ClCCoAOMT1 gene for regulating and controlling lignin content of Chinese fir and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a ClCCoAOMT1 gene for regulating and controlling the content of lignin in China fir and application of the ClCCoAOMT1 gene. The amino acid sequence of the protein coded by the ClCCoAOMT1 gene disclosed by the invention is as shown in SEQ ID NO. 2. The ClCCoAOMT1 gene for regulating and controlling the lignin content of the Chinese fir is found, the ClCCoAOMT1 gene is transferred into the Chinese fir through a transient expression method, the expression quantity of the ClCCoAOMT1 gene in a transient transfer strain is increased, the lignin content of the Chinese fir is increased, and the ClCCoAOMT1 gene has a wide application prospect in molecular breeding improvement of the Chinese fir. And a new gene resource is provided for cunninghamia lanceolata gene engineering.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a method for regulating the lignin content of Chinese fir. ClCCoAOMT1 Genes and their applications. Background Art

[0002] Chinese fir( Cunninghamia lanceolata Cunninghamia lanceolata, also known as sandwood, belongs to the Cupressales family and the family Caprifoliaceae. It is characterized by rapid growth, good wood quality, high yield, and a wide range of uses. Its roots, bark, wood, leaves, and seeds all have medicinal value. Currently, there are no reports on genetic transformation of Cunninghamia lanceolata seedlings.

[0003] Lignin, a major component of Chinese fir cell walls, possesses strong mechanical strength and durability, making it resistant to splitting and compression. Lignin contributes to Chinese fir's high density and hardness, making it widely used in construction, furniture, and other fields. By identifying genes that regulate Chinese fir's lignin content and increasing its content through transgenic or transient expression, the performance of Chinese fir can be improved. However, there is currently a lack of genetic resources for regulating Chinese fir's lignin content. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for regulating the lignin content of Chinese fir. ClCCoAOMT1 Gene and its application. The present invention has discovered a gene for regulating the lignin content of Chinese fir. ClCCoAOMT1 Gene, through transient expression method, can improve the ClCCoAOMT1 The expression of the gene increases the lignin content of Chinese fir, has broad application prospects in the molecular breeding and improvement of Chinese fir, and provides a new gene resource for Chinese fir genetic engineering.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for regulating lignin content ClCCoAOMT1 gene, the ClCCoAOMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0006] Preferably, the ClCCoAOMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The present invention provides ClCCoAOMT1 Genes and / or containing ClCCoAOMT1 Genetic biomaterials in improving Chinese fir ( Cunninghamia lanceolata ) lignin content and / or the use in cultivating high lignin content fir, the ClCCoAOMT1 The gene is as described in the above technical solution ClCCoAOMT1 Gene.

[0008] Preferably, the application is to increase the ClCCoAOMT1 The expression level of the gene increases the lignin content of Chinese fir.

[0009] Preferably, the increasing of the lignin content of Chinese fir is increasing the lignin content of Chinese fir roots.

[0010] Preferably, the ClCCoAOMT1 Genetic biological material includes ClCCoAOMT1 One or more of a gene expression cassette, a recombinant expression vector, a cloning vector and an engineered bacterium.

[0011] Preferably, containing ClCCoAOMT1 The recombinant expression vector of the gene includes a plant expression vector and a ClCCoAOMT1 Gene; the plant expression vector includes pCAMBIASuper1300-GFP vector.

[0012] The present invention provides a method for transiently expressing the above technical solution in Chinese fir. ClCCoAOMT1 The genetic method comprises the following steps: The above technical solution ClCCoAOMT1 Inserting the gene into a plant expression vector to obtain a recombinant expression vector; and transferring the recombinant expression vector into Agrobacterium to obtain an engineered bacterium; The roots of the Chinese fir seedlings were removed by 1-3 cm to obtain the Chinese fir seedlings to be infected; The roots of the Chinese fir seedlings to be infected are immersed in the infection solution containing the engineered bacteria, and the infection treatment is carried out under a vacuum condition of ≤-0.08 MPa to obtain instantaneous transformation. ClCCoAOMT1 Gene-containing fir seedlings; the OD value of the engineered bacteria in the infection solution containing the engineered bacteria 600 The value is 1.0~1.5.

[0013] Preferably, the plant expression vector is a pCAMBIASuper1300-GFP vector; the Agrobacterium includes GV3101 Agrobacterium; and the Chinese fir seedlings are 15 to 18 days old.

[0014] Preferably, the method further comprises: converting the instantaneous ClCCoAOMT1 The gene was transferred into the soil of Chinese fir seedlings, cultured in the dark for 45-50 h and then cultured in the light to obtain transient expression. ClCCoAOMT1 The light intensity of the light culture is 200~250 μmo1·m -2 ·s -1 , the photoperiod is 12 h:12 h, and the relative humidity is 65%~75%.

[0015] Beneficial effects: The present invention provides a method for regulating lignin content ClCCoAOMT1 gene, the ClCCoAOMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2. The present invention has discovered a method for regulating the lignin content of Chinese fir. ClCCoAOMT1 gene, the amino acid sequence encoded by this gene is similar to that of rice ( Oryza sativa ) has a high consistency (75%); protein domain analysis shows that the protein has a typical CCoAOMT conserved domain (PF01596). The present invention uses a transient expression method to ClCCoAOMT1 The gene was transferred into Chinese fir, which not only improved the ClCCoAOMT1 It can reduce the expression of genes and increase the lignin content of Chinese fir. It has broad application prospects in molecular breeding and improvement of Chinese fir and provides new gene resources for Chinese fir genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0017] Figure 1 This is the agarose gel electrophoresis diagram of the recombinant expression vector Super1300-ClCCoAOMT1 double enzyme digestion; Figure 2 This is the PCR electrophoresis diagram of the monoclonal colony after Agrobacterium was transformed with the recombinant expression vector Super1300-ClCCoAOMT1; Figure 3 This is a flowchart of the transient overexpression infection of Chinese fir; Figure 4 For transient overexpression in Chinese fir plants ClCCoAOMT1 PCR test results; Figure 5 for ClCCoAOMT1 Transient conversion GFP fluorescence detection diagram; Figure 6 This is the gene expression profile of ClCCoAOMT1 transient transformation; Figure 7 This is a microscopic observation of the roots of ClCCoAOMT1 transiently transformed plants; Figure 8 for ClCCoAOMT1 Figure 2. Determination of lignin monomer content in roots of transient transformants; Figure 9 The results show the effects of two different plant expression vectors on the transformation efficiency of Chinese fir. DETAILED DESCRIPTION

[0018] The present invention provides a method for regulating lignin content ClCCoAOMT1gene, the ClCCoAOMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, which is as follows: MASSEAPKDSAQQVSRHQEVGHKSLLQSDALYQYILETSVYPREPEPMRELREITAKHPWNLMTSADEGQFLNLLLKLINAKNTMEIGVYTGYSLLSTALALPDDGKILAMDINRENYELGLPVIQKAGVAHKI DFREGPALPVLDQMLENKEMHGSFDFIFVDADKDNYLNYHKRLIDLVKIGGVIGYDNTLWNGSVVAPPDAPLRKYVRYYRDFVIELNKALAADPRIEISPNSSRRWHHSLQKGYLKELKFSGFGLLCIVLWFYV.

[0019] As an embodiment, the ClCCoAOMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and is as follows: 5'--3'.

[0020] The present invention provides ClCCoAOMT1 A gene that regulates the lignin content of Chinese fir, the amino acid sequence encoded by this gene is similar to that of rice ( Oryza sativa ) has a high consistency (75%); protein domain analysis shows that the protein has a typical CCoAOMT conserved domain (PF01596). The present invention uses a transient expression method to ClCCoAOMT1The gene was transferred into Chinese fir, which not only improved the ClCCoAOMT1 The present invention also measured the root lignin content of the transiently transformed strains by gas chromatography-mass spectrometry and found that the main reason for the increase in the lignin content of Chinese fir was the increase in the content of G-lignin monomers. ClCCoAOMT1 The gene has broad application prospects in the molecular breeding and improvement of Chinese fir, and provides a new gene resource for Chinese fir genetic engineering.

[0021] Based on the above advantages, the present invention provides ClCCoAOMT1 Genes and / or containing ClCCoAOMT1 Application of biomaterials containing genes in increasing the lignin content of fir and / or cultivating fir with high lignin content, ClCCoAOMT1 The gene is as described in the above technical solution ClCCoAOMT1 Gene.

[0022] As an embodiment, the application is to increase the ClCCoAOMT1 The expression level of the gene increases the lignin content of Chinese fir.

[0023] As an embodiment, the increasing of the lignin content of Chinese fir is increasing the lignin content of Chinese fir roots.

[0024] As an embodiment, the ClCCoAOMT1 Genetic biological material includes ClCCoAOMT1 One or more of a gene expression cassette, a recombinant expression vector, a cloning vector and an engineered bacterium.

[0025] As an embodiment, ClCCoAOMT1 The recombinant expression vector of the gene includes a plant expression vector and a ClCCoAOMT1 gene; the plant expression vector includes a pCAMBIASuper1300-GFP vector. In one embodiment, the backbone vector of the cloning vector may be a pTOPO-Blunt vector. In one embodiment, the starting strain of the engineered bacteria may be Agrobacterium; in one embodiment, the Agrobacterium may be Agrobacterium tumefaciens; in one embodiment, the Agrobacterium may be Agrobacterium GV3101.

[0026] The present invention also provides a method for transiently expressing the above technical solution in Chinese fir. ClCCoAOMT1 The genetic method comprises the following steps: The above technical solution ClCCoAOMT1 Inserting the gene into a plant expression vector to obtain a recombinant expression vector; and transferring the recombinant expression vector into Agrobacterium to obtain an engineered bacterium; The roots of the Chinese fir seedlings were removed by 1-3 cm to obtain the Chinese fir seedlings to be infected; The roots of the Chinese fir seedlings to be infected are immersed in the infection solution containing the engineered bacteria, and the infection treatment is carried out under a vacuum condition of ≤-0.08 MPa to obtain instantaneous transformation. ClCCoAOMT1 Gene-containing fir seedlings; the OD value of the engineered bacteria in the infection solution containing the engineered bacteria 600 The value is 1.0~1.5.

[0027] As an embodiment, the plant expression vector is pCAMBIASuper1300-GFP vector; the Agrobacterium includes GV3101 Agrobacterium; the age of the Chinese fir seedlings is 15 to 18 days. ClCCoAOMT1 Insert the gene into the pCAMBIASuper1300-GFP vector Xbal I and Kpn The present invention found that the pCAMBIASuper1300-GFP vector is an expression vector suitable for infection of Chinese fir, with a conversion rate of 46.5%.

[0028] As an embodiment, the infection solution uses 1 / 2 MS culture medium as the infiltration solution and further includes the following components at final concentrations: surfactant 0.05 wt.%, dithiothreitol (DTT) 0.001 wt.%, acetosyringone 150 μM and a concentration of OD 600 =1.0~1.5. As an embodiment, the pH of the infection solution is 5.6; and the surfactant can be Silwet L-77.

[0029] As an embodiment, the method of removing 1 to 3 cm of the root system of the Chinese fir seedling may be cutting.

[0030] As an embodiment, the method further comprises: converting the instantaneous ClCCoAOMT1 The gene was transferred into the soil of Chinese fir seedlings, cultured in the dark for 45-50 h (recovery culture), and then cultured in the light to obtain transient expression. ClCCoAOMT1 The light intensity of the light culture is 200~250 μmo1·m -2 ·s -1 The light cycle is 12 h:12 h, and the relative humidity is 65% to 75%. As an embodiment, the dark culture time can be 48 h; the light intensity of the light culture can be 220 to 230 μmo1·m -2 ·s -1 The day and night temperatures of the light culture can be 25°C and 21°C, and the relative humidity can be 70%.

[0031] Chinese fir seedlings are difficult to transform as transformation materials. The present invention adopts Agrobacterium-mediated transformation combined with vacuum infiltration technology. By removing 1 to 3 cm of the roots of the Chinese fir seedlings, the permeability of the roots is improved and foreign genes are introduced, thereby achieving transient expression or long-term expression to obtain transgenic cell clusters or transgenic plants.

[0032] In order to further illustrate the present invention, the method for regulating the lignin content of Chinese fir provided by the present invention is described below with reference to the embodiments and the accompanying drawings. ClCCoAOMT1 The genes and their applications are described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0033] Example 1 RNA was extracted from Chinese fir plants and reverse transcribed into cDNA as a template. PCR amplification products were obtained using primers designed based on the predicted gene transcript_HQ_QB_5_Xylem_transcript73470_f2p0_1177 in Chinese fir. The primer sequences are as follows: Upstream primer: 5′-ATGGCAAGCTCAGAGGCTCCCA-3′ (SEQ ID NO. 3); Downstream primer: 5′-TACGTAAAACCATAAAACAATG-3′ (SEQ ID NO. 4); The PCR amplification product was detected by agarose gel electrophoresis, and the target band was excised and purified. The recovered DNA fragment was cloned into the pTOPO-Blunt vector and transformed into Escherichia coli DH5α competent cells. The positive clone plasmid was extracted and analyzed by enzyme digestion pattern. Single clone sequencing was performed, and the insert fragment was 807 bp, and the nucleotide sequence was shown in SEQ ID NO.1.

[0034] Using Blast online software to compare the full-length transcriptome of Chinese fir, it was found that SEQ ID NO.1 was completely consistent with the sequence of the predicted gene transcript_HQ_QB_5_Xylem_transcript73470_f2p0_1177.

[0035] Further online comparative analysis using BlastP software revealed that the amino acid sequence encoded by this gene (SEQ ID NO. 2) was similar to that of rice ( Oryza sativa ) has a high consistency (75%) with OsCCoAOMT; protein domain analysis showed that the protein has a typical CCoAOMT conserved domain (PF01596).

[0036] The predicted gene transcript_HQ_QB_5_Xylem_transcript73470_f2p0_1177 was named ClCCoAOMT1 Gene.

[0037] Example 2 Using Chinese fir cDNA as template, according to ClCCoAOMT1 Gene design primers, introduce Xbal I and Kpn I restriction enzyme cutting site, the primer sequences are as follows: Upstream primer: 5′-tctagaATGGCAAGCTCAGAGGCTCCCA-3′ (SEQ ID NO. 5); Downstream primer: 5′-ggtaccTACGTAAAACCATAAAACAATG-3′ (SEQ ID NO. 6); Among them, 5'-tctaga-3' is Xbal I restriction enzyme site sequence, 5'-ggtacc-3' Kpn I restriction enzyme cutting site sequence.

[0038] The PCR amplification products were detected by agarose gel electrophoresis, the target band was cut out and purified, the recovered DNA fragment was connected to the pTOPO-Blunt vector, transformed into Escherichia coli DH5α competent cells, the positive clone plasmid was extracted, and after the single clone was sequenced correctly, the plasmid containing ClCCoAOMT1 The plasmid pTOPO-ClCCoAOMT1 gene was used.

[0039] Plasmid pTOPO-ClCCoAOMT1 and expression vector pCAMBIASuper1300-GFP (denoted as pCAMBIASuper1300-eGFP) were cultured at 37°C. Xba I and Kpn I double enzyme digestion for 3 h, the digestion products were analyzed by 1% agarose gel electrophoresis, and the ClCCoAOMT1 The gene fragment and the linearized pCAMBIASuper1300-GFP vector were ligated with T4 DNA ligase to form the recovered vector containing restriction enzyme sites. ClCCoAOMT1 The gene fragment and the linearized pCAMBIASuper1300-GFP vector were ligated overnight at 16°C to obtain a ligation product. The ligation product was transformed into DH5α competent cells, and single colonies grown on kanamycin-resistant (50 mg / L) plates were picked. The plasmid was extracted and analyzed by enzyme digestion pattern identification and sequencing. The enzyme digestion pattern identification results are shown in Figure 1, where M is a DNA molecular weight marker (DL15000); lanes 1-2 are enzyme-digested plasmid fragments. The resulting recombinant expression vector was named Super1300-ClCCoAOMT1.

[0040] The recombinant expression vector Super1300-ClCCoAOMT1 was transformed into Agrobacterium ( Agrobacterium tumefaciens ) strain GV3101 competent cells, pick single colonies grown on kanamycin resistance (50 mg / L) plates for PCR identification. ClCCoAOMT1 The monoclonal colonies formed after transformation of the gene recombinant expression vector were used as templates, and PCR detection was performed using the primers shown in SEQ ID NO.5 and SEQ ID NO.6. At the same time, the recombinant expression vector Super1300-ClCCoAOMT1 was used as a positive control and water was used as a negative control. Figure 2 , where M: DNA molecular weight marker (DL15000); 1-4: monoclonal colonies transformed with Super1300-ClCCoAOMT1; 5: negative control; 6: positive control.

[0041] The results showed that the monoclonal colonies contained ClCCoAOMT Gene fragments were obtained by shaking the bacteria to obtain Agrobacterium monoclonal bacterial liquid, which can be used for infection and transformation experiments.

[0042] Example 3 The Agrobacterium monoclonal solution obtained in Example 2 was used to transform Chinese fir using the vacuum infiltration method. ClCCoAOMT Is the gene overexpressed in Chinese fir plants? The flowchart of transient overexpression infection in Chinese fir is shown in Figure 3 , where the device with the pressure gauge is a commercially available vacuum device. The specific steps are as follows: (1) Add the following components to the 1 / 2 MS culture medium (pH = 5.6) at the final concentration: Silwet L-77 0.05wt.%, dithiothreitol (DTT) 0.001wt.% and acetosyringone 150 μM, mix well and add ClCCoAOMT The Agrobacterium with the gene was resuspended to prepare an Agrobacterium infection solution; the concentration of the recombinant bacteria in the Agrobacterium infection solution was OD 600 =1.0; (2) Select 15-18-day-old Chinese fir seedlings, cut off part of the young roots (2 cm in length), immerse the roots in Agrobacterium infection solution, and treat them under a vacuum condition of -0.08 MPa for 20 min; (3) The Chinese fir plants were removed from the Agrobacterium infection solution, and the residual infection solution was washed away and then replanted in the soil. They were first cultured in a dark environment for 2 days, and then continued to be cultured under the conditions of a temperature of 23°C, a humidity of 70%, and a photoperiod of light / darkness = 16 / 8 h. The target gene was introduced into the transformed Chinese fir seedlings, and the target gene was transiently overexpressed in the plants (referred to as transiently transformed strains).

[0043] RNA was extracted from the transiently transfected lines and wild-type Chinese fir plants, and the qRT-PCR method was used to analyze the RNA sequences shown in SEQ ID NO.5 and SEQ ID NO.6. ClCCoAOMT1 Gene expression was detected using the recombinant expression vector Super1300-ClCCoAOMT1 as a positive control and water as a negative control. The sequences of the internal reference primers used are as follows: Internal reference upstream primer: 5′-GATCCAACTGGTGCCAAGGTAAC-3′ (SEQ ID NO. 7); Internal reference downstream primer: 5′-GCCACCGTCTATCAAGAACCCAG-3′ (SEQ ID NO. 8); The results are as follows Figure 4 As shown, where M1: DNA molecular weight marker (DL2000); 1-4: transient overexpression ClCCoAOMT The results showed that transient overexpression of ClCCoAOMT In the Chinese fir plants with the gene ClCCoAOMT1 The gene expression level was significantly higher than that of uninfected wild-type plants, indicating that ClCCoAOMT1 Overexpression has been achieved in Chinese fir.

[0044] The roots of the transiently transgenic lines were tested for fluorescence, the roots of the Chinese fir were sliced ​​and observed using an oscillating slicer, and the lignin content of the transgenic Chinese fir was determined using a gas chromatography-mass spectrometry (GC-MS). Figures 5 - 8 , where WT: wild-type Chinese fir; ClCCoAOMT1, OE or ClCCoAOMT1: transient overexpression ClCCoAOMT Gene of fir.

[0045] The results showed that compared with the wild-type Chinese fir (WT), the roots of the transiently transfected lines had obvious fluorescence ( Figure 5 ),and ClCCoAOMT The gene expression level was significantly upregulated, which was 7.38 times that of the wild type ( Figure 6 ), through vibration section combined with phloroglucinol staining analysis, it was found that the lignin content of the roots of the transiently transformed strains increased ( Figure 7), the root lignin content of the transiently transformed strains was determined by gas chromatography-mass spectrometry, and it was found that the content of lignin monomers in the roots of Chinese fir increased, and the main reason for the increase was the increase in the content of G-lignin monomers (the increase reached 95.89%) ( Figure 8 ).

[0046] Comparative Example 1 An Agrobacterium monoclonal bacterial solution was prepared in a similar manner to that in Example 2, except that the expression vector pCAMBIASuper1300-GFP was replaced with the pK2GW7-eYGFPuv-3xFLAG vector (disclosed in the literature [Wang L, Wang Q, Fu N, Song M, Han X, Yang Q, Zhang Y, Tong Z, Zhang J. Cyanidin-3-O-glucoside Contributes to Leaf Color Change by Regulating Two bHLHTranscription Factors in Phoebe bournei. Int J Mol Sci. 2023 Feb 14;24(4):3829. doi: 10.3390 / ijms24043829. PMID: 36835240; PMCID: PMC9960835.]), and the expression vector pCAMBIASuper1300-GFP was amplified. ClCCoAOMT The primer sequences of the genes are as follows: Upstream primer: 5′-actagtATGGCAAGCTCAGAGGCTCCCA-3′ (SEQ ID NO. 9); Downstream primer: 5′-gtttaaacTACGTAAAACCATAAAACAATG-3′ (SEQ ID NO. 10); Among them, 5'-actagt-3' is Spe I restriction enzyme site sequence, 5'-gtttaaac-3' Pme I restriction enzyme cutting site sequence.

[0047] ClCCoAOMT Insert the gene into the pK2GW7-eYGFPuv-3xFLAG vector Spe I and Pme I restriction enzyme cutting sites.

[0048] Comparative Example 2 A method for transient overexpression infection of Chinese fir is similar to that of Example 3, except that the Agrobacterium monoclonal bacterial solution obtained in Example 2 is replaced by the Agrobacterium monoclonal bacterial solution obtained in Comparative Example 1.

[0049] Determine the transformation rate of different Agrobacterium monoclonal bacterial solutions, transformation rate = measured fluorescence number / total number of infected Chinese fir plants × 100%, the results are shown in Figure 9 .

[0050] The results showed that the conversion rate of pCAMBIASuper1300-GFP vector (46.5%) was significantly higher than that of pK2GW7-eYGFPuv-3xFLAG vector (7.0%), indicating that pCAMBIASuper1300-GFP vector is an expression vector suitable for infection of Chinese fir.

[0051] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for regulating lignin content ClCCoAOMT1 A gene characterized by described ClCCoAOMT1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. according to claim 1 ClCCoAOMT1 A gene characterized by described ClCCoAOMT1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. ClCCoAOMT1 Genes and / or containing ClCCoAOMT1 Genetic biomaterials in improving Chinese fir ( Cunninghamia lanceolata ) lignin content and / or the use in cultivating high lignin content fir, the ClCCoAOMT1 The gene is as described in claim 1 or 2 ClCCoAOMT1 Gene.

4. The use according to claim 3, characterized in that The application is to increase the ClCCoAOMT1 The expression level of the gene increases the lignin content of Chinese fir.

5. The use according to claim 3 or 4, characterized in that The increasing of the lignin content of Chinese fir is to increase the lignin content of Chinese fir root system.

6. The use according to claim 3, characterized in that The said ClCCoAOMT1 Genetic biological material includes ClCCoAOMT1 One or more of a gene expression cassette, a recombinant expression vector, a cloning vector and an engineered bacterium.

7. The use according to claim 6, characterized in that contain ClCCoAOMT1 The recombinant expression vector of the gene includes a plant expression vector and a ClCCoAOMT1 Gene; the plant expression vector includes pCAMBIASuper1300-GFP vector.

8. A method for transiently expressing the protein of claim 1 or 2 in Chinese fir ClCCoAOMT1 A genetic method, characterized in that The following steps are involved: The claim 1 or 2 ClCCoAOMT1 Inserting the gene into a plant expression vector to obtain a recombinant expression vector; and transferring the recombinant expression vector into Agrobacterium to obtain an engineered bacterium; The roots of the Chinese fir seedlings were removed by 1-3 cm to obtain the Chinese fir seedlings to be infected; The roots of the Chinese fir seedlings to be infected are immersed in the infection solution containing the engineered bacteria, and the infection treatment is carried out under a vacuum condition of ≤-0.08 MPa to obtain instantaneous transformation. ClCCoAOMT1 Gene-containing fir seedlings; the OD value of the engineered bacteria in the infection solution containing the engineered bacteria 600 The value is 1.0~1.

5.

9. The method according to claim 8, characterized in that The plant expression vector is a pCAMBIASuper1300-GFP vector; the Agrobacterium includes GV3101 Agrobacterium; and the age of the Chinese fir seedlings is 15 to 18 days.

10. The method according to claim 8 or 9, characterized in that The method further comprises: converting the instantaneous ClCCoAOMT1 The gene was transferred into the soil of Chinese fir seedlings, cultured in the dark for 45-50 h and then cultured in the light to obtain transient expression. ClCCoAOMT1 The light intensity of the light culture is 200~250 μmo1·m -2 ·s -1 , the photoperiod is 12 h:12 h, and the relative humidity is 65%~75%.

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