Instantaneous conversion method for machilus nigrum

Abstract

The invention belongs to the technical field of garden plant breeding, and particularly relates to an instantaneous transformation method for woody evergreen broad-leaved arbor machilus megaeri. The method comprises the following steps: preparing a recombinant plasmid expression vector, preparing transfection liquid, infecting and transforming and the like. In the application, on the basis of analysis requirements of the genetic transformation mechanism of the machilus megaeri, the inventor preliminarily discusses an instantaneous genetic transformation system of the machilus megaeri. Preliminary test results show that the heredity transformation effect is influenced differently when the concentration of the thalli in the infection liquid is different in the phoebe nigrum materials at different growth stages. Based on the result, a certain technical foundation can be laid for follow-up identification and analysis of functional genes related to the machilus megaeri and cultivation of new varieties in the future, and therefore the method has good scientific and technological practical significance.

Description

Technical Field

[0001] The present application belongs to the technical field of garden plant breeding, and specifically relates to an instantaneous transformation method for the woody evergreen broad-leaved tree Machilus macholii. Background Art

[0002] Black shell nanmu Linderamegaphylla Hemsl.) is an evergreen broad-leaved large tree of the genus Piper in the Lauraceae family. Because of its beautiful tree shape, straight trunk and round crown, strong ornamental value, good stress resistance, and certain cold resistance, it has been increasingly widely used in landscaping in recent years. In addition, because its leaves and fruits are rich in aromatic oils, and its roots and bark are rich in bioactive substances, black shell nanmu also has broad application prospects in the fields of industry and medicine. It is precisely based on these uses that in the existing technology, the research on black shell nanmu is currently mainly focused on its physiological response mechanism, the separation and identification of unique chemical components, and the research on reproduction and cultivation technology. However, in combination with the actual development of biological technology and the current status of technology, an in-depth analysis of its growth and development, metabolism and genetic molecular mechanisms based on molecular biology has very important technical significance for further developing the biological utilization value of black shell nanmu and the cultivation of new black shell nanmu varieties. Summary of the invention

[0003] Based on the need for in-depth research on the genetic transformation mechanism of Phoebe machoga, the purpose of this application is to provide an instantaneous transformation method for Phoebe machoga, thereby laying a certain technical foundation for in-depth research on the genetic transformation mechanism of Phoebe machoga and future breeding of new varieties.

[0004] The specific technical solutions adopted in this application are as follows.

[0005] A method for instantaneous transformation of Phoebe nigromaculata comprises the following steps: 1. Preparation of recombinant plasmid expression vector Based on the target gene, a suitable plasmid is selected to prepare a recombinant plasmid expression vector; The target gene is, for example, RUBY Phoebe nigromaculata LmNAC90 Gene; Based on this target gene, the recombinant plasmid expression vector, for example, contains RUBY 35S:RUBY plasmid vector or containing LmNAC90 PCAMBIA2300- LmNAC90 -GFP plasmid vector; Phoebe nigromaculata LmNAC90 The gene sequence is shown in SEQ ID No. 1 (768 bp), and is as follows: ATGAGCAACCCCAACCTTCCACCAGGATATCGTTTCTTCCCAACAGAGGAAGAGCTTGTGGGATTTTATCTTCGTAGCAAGCTGGACATGAGGAGGGTCGAGTTGGATCGTGTGATTCCTGTCGTCGACATTTACAGCCTTGATCCGTGGGAGCTGCCTAGAAAATCAGGAGAGTTGTGCATTGGAGATGAGCAATGGTTCTTCTTTAGTCCTAGGCAAGAGAGAGAAGCCAATGGAGGGAGACCAAATCGGATCACGCCCTCGGGCTACTGGAAGGCCACAGGCATTCCTGGTTATGTTTTTTCCTCTGATAACCACATAATCGGTCTGAAAAGAACCATGGTTTTCTATCGAGGAAAAGCTCCTACAGGAACTAAAACTAAATGGAAGGTGAACGAGTACAAAGCCATTGAAGAAGCTACCACATCAGCTGCCTCTCTAAAGGTTCGACATGAAGTCAACTTATGTCGAGTATATGTAACATCGGGAAACCTGCGATCTTTTGATCGTCGTCCATTGGTTGCAGCGCCAAGAGAGATTTTAGAAAGAGCAGACAGAAGAGCAGCAATGCCTACTTTTGATTCTTGTAACAATATTGAAAATGCAAGTTGCAGGAATTTCGGTGAATCGGCTCATGGGAATAATCCTTCAATGGAGGAGCATACTGGCCATCTCTACAGCTCTCTAAAAGAAGAGACCGGCAATGACTTGAATATGATCAATGATATGGATGCTTTGTGGAATTCAATTATTGGGGCTGATTACTAG; Correspondingly, the Lindera megaphylla LmNAC90 protein encoded by the gene is as follows: MSNPNLPPGYRFFPTEEELVGFYLRSKLDMRRVELDRVIPVVDIYSLDPWELPRKSGELCIGDEQWFFFSPRQEREANGGRPNRITPSGYWKATGIPGYVFSSDNHIIGLKRTMVFYRGKAPTGTKTK WKVNEYKAIEEATTSAASLKVRHEVNLCRVYVTSGNLRSFDRRPLVAAPREILERADRRAAMPTFDSCNIENASCRNFGESAHGNNPSMEEHTGHLYSSLKEETGNDLNMINDMDALWNSIIGADY; 2. Preparation of transfection solution The recombinant plasmid expression vector in step (I) is transformed into Agrobacterium strains, and the correct transformed strains are screened; after the correct transformed strains are cultured and amplified, the cells are centrifuged and collected, and the cell concentration OD is adjusted with a suspension. 600 = 0.8~1.0, placed in dark place at room temperature (about 18~30℃) for 2h as infection solution; The suspension is: MS medium + 200 μM acetosyringone + 0.02% (mass ratio) Tween-20, pH=5.6; 3. Infection and transformation The vacuum penetration infection method is used to infect the black shell nanmu in the germination or seedling stage; The germination stage black nanmu refers to the germination stage black nanmu with a radicle length of about 3 cm and a young stem length of about 5 cm after being stored in sand and then sown and germinated for 21-30 days. 600= 0.8~1.0, under vacuum penetration conditions, infection for 20~30min; The black nanmu seedlings at the seedling stage refer to black nanmu seedlings that have grown to two or three true leaves after being stored in sand for 30-35 days and then sown. 600= 0.8~1.0, under vacuum infiltration conditions, the infection time is until the infection liquid penetrates about 50% of the leaf area (about 5~50min); After infection, the infected material was transplanted into the cultivation medium and cultured in the dark overnight (about 8-12 h). The culture medium was then cultured at 25 °C, 14 h / 10 h (day / night), and a light intensity of 200 μmol·m -2 ·s -2 , further culture, observation and identification of infection and transformation under conditions of relative humidity of about 60%.

[0006] Due to factors such as slow growth, long growth cycle, and high heterozygosity, when using molecular biology techniques to study woody plant materials, there are often unfavorable factors such as low genetic transformation efficiency and difficult regeneration process, which in turn causes great trouble for the application of molecular biology techniques in woody plants.

[0007] In this application, based on the need to analyze the genetic transformation mechanism of Phoebe nigromaculata, the inventors conducted a preliminary study on the transient genetic transformation system of Phoebe nigromaculata. Preliminary experimental results show that Phoebe nigromaculata materials at different growth stages and different concentrations of bacteria in the infection solution have different effects on the genetic transformation effect. Based on this result, a certain technical foundation can be laid for the subsequent identification and analysis of Phoebe nigromaculata-related functional genes and the cultivation of new varieties in the future, so it has good scientific and technological practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of PCR detection of positive monoclonal bacterial solution after plasmid transformation into Agrobacterium; in the figure: M is Marker, CK is 35S:RUBY plasmid; lanes 1-9 are the results of positive monoclonal identification after transformation; Figure 2 To use OD 600 =1.0 infection solution infection transformation test results of black shell nanmu in germination period; in the figure: the control group was infected with empty rhizobacterium, and the experimental group was the phenotypic control results of some materials infected with 35S:RUBY Agrobacterium and then cultured normally for 20 days; Figure 3 To use OD 600 =1.0 infection solution after infection of three-leaf stage black shell nanmu; in the figure: the control group was infected with empty rhizobacterium, and the experimental group was infected with 35S:RUBY Agrobacterium and then cultured normally; a is the immediate phenotype at 0 days after infection, b is the phenotype control after infection and normal culture for 3 days, c is the phenotype control after infection and normal culture for 5 days, d is the phenotype control after infection and normal culture for 7 days, and e is the phenotype control after infection and normal culture for 20 days; the phenotype controls in the figure are only photos of typical materials randomly selected; Figure 4 for LmNAC90 RT-qPCR analysis of transiently transformed P. maculatum; Figure 5 for LmNAC90 Analysis of relative contents of total terpenes and copaene in transiently transformed Pseudostellaria baicalensis. DETAILED DESCRIPTION

[0009] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0010] Biomaterials: Black shell nanmu Linderamegaphylla Hemsl.) seeds, provided by the Xixia County Forest Pest Control and Quarantine Station; During cultivation and management, the seeds of Phoebe macranthoides were sown in a culture tray containing substrate soil (peat soil: perlite: vermiculite = 3:1:1, volume ratio) at 25°C, 14 h / 10 h (day / night), and a light intensity of 200 μmol·m -2 ·s -2 , cultured under the condition of relative humidity of about 60%; Competent cells of Agrobacterium rhizogenes strain (Ar.Qual) were purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; Recombinant expression vector containing RUBY 35S: RUBY , the vector uses 35S as the promoter, carries the RUBY reporter gene (the expression of which can convert tyrosine into red beet pigment visible to the naked eye) and the spectinomycin (Spec) resistance marker; DR5 was kindly donated by Nanjing Agricultural University and Professor Yu Bing's research group: RUBY The carrier was later transformed into 35S by the research group of Associate Professor Cheng Jun of the College of Horticulture of Henan Agricultural University: RUBY Gift; Main reagents: Spectinomycin (Spec) (50 mg / mL), streptomycin (Strp) (50 mg / mL), acetosyringone (AS) (0.0981 g acetosyringone powder dissolved in 5 mL DMSO, sterilized by sterile filter), MS medium (MS powder (4.43 g / L) + sucrose (30 g / L)), refer to the existing technical operation routine preparation and sterilization; TY liquid medium for culturing Agrobacterium rhizogenes: 3 g / L yeast extract + 5 g / L tryptone + 10mM CaCl2, conventional preparation and sterilization operations (TY solid medium, add 15 g / L agar to the above liquid medium).

[0011] Example 1 Transient transformation (transient transfection) is a commonly used method in molecular biology research and an important technical prerequisite for analyzing gene (protein) functions and building a stable transformation system. However, in actual technical operations, different plant physiological characteristics, different transfection systems, and differences in transfection methods will lead to significant differences in actual transformation effects. Since the prior art lacks in-depth research and discussion on the transformation system of Phoebe nigromaculata, this application uses 35S: RUBY Taking plasmid vector as an example, the main purpose is to conduct a preliminary technical discussion on the transient transformation method of Phoebe nigromaculata, which can lay a certain technical foundation for the subsequent construction of a stable transformation system for Phoebe nigromaculata and the study of the genetic transformation mechanism of Phoebe nigromaculata. The following is a brief introduction to the relevant experimental process.

[0012] 1. Transformation of Agrobacterium This application uses 35S:RUBY plasmid vector (ie: RUBY As an example, a gene is equivalent to the target gene, and it is transformed into Agrobacterium rhizogenes Ar.Qual and a transfection solution is prepared for subsequent transient transformation. The specific transformation operation is as follows.

[0013] First, add 1 μL of 35S to 100 μL of Agrobacterium rhizogenes Ar.Qual competent cells thawed on ice: RUBY Plasmid vector, mix thoroughly and place on ice for 5 min; Afterwards, after the ice bath, the samples were quickly placed in liquid nitrogen for 5 min, then in a 37°C water bath for 5 min, and then in an ice bath for another 5 min; Then, add 800 μL of TY liquid medium to the mixture after the ice bath, shake and culture at 28°C and 200 rpm / min for about 2 hours, centrifuge at 5000 rpm for 1 min (retain about 100 μL of supernatant, then gently pipette and mix to resuspend the bacterial blocks), spread the bacterial liquid on TY solid medium (Spec 50 mg / L+Strp 50 mg / L), and culture at 28°C and invert for about 2 days; Finally, pick the positive monoclonal colonies and further inoculate them into TY liquid culture medium (Spec 50 mg / L+Strp50 mg / L, 28℃, 200rpm / min shaking culture for about 1 day). After amplification and culture, perform PCR detection on the bacterial liquid to ensure the correct transformation of the plasmid. For the strains with the correct transformation of the plasmid, further amplification is carried out and used directly for the subsequent transfection solution preparation or stored for later use.

[0014] In the above process, when testing bacterial liquid PCR, the reference design of the 20μl reaction system is as follows: 1.1×T3 Super PCR Mix (purchased from Beijing Qingke Biotechnology Co., Ltd.), 17 μl; Primers RUBY -F, 1 μl; Primers RUBY -R, 1 μl; Template (bacterial solution), 1 μl; PCR reaction program (T100 TM Thermal Cycler, BIO-RAD product): 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 10 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 2 min. After PCR, the amplified product was detected by agarose gel electrophoresis.

[0015] The primer sequences are specifically designed as follows: RUBY -F:5'-TTTACGACCAGCCTCAACCTCCTCA-3', RUBY -R: 5'-CTTGATTTCCTCTTGGGCCTTTTCC-3'.

[0016] The electrophoresis results after PCR were as follows: Figure 1 As shown in the figure, the target band is clear and consistent with the target fragment length, which indicates that the plasmid has been successfully transformed into Agrobacterium and subsequent experiments can be carried out.

[0017] 2. Preparation of transfection solution After the correct strain transformed in step (I) is amplified, centrifuge and collect the cells, and use the suspension to adjust the cell OD 600 to a suitable value (in the subsequent experiments, to determine the appropriate OD 600 Value, adjust OD 600 = 0.6, 0.8, 1.0), and placed in the dark at room temperature (about 25°C) for 2 h before use for subsequent transfection; The suspension is: 4.43 g / L MS + 30 g / L sucrose + 200 μM acetosyringone + 0.02% (mass ratio) Tween-20, pH=5.6; 3. Infection and transformation Since the transfection efficiency of plants at different growth stages varies in actual transfection, the inventors selected the germination stage (after the seeds extend the radicle and plumule for infection) and seedling stage (seedlings grown to 2 or 3 true leaves after germination) of Phoebe chinensis for infection and transformation experiments. The vacuum infiltration method was used for infection and transformation. The specific operation can be referred to as follows.

[0018] When the infection and transformation is performed on the budding stage of Phoebe macranthoides, firstly, a cotyledon of the budding stage Phoebe macranthoides is removed (removing the cotyledon does not affect the further growth of the subsequent embryo and is also convenient for the subsequent infection), and then the root and stem are gently pierced with a needle without affecting the subsequent growth; after the piercing, the entire budding stage Phoebe macranthoides is placed in the infection solution prepared in step (ii), and after vacuuming to about -0.7 MPa, the negative pressure is maintained for about 25 minutes (at this time, the bubble exudation speed of the liquid surface contacting the Phoebe macranthoides rhizomes is significantly reduced); after the infection is completed, the normal atmospheric pressure is restored; Finally, the infected black-shelled nanmu was taken out and transplanted into a conventional cultivation medium. After being cultured in the dark overnight (about 12 h), the medium was heated at 25 °C, 14 h / 10 h (day / night), and a light intensity of 200 μmol·m -2 ·s -2 , cultivate and observe the growth of plants under the condition of relative humidity of about 60%; When the black shell macaw seedlings are infected and transformed, the true leaves of the black shell macaw seedlings are gently pierced with a needle (for black shell macaw seedlings with 2 or 3 true leaves, each true leaf is pierced separately, and the needle does not penetrate the back of the black shell macaw leaves). After the piercing, the entire black shell macaw seedling is placed in the infection solution prepared in step (ii), and after vacuuming to about -0.7 MPa, the negative pressure time is until the infection solution penetrates the leaf area to about 50% (about 5 to 50 minutes). After the infection is completed, the normal atmospheric pressure is restored; Finally, the infected Phoebe macranthoides seedlings were taken out and transplanted into conventional culture medium. After dark culture overnight (about 12 h), the medium was heated at 25 °C, 14 h / 10 h (day / night), and a light intensity of 200 μmol·m -2 ·s -2 , cultivate and observe the growth of plants under the condition of relative humidity of about 60%; During the above experimental operation, synchronously, an infection solution prepared only from Agrobacterium rhizogenes Ar.Qual without transformation plasmid was used for infection and transformation as a blank control.

[0019] As mentioned above, considering that the receptor materials at different growth stages and the different concentrations of bacteria in the infection solution have a significant impact on the infection and transformation efficiency, based on the above experimental description, the inventors conducted different experimental design combinations and directly determined whether the transformation was successful based on the intuitive phenotypic changes after transformation. The specific experimental design and experimental results are briefly described as follows.

[0020] 1. Experimental design and results for the germination period of Phoebe macranthoides The black shell nanmu seeds at the germination stage about 21 days after sowing were used as the recipient material (at this time, the radicle was about 3 cm long and the young stem was about 5 cm long). 600 = 0.6, 0.8, 1.0 infection solution, transplant and plant about 20 days after infection (preliminary observation can be made about 3 days after transplanting, but to ensure the accuracy of the observation results, 10 days after transplanting is used as the observation date) to determine whether the transformation is successful by observing the color of the stem base (the base of the stem that is successfully infected and transformed will show obvious red color visible to the naked eye, some experimental results refer to Figure 2 The results of transfection success rate under different bacterial concentrations are shown in Table 1 below.

[0021] Table 1. Effects of different bacterial concentrations on the infection conversion rate of Phoebe macranthoides at the germination stage From the results in the above table, it can be seen that for the germination period of Phoebe macranthoides, appropriately increasing the concentration of the bacterial solution can significantly improve the transformation success rate.

[0022] 2. Experimental design and results of infection and transformation of Phoebe macranthoides seedlings When genetic transformation was performed using seedlings at the two-leaf stage (about 30 days after seed sowing) and the three-leaf stage (about 35 days after seed sowing), OD 600 = 0.8, 1.0 infection solution, and the design considered the effect of different infection time on transformation efficiency. After infection, the transplanted plants were planted for about 20 days (red spots appeared around 3 days after transplanting, but to ensure the accuracy of the observation results, 20 days after transplanting was used as the observation date). The surface color of the leaves (obvious dense red spots or large areas of red, some experimental results are shown in Figure 2) was observed. Figure 3 The results of transfection success rate under different bacterial concentrations and different infection times are shown in Table 2.

[0023] Table 2. Transfection success rate of Phoebe macranthoides seedlings at different bacterial concentrations and infection times From the results in the above table, it can be seen that for the black-shelled Macaoia seedlings, even a slightly lower bacterial solution concentration and a slightly shorter infection time can better ensure the genetic transformation efficiency.

[0024] Example 2 Based on Example 1, the inventor further used black shell nanmu LmNAC90 Taking genes as an example, LmNAC90 The gene was transformed and its function was verified. The specific experimental situation is briefly described as follows.

[0025] (I) Preparation of PCAMBIA2300- LmNAC90 -GFP recombinant vector Using PCAMBIA2300-GFP plasmid as a vector (a commonly used plant expression vector in the prior art, available from public channels), refer to conventional molecular biology techniques to recombinant LmNAC90 Gene, prepared to obtain PCAMBIA2300- LmNAC90 -GFP recombinant vector ( LmNAC90 When the gene is connected and recombined with the PCAMBIA2300-GFP plasmid, the restriction site is Bm I).

[0026] During the preparation process, PCR amplification LmNAC90 When the gene is detected (using Phoebe macranthoides leaves), the specific primers are designed as follows: LmNAC90 -F:5'-ATGAGCAACCCCAACCTTCC-3', LmNAC90 -R:5'-GTAATCAGCCCCAATAATTGAA-3'.

[0027] 2. Preparation of infection solution Refer to Example 1 and operate PCAMBIA2300- LmNAC90 -GFP recombinant vector Agrobacterium, and select the correct transformed strain for amplification (OD 600 =1.0) to prepare the infection solution.

[0028] 3. Infection, transformation and functional detection Refer to the operation of Example 1, use Phoebe nanmu in the germination stage as the test material, use the above step (ii) to prepare the infection solution for vacuum infection, and screen and identify the positive transformed seedlings.

[0029] For positive transformed seedlings, samples were taken on the 5th day after infection for relevant quantitative analysis and gas chromatography-mass spectrometry (GC-MS) detection. The results of RT-qPCR showed that (using young leaves of Phoebe macranthoides as samples): compared with the control group (EV) with no transformation, LmNAC90 Overexpression lines OE1, OE2, and OE3 LmNAC90 The relative expression levels of the genes were increased by 10.45 to 108.29 times, indicating that the transient transformation system using this method can be used for functional verification of related functional genes and is expected to be used for the construction and application of related stable genetic transformation systems. The headspace solid phase microextraction (HS-SPEM) method was further used to LmNAC90 The results of total terpenes (including β-phellandrene, copaene, α-pinene, β-pinene, β-myrcene, p-cymene, humulene and β-caryophyllene) and specific copaene content in the overexpression strains showed that: LmNAC90 After overexpression, the total terpene content in the overexpression strain increased significantly, which was 1.32-1.68 times that of the empty control group; the content of the main volatile compound Cubane was 1.67-2.25 times that of the empty control group; this result shows that: LmNAC90 It has a very important use in the metabolic synthesis of total terpenes of Pseudostellaria baicalensis (including β-phellandrene, copaene, α-pinene, β-pinene, β-myrcene, p-cymene, humulene and β-caryophyllene), especially copaene.

Claims

1. A method for instantaneous transformation of Phoebe nigromaculata, characterized in that: The method comprises the following steps:

1. Preparation of recombinant plasmid expression vector Based on the target gene, a suitable plasmid is selected to prepare a recombinant plasmid expression vector; 2. Preparation of transfection solution Transform the recombinant plasmid expression vector in step (i) into Agrobacterium strains, and screen to obtain the correct transformed strains; After the transformed strains were cultured and amplified, the cells were collected by centrifugation and the cell concentration was adjusted by OD 600 =0.8~1.0, 18~30℃, place in dark place and use as infection solution; 3. Infection and transformation The vacuum penetration infection method is used to infect the black shell nanmu in the germination or seedling stage; The germination stage black shell nanmu refers to the germination stage black shell nanmu that is stored in sand and then sown and germinated for 21-30 days. When infecting, the infection liquid OD 600= 0.8~1.0, under vacuum penetration conditions, infection for 20~30min; The black nanmu seedling stage refers to the black nanmu seedlings that have grown to two or three true leaves after being stored in sand and then sown. When infected, the infection liquid OD 600= 0.8~1.0, under vacuum infiltration conditions, infect until the infection solution penetrates 50% of the leaf area; After infection, the infected material is transplanted into the cultivation medium, cultured, observed and identified for infection transformation.

2. The instantaneous transformation method of Phoebe nanmu as claimed in claim 1, characterized in that: In step (a), the target gene is RUBY Phoebe nigromaculata LmNAC90 Gene; Based on this target gene, the recombinant plasmid expression vector contains RUBY 35S:RUBY plasmid vector or containing LmNAC90 PCAMBIA2300- LmNAC90 -GFP plasmid vector; Phoebe nigromaculata LmNAC90 Gene, the sequence is shown as SEQ ID No.

1.

3. The instantaneous transformation method of Phoebe nanmu as claimed in claim 1, characterized in that: In step (iii), after transplanting to the cultivation medium, first culture in the dark overnight, then at 25°C, 14 h day / 10 h night, and light intensity of 200 μmol·m -2 ·s -2 , culture under relative humidity of 60%.

4. The instantaneous transformation method of Phoebe nanmu as claimed in claim 1, characterized in that: In step (ii), when preparing the infection solution, the suspension was used to adjust the bacterial concentration OD 600 =0.8~1.0; The suspension is: MS medium + 200 μM acetosyringone + 0.02% (mass ratio) Tween-20, pH=5.

6.

5. A black shell nanmu LmNAC90 A gene characterized by Phoebe nigromaculata LmNAC90 Gene, the sequence is shown in SEQ ID No.

1.

6. The black shelled nanmu described in claim 5 LmNAC90 A primer pair for PCR amplification of a gene, characterized in that: The specific primer pairs were designed as follows: LmNAC90 -F:5’-ATGAGCAACCCCAACCTTCC-3’, LmNAC90 -R: 5'-GTAATCAGCCCCAATAATTGAA-3'.

7. The black shelled nanmu according to claim 5 LmNAC90 The gene is used in the synthesis of plant secondary metabolites, characterized in that: LmNAC90 The gene is used for regulating the synthesis and metabolism of total terpenoid substances; the total terpenoid substances include: β-phellandrene, copaene, α-pinene, β-pinene, β-myrcene, p-cymene, humulene and β-caryophyllene.

8. Utilize the black shell nanmu described in claim 5 LmNAC90 The method for breeding black-shelled nanmu varieties with the gene is characterized by: Overexpression of Phoebe maculatum LmNAC90 After the gene was modified, a black-shelled nanmu variety with increased content of total terpenoids was cultivated.

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