Application of OpJRB1 transcription factor in improving the content of camptothecin in ophiorrhiza pumila

Abstract

This invention provides a bHLH transcription factor involved in the regulation of camptothecin synthesis in *Sedum pachycarpa*. OpJRB1 And its applications. OpJRB1 Transcription factors are induced by jasmonic acid, highly expressed in roots, and can bind to repressors in the jasmonic acid pathway. OpJAZ1 , OpJAZ5 and OpJAZ6 Interaction, fully explaining OpJRB1 Transcription factors are involved in the jasmonic acid signaling pathway. Knockout OpJRB1 The gene significantly reduces the camptothecin content in the hairy roots of *Hedyotis diffusa* and is overexpressed. OpJRB1 Genes can increase camptothecin content. OpJRB1 Transcription factors can transiently activate the camptothecin synthesis gene. OpTDC1 and OpLAMT1 Gene expression is enhanced, thereby increasing the accumulation of camptothecin. This invention provides a novel, high-quality drug source for the production of camptothecin with broad-spectrum anticancer efficacy, possessing significant theoretical importance and potential application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a... OpJRB1 Application of transcription factors in increasing camptothecin content in the hairy roots of *Hedyotis diffusa*. Background Technology

[0002] Ophiorrhiza pumila is a species of grass belonging to the Rubiaceae family ( Rubiaceae ) Snakeroot genus ( Ophiorrhiza Camptothecin, a perennial dicotyledonous herbaceous plant, has been found to contain high levels of the anticancer drug camptothecin in various tissues and hairy root culture systems, exhibiting advantages such as rapid growth, short growth cycle, and heritability. Camptothecin is an important natural product with broad-spectrum anticancer activity, and its derivatives are commonly used clinically to treat malignant tumors such as colon cancer, rectal cancer, and ovarian cancer. The monoculture planting model and scarcity of germplasm resources severely restrict the supply of camptothecin-based anticancer drugs, making it difficult to meet the growing market demand. Therefore, increasing camptothecin yield has gradually become a research hotspot in recent years. Utilizing metabolic engineering techniques to enhance the synthesis of plant secondary metabolites is a feasible technical approach. Transcription factors, as a recent research hotspot in the life sciences, have been extensively studied and reported to participate in the regulation of plant secondary metabolic processes, providing new insights for related research.

[0003] bHLH (basic helix-loop-helix) transcription factors, as one of the largest families of transcription factors in plants, play important roles in plant growth, development, physiological metabolism, and stress responses. Members of the bHLH family contain two highly conserved and functionally distinct domains: the basic region and the helix-loop-helix (HLH) region. The basic region, composed of 15-20 amino acids, is located at the N-terminus of the bHLH domain and serves as a DNA-binding region, recognizing both the E-box and G-box. The C-terminal HLH region relies on the interaction of hydrophobic amino acids to form homodimers or heterodimers of two HLH proteins, which then regulate the expression of downstream target genes. Members of the bHLH transcription factor family have been shown to synergistically control the biosynthesis of various secondary metabolites, mainly including terpenes, alkaloids, and phenylpropanoids. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a... OpJRB1 Application of transcription factors in increasing camptothecin content in the hairy roots of *Hedyotis diffusa*.

[0005] In a first aspect, the present invention provides a transcription factor of *Sophora japonica*. OpJRB1The transcription factor OpJRB1 The amino acid sequence is shown in SEQ ID NO. 2.

[0006] Secondly, the present invention provides the aforementioned *Syzygium buergerianum* transcription factor. OpJRB1 The encoding gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned coding gene.

[0008] Fourthly, the present invention provides a transgenic engineered bacterium containing the aforementioned recombinant expression vector.

[0009] Fifthly, the present invention provides an application of the aforementioned encoding gene, the aforementioned recombinant expression vector, or the aforementioned transgenic engineered bacteria in regulating the camptothecin content in *Hedyotis diffusa*.

[0010] Furthermore, the encoding gene was overexpressed in *Hedyotis diffusa*, increasing the camptothecin content in *Hedyotis diffusa*.

[0011] Sixthly, the present invention provides an application of the described encoding gene, the described recombinant expression vector, or the described transgenic engineered bacteria in activating the expression of the camptothecin synthesis gene in *Gnaphalium affine*, wherein the camptothecin synthesis gene is... OpTDC1 or OpLAMT1, The encoding gene is overexpressed in *Sedum pachyphyllum*.

[0012] In a seventh aspect, the present invention provides an application of the aforementioned coding gene, the aforementioned recombinant expression vector, or the aforementioned transgenic engineered bacteria in cultivating transgenic plants containing camptothecin, wherein the coding gene is overexpressed in the plant, and the plant is *Hedyotis diffusa*.

[0013] Furthermore, the application employs hairy root cultivation.

[0014] Eighthly, the present invention provides a method for increasing the camptothecin content in the hairy roots of *Sophora japonica*, comprising the following steps:

[0015] Step 1: Construct an overexpression vector for the encoded gene;

[0016] Step 2: Transform the overexpression vector into Agrobacterium rhizogenes;

[0017] Step 3: Infect *Hedyotis diffusa* with the *Agrobacterium rhizogenes* to obtain *Hedyotis diffusa* hairy root strains with increased camptothecin content.

[0018] The beneficial effects of this invention are:

[0019] (1) In this invention, the homology comparison method used in the prior art was not used for screening. Instead, the transcription factors of *Hedyotis diffusa* that are induced by MeJA and expressed in tissues and verified by yeast two-hybrid analysis were screened to obtain the transcription factors of *Hedyotis diffusa* that are induced by MeJA and are related to regulation. OpJRB1 .

[0020] (2) The short snake root transcription factor in this invention OpJRB1 It can activate the camptothecin synthesis gene in *Gnaphalium affine*. OpTDC1 and OpLAMT1 The expression of this substance promotes the accumulation of camptothecin in the hairy roots of *Hedyotis diffusa*, providing a novel and high-quality source for the production of camptothecin, which has broad-spectrum anticancer effects.

[0021] (3) The present invention has resolved the following: OpJRB1 The molecular mechanism by which transcription factors regulate the biosynthesis of camptothecin has important theoretical significance and potential application value. Attached Figure Description

[0022] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0023] Figure 1 Heatmap showing the differential changes in transcriptional expression levels of *Hedyotis diffusa* transcription factors after MEJA treatment;

[0024] Figure 2 The expression levels of candidate transcription factors induced by jasmonic acid in different tissues of *Hedyotis diffusa*.

[0025] Figure 3 for OpJRB1 and OpJAZs The results of the yeast two-hybrid validation;

[0026] Figure 4 To knock out OpJRB1 Results of determination of camptothecin content in the hairy roots of *Hedyotis diffusa* var. *short*;

[0027] Figure 5 For overexpression OpJRB1 Results of determination of camptothecin content in the hairy roots of *Hedyotis diffusa* var. *short*;

[0028] Figure 6 for OpJRB1 Transcription factors on the camptothecin biosynthesis gene OpTDC1 The activation ability test results;

[0029] Figure 7 for OpJRB1 Transcription factors on the camptothecin biosynthesis gene OpLAMT1 The activation ability test results. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in molecular cloning (Sambrook et al.), or according to the conditions recommended in the instructions accompanying the reagents or kits provided by the manufacturer.

[0031] Example 1: Short snake root grass OpJRB1 Gene screening

[0032] 1. MeJA-induced expression profile analysis

[0033] Plants of *Hedyotis diffusa* were treated with the plant hormone jasmonic acid (JA) at 100 µM, and their transcriptomes were sequenced. Transcriptome sequencing analysis was performed on samples of *Hedyotis diffusa* treated with JA at different time points (0, 3, 6, 9, 12, and 24 h). Materials treated for 0 h were used as the control group. Fifteen transcription factors that transiently responded to JA signaling were screened. Figure 1 )

[0034] Transcriptome analysis of *Sophora japonica* tissue

[0035] Combined with MeJA-induced transcriptome analysis, the expression levels of these transcription factors in different tissues (roots, stems, and leaves) were further analyzed, and seven transcription factors predominantly expressed in roots were identified. Among them, Op01g01318 is... OpJRB1 . ( Figure 2 )

[0036] Yeast two-hybrid experiment

[0037] (1) Carrier construction

[0038] Construct PGADT7-Op01g01318 and PGBKT7- OpJAZ1 -6 recombinant plasmid.

[0039] The plasmids of the constructed vectors were transferred into Y2H yeast competent cells for yeast two-hybrid experiments.

[0040] Yeast competent cells were prepared using the 1.1x LiAc / TE mixture.

[0041] 5 μL of PGADT7-Op01g01318 recombinant plasmid and PGADT7-EV plasmid were respectively mixed with PGBKT7- OpJAZ1 -6 recombinant plasmid, 100 μL of competent yeast cells, 10 μL of salmon sperm DNA, and 600 μL of PEG / LiAc mixture were sequentially added to 1.5 mL EP tubes for transformation. After transformation into yeast strain AH109, ​​the cells were plated on SD / -Leu / -Trp solid medium and incubated upside down at 30°C in the dark for 2-3 days. Single colonies were picked from a two-deficient plate and diluted with water. Then, 3 μL of the bacterial culture was spotted onto labeled SD / -Leu / -Trp / -His / -Ade medium plates and incubated at 30°C in the dark for 2-3 days before observing the results.

[0042] The results are as follows Figure 3 As shown, PGADT7-Op01g01318 and PGBKT7- OpJAZ1 PGBKT7- OpJAZ5 PGBKT7- OpJAZ6 The co-transfected yeast strains grew on SD / -Trp / -His / -Ade / -Leu solid medium. The remaining co-transfected yeast strains and the negative control did not grow on SD / -Trp / -His / -Ade / -Leu solid medium. In the figure, AD represents the transcription activation domain, and BD represents the DNA binding domain. This demonstrates the effectiveness of the yeast two-hybrid system. OpJAZ1 , OpJAZ5 and OpJAZ6 The proteins expressed by these three genes interact with the protein expressed by Op01g01318. These results suggest that the Op01g01318 bHLH gene may respond to jasmonic acid and participate in the regulation of camptothecin biosynthesis in *Hedyotis diffusa*. We named it JRB1 (Jasmonic acid regulation bHLH 1).

[0043] Example 2: Short snake root grass OpJRB1 Cloning of genes

[0044] 1. Extraction of total RNA from *Hedyotis diffusa*

[0045] Take a small amount of young leaves of *Hedyotis diffusa*, flash-freeze them in liquid nitrogen, crush them, and place them in a 1.5 mL Eppendorf (EP) centrifuge tube. Add 600 μL of lysis buffer and shake thoroughly. Extract total RNA according to the instructions of the TIANGEN kit. Detect RNA quality by agarose gel electrophoresis, and then determine RNA content using a spectrophotometer.

[0046] 2. Short snake root grass OpJRB1 Cloning of genes

[0047] Using the extracted total RNA as a template, cDNA was synthesized after reverse transcription; according to OpJRB1Gene-specific primers were designed based on the gene sequence, as shown in Table 1. The gene was amplified by PCR using TOYOBO's high-success-rate PCR enzyme KOD FX. The PCR reaction mixture consisted of: 1 μL of *Hedyotis diffusa* cDNA, 1.5 μL each of forward and reverse primers, 25 μL of 2×PCR buffer for KOD FX, 10 μL of 2 mM dNTPs, 1 μL of KOD FX, and double-distilled water to a total volume of 50 μL. PCR amplification was performed using a three-step method. The product was recovered and a pLB intermediate vector was constructed for DNA sequencing.

[0048] Table 1 PCR Primers

[0049]

[0050] Through the above steps, the full-length coding sequence (SEQ ID NO. 1) of the transcription factor in *Hedyotis diffusa* was obtained and its protein coding sequence (SEQ ID NO. 2) was deduced, wherein the start codon is ATG and the stop codon is TAA.

[0051] Example 3: Short-stemmed snake root OpJRB1 Functional verification of genes

[0052] 1. Including OpJRB1 Construction of plant gene knockout vectors

[0053] use BBs The intermediate vector pMD18T-CRISPR / Cas9 was digested with a single restriction endonuclease (I) to form a linearized vector with sticky ends. The target sequence primers of the OpJRB1 gene were annealed and then ligated to the vector fragment using a T4 enzyme to construct pMD18T-CRISPR / Cas9-OpJRB1sgRNA. Hindi III and EcoR I. Restriction endonuclease for the successfully constructed 18T- OpJRB1 - The Cas9 vector and the plant expression pCAMBIA2300 vector were double-digested and ligated using the T4 enzyme. The primers are shown in Table 2.

[0054] Table 2. PCR primers for constructing the pMD18T-CRISPR / Cas9-OpJRB1sgRNA vector

[0055]

[0056] This implementation example will use transcription factors. OpJRB1 The target sequence is linked to the expression regulatory sequence to construct a system containing... OpJRB1The plant knockout expression vector pCAMBIA2300 contains the gene target sequence and can be used to further validate gene function through metabolic engineering strategies.

[0057] 2. Including OpJRB1 Construction of plant overexpression vectors for genes

[0058] Will OpJRB1 The gene was constructed on the plant expression vector pHB-X-YFP. To facilitate the construction of the expression vector, [missing information - likely a specific primer or ingredient] was introduced into the forward primer. BamH The restriction site of I was introduced into the reverse primer. Spe The enzyme cleavage sites for I and the primers are shown in Table 3.

[0059] Table 3 pHB- OpJRB1 PCR primers constructed using the YFP vector

[0060]

[0061] This implementation example will involve transcription factors that regulate camptothecin synthesis. OpJRB1 It can be operatively linked to expression regulatory sequences to construct a system containing... OpJRB1 plant overexpression vector pHB- OpJRB1 -YFP, this expression vector can be used to increase the camptothecin content in *Hedyotis diffusa* through metabolic engineering strategies.

[0062] 3. Agrobacterium rhizogenes-mediated OpJRB1 Genetic transformation of *Sophora japonica* using knockout and overexpression vectors yielded transgenic hairy roots.

[0063] (1) Contains OpJRB1 Obtaining Agrobacterium rhizogenes engineered strains using gene knockout and overexpression vectors

[0064] will contain OpJRB1 Plant knockout expression vectors containing gene target sequences and OpJRB1 The plant expression vectors of the gene were transformed into Agrobacterium rhizogenes (such as C58C1, which is a commercially available biological material) using the freeze-thaw method, and then verified by PCR.

[0065] (2) Agrobacterium rhizogenes-mediated OpJRB1 Genetic transformation of short snake root grass

[0066] (2.1) Pre-culture of explants

[0067] Stem segments were cut from healthy, short, sterile snake root seedlings and placed on B5 medium for pre-culture, followed by dark culture at 25 ℃ for 2 days.

[0068] (2.2) Co-culture of Agrobacterium and explants

[0069] The short snake root stem explants were transferred into the activated snake root extract containing... OpJRB1 Soak the explants in a B5 culture medium suspension (OD value around 0.3) of Agrobacterium rhizogenes with gene knockout or overexpression vectors for 10 minutes, gently shake to ensure full contact between the explants and the bacterial suspension, and after infection, wipe the bacterial suspension off the surface of the stem segments and place them on the surface of B5 culture medium for dark incubation at 28 ℃ for 2 days.

[0070] (2.3) Induction and subculture of hairy roots

[0071] After two days of dark culture, the materials were transferred to primary sterile solid medium (B5 + Cb 300 mg / L) and cultured in the dark at 25°C, with the medium changed approximately every two weeks. After about 14-20 days, small callus tissue appeared at the wound sites of the explants, followed by the emergence of pale yellow hairy roots. At this point, the stem segments with hairy roots were transferred in batches to secondary sterile solid medium (B5 + Cb 200 mg / L), where the hairy roots grew rapidly. After 2-3 weeks, the hairy roots reached 3-4 cm in length and began to branch. At this stage, single colonies of well-grown, branched hairy roots were cut from the callus tissue and transferred to tertiary sterile solid medium (B5 + Cb 100 mg / L), still cultured in the dark at 25°C. After a period of cultivation, clones showing good growth without bacterial oozing were selected on a tertiary sterile solid medium. Newly grown hairy roots, approximately 2 cm in diameter, were cut from these clones and transferred to B5 solid medium for further cultivation at 25°C in the dark. After about two weeks of cultivation, the clones that did not exhibit bacterial oozing were individually numbered.

[0072] (3) PCR detection of hairy roots of transgenic snake root

[0073] As shown in Table 4, the RolB gene expressed in hairy roots was detected by PCR using upstream and downstream primers (SEQ ID NO. 9-10). The RolB gene was detected using pMD18T-CRISPR / Cas9. M13F (SEQ ID NO.11) and OpJRB1sgRNA-R (SEQ ID NO. 8) The target sequence was detected by PCR, and the positive lines were further sequenced for verification. The sequence was determined based on the 35S promoter region upstream of the expression cassette containing the target gene. OpJRB1Forward primers (SEQ ID NO. 12) and reverse primers (SEQ ID NO. 13) were designed to detect the transgenic lines overexpressing *Hedyotis diffusa* by PCR.

[0074] Table 4 OpJRB1 PCR primers for identifying transgenic hairy root lines

[0075]

[0076] In this embodiment, the plant expression vector was transformed into *Agrobacterium rhizogenes* to obtain a gene containing *Hedyotis diffusa* for transforming *Sophora japonica*. OpJRB1 Agrobacterium rhizogenes strains containing gene knockout and overexpression vectors were used to infect *Hedyotis diffusa* stem segments, resulting in transgenic *Hedyotis diffusa* hairy roots verified by PCR detection and sequencing, thus obtaining knockout lines. OpJRB1-KO1 , OpJRB1-KO23 , OpJRB1-KO36 ) and overexpression transgenic lines ( OpJRB1-OE3 , OpJRB1-OE6 , OpJRB1-OE41 The acquisition of transgenic short-root hairy roots of *Hedyotis diffusa* provides direct material for verifying gene function and screening for obtaining short-root hairy roots with higher camptothecin content.

[0077] 4. Determination of camptothecin content in transgenic short-rooted snakegrass lines

[0078] (1) Liquid culture of hairy roots

[0079] Select healthy hairy roots from Example 3, cut off the middle and tip sections, and inoculate them into 100 mL of B5 liquid culture medium. Then, expand the culture using a shaker at 25°C and 100 rpm in the dark. Remove the hairy roots from the culture container, blot dry the surface culture medium with absorbent paper, and cut three portions of hairy roots, each weighing approximately 0.2-0.6 g. Wrap them in aluminum foil, label them, and flash-freeze them in liquid nitrogen. Then, store them at -80°C for subsequent RNA extraction. Wrap the remaining hairy roots in absorbent paper, label them, and dry them in a 50°C incubator until the weight no longer changes. These are then used for subsequent extraction and content determination of camptothecin.

[0080] (2) Extraction of camptothecin from hairy roots

[0081] The dried rootlets were removed and ground into powder. 50 mg of the powder was weighed and placed into a 2 mL centrifuge tube. Approximately 1 mL of chromatographically pure methanol was added. The centrifuge tube was capped tightly and sealed with a sealing film. The tube was vortexed for 3 min to mix. The mixture was then placed in an ultrasonic water bath for 1 hour for extraction. The tube was then centrifuged at 12,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm organic phase filter membrane. The crude camptothecin extract was obtained and stored at -20℃ for later use.

[0082] (3) HPLC determination of camptothecin content in hairy roots

[0083] First, prepare the camptothecin standard to construct a standard curve. The preparation method for the camptothecin standard is as follows: Weigh 1 mg of camptothecin standard using an analytical balance and place it in a small beaker. Add 10 mL of methanol to the beaker, then sonicate the beaker in a water bath for 10-20 minutes until completely dissolved. This yields a standard solution with a final concentration of 100 μg / mL. When measuring the standard, dilute it to multiple concentration gradients to construct a standard curve.

[0084] 10 µL of each of the crude camptothecin extracts was injected into a high-performance liquid chromatograph (HPLC). The chromatographic column used was a C-18 reversed-phase silica column (Symmetry Shield™ C18, 5 μm, 250 x 4.6 mm, Waters); the chromatographic conditions were: mobile phase acetonitrile: 1‰ formic acid water = 30:70; column temperature 30℃; flow rate set to 1 mL / min; detection wavelength set to 254 nm. After detection, the peak area of ​​the camptothecin component in each sample was recorded in the HPLC. The peak area was substituted into the standard curve described above to calculate the camptothecin content in the sample. The results showed that the knockout strain ( OpJRB1-KO1 , OpJRB1-KO23 , OpJRB1- KO36 The content of camptothecin in the sample was lower than that in the control sample. 2300-EV ) significantly decreased, while overexpression of transgenic lines ( OpJRB1-OE3 , OpJRB1-OE6 , OpJRB1-OE41 The content of camptothecin was lower than that of the control ( 2306-EV Significantly improved (see) Figure 4 and Figure 5 Note: * indicates P<0.05 compared to the control group, and ** indicates P<0.01 compared to the control group.

[0085] Example 4: OpJRB1 Mechanism of action of transcription factors

[0086] Dual-LUC detection OpJRB1 The gene for the biosynthesis of camptothecin OpTDCActivation capability of promoters

[0087] 1. Vector construction and Agrobacterium-mediated transformation

[0088] Each OpTDC1 as well as OpLAMT1 The gene promoter was constructed into the pGreenII0800-fLUC expression vector, and OpJRB1 Gene construction to pHB-YFP The constructed plasmids were transformed into Agrobacterium GV3101 strain on the expression vector, and positive clones were obtained by bacterial PCR detection.

[0089] 2. Dual-luciferase reporter gene assay (Dual-LUC)

[0090] will contain pHB-YFP and pHB-OpJRB1-YFP GV3101 positive strains containing plasmids pTDC1-fLUC plasmids and pLAMT1-fLUC The GV3101 positive strain of plasmid was cultured at 28°C, the supernatant was removed by centrifugation, and the bacterial cells were resuspended in MS liquid medium to OD. 600 The concentration was 0.6. 0.1M acetylsalicylic acid and 0.5M MES solution (pH: 5.7) were added at 2 μL and 20 μL per milliliter, respectively. The mixture was thoroughly mixed and allowed to stand at room temperature for 3 h. The solution containing... pHB-YFP or pHB-OpJRB1-YFP GV3101 bacterial culture containing plasmid was respectively compared with... pTDC1-fLUC and pLAMT1-fLUC The plasmid was mixed 1:1 with GV3101 bacterial culture and briefly injected into tobacco. Two days later, samples were collected from tobacco leaves using a perforator and then rapidly frozen in liquid nitrogen. The LUC / REN ratio was determined using a Promega fluorescence detection kit and instrument. The results showed that the OpJRB1 transcription factor from *Hedyotis diffusa* can activate the camptothecin synthesis gene. OpTDC1 as well as OpLAMT1 The expression (see) Figure 6 , Figure 7 Note: ** indicates P < 0.01 compared to the control group.

[0091] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by this application.

Claims

1. A short shorthair transcription factor OpJRB1 characterized in that, The transcription factor OpJRB1 The amino acid sequence of the transcription factor is shown in SEQ ID NO.

2.

2. A gene encoding a transcription factor of Physcomitrella patens according to claim 1. OpJRB1 characterized in that The nucleotide sequence of the coding gene is shown as SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, The coding gene of claim 2.

4. A genetically engineered bacterium, characterized by comprising a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO:

1. The recombinant expression vector of claim 3.

5. The coding gene of claim 2, the recombinant expression vector of claim 3 or the genetically engineered bacteria of claim 4 in regulating the content of camptothecin in Ophiorrhiza pumila.

6. Use according to claim 5, characterized in that, The coding gene is overexpressed in Ophiorrhiza pumila to increase the content of camptothecin in Ophiorrhiza pumila.

7. The application of the encoding gene of claim 2, the recombinant expression vector of claim 3, or the transgenic engineered bacteria of claim 4 in activating the expression of the camptothecin synthesis gene in *Hedyotis diffusa*, characterized in that, The said camptothecine synthetic gene is OpTDC1 or OpLAMT1, The said coding gene is overexpressed in O. gracilis.

8. Use of the coding gene of claim 2, the recombinant expression vector of claim 3 or the genetically engineered bacteria of claim 4 in cultivating camptothecine drug source transgenic plants, characterized in that, The coding gene is overexpressed in plants, and the plants are Ophiorrhiza pumila.

9. Use according to any one of claims 5 to 8, characterized in that, Hairy roots are used for cultivation.

10. A method for increasing the content of camptothecin in hairy roots of Ophiorrhiza pumila, characterized in that, The following steps are used: Step 1, constructing an overexpression vector of the coding gene of claim 2; Step 2, transforming the overexpression vector into Agrobacterium rhizogenes; Step 3, infecting Ophiorrhiza pumila with the Agrobacterium rhizogenes to obtain Ophiorrhiza pumila hairy root lines with increased content of camptothecin.

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