analyzing the expression of a gene BnaC08-CYP705a12 related to cabbage type rape chlorophyll synthesis in the transgenic plants and the wild plants by means of qRT-PCR.

Has the advantages that:

the invention finds a gene BnaC08-CYP705a12 which is derived from rape and is related to the synthesis of cabbage type rape chlorophyll, the gene can reduce the synthesis of the chlorophyll in leaves of a plant after overexpression, so that the content of the chlorophyll in the leaves of the plant is reduced, and the yellowing of the leaves of the plant is finally caused, when the expression of the gene is interfered, the plant can recover the synthesis of the cabbage type rape chlorophyll which is involved in the plant, so that the content of the chlorophyll in the leaves of the plant is recovered, and the leaves of a yellowing material recover a green leaf phenotype.

The gene BnaC08-CYP705a12 is related to cabbage type rape chlorophyll synthesis. After the complete coding sequence (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(ZS11) in a conventional excellent variety of brassica napus, and the leaves of a transgenic plant are obviously yellowed compared with the leaves of a control plant; the constructed expression vector of the gene RNAi-BnaC08-CYP705a12 is transformed into the homozygous cabbage type rape yellowing mutant cde1, and the leaves of the transgenic plant can be restored to the green leaf phenotype. The BnaC08-CYP705a12 gene can participate in chlorophyll synthesis, so that the leaf traits of rape can be controlled.

Drawings

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

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

FIG. 3 is a schematic diagram of the construction of a plant overexpression vector PBI 121;

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

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

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

Detailed Description

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

Example 1

Clone of related gene BnaC08-CYP705a12 participating in cabbage type rape chlorophyll synthesis

The present study employed the lab-improved CTAB method for total DNA extraction. Extracting total RNA of fresh leaves of Brassica napus (Brassica napus) by using TRIZAL reagent and referring to kit instructions, then using Reverse Transcription kit of Takara company and according to kit instructions to reversely synthesize cDNA, using the synthesized cDNA as a template, and using the disclosed genome of the Brassica napus as a reference to design a primer, and adding the primer P1 (upstream primer): 5'-ATGGCAGCAATGATAGTTGA-3' and P2 (downstream primer): 5'-TTAAAGGCTGAGTCGAGAAA-3', PCR amplifying rape gene participating chlorophyll synthesis, after reaction, carrying out 1% agarose gel electrophoresis detection on PCR amplification product, adopting AXYGEN centrifugal column type gel recovery kit, recovering and purifying target band according to instructions, connecting the recovered product into Easy Blunt Simple, transforming Escherichia coli (E.coli) DH5 alpha competent cell by heat shock method. And (3) screening positive clones by using the blue-white spots, inoculating the positive clones into a kanamycin-containing LB liquid culture medium, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and indicating that the amplified fragments have the nucleotide sequences shown in SEQ ID NO: 2 consisting of 1479 bases, the coding sequence site of which is the 1 st to 1479 th bases from the 5' end, and codes the nucleotide sequence with the sequence table of SEQ ID NO: 1, wherein bases 43 to 1473 from the 5' -end encode a conserved sequence. The genome gene of the gene has the nucleotide sequence shown in SEQ ID NO: 3 consisting of 1896 bases, wherein the 1 st to 672 th bases from the 5 ' end are the first exon of the genomic gene, the 673 th and 699 th bases from the 5 ' end are the first intron of the genomic gene, the 700 th and 907 th bases from the 5 ' end are the second exon of the genomic gene, the 908 th and 1297 th bases from the 5 ' end are the second intron of the genomic gene, and the 1298 th and 1479 th bases from the 5 ' end are the third exon of the genomic gene. The structural frame of the gene is shown in FIG. 1.

Example 2

Acquisition of BnaC08-CYP705a12 overexpression transgenic plant

Construction of plant over-expression vector containing BnaC08-CYP705a12 gene

Adding Xba I restriction endonuclease site in the CDS sequence upstream of BnaC08-CYP705a12 gene, and adding an upstream primer P3: 5'-GCTCTAGAATGGCAGCAATGATAG-3', adding a Sma I restriction endonuclease site downstream primer P4 to the CDS sequence of the BnaC08-CYP705a12 gene: 5'-TCCCCCGGGTTAAAGGCTGAGTCGA-3' are provided. Primers P3 and P4 were used to determine the nucleotide sequence of SEQ ID NO: 2, carrying out PCR amplification, carrying out 1% agarose gel electrophoresis detection on a PCR amplification product after the reaction is finished, adopting an AXYGEN centrifugal column type gel recovery kit, recovering a target band according to instructions, purifying the target band, connecting the recovered product into an Easy Blunt Simple carrier, and transforming an escherichia coli (E.coli) DH5 alpha competent cell by a heat shock method. And (3) screening positive clones by using the blue-white spots, inoculating the positive clones into a kanamycin-containing LB liquid culture medium, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and indicating that the amplified fragments have the nucleotide sequences shown in SEQ ID NO: 2 adding nucleotide sequences of restriction endonucleases Xba I and Sma I sites, carrying out enzyme digestion on the constructed plasmid containing the BnaC08-CYP705a12 gene by using the restriction endonucleases Xba I and Sma I, carrying out 1% agarose gel electrophoresis detection on the digestion product, recovering a BnaC08-CYP705a12 gene fragment with the length of about 1496bp (adding the enzyme digestion sites) and purifying the gene fragment, connecting the recovered fragment with a vector PBI121 subjected to the same enzyme digestion by using T4 DNA ligase (Takara), transforming an escherichia coli (E.coli) DH5 alpha competent cell by using a heat shock method, screening positive clones, inoculating the positive clones into an LB liquid culture medium containing kanamycin, culturing at 37 ℃ and 200rpm, extracting plasmids, carrying out enzyme digestion identification on recombinant plasmids by using the restriction endonucleases Xba I and Sma I, conforming to the expected result, and further carrying out PCR identification by using primers P3 and P4, the result is amplified by PCR to obtain a 1496bp DNA fragment, which is consistent with the expected result, and shows that the plant expression vector containing the BnaC08-CYP705a12 with correct insertion sequence and position is obtained and is named as PBI121-BnaC08-CYP705a 12.

Secondly, obtaining PBI121-BnaC08-CYP705a12 transgenic rape

Transforming Agrobacterium EHA105 competent cells by a heat shock method with the plant expression vector PBI121-BnaC08-CYP705a12 constructed in the first step, spreading the competent cells on LB resistant plates containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm, picking out single colonies of the Agrobacterium grown, inoculating the single colonies into 20ml LB liquid medium containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm for 2 days, inoculating the single colonies into 300ml LB liquid medium containing kanamycin and rifampicin according to 2% of inoculum concentration, and culturing at 28 ℃ and 150rpm for 16-18 hours. After completion of the culture, the cells were centrifuged at 5000rpm for 20 minutes, and the cells were collected and suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. And finally, transferring the bacterial liquid into a 250ml beaker, removing the cabbage type rape flowers after pollination, and pouring the plants into the beaker to ensure that the inflorescences of the plants completely invade the bacterial liquid, wherein the steps are repeated after one week in order to improve the transformation efficiency. And (3) carrying out conventional culture on the transformed plant, harvesting seeds, and identifying and screening the obtained seeds by kanamycin and PCR to obtain a BnaC08-CYP705a12 transgenic plant.

Thirdly, PBI121-BnaC08-CYP705a12 transgenic plant phenotype observation

Collecting seeds of the positive transgenic plants over-expressing BnaC08-CYP705a12 obtained in the third step, planting the seeds under the same condition with wild type, and using wild type of Brassica napus as a control. The growth conditions of BnaC08-CYP705a12 overexpression plants and wild-type plants are observed, the phenotype of BnaC08-CYP705a12 overexpression plants grown under natural conditions is shown in figure 5, and the leaves of BnaC08-CYP705a12 overexpression plants are yellowed.

Example 3

Obtaining of RNAi-BnaC08-CYP705a12 transgenic plant

Construction of plant PFGC5941 vector containing BnaC08-CYP705a12 gene

The interference fragment 170bp (SEQ ID NO: 4) was selected based on the conserved sequence of BnaC08-CYP705a12 gene, and the sequence shown in SEQ ID NO: 4 plus Asc I restriction endonuclease site, upstream primer P5: 5'-TTACAATTACCATGGGGCGCGCCATCCCTCATCACACCATA-3', in SEQ ID NO: 4, adding a Swa I restriction endonuclease site downstream of the sequence, wherein a downstream primer P6: 5'-TTAAATCATCGATTGGGCGCGCATCTACCAAACCTCCCTT-3' are provided. Primers P5 and P6 were used to determine the nucleotide sequence of SEQ ID NO: 2, and the amplification product is the sense strand of the BnaC08-CYP705a12 gene. In SEQ ID NO: 4 plus a Sma I restriction endonuclease site upstream of the sequence, upstream primer P7: 5'-GGACTCTAGAGGATCCCCGGGATCCCTCATCACACCATA-3', in SEQ ID NO: 4, adding a BamH I restriction endonuclease site downstream, and adding a downstream primer P8: 5'-ATAAGGGACTGACCACCCGGGATCTACCAAACCTCCCTT-3' are provided. Primers P7 and P8 were used to determine the nucleotide sequence of SEQ ID NO: 2, and the amplification product is the antisense chain of the BnaC08-CYP705a12 gene. After the PCR reaction is finished, 1% agarose gel electrophoresis detection is respectively carried out on the PCR amplification products, an AXYGEN centrifugal column type gel recovery kit is adopted, the target band is recovered and purified according to the instructions, the recovered products are connected into a carrier Easy Blunt Simple, and escherichia coli (E.coli) DH5 alpha competent cells are transformed through a heat shock method. And (3) screening positive clones by using a blue-white spot, inoculating the positive clones into a kanamycin-containing LB liquid culture medium, culturing at 37 ℃ and 200rpm, extracting plasmids, respectively sequencing the plasmids, wherein the sequencing result shows that the P5 nuclear P6 amplified fragment has the nucleotide sequence shown in SEQ ID NO: 4 plus sense strand sequence of Asc I and Swa I sites of restriction enzymes, and the amplified fragments of P7 and P8 have the nucleotide sequences shown in SEQ ID NO: 4 plus the antisense strand sequence of restriction endonuclease Sma I and BamH I sites. To construct a gene silencing vector, the interfering sense strand was first ligated to the PFGC5941 expression vector, and the dna containing SEQ ID NO: 4, carrying out enzyme digestion on the sense strand plasmid with the sequence of 4, carrying out 1% agarose gel electrophoresis detection on the enzyme digestion product, and recovering a nucleotide sequence which is about 170bp and contains SEQ ID NO: 4 and purifying it, and ligating the recovered fragment with the same digested vector PFGC5941 using T4 DNA ligase (Takara). And then the DNA containing the nucleotide sequence of SEQ ID NO: 4, carrying out enzyme digestion on the antisense strand plasmid with the sequence of 4, carrying out 1% agarose gel electrophoresis detection on the enzyme digestion product, and recovering a plasmid with the length of about 170bp containing SEQ ID NO: 4 and purified, the recovered fragment was ligated with the vector PFGC5941, which had been digested with the same enzyme and to which the sense strand had been ligated, using T4 DNA ligase (Takara). And transforming the ligation product into escherichia coli (e.coli) DH5 alpha competent cells by a heat shock method, screening positive clones, inoculating the positive clones into an LB liquid medium containing kanamycin, culturing at 37 ℃ and 200rpm, extracting plasmids, performing PCR identification by using primers P5/P6 and P7/P8, and indicating that the sequences of the inserted sense strand and antisense strand and the nucleotide sequences containing SEQ ID NOs: 4 sequence, namely RNAi-BnaC08-CYP705a 12.

II, obtaining RNAi-BnaC08-CYP705a12 transgenic rape

Transforming Agrobacterium EHA105 competent cells by a heat shock method by using the plant RNAi-BnaC08-CYP705a12 vector constructed in the step one, coating the competent cells on an LB resistant plate containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm, picking out a single colony of the grown Agrobacterium, inoculating the single colony in 20ml of LB liquid culture medium containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm for 2 days, then inoculating the bacterial liquid in 300ml of LB liquid culture medium containing kanamycin and rifampicin according to 2% of inoculum concentration, and culturing at 28 ℃ and 150rpm for 16-18 hours. After completion of the culture, the cells were centrifuged at 5000rpm for 20 minutes, and the cells were collected and suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. And finally, transferring the bacterial liquid into a 250ml beaker, removing the cabbage type rape flowers of the cde1 material after pollination, and pouring the plants into the beaker to ensure that the inflorescences of the plants completely invade the bacterial liquid, wherein the steps are repeated after one week in order to improve the transformation efficiency. And (3) carrying out conventional culture on the transformed plant, harvesting seeds, and identifying and screening the obtained seeds by kanamycin and PCR to obtain the RNAi-BnaC08-CYP705a12 transgenic plant.

Third, RNAi-BnaC08-CYP705a12 transgenic plant phenotype observation

Collecting seeds of the RNAi-BnaC08-CYP705a12 vector positive transgenic plant obtained in the step two, planting the seeds and the yellowing mutant of the cabbage type rape under the same condition, and taking the yellowing mutant of the cabbage type rape as a control. The growth conditions of the RNAi-BnaC08-CYP705a12 transgenic plant and the control group are observed, the phenotype of the RNAi-BnaC08-CYP705a12 transgenic plant grown under natural conditions is shown in figure 5, and the etiolating mutant cde1 plant restores the phenotype of green leaves of leaves.

Example 4

BnaC08-CYP705a12 subcellular localization

Construction of fusion expression vector containing BnaC08-CYP705a12 gene

This laboratory is usedThe subcellular localization fusion vector is pA7-GFP, two enzyme cutting sites of Xho I and Sal I are selected for carrying out vector enzyme cutting, and the vector enzyme cutting system (50 mu l system): mu.l of each of the two enzymes, 1 XH buffer, 16. mu.l of vector, ddH₂And supplementing O to a 50 mu l system, carrying out enzyme digestion in water bath at 37 ℃ for 3h, immediately carrying out inactivation in water bath at 60 ℃ for 15 min after enzyme digestion, and then storing at-20 ℃ or 4 ℃. Using CDS obtained by cloning as a template, utilizing a pair of primers added with corresponding enzyme cutting sites (Xho I and Sal I), wherein the upstream primer is a vector sequence of 15bp + enzyme cutting sites +19bp gene sequence (19 bp from the front of ORF), the sequence is CATTTACGAACGATA + CTCGAG + ATGGCAGCAATGATAG, the downstream primer is a 19bp gene sequence (19 bp from the back of the gene) + enzyme cutting sites + vector sequence of 15bp, the sequence is + GTCGAC + TTCTCGACTCAGCCTT, and then carrying out reverse complementation. PCR amplification was performed using the above primers, and then gel recovery was performed. And connecting the target fragment with the enzyme digestion vector. Ligation system (10 μ l): mu.l of homologous recombinase, 2. mu.l buffer, 3. mu.l digestion vector and 4. mu.l of target fragment were ligated in a water bath at 37 ℃ for 30 minutes, and then placed on ice for 5 minutes to terminate the reaction. Transforming the ligation product into escherichia coli (E.coli) DH5 alpha competent cells by a heat shock method, screening positive clones, inoculating the positive clones into LB liquid culture medium of ampicillin, culturing at 37 ℃ and 200rpm, extracting plasmids, carrying out enzyme digestion identification on recombinant plasmids by using restriction enzymes Xho I and Sal I, and according with expected results, indicating that a subcellular fusion vector containing BnaC08-CYP705a12 with correct insertion sequence and position is obtained and named as BnaC08-CYP705a 12-GFP.

Second, Agrobacterium-mediated transient transformation of tobacco

The fusion expression vector BnaC08-CYP705a12-GFP constructed in the step one is used for transforming agrobacterium EHA105 competent cells by a heat shock method, the competent cells are coated on an LB resistance plate containing ampicillin and rifampicin, and activated agrobacterium monoclonal is inoculated into 50ml of culture solution containing corresponding antibiotics at 28 ℃ and 200rpm overnight. Bacteria solution OD₆₀₀When the value is between 0.6 and 1.0, carrying out centrifugation at 5000rpm for 5min to collect the agrobacterium. With heavy suspension (MgCL)₂·6H₂O,10 mM; MES,10 μ M, PH 0.7; AS, 100. mu.M) was washed twice with 10ml each. The resuspension was diluted to OD₆₀₀The value is between 0.6 and 0.8. Standing at 25 deg.C for 3 hr, injecting, and darkeningCulturing for 12-16 hr, culturing under light, and observing after 3 days. Fluorescence was observed under a confocal laser microscope, and the results showed that fluorescence of the BnaC08-CYP705a 12-GFP-expressed GFP protein had clear signals on cell membranes (FIG. 6), indicating that the BnaC08-CYP705a12 protein was co-localized in the cell membranes and functions in the cell membranes.

Sequence listing

<110> Nanjing university of agriculture

<120> one gene BnaC08-CYP705a12 related to 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 (10)

1. A related gene participating in chlorophyll synthesis of cabbage type rape, which is characterized in that the gene is one of the following amino acid residue sequences:

(1) SEQ ID NO: 1;

(2) and (3) mixing the amino acid sequence shown in SEQ ID NO: 1, has a function of participating in the synthesis of cabbage type rape chlorophyll by substituting and/or deleting and/or adding one to ten amino acid residues;

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

2. The gene involved in the synthesis of chlorophyll of Brassica napus according to claim 1, wherein said amino acid residue is an amino acid residue in a non-structural domain, and wherein said alteration does not affect the function of said protein.

3. The gene involved in the synthesis of chlorophyll of Brassica napus according to claim 1, wherein the cDNA is one of the following nucleotide sequences:

(1) SEQ ID NO: 2;

(2) encoding the amino acid sequence shown in SEQ ID NO: 1;

(3) and SEQ ID NO: 2 has more than 90% of homology and has nucleotide sequence related to the synthesis of the chlorophyll of the cabbage type rape;

(4) can be combined with the sequence shown in SEQ ID NO: 2 to the defined DNA sequence;

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

4. The gene related to cabbage type rape chlorophyll synthesis according to claim 3, wherein the amino acid sequence of SEQ ID NO: 2 consists of 1479 bases, the coding sequence of the coding sequence is the 1 st to 1479 th bases at the 5' end, and the coding sequence has the nucleotide sequence shown in SEQ ID NO: 1, and a conserved sequence encoded by bases 43 to 1473 from the 5' end.

5. The gene related to the synthesis of chlorophyll of Brassica napus according to claim 1, wherein the genomic gene is one of the following nucleotide sequences:

(1) SEQ ID NO: 3 in the sequence listing;

(2) and SEQ ID NO: 3, the DNA sequence has more than 90% of homology and has a nucleotide sequence which is related to the synthesis of the chlorophyll of the cabbage type rape;

(3) can be combined with the sequence shown in SEQ ID NO: 3 a nucleotide sequence to which the defined DNA sequence hybridizes;

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

6. The gene related to cabbage type rape chlorophyll synthesis according to claim 5, wherein the amino acid sequence of SEQ ID NO: 3 consists of 1896 bases, the 1 st to 672 th bases from the 5 ' end are the first exon of the genome gene, the 673 th and 699 th bases from the 5 ' end are the first intron of the genome gene, the 700 th and 907 th bases from the 5 ' end are the second exon of the genome gene, the 908 th and 1297 th bases from the 5 ' end are the second intron of the genome gene, and the 1298 th and 1896 th bases from the 5 ' end are the third exon of the genome gene.

7. Expression vectors, transgenic cell lines, host bacteria comprising the gene of any one of claims 1 to 6 and proteins encoded thereby.

8. A primer and a primer pair for any fragment of the gene of any one of claims 1 to 6.

9. Use of the gene of any one of claims 1-6 for leaf breeding of brassica napus.

10. The application of claim 9, the operation steps are as follows:

(1) introducing the gene of claim 1 into a brassica napus receptor by an agrobacterium-mediated transgenic method to obtain a transformed plant;

(2) analyzing and identifying the positive transgenic plant by means of a PCR method;

(3) planting the transgenic plant and observing the character of the transgenic plant;

(4) and analyzing the expression of genes related to the cabbage type rape chlorophyll synthesis in the transgenic plants and the wild plants by means of qRT-PCR.

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