CN114507679B - Pinus massoniana terpenoid synthesis related enzyme gene PmDXR and application of promoter thereof - Google Patents

Abstract

The invention discloses an enzyme gene PmDXR related to pinus massoniana terpenoid synthesis and application of a promoter thereof, belonging to the technical field of plant genetic engineering. The invention converts the Pinus massoniana PmDXR gene into Arabidopsis thaliana by using agrobacterium mediation, and the DXR enzyme activity, chlorophyll a, chlorophyll b and carotenoid content of the transgenic Arabidopsis thaliana are all higher than those of the wild Arabidopsis thaliana. And a promoter of the PmDXR gene is cloned from Pinus massoniana, the nucleotide sequence is shown as SEQ ID NO.1, the transient transformation method infects Nicotiana benthamiana by constructing a pBI121-ProDXR vector, the PmDXR gene promoter can drive GUS gene to express in roots, stems and leaves of Nicotiana benthamiana, and the promoter has no obvious tissue specificity. The invention has important research value and application prospect in improving the yield traits of pine resin by using genetic engineering technology.

Description

Pinus massoniana terpenoid synthesis related enzyme gene PmDXR and application of promoter thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an enzyme gene PmDXR related to synthesis of pinus massoniana terpenoid and application of a promoter thereof.

Background

Pinus massoniana (Pinus massoniana) belongs to Pinaceae (Pinaceae) Pinus genus double-vascular bundle Pinus, the trunk is straight, the bark is reddish brown, and the bark is split into irregular squamous pieces; the branches are flat or inclined; needle She Duowei, one bundle of needles, 3 bundles of thin needles, length 12-20cm; the leaf sheath is initially brown and then gradually changed to gray-black to lodge. The male bulbil takes a shape of a cylinder and gathers on the armpit of the lower part bud of the new branch; single female bulbil or 2-4 female bulbil gathers on the near top of new branches; the cones are oval or cone-shaped oval, the color of the cones is changed before and after the cones are ripe, the cones are green before the cones are ripe, and the cones are chestnut brown when the cones are ripe. The pinus massoniana is widely distributed in the pine tree species in China, and 17 provinces (autonomous region, direct jurisdiction and city) from Qinling, huaihe and Nan and Yun Guigao are distributed, so that the pinus massoniana has the characteristics of strong adaptability, high economic value and the like. The economic value of the pinus massoniana is not only reflected in the aspect of material utilization, but also brings great economic benefit to China in the aspect of forest product processing. Pinus massoniana can secrete a large amount of secondary metabolites mainly containing terpenoid, and the secondary metabolites are called rosin, which is a basic raw material of rosin and turpentine industries and is widely used in industrial biological products such as solvents, disinfectants, cleaning products, fragrances, paints and the like.

Terpenoid components in pinus massoniana rosin mainly include monoterpenes, sesquiterpenes and diterpenes, which are generally present in the roots, stems, leaves and cones of pine. The rosin not only brings great economic benefit, but also plays an important role in the defense system of conifer. After conifers are subjected to biological or non-biological stimuli, rosin can be released from the resin tract of the tree, and new rosin can be induced to synthesize. At present, 6 pine trees in China can be used for cutting pine resin, but only Pinus massoniana (Pinus elliottii), pinus massoniana (Pinus yunnanensis) and Pinus massoniana (Pinus kesiya var. Langbianensis) are used for large-scale oil extraction, wherein 90% of the pine resin is extracted from Pinus massoniana. The rosin production area in China is mainly in Guangxi provinces, and researches show that the pinus massoniana rosin yield in the Guangxi province in 2010 can account for about 42% of the rosin yield in China, and the total yield is about 30 ten thousand tons. In order to promote the development of the rosin industry, advanced technology is adopted to screen and cultivate excellent germplasm materials of high-yield grease, and the construction of high-yield grease industrial raw material forest is an effective way for improving the utilization rate of pine forest resources.

1-Deoxy-D-xylulose 5-phosphate reductoisomer (1-Deoxy-D-xylulose-5-phosphate reductase DXR) is a key rate limiting enzyme in the MEP pathway, catalyzing the 2 nd reaction of the MEP pathway, which requires the cofactors Nicotinamide Adenine Dinucleotide Phosphate (NADPH), mn ²⁺ 、Co ²⁺ Or Mg (Mg) ²⁺ The participation in the conversion of 1-deoxy-D-xylulose 5-phosphate synthase (DXP) to 2-methylerythritol 4-phosphate (MEP), an important precursor for terpenoid synthesis.The MEP pathway is localized in plastids and is primarily involved in the biosynthesis of monoterpenes, diterpenes and tetraterpenoids. The MEP pathway processes to produce prenyl pyrophosphoric acid (IPP) and dimethylpropenyl Diphosphate (DMAPP) are largely divided into 7 steps: first, pyruvic acid and 3-phosphate-glyceraldehyde are catalyzed to generate DXP by 1-deoxy-D-xylulose 5-phosphate synthase (DXS); secondly, carrying out reduction reaction on DXP by the catalysis of 1-deoxy-D-xylulose 5-phosphate reductase (DXR) to generate MEP; third, MEP is catalyzed by 2-methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) to generate 4- (5' -cytidine pyrophosphate) -2-C-methyl-D-erythritol (CDP-ME) in the presence of NADPH; fourth, CDP-ME is phosphorylated by 4- (5 '-cytidine pyrophosphate) -2-C-methyl-D-erythritol kinase (CMK) to form 4- (5' -cytidine pyrophosphate) -2-C-methyl-D-erythritol-2-phosphate (CDP-MEP); fifthly, the CDP-MEP generates 2-methyl-D-erythritol-2, 4-cyclic pyrophosphate (MEcPP) under the action of 2-methyl-D-erythritol-2, 4-cyclic pyrophosphate synthase (MDS); sixthly, forming 1-hydroxy-2-methyl-2-E-butenyl-4-pyrophosphoric acid (HMBPP) by MEcPP under the action of 1-hydroxy-2-methyl-2-E-butenyl-4-pyrophosphoric acid synthase (HDS); finally, the HMBPP is catalyzed by 1-hydroxy-2-methyl-2-E-butenyl-4-pyrophosphoric acid reductase (HDR) to form a terpenoid synthesis precursor substance IPP. Therefore, the expression of DXR gene of Pinus massoniana can improve the content of downstream target products, and has important significance for engineering breeding of high-yield lipid genes of Pinus massoniana.

Disclosure of Invention

Aiming at the problems existing in the prior art, the technical problem to be solved by the invention is to provide an application of a pinus massoniana terpenoid synthesis related enzyme gene PmDXR and a promoter thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of pinus massoniana terpenoid synthesis related enzyme gene PmDXR or a promoter of pinus massoniana terpenoid synthesis related enzyme gene PmDXR in engineering breeding of pinus massoniana high-yield lipid genes.

The nucleotide sequence of the enzyme gene PmDXR related to the synthesis of pinus massoniana terpenoid is shown in GenBank: MK969119.1.

DXR is a key rate-limiting enzyme in the MEP pathway, catalyzes the 2 nd step reaction of the MEP pathway, and is mainly involved in biosynthesis of monoterpenes, diterpenes and tetraterpenoids. The terpenoid synthesized by cloning from Pinus massoniana is a key speed-limiting enzyme gene named PmDXR.

The upstream sequence 1600bp of the ATG of the initiation codon of the PmdXR gene is obtained by taking the genomic DNA of Pinus massoniana as a template for amplification, namely the promoter of the enzyme gene PmdXR related to Pinus massoniana terpenoid synthesis, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

The application of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR or a promoter of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR in improving the photosynthetic pigment content of plants.

The application of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR or a promoter of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR in improving the activity of plant DXR enzyme is provided.

Further, the plant is Pinus massoniana or Arabidopsis thaliana.

The application takes model plant Arabidopsis thaliana as a receptor plant, successfully constructs a pCAMBIA1302-PmDXR overexpression vector, utilizes agrobacterium tumefaciens to mediate transformation into Arabidopsis thaliana, cultures the Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana, and then determines DXR enzyme activity and photosynthetic pigment content of the transgenic Arabidopsis thaliana.

The promoter of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR is used for driving GUS gene to express in plants.

Further, the plant is Pinus massoniana or Nicotiana tabacum.

The promoter of the enzyme gene PmDXR related to the synthesis of pinus massoniana terpenoid is inserted into an expression vector pBI121 containing GUS genes to replace the existing promoter in the expression vector, a pBI121-ProDXR vector is constructed, the tobacco with the formula is taken as an instantaneous transformation object, the cloned pBI121-ProDXR vector is transferred into the tobacco by an instantaneous transformation method and is cultivated, and the PmDXR gene promoter can drive the GUS genes to express in roots, stems and leaves of the Nicotiana benthamiana.

Compared with the prior art, the invention has the beneficial effects that:

the invention takes model plants of tobacco and arabidopsis as receptor plants, converts the Pinus massoniana PmDXR gene into the arabidopsis by using agrobacterium mediation, and obtains transgenic arabidopsis through cultivation, wherein DXR enzyme activity, chlorophyll a, chlorophyll b and carotenoid content are higher than those of wild arabidopsis, and morphological observation shows that the transgenic of normal growth for 30d and the wild arabidopsis have difference in the growth of a young shaft. And constructing a pBI121-ProDXR vector, infecting Nicotiana benthamiana by a transient transformation method, and carrying out GUS histochemical staining analysis. The result shows that the PmdXR gene promoter can drive GUS gene to express in root, stem and leaf of Nicotiana benthamiana, and the promoter has no obvious tissue specificity. Has good practicability and has good application prospect in plant breeding and stress resistance research.

Drawings

FIG. 1 is a diagram showing tissue-specific expression of the PmdXR gene in Pinus massoniana; in the figure, R: root; f: flower; NS: young stems; OS: old stems; NL: new leaves; ML: mature leaves; x: a wood part; p: phloem;

FIG. 2 is a graph showing the expression of the PmdXR gene in different treated Pinus massoniana seedlings; in the figure, (A) is mechanical injury treatment, (B) is 15% PEG6000 osmotic stress treatment, (C) is 0mM H ₂ O ₂ Stress treatment, (D) 500. Mu.M ETH treatment, (E) 1mM SA treatment; (F) MeJA treatment at 100. Mu.M;

FIG. 3 is an identification map of transgenic Arabidopsis; in the figure, M is normalRun ^TM 250bp-II DNA ladder;1-14 is transgenic arabidopsis thaliana; 15 is wild type arabidopsis; 16 is the positive control pCAMBIA1302-PmDXR plasmid;

FIG. 4 is a graph showing the relative expression level of the transgenic Arabidopsis PmdXR gene; in the figure, WT is wild type arabidopsis; R1-R12 are transgenic Arabidopsis thaliana;

FIG. 5 is a graph of DXR enzyme activity assay; in the figure, WT is wild type arabidopsis; R1-R12 are transgenic Arabidopsis thaliana;

FIG. 6 is a graph showing the analysis of photosynthetic pigment content of transgenic plants and wild type plants; in the figure, WT is wild type arabidopsis; R1-R12 are transgenic Arabidopsis thaliana;

FIG. 7 is a diagram of phenotype observations of transgenic plants and wild type plants; in the figure, (A) is Arabidopsis rosette; (B) is an arabidopsis thaliana bolt shaft; (C) drought-treated arabidopsis; WT is wild type Arabidopsis, DXR is Arabidopsis over-expressing PmDXR; a is that 20d arabidopsis thaliana grows in soil before drought treatment, b is that 15d arabidopsis thaliana is drought treated, c is that arabidopsis thaliana is rehydrated for 3 days;

FIG. 8 is a map of the amplification products of the PmdXR promoter; in the figure, M is DNAMaroer, and PRO is PmDXR promoter;

FIG. 9 is an electrophoresis detection diagram of recombinant plasmids;

FIG. 10 is a graph of GUS staining of different tissues;

FIG. 11 is a graph of hormone stress response analysis of the PmDXR promoter;

FIG. 12 is a map of PmdXR protein subcellular localization; in the graph, A is a green fluorescence effect graph, B is a chloroplast autofluorescence effect graph, and C is a bright field effect graph; d is an superimposed field effect diagram.

Detailed Description

The invention is further described below in connection with specific embodiments. All primer sequences described below are 5 'to 3' in sequence.

EXAMPLE 1 analysis of expression Pattern of the Pinus massoniana PmdXR Gene (GenBank: MK 969119.1)

1. Tissue-specific expression of PmdXR gene in Pinus massoniana

The expression of the PmDXR gene in different tissues (root, young stem, old stem, new leaf, mature leaf, flower, xylem and phloem) of Pinus massoniana was analyzed by qRT-PCR (FIG. 1). The result shows that the expression level of PmDXR in young stem is 1, the PmDXR is expressed in all tissues, the expression of PmDXR in different tissues is different, the expression of PmDXR in xylem is highest, the expression level of PmDXR in mature leaves and roots is not obviously different, and the expression level of PmDXR in other tissues is obviously higher than that of PmDXR in other tissues. Overall, xylem > root > mature leaf > new leaf > phloem > old stem > young stem > flower.

qRT-PCR reaction System (20. Mu.l): 2 μl cDNA;10 mu l SYBR Green Master Mix;0.4 μl qPmDXR-F;0.4 μl qPmDXR-R;7.2 μl ddH ₂ O。

The reaction procedure was as follows: 95 ℃;2min;95 ℃;10sec;55 ℃;30sec;72 ℃;30sec;40 cycles.

2. Expression of PmdXR Gene in different treated Pinus massoniana seedlings

Adversity stress (mechanical injury, 15% PEG6000 osmotic stress, 10mM H) is applied to two-year-old Pinus massoniana seedlings ₂ O ₂ ) And hormone treatment (500. Mu.M ethephon, 1mM SA salicylate and 100. Mu.M methyl jasmonate MeJA), and samples were taken at different time periods. The qRT-PCR results under the stress show that (A) - (C) in FIG. 2) after mechanical injury treatment, the expression level of the PmDXR gene is up-regulated to different degrees at other treatment time except for 3 hours, and the expression level is highest at 6 hours of treatment and is 2.92 times that of a control group. In PEG6000 and H ₂ O ₂ After treatment, the expression level of the PmdXR gene was decreased at each treatment time point. The results of qRT-PCR under hormone induction treatment showed that (FIGS. 2 (D) - (F)) PmdXR was slightly up-regulated at 6h after ETH treatment. After SA treatment, the expression level of PmDXR was up-regulated at 6h, and the expression level was decreased at the other treatment time points. After the MeJA treatment, the expression level of the PmDXR gene was decreased at each treatment time point.

EXAMPLE 2 construction of Pinus massoniana PmdXR Gene expression vector and transformation of Arabidopsis thaliana

1. Construction of vectors

1ml of colibacillus liquid containing pCAMBIAl302 vector stored at-80 ℃ is taken for activation, 1-4ml of overnight cultured bacterial liquid is taken, and plasmids are extracted by using a plasmid small extraction kit. Taking 1 mug of the extracted pCAMBIA1302 plasmid, carrying out single enzyme digestion by using Nco I restriction enzyme, designing recombinant primers according to the sequences at the two ends of the pCAMBIA1302 carrier after single enzyme digestion and the ORF sequence of PmDXR, and carrying out PCR amplification.

The connection system is as follows: adding linearization vector of 0.02 Xcloning vector base pair number (ng); 0.04 x insert base pair number (ng); 4 μl of 5 XCE II Buffer;2 μl Exnase II; adding ddH ₂ O to 20. Mu.l.

The mixture was placed in a PCR apparatus and reacted at 37℃for 30 minutes, and then immediately placed on ice.

The successfully constructed recombinant vector plasmid is transformed into an agrobacterium competent cell GV3101 by the following method:

(1) 10 μl of recombinant plasmid is added into 100 μl of agrobacteria competence in ice water mixing state, the mixture is gently stirred at the bottom of the tube and mixed, and sequentially placed on ice, liquid nitrogen, 37 ℃ water bath and ice bath for 5min each.

(2) Mu.l of LB liquid medium was added and the mixture was placed on a shaking table at a constant temperature of 28℃for 2.5 hours.

(3) The supernatant was applied to a sample containing 50 mg.L by pipetting about 100. Mu.l ^-1 Kanamycin (Kan), 25 mg.L ^-1 On LB plates of Rif, the plates were inverted and incubated in an incubator at 28℃for 2d.

(4) And (3) selecting a monoclonal colony with good growth state for PCR detection, performing amplification culture on the monoclonal colony with positive detection, adding 50% glycerol with equal volume into bacterial liquid, and storing at-80 ℃ for subsequent experiments.

2. Sowing and culturing of arabidopsis thaliana

(1) Sterilizing Arabidopsis seeds: an appropriate amount of Arabidopsis seeds are placed in a 1.5ml centrifuge tube, 1ml of 75% ethanol is added into the centrifuge tube, the centrifuge tube is turned upside down for 45sec, 1ml of 20% sodium hypochlorite is added after the centrifuge tube is washed by sterile water, the centrifuge tube is turned upside down for 5min, and the centrifuge tube is repeatedly washed by sterile water for 5-6 times.

(2) Sowing: the sterilized Arabidopsis seeds were inoculated on a 1/2MS medium with a 1ml pipette.

(3) Culturing Arabidopsis thaliana: sealing the culture medium of the sown arabidopsis seeds, culturing for 2d under the dark condition at the temperature of 4 ℃, and then placing the culture medium in an artificial climate incubator until the culture medium germinates and grows. After about one week, the well-grown Arabidopsis seedlings in the medium were transferred to nutrient soil (black soil: vermiculite: perlite=6:2:1) for continued cultivation, covered with preservative film, and the preservative film was removed the third day.

3. Inflorescence dip-dyeing method for transforming arabidopsis thaliana

(1) Culturing agrobacterium solution containing pCAMBIAl302-PmDXR recombinant plasmid in a super clean bench by streak culture in a culture medium containing 50mg.L ^-1 Kan and 25 mg.L ^-1 Rif LB plates.

(2) Picking single colony with good growth state, adding 5ml containing 50mg.L ^-1 Kan and 25 mg.L ^-1 LB liquid medium of Rif at 28 DEG CCulturing overnight in a constant temperature shaker at 200 rpm.

(3) 1ml of the overnight cultured bacterial liquid is inoculated into 50ml of a bacterial liquid containing 50 mg.L ^-1 Kan and 25 mg.L ^-1 Shake culturing in LB liquid medium of Rif to OD ₆₀₀ =0.8 or so.

(4) The bacterial liquid is poured into a 50ml sterile centrifuge tube, centrifuged at 5000rpm for 10min to collect bacterial bodies, and 50ml of osmotic buffer prepared in advance is added to suspend bacterial body sediment.

(5) Selecting an arabidopsis plant which grows for about 4 weeks and is bolting, removing the flower buds which are already opened, soaking inflorescences in the infection liquid for 30sec, and covering preservative films after the completion of the soaking.

(6) Dark culturing for 18-20h, washing with clear water, and culturing in incubator.

(7) After 7-10d, repeating 1-6 steps to infect again.

(8) And harvesting transgenic T0 generation seeds after the pods of the arabidopsis plants turn yellow, and properly controlling the watering times when the seeds are about to mature in order to promote the seed maturation.

4. Resistance selection of transgenic Arabidopsis plants

(1) Taking a proper amount of harvested T0 generation seeds into a 1.5ml centrifuge tube, adding 1ml of 75% ethanol into the centrifuge tube, turning over for 45sec up and down, washing with sterile water, adding 1ml of 20% sodium hypochlorite, turning over for 5min up and down, and repeatedly washing with sterile water for 5-6 times.

(2) The seeds were evenly spot-sown in a 1ml pipette containing 30 mg.L ^-1 On a 1/2MS medium of hygromycin (Hyg), the culture is carried out for 2 days at the temperature of 4 ℃ in a dark culture way, and then the culture is carried out in an artificial climate incubator.

(3) After about 2 weeks of culture, transplanting 2 pieces of green true She Ju-root-system normally-grown resistant seedlings in the culture medium into nutrient soil, covering the seedlings with preservative film, and removing the preservative film in the third day.

(4) After the seeds are mature, the T1 generation seeds are separately harvested by a single plant, and sowing and screening are continued until the T2 generation seeds are harvested for the operation of the subsequent embodiment.

5. Transgenic arabidopsis molecular level detection

Preliminary resistance selection was performed on 1/2MS (containing Hyg) medium, and negative plants that did not successfully transform could not grow normally, while positive transgenic plants could grow normally. And transferring the Arabidopsis seedlings which can grow normally into nutrient soil for continuous culture, and extracting genome DNA and RNA by taking leaves as materials. Firstly, carrying out PCR amplification by taking genomic DNA of wild type and transgenic Arabidopsis thaliana as a template, wherein the Arabidopsis thaliana successfully transferred into the PmdXR gene can amplify a band with the same size as a plasmid amplification product (figure 3); then, the transgenic lines successfully identified in gene level are identified in transcription level (figure 4), and the result shows that the expression levels of different transgenic plants are different, and the expression level of the Arabidopsis plants numbered R8 is the highest in the 12 detected transgenic Arabidopsis plants numbered R1-R12, and then R12 and R3.

The specific implementation steps are as follows:

(1) Fresh leaves of Arabidopsis thaliana were cut, and Arabidopsis thaliana genomic DNA was extracted using DNAsecure novel plant genomic DNA extraction kit (TIANGEN), and the extraction procedure was strictly according to the specification. The concentration of the extracted Arabidopsis gDNA and the OD260/280 ratio were measured by an ultra-micro spectrophotometer.

(2) PCR detection is carried out by taking gDNA obtained in the first step as a template, 1302-CheckF as a front primer and 1302-PmDXR-R as a rear primer.

The primer has the following sequence:

1302-CheckF：ACAGTCTCAGAAGACCAAAGGGCA；

1302-PmDXR-R：ACTAGTCAGATCTACCATGGTCAGACTGTGGCAGGCTCCAAG。

PCR amplification reaction System (50. Mu.l): 2 μl of Arabidopsis gDNA;25 μl 2×

Master Mix；2μl 1302-CheckF；2μl 1302-PmDXR-R；19μl ddH ₂ O。

The reaction procedure: 98 ℃ for 3min;98℃15sec,55℃15sec,72℃1min,35 cycles; 72 ℃ for 5min; maintained at 4 ℃.

(3) Extracting DNA level to detect total RNA of positive plants, reversely transcribing the total RNA into cDNA, and detecting the relative expression quantity of PmDXR genes in transgenic arabidopsis through qRT-PCR, wherein an internal reference gene is an action 2.

Extracting total RNA by using a polysaccharide polyphenol plant total RNA extraction kit (Tiangen Biochemical technology Co., ltd.) by taking Pinus massoniana seedlings of positive plants as materials; when the agarose gel electrophoresis diagram of 1% of total RNA of masson pine shows clear bands and OD260/280 and OD260/230 are between 1.8 and 2.1, the method can be used for gene cloning.

Using the extracted RNA as a template, using

The One-Step gDNA Removal and cDNA Synthesis SuperMix kit synthesizes the first strand of cDNA according to the instruction manual.

qRT-PCR System and procedure set-up reference example 1.

6. Determination of transgenic Arabidopsis DXR enzyme Activity and photosynthetic pigment content

The transgenic Arabidopsis seedlings and the wild Arabidopsis seedlings are simultaneously cultured in an artificial climate incubator. The DXR enzyme activity, chlorophyll a, chlorophyll b and carotenoid content of transgenic Arabidopsis thaliana were measured by Shanghai Huding Biotech Co.Ltd. As shown in FIG. 5, the DXR enzyme activity of the transgenic Arabidopsis thaliana was higher than that of the wild type Arabidopsis thaliana, and the DXR enzyme activity of R5, R11 and R12 was significantly higher than that of the wild type Arabidopsis thaliana. The enzyme activity of R5 is highest and reaches 1.7 times that of the wild type, and the DXR enzyme activity of R11 and R12 is about 1.5 times that of the wild type. These results indicate that the enzyme activity of DXR is enhanced after transformation of arabidopsis thaliana with the PmDXR gene. As shown in figure 6, the content of chlorophyll a, chlorophyll b and carotenoid in transgenic Arabidopsis are all improved compared with that in wild type. The chlorophyll a content of the transgenic arabidopsis is 1.1-1.7 times of that of the wild type, wherein the chlorophyll a content of R1, R6, R7, R8 and R12 is obviously higher than that of the wild type, the highest chlorophyll a content of R12 is 1.7 times of that of the wild type, and the chlorophyll a content of the transgenic arabidopsis is 1.4 times of that of the wild type. The chlorophyll b content of the transgenic arabidopsis is 1.3-2.0 times of that of the wild type, except R5 and R11, the chlorophyll b content of the rest transgenic plants is obviously or extremely obviously higher than that of the wild type, and the chlorophyll b content of R4 is the highest. All transgenic arabidopsis have a significantly or very significantly higher carotenoid content than the wild type, and their carotenoid content is 1.2-1.4 times that of the wild type, wherein the carotenoid content of R1 is highest, up to 210.4pg/ml.

7. Transgenic Arabidopsis phenotype observations

Transgenic arabidopsis and wild arabidopsis were cultured under the same culture conditions, and after normal growth for 30d, differences in rosette and bolt axis growth were observed (fig. 7). Phenotypic observation shows that the arabidopsis thaliana over-expressing the PmDXR has obvious difference with the rosette growth of the wild type arabidopsis thaliana, but the difference exists in the shoot axis growth, and the shoot axis of the transgenic arabidopsis thaliana is obviously lower than that of the wild type arabidopsis thaliana. After the arabidopsis thaliana is subjected to drought treatment for 15d, the transgenic arabidopsis thaliana has better growth condition than the wild type when the transgenic arabidopsis thaliana lacks water for 15d, and after rehydration for 3d, the transgenic arabidopsis thaliana and the wild type arabidopsis thaliana can be gradually recovered.

EXAMPLE 3 cloning of the Pinus massoniana PmdXR promoter fragment and transient expression in tobacco

1. Extraction of Pinus massoniana gDNA

Extracting genomic DNA of Pinus massoniana seedlings by using DNAsecure novel plant genomic DNA extraction kit (TIANGEN), wherein the specific operation steps are strictly carried out according to the specification. After the extraction is completed, the concentration of the masson pine gDNA and the OD260/280 ratio are detected by an ultra-micro spectrophotometer.

2. Pmdxr gene promoter primer design

According to the PmDXR gene sequence, 3 specific downstream primers of PmDXR-SP1, PPmDXR-SP2 and PPmDXR-SP3 with higher annealing temperature are designed, wherein the PPmDXR-SP2 is positioned at the inner side of the PmDXR-SP1, and the PmDXR-SP3 is positioned at the inner side of the PmDXR-SP 2. Specific primers pPmDXR-F and pPmDXR-R are designed according to flanking sequences obtained from a sequencing result.

The primer sequences were as follows:

pPmDXR-SP1：GTGAAGATGGCAGAGTCGCAGGAA；

pPmDXR-SP2：GCGAGTGTAGGGTGGAGGCTTATT；

pPmDXR-SP3：GGGCGGAGGATAAGACAAAGAAGA；

pPmDXR-F：TGGTAATGCAATGAAGTTGGGAGG；

pPmDXR-R：GGGGTGGAAAGGGGCGGAGGATAA。

3 rounds of PCR amplification were performed with reference to Genome Walking kit instructions, and specific PCR reaction systems and procedures were set as follows:

(1) The first round PCR reaction (50. Mu.l) was prepared as follows: 4 μl gDNA;8 μl dNTP mix (2.5 mM each); mu.l 10 XLA PCR Buffer II (Mg ²⁺ plus)；0.5μl LA Taq；1μl AP1 Primer；1μl SP1 Primer；30.5μl ddH ₂ O。

(2) The first round PCR reaction conditions were as follows: 94 ℃ for 1min;98 ℃ for 1min;94 ℃ for 30sec,64 ℃ for 1min and 72 ℃ for 2min, and 5 cycles; 94 ℃ for 30sec,25 ℃ for 3min and 72 ℃ for 2min;94℃30sec,64℃1min,72℃2min,94℃30sec,44℃1min,72℃2min for a total of 15 cycles; and at 72℃for 10min.

(3) The second round PCR reaction (50. Mu.l) was prepared as follows: 1 μl of the first round PCR reaction; 8 μl dNTP mix (2.5 mM each); mu.l 10 XLA PCR Buffer II (Mg ²⁺ plus)；0.5μl LA Taq；1μl AP1 Primer；1μl SP2 Primer；33.5μl ddH ₂ O。

(4) The second round of PCR reaction conditions were as follows: 94℃30sec,64℃1min,72℃2min,94℃30sec,44℃1min,72℃2min for a total of 15 cycles; and at 72℃for 10min.

(5) The third round of PCR reaction (50. Mu.l) was prepared as follows: 1 μl of the second round PCR reaction; 8 μl dNTP mix (2.5 mM each); mu.l 10 XLA PCR Buffer II (Mg ²⁺ plus)；0.5μl LA Taq；1μl AP1 Primer；1μl SP3 Primer；33.5μl ddH ₂ O。

(6) The third round of PCR reaction conditions were as follows: 94℃30sec,641min,72℃2min,94℃30sec,64℃1min,72℃2min,94℃30sec,44℃1min,72℃2min for a total of 15 cycles; and at 72℃for 10min.

(7) Gel electrophoresis detection of PCR products, gel cutting and recovery of target strips, connection transformation and positive detection referring to the operation process of the embodiment, the bacterial liquid positive in bacterial detection is sent to Beijing qingke biological science and technology Co., ltd for sequencing.

3. Verification of Pinus massoniana PmdXR promoter sequence

PCR amplification the PCR reaction system (50. Mu.l) verifying the full length of the promoter was as follows: 2 μl gDNA;25 μl 2×

Master Mix；2μl pPmDXR-F；2μl pPmDXR-R；19μl ddH ₂ O。

The PCR procedure was set as follows: 98 ℃ for 3min;98℃15sec,55℃15sec,72℃1min for a total of 35 cycles; 72 ℃ for 5min; maintained at 4 ℃.

The 1.2% agarose gel electrophoresis of the amplified product of the PmDXR gene promoter is shown in figure 8, and the nucleotide sequence is shown as SEQ ID NO. 1.

4. Construction of pBI121-ProDXR recombinant vector

Amplifying the pBI121-ProDXR-F and pBI121-ProDXR-R (ProDXR refers to the promoter of the PmDXR gene) to obtain a PmDXR promoter fragment with enzyme cutting sites at two ends, carrying out double enzyme cutting on the pBI121 plasmid by using Hind III and Xba I restriction enzymes, and connecting the enzyme cutting products with the inserted fragment to convert competent cells Trelief of the escherichia coli ^TM 5d, monoclonal colonies were detected by PCR (FIG. 9). And then, carrying out sequencing verification on the bacterial liquid with positive detection, wherein the sequencing result shows that the pBI121-ProDXR vector is successfully constructed.

The primer sequences were as follows:

pBI121-ProDXR-F：GACCATGATTACGCCAAGCTTTGGTAATGCAATGAAGTTGGGA；

pBI121-ProDXR-R：ACCACCCGGGGATCCTCTAGAGGGGTGGAAAGGGGCGGA。

the plasmid cleavage reaction (50 μl) was as follows: 1 μg pBI121 plasmid; 5 μl of 10 XQuickCut Buffer;1 μl HindIII; 1 μl Xba I; adding ddH ₂ O to 50. Mu.l.

5. Transient transformation of tobacco by agrobacterium-mediated method

The pBI121-ProDXR recombinant plasmid which is successfully constructed is transformed into an agrobacterium competent cell EHAl05, and single colony is selected for bacterial liquid PCR identification. Amplifying and culturing the monoclonal colony with positive detection to OD ₆₀₀ After about=0.8, transformed nicotiana benthamiana, the specific method is as follows:

(1) The cells were collected by centrifugation at 5000rpm for 10min, 50ml of transient transformation resuspension was added to suspend the cell pellet, and the pellet was allowed to stand in the dark for 3h.

(2) Leaf discs are made from the strong leaf blades of the tissue culture seedlings of the Nicotiana benthamiana, roots and stems of the Nicotiana benthamiana are cut into small sections, and the prepared leaf blades, stems and roots are placed in sterile water for standby in order to avoid wilting due to water loss.

(3) The water is sucked on sterile filter paper, the prepared tobacco leaves, roots and stems are placed in heavy suspension, the tobacco leaves, roots and stems are gently shaken for about 10min, taken out by forceps, sucked by the sterile filter paper, and then the infected tobacco leaves, roots and stems are placed on a tobacco co-culture medium and are cultured for 24h under dark conditions.

6. Tobacco leaf treated with different hormones

After 24 hours of dark culture, the tobacco leaves were transferred to a medium containing 100. Mu. Mol.L each ^-1 MeJA and 100. Mu. Mol.L ^-1 ABA、1mmol·L ^-1 Culturing GA in MS culture medium for 36 hr to allow it to stand in MS ₀ Leaves on the medium served as controls.

7. Histochemical staining of beta-Glucuronidase (GUS)

Respectively placing leaves, roots and stems of Nicotiana benthamiana into a 5ml centrifuge tube, adding a proper amount of GUS staining solution, adding GUS staining solution which is required to be soaked in experimental materials, staining for 16 hours at 37 ℃ in a dark place, pouring out the staining solution, adding 70% ethanol for decoloring, changing the ethanol once every 3 hours, observing the staining condition of the Nicotiana benthamiana under a stereoscopic microscope after decoloring, and photographing and recording. The results of GUS histochemical staining analysis showed that the GV3101 null negative control had no GUS blue signal, the GUS staining color of roots, stems and leaves of pBI121 transiently transformed tobacco was the darkest, and roots, stems and leaves of pBI121-ProDXR transiently transformed tobacco were blue after GUS staining, but the staining color was lighter than that of positive control pBI 121. Thus, the ProPmDXR can drive GUS gene to express in tobacco roots, stems and leaves.

Transfer the tobacco leaves co-cultured for 24 hours to the medium containing 100. Mu. Mol.L respectively ^-1 MeJA、100μmol·L ^-1 ABA、1mmol·L ^-1 Treating on MS culture medium of GA for 36h, observing dyeing result after GUS dyeing, and analyzing influence of different hormones on PmDXR promoterAnd (5) sounding. The results showed (FIG. 11) that the GUS staining effect driven by ProDXR was stronger after ABA and MeJA treatment than that of the control, the color was darker than that of the untreated tobacco leaves, and the GUS staining effect after GA treatment was slightly weaker than that after untreated.

Example 4 subcellular localization of Pinus massoniana PmdXR protein

The pBI121-GFP vector contains green fluorescent protein gene, and can be used as marker gene to connect and express with target gene. Recombinant primers were designed based on the pBI121-GFP vector sequence and the ORF sequence (deletion stop codon) of the PmdXR gene. The PmDXR-GFP was transformed into Nicotiana benthamiana leaves by transient transformation, dark-cultured in an artificial climatic incubator for 2d, and the expression position of fluorescent GFP in cells was examined by a laser confocal microscope. The results showed that pBI121-GFP empty vector was localized in the whole tobacco leaf epidermal cells and PmdXR-GFP was localized in chloroplasts of tobacco leaf epidermal cells (FIG. 12).

The specific operation is as follows:

(1) In the presence of 50 mg.L ^-1 Kan and 25 mg.L ^-1 Agrobacteria containing the PmdXR-GFP recombinant plasmid are streaked on LB plates of Rif.

(2) Picking single colony with good growth state, and adding the single colony to 20ml containing 50 mg.L ^-1 Kan and 25 mg.L ^-1 Shake culturing in LB liquid medium of Rif to OD ₆₀₀ About=0.8 (P19 strain was simultaneously cultured).

(3) The bacterial liquid was transferred to a 50ml sterile centrifuge tube and centrifuged at 5000rpm for 10min at 4℃in a high-speed refrigerated centrifuge to collect the bacterial cells.

(4) Adding the instant transformation heavy suspension with the same volume as the bacterial liquid into a centrifuge tube, mixing uniformly by vortex, mixing with the P19 heavy suspension with the same volume, and standing for 3 hours at room temperature in a dark place.

(5) The resuspension was injected into the leaf of Nicotiana benthamiana and incubated in dark at room temperature for 48h.

(6) Transient expression of GFP fusion protein in leaf epidermal cells of nicotiana benthamiana was observed with laser confocal microscope LSM 710.

Sequence listing

<110> university of Nanjing forestry

<120> application of Pinus massoniana terpenoid synthesis related enzyme gene PmDXR and promoter thereof

<130> 1

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1600

<212> DNA

<213> PmDXR Gene promoter (Artificial)

<400> 1

tggtaatgca atgaagttgg gagggggagg tcacgggttc gacctgccct ccgcccatcc 60

cttaatacat ttttccgccc attgaatttt ccattcaatt atttacatat atggggattt 120

ggatatagtt caatgccttt ggatttacta tggaattata tatatttcta aaaaatacaa 180

tttccacata atattcatca tgaacccata attccacctt ggaggacatt gggcaatttc 240

accttcacac agccaaattg gcaaatccta aagtgacact tgtcacaacc ttattggaaa 300

atactaaaga agattcccat catgctcact gtactgccat gtcatcattc tgtcctgttt 360

agaaaaacga acaacaggca ttttctcctt catccaaact cggtttttag tgaaacgaat 420

tgcattgtga tcgtcttgac gagacagaca cagtcgtaca tgtttcgagc caatacaaga 480

actttgattt tttgatgccc taaatttctc aacataacgc acccattccc aaagcacaaa 540

tgccaatggt caaaacccat taaaactaaa agtaatggaa tgctgcatat agtctatgcc 600

aaaatttttg gattaagcat gatccacttt aatagatgga atatcttctg cctgaaccac 660

aatgaagacc gactctaaga gatctttaag agtaagatgt gagagccatg ggtgtagggc 720

atccttaaag gatctagact gagaggaaga gggcacaccc cagcctttgg ctccatcgtt 780

taatcttgaa attaaaaagc ttgaagcatc cttggttatg ccataatcat cgagcaaatt 840

tctttcttga agctacctac cactgtgaag gagttgcata aacatggagg ggattcccat 900

ccgatcttga attctagcta cgagtgaaga gaccatgtta gcgaatttca cctaaatata 960

taaaataccc cctgagattg gtctcacaaa gaccaaaatg ttcaagaggt ttctgtcctc 1020

agcttgaatg catgaagaac cctatgacaa aagaagttcc ccttgatttc aatgatctac 1080

aacaattaca ccaagatgag ggggattcaa acaaaatgtc gagtcttgaa gcaaaaaatc 1140

ttccacaaac agggtggcag tggctcaagg cattagggtt tccatcgcga taactaaaaa 1200

cttcgattta atttttcata tttttattta tgaaaactac tcttaaaata aatctcaatt 1260

ctttatttct aataattatt aaaaatattt tgaaattatt aaattattaa aatattcggt 1320

tgcctggctc tctaggcaag gccccctcaa acgcacttta ctattatcaa gtcaacatca 1380

ttatcgagtc aacaccatta gttagttata tgtatagaag tgacacatgt acaacgggac 1440

atgaaaatta ttgacacagt ggaaatgggt agccgtggga aagatacccc tgtattttgg 1500

agtttagcgg aggaacgcaa atggcattcc gcatggtgtc caattccact actacattgc 1560

ttgattcttc tttgtcttat cctccgcccc tttccacccc 1600

<210> 2

<211> 24

<212> DNA

<213> 1302-CheckF(Artificial)

<400> 2

acagtctcag aagaccaaag ggca 24

<210> 3

<211> 42

<212> DNA

<213> 1302-PmDXR-R(Artificial)

<400> 3

actagtcaga tctaccatgg tcagactgtg gcaggctcca ag 42

<210> 4

<211> 24

<212> DNA

<213> pPmDXR-SP1(Artificial)

<400> 4

gtgaagatgg cagagtcgca ggaa 24

<210> 5

<211> 24

<212> DNA

<213> pPmDXR-SP2(Artificial)

<400> 5

gcgagtgtag ggtggaggct tatt 24

<210> 6

<211> 24

<212> DNA

<213> pPmDXR-SP3(Artificial)

<400> 6

gggcggagga taagacaaag aaga 24

<210> 7

<211> 24

<212> DNA

<213> pPmDXR-F(Artificial)

<400> 7

tggtaatgca atgaagttgg gagg 24

<210> 8

<211> 24

<212> DNA

<213> pPmDXR-R(Artificial)

<400> 8

ggggtggaaa ggggcggagg ataa 24

<210> 9

<211> 43

<212> DNA

<213> pBI121-ProDXR-F(Artificial)

<400> 9

gaccatgatt acgccaagct ttggtaatgc aatgaagttg gga 43

<210> 10

<211> 39

<212> DNA

<213> pBI121-ProDXR-R(Artificial)

<400> 10

accacccggg gatcctctag aggggtggaa aggggcgga 39

Claims (2)

1. Application of a pinus massoniana terpenoid synthesis related enzyme gene PmDXR in improving the photosynthetic pigment content of arabidopsis thaliana, wherein the sequence of the pinus massoniana terpenoid synthesis related enzyme gene PmDXR is as GenBank: MK969119.1.

2. The application of a promoter of a Pinus massoniana terpenoid synthesis related enzyme gene PmDXR in driving GUS gene expression in tobacco is provided, and the sequence of the Pinus massoniana terpenoid synthesis related enzyme gene PmDXR promoter is shown as SEQ ID NO. 1.

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