CN114507679A - A kind of masson pine terpenoid synthesis related enzyme gene PmDXR and the application of its promoter - Google Patents

Abstract

The invention discloses the application of an enzyme gene PmDXR related to the synthesis of masson terpenoid substances and a promoter thereof, and belongs to the technical field of plant genetic engineering. In the present invention, the PmDXR gene of Pinus massoniana is transformed into Arabidopsis thaliana mediated by Agrobacterium, and the DXR enzyme activity, chlorophyll a, chlorophyll b and carotenoid contents of the transgenic Arabidopsis are higher than those of the wild type Arabidopsis. And clone the promoter of PmDXR gene from Pinus massoniana, the nucleotide sequence is as shown in SEQ ID NO.1, by constructing pBI121-ProDXR carrier, the transient transformation method infects Nicotiana benthamiana, PmDXR gene promoter can drive GUS gene in It is expressed in N. benthamiana roots, stems and leaves, and the promoter has no obvious tissue specificity. The invention has important research value and application prospect in improving pine resin yield traits by using genetic engineering technology.

Description

A kind of masson pine terpenoids synthesis related enzyme gene PmDXR and its promoter application

technical field

The invention belongs to the technical field of plant genetic engineering, in particular to the application of PmDXR, an enzyme gene related to the synthesis of masson pine terpenoid substances, and a promoter thereof.

Background technique

Pinus massoniana (Pinus massoniana) belongs to the Pinaceae (Pinaceae) genus and subgenus Bivascular Pinus, with a straight trunk, reddish-brown bark, and cracked into irregular scaly pieces; the branches are flat or oblique; there are many needles. 2-needle bundle, 3-needle bundle, 12-20cm long; leaf sheath is brown at first, then gradually becomes gray-black and persists. The male cones are cylindrical and clustered in the bud axil at the lower part of the new branch; the female cones are solitary or 2-4 clustered near the top of the new branch; the cones are oval or conical-oval, and the color will change before and after maturity , green before maturity, chestnut brown when mature. Pinus massoniana is widely distributed among the Pinus species in my country. It is distributed in 17 provinces (autonomous regions and municipalities directly under the Central Government) from the south of the Qinling Mountains and the Huaihe River to the east of the Yunnan-Guizhou Plateau. It has the characteristics of strong adaptability and high economic value. The economic value of masson pine is not only reflected in the use of timber, but also in the processing of forest products, which has brought huge economic benefits to our country. Masson pine can secrete a large number of secondary metabolites, mainly terpenoids. These secondary metabolites are called turpentine, which is the basic raw material of rosin and turpentine industry and is widely used in solvents, disinfectants, cleaning products, fragrances, coatings. and other industrial biological products.

The terpenoids in masson pine resin mainly include monoterpenes, sesquiterpenes and diterpenes, which generally exist in the roots, stems, leaves and cones of pine trees. Not only does turpentine bring great economic benefits, its components also play an important role in the defense system of conifers. After conifers are subjected to biotic or abiotic stimuli, turpentine can be released from the tree's resin canal, and new turpentine can be induced to synthesize. At present, there are mainly 6 kinds of pine trees in my country that can be harvested for pine resin, but only Masson pine, Pinus elliottii (Pinus elliottii), Pinus yunnanensis (Pinus yunnanensis) and Simao pine (Pinus kesiyavar. Pine resin is collected from Masson pine. my country's pine resin production areas are mainly in Guangxi. The survey shows that in 2010, the masson pine pine resin production in Guangxi production areas accounted for about 42% of the national pine resin production, with a total output of about 300,000 tons. In order to promote the development of the pine resin industry, it is an effective way to improve the utilization rate of pine forest resources by using advanced technology to screen and cultivate high-quality germplasm materials with high lipid yield and create high-yield industrial raw material forests.

1-Deoxy-D-xylulose-5-phosphate reductase DXR (1-Deoxy-d-xylulose-5-phosphate reductase DXR) is a key rate-limiting enzyme in the MEP pathway, catalyzing the second step of the MEP pathway. The cofactor nicotinamide adenine dinucleotide phosphate (NADPH), Mn ²⁺ , Co ²⁺ or Mg ²⁺ is required to participate in the conversion of 1-deoxy-D-xylulose-5-phosphate synthase (DXP) into An important precursor for terpenoid synthesis, 2-methylerythritol-4-phosphate (MEP). The MEP pathway is localized in plastids and is mainly involved in the biosynthesis of monoterpenes, diterpenes and tetraterpenoids. The process of generating isoprenyl pyrophosphate (IPP) and dimethylpropenyl diphosphate (DMAPP) by MEP pathway is mainly divided into 7 steps: in the first step, pyruvate and 3-phosphate-glyceraldehyde are converted from 1-deoxygenated -D-xylulose-5-phosphate synthase (DXS) catalyzes the formation of DXP; in the second step, DXP undergoes a reduction reaction catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) Generate MEP; in the third step, in the presence of NADPH, MEP is catalyzed by 2-methyl-D-erythritol-4-phosphocytidyltransferase (MCT) to generate 4-(5′-cytidine pyrophosphate)-2 -C-methyl-D-erythritol (CDP-ME); in the fourth step, CDP-ME is treated with 4-(5′-cytidine pyrophosphate)-2-C-methyl-D-erythritol kinase The phosphorylation of (CMK) generates 4-(5′-cytidine pyrophosphate)-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP); in the fifth step, CDP-MEP is 2-Methyl-D-erythritol-2,4-cyclic pyrophosphate synthase (MDS) produces 2-methyl-D-erythritol-2,4-cyclic pyrophosphate (MEcPP); sixth In the next step, MEcPP forms 1-hydroxy-2-methyl-2-E-butenyl- 4-pyrophosphate (HMBPP); finally, HMBPP is catalyzed by 1-hydroxy-2-methyl-2-E-butenyl-4-pyrophosphate reductase (HDR) to form terpenoid synthesis precursor IPP. Therefore, the expression of DXR gene of Pinus massoniana to increase the content of downstream target products is of great significance to the genetic engineering breeding of Pinus massoniana for high lipid production.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of a masson pine terpenoid synthesis-related enzyme gene PmDXR and its promoter.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is as follows:

The application of a masson pine terpenoid synthesis-related enzyme gene PmDXR or the promoter of the masson pine terpenoid synthesis-related enzyme gene PmDXR in masson pine high-fat-yielding genetic engineering breeding.

Masson pine terpenoid synthesis related enzyme gene PmDXR, the nucleotide sequence of which can be found in GenBank: MK969119.1.

DXR is a key rate-limiting enzyme in the MEP pathway, which catalyzes the second step of the MEP pathway and is mainly involved in the biosynthesis of monoterpenes, diterpenes and tetraterpenoids. The key rate-limiting enzyme gene for terpenoid synthesis cloned from Masson pine in the present application is named PmDXR.

The 1600bp upstream sequence of the initiation codon ATG of the PmDXR gene was amplified by using the masson pine genomic DNA as the template, which is the promoter of the masson pine terpenoid synthesis-related enzyme gene PmDXR, and its nucleotide sequence is shown in SEQ ID NO.1.

Application of the promoter of masson pine terpenoid synthesis related enzyme gene PmDXR or masson pine terpenoid synthesis related enzyme gene PmDXR in improving the content of photosynthetic pigments in plants.

The application of the promoter of masson pine terpenoid synthesis related enzyme gene PmDXR or masson pine terpenoid synthesis related enzyme gene PmDXR in improving plant DXR enzyme activity.

Further, the plant is Masson pine or Arabidopsis.

In this application, the model plant Arabidopsis thaliana was used as the recipient plant, and the pCAMBIA1302-PmDXR overexpression vector was successfully constructed, and transformed into Arabidopsis thaliana mediated by Agrobacterium, and the transgenic Arabidopsis was cultivated to obtain the transgenic Arabidopsis thaliana, and then the DXR of the transgenic Arabidopsis was determined. Enzyme activity and photosynthetic pigment content.

Application of the promoter of masson pine terpenoid synthesis-related enzyme gene PmDXR in driving GUS gene expression in plants.

Further, the plant is masson pine or tobacco.

In the present application, the promoter of the PmDXR gene of masson terpenoid synthesis-related enzymes was inserted into the expression vector pBI121 containing the GUS gene to replace the existing promoter in the expression vector, and the pBI121-ProDXR vector was constructed, and the present type of tobacco was used for transient transformation. The subject, the cloned pBI121-ProDXR vector was transformed into tobacco by transient transformation and cultivated. The PmDXR gene promoter can drive the expression of GUS gene in the roots, stems and leaves of N. benthamiana.

Compared with the prior art, the beneficial effects of the present invention are:

In the present invention, the model plants Nicotiana benthamiana and Arabidopsis thaliana are used as recipient plants, the PmDXR gene of Pinus massoniana is transformed into Arabidopsis thaliana mediated by Agrobacterium, and the transgenic Arabidopsis thaliana is obtained through cultivation, wherein the DXR enzyme activity, chlorophyll a, chlorophyll a, The contents of chlorophyll b and carotenoid were higher than those of wild-type Arabidopsis. Morphological observation found that there were differences in the growth of sprouts between the transgenic and wild-type Arabidopsis that grew normally for 30 days. The pBI121-ProDXR vector was constructed, and the transient transformation method was used to infect N. benthamiana, and GUS histochemical staining was performed. The results showed that the PmDXR gene promoter could drive the expression of GUS gene in N. benthamiana roots, stems and leaves, and the promoter had no obvious tissue specificity. It has good practicability and will have good application prospects in plant breeding and stress resistance research.

Description of drawings

Figure 1 is the tissue-specific expression map of PmDXR gene in Pinus massoniana; in the figure, R: root; F: flower; NS: young stem; OS: old stem; NL: new leaf; ML: mature leaf; X: xylem; P: Phloem;

Figure 2 is the expression map of PmDXR gene in different treatments of Pinus massoniana seedlings; in the figure, (A) is mechanical damage treatment, (B) is 15% PEG6000 osmotic stress treatment, (C) is 0 mM H ₂ O ₂ stress treatment, (D) is treated with 500 μM ETH, (E) is treated with 1 mM SA; (F) is treated with 100 μM MeJA;

Figure 3 is the identification map of transgenic Arabidopsis; in the figure, M is NormalRun ^TM 250bp-II DNA ladder; 1-14 are transgenic Arabidopsis; 15 is wild-type Arabidopsis; 16 is the positive control pCAMBIA1302-PmDXR plasmid;

Figure 4 is a graph showing the relative expression of PmDXR gene in transgenic Arabidopsis; in the figure, WT is wild-type Arabidopsis; R1-R12 are transgenic Arabidopsis;

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

Figure 6 is an analysis diagram of photosynthetic pigment content in transgenic plants and wild-type plants; in the figure, WT is wild-type Arabidopsis; R1-R12 are transgenic Arabidopsis;

Figure 7 is a phenotype observation diagram of transgenic plants and wild-type plants; in the figure, (A) is Arabidopsis rosette; (B) is Arabidopsis scabbard; (C) is drought-treated Arabidopsis; WT is wild-type Arabidopsis thaliana, DXR is Arabidopsis thaliana overexpressing PmDXR; a is Arabidopsis thaliana grown in soil before drought treatment for 20 days, b is Arabidopsis thaliana after drought treatment for 15 days, and c is Arabidopsis thaliana that has been rehydrated for 3 days;

Figure 8 is a diagram of the amplification product of the PmDXR promoter; in the figure, M is the DNAMarker, and PRO is the PmDXR promoter;

Fig. 9 is the electrophoresis detection figure of recombinant plasmid;

Figure 10 is a graph of GUS staining of different tissues;

Figure 11 is an analysis diagram of PmDXR promoter hormone stress response;

Figure 12 is the subcellular localization map of PmDXR protein; A is the green fluorescence effect map in the figure, B is the chloroplast autofluorescence effect map, C is the bright field effect map; D is the superimposed field effect map.

Detailed ways

The present invention will be further described below with reference to specific embodiments. All primer sequences described below are oriented 5' to 3'.

Example 1 Expression pattern analysis of PmDXR gene (GenBank: MK969119.1) of Pinus massoniana

1. Tissue-specific expression of PmDXR gene in Masson pine

The expression of PmDXR gene in different tissues of Pinus massoniana (root, young stem, old stem, new leaf, mature leaf, flower, xylem and phloem) was analyzed by qRT-PCR technology (Figure 1). The expression level in young stems was set to 1. The results showed that PmDXR was expressed in all tissues, and the expression in different tissues was different. PmDXR had the highest expression in xylem, which was closely related to the expression of PmDXR in mature leaves and roots. There was no significant difference in the amount of PmDXR, but it was significantly higher than the expression of PmDXR in other tissues. Overall, xylem>root>mature leaf>new leaf>phloem>old stem>young stem>flower.

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

The reaction program was as follows: 95 °C; 2 min; 95 °C; 10 sec; 55 °C; 30 sec; 72 °C; 30 sec; 40 cycles.

2. Expression of PmDXR gene in different treatments of Masson pine seedlings

Biennial masson pine seedlings were subjected to stress stress (mechanical injury, 15% PEG6000 osmotic stress, 10 mM H ₂ O ₂ ) and hormone treatments (500 μM ethephon ETH, 1 mM salicylic acid SA and 100 μM methyl jasmonate MeJA), respectively, and were treated with Sampling at different time periods. The results of qRT-PCR under stress showed ((A)-(C) in Figure 2) that after mechanical injury, except for 3h, the expression of PmDXR gene was up-regulated to varying degrees at other treatment times. The expression level was the highest at 6h, which was 2.92 times that of the control group. After PEG6000 and H ₂ O ₂ treatment, the expression level of PmDXR gene decreased at each treatment time point. The results of qRT-PCR under hormone-induced treatment showed ((D)-(F) in Figure 2) that PmDXR was slightly up-regulated at 6 h after ETH treatment. After SA treatment, the expression of PmDXR was up-regulated at 6 h, and the expression was decreased at other treatment time points. After MeJA treatment, the expression of PmDXR gene decreased at each treatment time point.

Example 2 Construction of Masson pine PmDXR gene expression vector and transformation of Arabidopsis thaliana

1. Construction of the vector

Take 1 ml of Escherichia coli bacteria liquid containing pCAMBIAl302 vector stored at -80°C for activation, take 1-4 ml of bacterial liquid cultured overnight, and extract plasmids using a plasmid mini-extraction kit. Take 1 μg of the extracted pCAMBIA1302 plasmid, use Nco I restriction endonuclease for single digestion, and design recombinant primers according to the sequence of the two ends of the pCAMBIA1302 vector after single digestion and the ORF sequence of PmDXR, and carry out PCR amplification.

The ligation system is as follows: add 0.02×cloning vector base pairs (ng) of linearized vector; 0.04×insert base pair (ng) insert; 4μl 5×CE II Buffer; 2μl Exnase II; add ddH ₂ 0 to 20 μl.

Placed in a PCR machine at 37 °C for 30 min and immediately placed on ice.

The constructed recombinant vector plasmid was transformed into Agrobacterium competent cell GV3101, and the transformation method was as follows:

(1) Take 10 μl of the recombinant plasmid and add it to 100 μl of Agrobacterium competent bacteria mixed with ice and water. Gently dial the bottom of the tube to mix evenly, and place it on ice, in liquid nitrogen, in a 37°C water bath, and in an ice bath for 5 minutes each.

(2) Add 700 μl of LB liquid medium and place it at 28°C for 2.5 hours with shaking on a constant temperature shaker.

(3) Pipette about 100 μl of the supernatant and spread it on the LB plate containing 50 mg·L ^-1 kanamycin (Kan) and 25 mg·L ^-1 rifampicin (Rif), and place it upside down in a 28°C incubator for 2 days. .

(4) Picking monoclonal colonies with good growth conditions for PCR detection, expanding and culturing positive monoclonal colonies, adding an equal volume of 50% glycerol to the bacterial solution, and storing at -80°C for subsequent experiments.

2. Sowing and Cultivation of Arabidopsis

(1) Sterilization of Arabidopsis thaliana seeds: put an appropriate amount of Arabidopsis thaliana seeds in a 1.5ml centrifuge tube, add 1ml of 75% ethanol to the centrifuge tube, turn upside down for 45sec, wash with sterile water and add 1ml of 20% sodium hypochlorite, Flip up and down for 5 minutes, and wash with sterile water 5-6 times.

(2) Seeding: The sterilized Arabidopsis thaliana seeds were seeded on 1/2 MS medium with a 1 ml pipette.

(3) Arabidopsis thaliana culture: Seal the medium on which Arabidopsis thaliana seeds were sown, cultured in the dark at 4°C for 2 days, and then placed in an artificial climate incubator for germination and growth. After about a week, the Arabidopsis seedlings in good growth condition in the medium were transferred to nutrient soil (black soil: vermiculite: perlite = 6: 2: 1) for further cultivation, and covered with plastic wrap, and the fresh-keeping was removed on the third day. membrane.

3. Transformation of Arabidopsis thaliana by inflorescence dip method

(1) In an ultra-clean workbench, streak the Agrobacterium liquid containing the pCAMBIAl302-PmDXR recombinant plasmid on an LB plate containing 50 mg·L ^-1 Kan and 25 mg·L ^-1 Rif.

(2) Pick a single colony with a good growth state, add 5 ml of LB liquid medium containing 50 mg·L ^-1 Kan and 25 mg·L ^-1 Rif, and cultivate overnight at 200 rpm in a constant temperature shaker at 28°C.

(3) Inoculate 1 ml of overnight cultured bacterial solution into 50 ml of LB liquid medium containing 50 mg·L ^-1 Kan and 25 mg·L ^-1 Rif, and shake the culture to OD ₆₀₀ =0.8.

(4) Pour the bacterial liquid into a 50 ml sterile centrifuge tube, centrifuge at 5000 rpm for 10 min to collect the bacterial cells, and add 50 ml of the osmotic buffer prepared in advance to suspend the bacterial cell precipitation.

(5) Select Arabidopsis thaliana plants that have been bolted for about 4 weeks, remove the flower buds that have opened, soak the inflorescence in the infection solution for 30 sec, and cover with plastic wrap after the soaking is completed.

(6) Incubate in the dark for 18-20h, rinse with clean water and continue to cultivate in the incubator.

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

(8) Harvest the transgenic T0 generation seeds after the seed pods of the Arabidopsis thaliana plants turn yellow. In order to promote the maturity of the seeds, the watering frequency should be appropriately controlled when the seeds are about to mature.

4. Resistance screening of transgenic Arabidopsis plants

(1) Take an appropriate amount of harvested T0 generation seeds into a 1.5ml centrifuge tube, add 1ml of 75% ethanol to the centrifuge tube, invert up and down for 45sec, wash with sterile water, add 1ml of 20% sodium hypochlorite, invert up and down for 5min, use sterile Wash with water 5-6 times.

(2) Seeds were evenly sown on 1/2 MS medium containing 30 mg·L ^-1 hygromycin (Hyg) with a 1 ml pipette, cultured in the dark at 4°C for 2 days and then placed in an artificial climate incubator.

(3) After about 2 weeks of culture, transplant the resistant seedlings with 2 green true leaves and normal root growth in the medium into nutrient soil, cover with plastic wrap, and remove the plastic wrap on the third day.

(4) After the seeds are mature, the seeds of the T1 generation are harvested separately for each individual plant, and the sowing and screening are continued until the seeds of the T2 generation are harvested for subsequent example operations.

5. Molecular level detection of transgenic Arabidopsis

Preliminary resistance screening was carried out on 1/2MS (containing Hyg) medium, and the negative plants that were not successfully transformed could not grow normally, while the positive transgenic plants could grow normally. The Arabidopsis thaliana seedlings that could grow normally were transferred to nutrient soil for further cultivation, and genomic DNA and RNA were extracted from leaves. First, PCR amplification was carried out using the genomic DNA of wild-type and transgenic Arabidopsis as templates. Arabidopsis successfully transformed with PmDXR gene could amplify the band with the same size as the plasmid amplification product (Figure 3). The transgenic lines that were successfully identified were identified at the transcription level (Fig. 4). The results showed that there were differences in the expression levels of different transgenic plants. Among the 12 transgenic Arabidopsis tested (the numbers were named R1-R12), the numbers were The Arabidopsis plant of R8 had the highest expression level, followed by R12 and R3.

The specific implementation steps are as follows:

(1) Cut the fresh leaves of Arabidopsis thaliana, and use the DNAsecure new plant genomic DNA extraction kit (TIANGEN) to extract the genomic DNA of Arabidopsis thaliana. The extraction steps are carried out in strict accordance with the instructions. The concentration and OD260/280 ratio of the extracted Arabidopsis gDNA were detected by ultra-micro spectrophotometer.

(2) Using the gDNA obtained in the first step as a template, 1302-CheckF as a pre-primer, and 1302-PmDXR-R as a post-primer, PCR detection was performed.

The primers are sequenced as follows:

1302-CheckF: ACAGTCTCAGAAGACCAAAGGGCA;

1302-PmDXR-R: ACTAGTCAGATCTACCATGGTCAGACTGTGGCAGGCTCCAAG.

Master Mix；2μl1302-CheckF；2μl 1302-PmDXR-R；19μl ddH₂O。PCR amplification reaction system (50 μl): 2 μl Arabidopsis gDNA; 25 μl 2×

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

Reaction program: 98 °C for 3 min; 98 °C for 15 sec, 55 °C for 15 sec, 72 °C for 1 min, 35 cycles; 72 °C for 5 min; 4 °C hold.

(3) The total RNA of the plants with positive DNA level detection was extracted and reverse transcribed into cDNA, and the relative expression of PmDXR gene in transgenic Arabidopsis was detected by qRT-PCR, wherein the internal reference gene was Actin2.

Using the masson pine seedlings of the positive plants as the material, the total RNA was extracted by the polysaccharide polyphenol plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.); when the 1% agarose gel electrophoresis of the masson pine total RNA, the bands were clear. , and when OD260/280 and OD260/230 are between 1.8-2.1, it can be used for gene cloning.

One-Step gDNA Removal and cDNASynthesis SuperMix试剂盒，按照说明书操作步骤合成cDNA第一链。Using the extracted RNA as a template,

One-Step gDNA Removal and cDNASynthesis SuperMix kit, according to the instructions to synthesize the first strand of cDNA.

Refer to Example 1 for the qRT-PCR system and program settings.

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

The transgenic Arabidopsis seedlings and wild-type Arabidopsis seedlings were simultaneously cultivated in an artificial climate incubator. Using Arabidopsis leaves as materials, the DXR enzyme activity, chlorophyll a, chlorophyll b and carotenoid contents of transgenic Arabidopsis were determined by Shanghai Huding Biotechnology Co., Ltd. The results of enzyme activity assay are shown in Figure 5. The DXR enzyme activities of transgenic Arabidopsis were higher than those of wild-type Arabidopsis, and the DXR enzyme activities of R5, R11 and R12 were significantly higher than those of wild-type Arabidopsis. The enzyme activity of R5 was the highest, reaching 1.7 times that of the wild type, and the DXR enzyme activities of R11 and R12 were about 1.5 times that of the wild type. These results indicated that PmDXR gene transformed Arabidopsis thaliana to enhance the enzymatic activity of DXR. The results of the determination of photosynthetic pigment content are shown in Figure 6. The contents of chlorophyll a, chlorophyll b and carotenoid in transgenic Arabidopsis were all higher than those of the wild type. The chlorophyll a content of transgenic Arabidopsis was 1.1-1.7 times that of the wild type, and the chlorophyll a content of R1, R6, R7, R8, and R12 was significantly higher than that of the wild type, and the chlorophyll a content of R12 was the highest, which was 1.7 times that of the wild type. , followed by R7, the content of chlorophyll a was 1.4 times that of the wild type. The chlorophyll b content of transgenic Arabidopsis was 1.3-2.0 times that of the wild type. Except for R5 and R11, the chlorophyll b content of other transgenic plants was significantly or extremely significantly higher than that of the wild type, and the chlorophyll b content of R4 was the highest. The carotenoid content of all transgenic Arabidopsis was significantly or extremely significantly higher than that of the wild type, and their carotenoid content was 1.2-1.4 times that of the wild type. The carotenoid content of R1 was the highest, reaching 210.4pg/ml.

7. Phenotype observation of transgenic Arabidopsis

Transgenic Arabidopsis and wild-type Arabidopsis were cultured under the same culture conditions, and the differences in the growth of rosettes and sprouts were observed after 30 days of normal growth (Fig. 7). Phenotypic observation did not reveal significant difference in rosette growth between PmDXR-overexpressing Arabidopsis thaliana and wild-type Arabidopsis thaliana, but there were differences in scabbard growth. After drought treatment of Arabidopsis for 15 days, it was found that the growth of transgenic Arabidopsis was better than that of the wild type when dehydrated for 15 days. After 3 days of rehydration, both the transgenic and wild type Arabidopsis could gradually recover.

Example 3 Cloning of PmDXR promoter fragment of Pinus massoniana and transient expression in tobacco

1. Extraction of masson pine gDNA

The genomic DNA of Masson pine seedlings was extracted using the DNAsecure new plant genomic DNA extraction kit (TIANGEN). After extraction, the concentration and OD260/280 ratio of Masson pine gDNA were detected by ultra-micro spectrophotometer.

2. Design of PmDXR Gene Promoter Primer

According to the PmDXR gene sequence, three specific downstream primers with higher annealing temperature, pPmDXR-SP1, pPmDXR-SP2 and pPmDXR-SP3, were designed, where pPmDXR-SP2 was located inside pPmDXR-SP1, and pPmDXR-SP3 was located inside pPmDXR-SP2. Specific primers pPmDXR-F and pPmDXR-R were designed according to the flanking sequences obtained from the sequencing results.

The primer sequences are as follows:

pPmDXR-SP1: GTGAAGATGGCAGAGTCGCAGGAA;

pPmDXR-SP2:GCGAGTGTAGGGTGGAGGCTTATT;

pPmDXR-SP3:GGGCGGAGGATAAGACAAAGAAGA;

pPmDXR-F:TGGTAATGCAATGAAGTTGGGAGG;

pPmDXR-R: GGGGTGGAAAGGGGCGGAGGATAA.

Carry out 3 rounds of PCR amplification according to the instructions of the Genome Walking kit. The specific PCR reaction system and program settings are as follows:

(1) The first-round PCR reaction solution (50 μl) was prepared as follows: 4 μl gDNA; 8 μl dNTP Mixture (2.5 mMeach); 5 μl 10×LA PCR Buffer II (Mg ²⁺ plus); 0.5 μl LA Taq; 1 μl AP1 Primer; 1 μl SP1 Primer; 30.5 μl ddH ₂ O.

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

(3) The second round PCR reaction solution (50 μl) was prepared as follows: 1 μl first round PCR reaction solution; 8 μl dNTP Mixture (2.5mM each); 5 μl 10×LA PCR Buffer II (Mg ²⁺ plus); 0.5 μl LA Taq ; 1 μl AP1 Primer; 1 μl SP2 Primer; 33.5 μl ddH ₂ O.

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

(5) The third round PCR reaction solution (50 μl) was prepared as follows: 1 μl second round PCR reaction solution; 8 μl dNTP Mixture (2.5mM each); 5 μl 10×LA PCR Buffer II (Mg ²⁺ plus); 0.5 μl LA Taq ; 1 μl AP1 Primer; 1 μl SP3 Primer; 33.5 μl ddH ₂ O.

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

(7) Gel electrophoresis detects PCR products, gel cutting to recover the target band, ligation transformation and positive detection Referring to the operation process of the above-mentioned embodiment, the bacterial liquid with positive bacterial detection is sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

3. Validation of PmDXR promoter sequence of Masson pine

Master Mix；2μl pPmDXR-F；2μl pPmDXR-R；19μl ddH₂O。The PCR reaction system (50 μl) to verify the full length of the promoter by PCR amplification is as follows: 2 μl gDNA; 25 μl 2×

Master Mix; 2 μl pPmDXR-F; ₂ μl pPmDXR-R; 19 μl ddH2O.

The PCR program was set as follows: 98°C for 3 min; 98°C for 15 sec, 55°C for 15 sec, 72°C for 1 min, a total of 35 cycles; 72°C for 5 min; 4°C hold.

1.2% agarose gel electrophoresis of the PmDXR gene promoter amplification product is shown in Figure 8, and the measured nucleotide sequence is shown in SEQ ID NO.1.

4. Construction of pBI121-ProDXR recombinant vector

Using pBI121-ProDXR-F and pBI121-ProDXR-R (ProDXR refers to the promoter of the PmDXR gene) as primers, amplify the PmDXR promoter fragment with restriction sites at both ends, and use Hind III and Xba I for restriction endonucleation The pBI121 plasmid was digested with double enzymes, and the digested product was ligated with the inserted fragment and transformed into E. coli competent cells Trelief ^TM 5d, and the single clone colonies were detected by PCR (Fig. 9). The positive bacterial liquid was then verified by sequencing, and the sequencing results showed that the pBI121-ProDXR vector was successfully constructed.

The primer sequences are as follows:

pBI121-ProDXR-F:GACCATGATTACGCCAAGCTTTGGTAATGCAATGAAGTTGGGA;

pBI121-ProDXR-R: ACCACCCGGGGATCCTCTAGAGGGGTGGAAAGGGGCGGA.

The plasmid digestion reaction system (50 μl) is as follows: 1 μg pBI121 plasmid; 5 μl 10×QuickCut Buffer; 1 μl Hind III; 1 μl Xba I; add ddH ₂ O to 50 μl.

V. Agrobacterium-mediated transient transformation of tobacco

The successfully constructed pBI121-ProDXR recombinant plasmid was transformed into Agrobacterium competent cell EHA105, and a single colony was picked for PCR identification. The positive monoclonal colonies are expanded and cultured to about OD ₆₀₀ =0.8 and then transformed into Nicotiana benthamiana. The specific method is as follows:

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

(2) The leaves of the tissue cultured N. benthamiana seedlings with strong growth are made into leaf discs, and the roots and stems of the tobacco are cut into small sections. In order to avoid dehydration and wilting, the prepared leaves, stems and roots are placed in sterile water. stand-by.

(3) Drain the water on sterile filter paper, place the prepared tobacco leaves, roots and stems in the re-suspension, shake gently for about 10 minutes, take out with tweezers, and blot dry with sterile filter paper, and then the infected The treated tobacco leaves, roots and stems were placed on tobacco co-cultivation medium and cultured in the dark for 24 h.

6. Treatment of tobacco leaves with different hormones

After 24 hours of dark culture, tobacco leaves were transferred to MS medium containing 100 μmol·L ^-1 MeJA, 100 μmol·L ^-1 ABA, and ^{1 mmol·L -1} GA, respectively, for 36 hours to place leaves on MS ₀ medium. as comparison.

Seven, β-glucuronidase (GUS) histochemical staining

Put the leaves, roots and stems of Nicotiana benthamiana into a 5ml centrifuge tube respectively, add an appropriate amount of GUS staining solution, the GUS staining solution added must be less than the experimental material, dye at 37°C for 16h in the dark, pour out the staining solution and add 70% Ethanol was used for decolorization, and the ethanol was changed every 3 h. After the decolorization was completed, the staining of tobacco was observed under a stereomicroscope and photographed for recording. The results of GUS histochemical staining analysis (Fig. 10) showed that the negative control of GV3101 empty strain had no GUS blue signal, and the GUS staining of tobacco roots, stems and leaves transiently transformed with pBI121 was the darkest. The roots, stems and leaves were all blue, but the staining color was lighter than that of the positive control pBI121. This indicates that proPmDXR can drive the expression of GUS gene in tobacco roots, stems and leaves.

Tobacco leaves co-cultured for 24 h were transferred to MS medium containing 100 μmol·L ^-1 MeJA, 100 μmol·L ^-1 ABA, and ^{1 mmol·L -1} GA for 36 h. The staining results were observed after GUS staining, and the effect of different hormones on the effects of different hormones was analyzed. Effects of the PmDXR promoter. The results showed (Fig. 11) that after ABA and MeJA treatment, the GUS staining effect driven by ProDXR was stronger than that of the control, and the color was darker than that of untreated tobacco leaves, and the GUS staining effect after GA treatment was slightly weaker than that of untreated tobacco leaves.

Example 4 Subcellular localization of PmDXR protein of Pinus massoniana

The pBI121-GFP vector contains the green fluorescent protein gene, which can be used as a marker gene to link with the target gene for expression. Recombination primers were designed according to the pBI121-GFP vector sequence and the ORF sequence of the PmDXR gene (stop codon deleted). Transient transformation method was used to transform PmDXR-GFP into N. benthamiana leaves, which were cultured in the dark in an artificial climate incubator for 2 days, and the expression position of fluorescent GFP in cells was detected by laser confocal microscope. The results showed that the pBI121-GFP empty vector was localized in the entire tobacco leaf epidermal cells, and PmDXR-GFP was localized in the chloroplasts of the tobacco leaf epidermal cells (Fig. 12).

The specific operations are as follows:

(1) Agrobacterium containing PmDXR-GFP recombinant plasmid was streaked on LB plates containing 50 mg·L ^-1 Kan and 25 mg·L ^-1 Rif.

(2) Pick a single colony with good growth state in 20ml of LB liquid medium containing 50mg·L ^-1 Kan and 25mg·L ^-1 Rif, and shake to culture to about OD ₆₀₀ =0.8 (culture P19 strain at the same time).

(3) Transfer the bacterial liquid to a 50 ml sterile centrifuge tube, and centrifuge in a high-speed refrigerated centrifuge at 4°C and 5000 rpm for 10 min to collect the bacterial cells.

(4) Add an equal volume of transient transformation resuspension to the centrifuge tube, vortex and mix, mix with an equal volume of P19 resuspension, and place at room temperature for 3 hours in the dark.

(5) Inject the resuspended liquid into N. benthamiana leaves with a disposable syringe, and incubate in the dark for 48 hours at room temperature.

(6) The transient expression of GFP fusion protein in N. benthamiana leaf epidermal cells was observed by laser confocal microscope LSM710.

sequence listing

<110> Nanjing Forestry University

<120> A kind of masson pine terpenoid synthesis-related enzyme gene PmDXR and the application of its promoter

<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 tattatttcta 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 ttttttattta tgaaaactac tcttaaaata aatctcaatt 1260

ctttatttct aataattatt aaaaatattt tgaaattatt aaattattaa aatattcggt 1320

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atgaaaatta ttgacacagt ggaaatgggt agccgtggga aagatacccc tgtattttgg 1500

agtttagcgg aggaacgcaa atggcattcc gcatggtgtc caattccact actacattgc 1560

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Claims (10)

1. An application of masson pine terpenoid substance synthesis related enzyme gene PmDXR in masson pine high-lipid-production gene engineering breeding.

2. An application of masson pine terpenoid substance synthesis related enzyme gene PmDXR in improving plant photosynthetic pigment content is provided.

3. An application of a pinus massoniana terpenoid substance synthesis related enzyme gene PmDXR in improving the activity of plant DXR enzyme.

4. Use according to any one of claims 1 to 3, wherein the plant is Pinus massoniana or Arabidopsis thaliana.

5. The application of the promoter of pinus massoniana terpene substance synthesis related enzyme gene PmDXR with the nucleotide sequence shown as SEQ ID NO.1 in pinus massoniana high-yield genetic engineering breeding.

6. The application of the promoter of masson pine terpene substance synthesis related enzyme gene PmDXR with the nucleotide sequence shown as SEQ D NO.1 in improving the content of plant photosynthetic pigments.

7. The application of the promoter of masson pine terpene substance synthesis related enzyme gene PmDXR with the nucleotide sequence shown as SEQ ID NO.1 in improving the activity of plant DXR enzyme.

8. Use as claimed in any one of claims 5 to 7 wherein the plant is Pinus massoniana or Arabidopsis thaliana.

9. The application of the promoter of the masson pine terpene substance synthesis related enzyme gene PmDXR in driving GUS gene to plant expression.

10. Use according to claim 9, wherein the plant is masson pine or tobacco.

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