Application of OpJRB1 transcription factor in increasing camptothecin content in ophiorrhiza pumila

Abstract

The invention provides a bHLH transcription factor OpJRB1 participating in regulation and control of synthesis of ophiorrhiza pumila camptothecin and application of the bHLH transcription factor OpJRB1. An OpJRB1 transcription factor is induced by jasmonic acid, is highly expressed in roots and can interact with inhibitory factors OpJAZ1, OpJAZ5 and OpJAZ6 in a jasmonic acid pathway, which fully shows that the OpJRB1 transcription factor participates in a jasmonic acid signal pathway. Knockout of the OpJRB1 gene significantly reduces the camptothecin content of hairy roots of ophiorrhiza pumila, and overexpression of the OpJRB1 gene can increase the camptothecin content. The OpJRB1 transcription factor can instantly activate the expression of the gene OpTDC1 and the gene OpLAMT1 for synthesizing the camptothecin, so that the accumulation of the camptothecin is improved. The invention provides a novel high-quality medicine source for producing camptothecin with a broad-spectrum anti-cancer curative effect, and has important theoretical significance and potential application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and relates to a kind of OpJRB1 Application of transcription factor in improving camptothecin content of Ophiorrhiza pumila hairy root. BACKGROUND

[0002] Ophiorrhiza pumila is a perennial dicotyledonous herb of Rubiaceae ( Rubiaceae ) Ophiorrhiza Ophiorrhiza ) genus, and a high content of anticancer drug camptothecin can be detected in each tissue and hairy root culture system thereof, which has the advantages of fast growth, short cycle and heritable operation. Camptothecin is an important natural product with broad-spectrum anticancer activity, and its derivatives are often used in clinical treatment of malignant tumors such as colon cancer, rectal cancer and ovarian cancer. The single planting mode and the scarcity of germplasm resources seriously restrict the supply of camptothecin anticancer drugs, which is difficult to meet the growing market demand. Therefore, improving the yield of camptothecin has gradually become a research hotspot in recent years. Using metabolic engineering technology to enhance the synthesis of plant secondary metabolites is a feasible technical path. As a research hotspot in the field of life sciences in recent years, a large number of research reports have confirmed that transcription factors can participate in the regulation of plant secondary metabolism, providing a new idea for related research.

[0003] bHLH (basic helix-loop-helix, basic helix-loop-helix) transcription factor is one of the largest transcription factor families in plants, and plays an important role in plant growth and development, physiological metabolism and stress response. The bHLH family member contains two highly conserved and functionally different domains: basic region and helix-loop-helix (HLH) region. The basic region consists of 15-20 amino acids, located at the N terminus of the bHLH domain, as a DNA binding region, which can recognize E-box and G-box; the HLH region at the C terminus depends on the interaction of hydrophobic amino acids to form homodimer or heterodimer of two HLH proteins, which then regulates the expression of downstream target genes. The bHLH transcription factor family members have been shown to cooperatively control the biosynthesis of a variety of secondary metabolites, mainly including terpenes, alkaloids, phenylpropanoids and the like. SUMMARY

[0004] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide a kind of OpJRB1 Application of transcription factor in improving camptothecin content of Ophiorrhiza pumila hairy root.

[0005] In a first aspect, the present application provides a kind of Ophiorrhiza pumila transcription factor OpJRB1, the amino acid sequence of the transcription factor OpJRB1 is shown as SEQ ID NO. 2.

[0006] In a second aspect, the present application provides a coding gene of the transcription factor of the short small snake grass OpJRB1 , the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

[0007] In a third aspect, the present application provides a recombinant expression vector comprising the coding gene.

[0008] In a fourth aspect, the present application provides a genetically engineered bacteria comprising the recombinant expression vector.

[0009] In a fifth aspect, the present application provides the coding gene, the recombinant expression vector or the genetically engineered bacteria in regulating the content of camptothecin in the short small snake grass. Further, the coding gene is overexpressed in the short small snake grass to increase the content of camptothecin in the short small snake grass.

[0010] In a sixth aspect, the present application provides the coding gene, the recombinant expression vector or the genetically engineered bacteria in activating the expression of camptothecin synthesis genes in the short small snake grass. OpTDC1 or OpLAMT1, The coding gene is overexpressed in the short small snake grass.

[0011] In a seventh aspect, the present application provides the coding gene, the recombinant expression vector or the genetically engineered bacteria in cultivating camptothecin source transgenic plants, the coding gene is overexpressed in the plants, and the plants are short small snake grass.

[0012] Further, the application adopts hairy roots for cultivation.

[0013] In an eighth aspect, the present application provides a method for increasing the content of camptothecin in the hairy roots of the short small snake grass, comprising the following steps: Step 1, constructing an overexpression vector of the coding gene; Step 2, transforming the overexpression vector into Agrobacterium rhizogenes; Step 3, infecting the short small snake grass with the Agrobacterium rhizogenes to obtain a short small snake grass hairy root strain with increased content of camptothecin.

[0014] The present application has the following beneficial effects: (1) The short snake root transcription factor in the present application is screened by MeJA induced transcriptome and tissue expression transcriptome analysis and yeast two-hybrid verification, instead of using the homologous alignment method in the prior art OpJRB1 .

[0015] (2) The short snake root transcription factor OpJRB1 in the present application can activate the expression of camptothecin synthesis genes in short snake root OpTDC1 and OpLAMT1 , and promote the accumulation of camptothecin in short snake root hairy roots, thereby providing a new high-quality drug source for producing camptothecin with broad-spectrum anticancer efficacy.

[0016] (3) The molecular mechanism of the transcription factor regulating camptothecin biosynthesis is analyzed in the present application, which has important theoretical significance and potential application value. OpJRB1 BRIEF DESCRIPTION OF DRAWINGS

[0017] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the accompanying drawings: Figure 1 is a heat map of the transcriptional level expression difference change of the short snake root transcription factor after MEJA treatment; Figure 2 is the expression level of the candidate transcription factor induced by jasmonic acid in different tissues of short snake root; Figure 3 is the yeast two-hybrid verification result of OpJRB1 and OpJAZs ; Figure 4 is the content determination result of camptothecin in short snake root hairy roots with knockout OpJRB1 gene; Figure 5 is the content determination result of camptothecin in short snake root hairy roots with overexpression of OpJRB1 gene; Figure 6 is the activation ability detection result of the short snake root transcription factor on camptothecin biosynthesis genes OpJRB1 ; OpTDC1 Figure 7 is the activation ability detection result of the short snake root transcription factor on camptothecin biosynthesis genes OpJRB1 . OpLAMT1 DETAILED DESCRIPTION

[0018] ​​​The application will be described in greater detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are intended to illustrate the application and not to limit the scope of the application. Furthermore, it should be understood that after reading the present disclosure, those skilled in the art will be able to make various modifications or alterations to the application, and these equivalent forms should also fall within the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out under conventional conditions, for example, the conditions described in Molecular Cloning (Sambrook et al.) or the conditions recommended in the instructions provided by the manufacturers of the reagents or kits.

[0019] Example 1: Ophiorrhiza pumila OpJRB1 Screening of genes 1. MeJA-induced expression profile analysis Ophiorrhiza pumila plants were treated with 100 µM of the plant hormone jasmonic acid (JA) and subjected to transcriptome sequencing. Samples of O. pumila treated with JA at different times (0, 3, 6, 9, 12, and 24 h) were subjected to transcriptome sequencing analysis. The material at 0 h of treatment was used as the Control group, and 15 transcription factors transiently responsive to JA signaling were screened. Figure 1 < / s> Transcriptome analysis of O. pumila tissues Based on the MeJA-induced transcriptome analysis, the expression levels of these transcription factors in different tissues (roots, stems, and leaves) were further analyzed, and 7 transcription factors that were predominantly expressed in roots were screened. Among them, Op01g01318 is OpJRB1 < / s> Figure 2 < / s> Yeast two-hybrid experiment (1) Vector construction Construction of PGADT7-Op01g01318 and PGBKT7- OpJAZ1 -6 recombinant plasmids.

[0020] The plasmids of the above-constructed vectors were transformed into Y2H yeast competent cells, and yeast two-hybrid experiments were performed.

[0021] Yeast competent cells were prepared according to the prepared 1.1x LiAc / TE mixture.

[0022] 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.

[0023] 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).

[0024] Example 2: Short snake root grass OpJRB1 Cloning of genes 1. Extraction of total RNA from *Hedyotis diffusa* 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.

[0025] 2. Short snake root grass OpJRB1 Cloning of genes 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.

[0026] Table 1 PCR Primers

[0027] 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.

[0028] Example 3: Short-stemmed snake root OpJRB1 Functional verification of genes

[0029] 1. Including OpJRB1 Construction of plant gene knockout vectors

[0030] 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. Hind 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. Table 2. PCR primers for constructing the pMD18T-CRISPR / Cas9-OpJRB1sgRNA vector

[0031] This implementation example will use transcription factors. OpJRB1 The target sequence is linked to the expression regulatory sequence to construct a system containing... OpJRB1 The plant knockout expression vector pCAMBIA2300 contains the gene target sequence and can be used to further validate gene function through metabolic engineering strategies.

[0032] 2. The plant overexpression vector containing OpJRB1 Construction of plant overexpression vector of gene

[0033] The OpJRB1 gene was constructed in plant expression vector pHB-X-YFP, in order to facilitate the construction of expression vector, the restriction sites of BamH I were introduced in the forward primers, and the restriction sites of Spe I were introduced in the reverse primers, the primers are shown in Table 3; Table 3 PCR primers for construction of pHB- OpJRB1 -YFP vector

[0034] In this embodiment, the transcription factor involved in regulating the synthesis of camptothecin OpJRB1 was operably linked to the expression regulatory sequence, and a plant overexpression vector pHB- OpJRB1 containing the gene was constructed, which can be used to improve the content of camptothecin in O. cummingsii by metabolic engineering strategy. OpJRB1

[0035] 3. Agrobacterium rhizogenes-mediated OpJRB1 knockout vector and overexpression vector genetic transformation of O. cummingsii to obtain transgenic hairy roots (1) Obtaining of Agrobacterium rhizogenes engineering bacteria containing OpJRB1 gene knockout vector and overexpression vector The plant knockout expression vector containing the target sequence of OpJRB1 gene and the plant expression vector containing OpJRB1 gene were respectively transformed into Agrobacterium rhizogenes (such as C58C1, which is a publicly sold biological material in the market) by freeze-thaw method, and PCR verification was performed.

[0036] (2) Agrobacterium rhizogenes-mediated OpJRB1 gene genetic transformation of O. cummingsii (2.1) Pre-culture of explants The stem segments were cut from healthy O. cummingsii sterile seedlings and placed on B5 medium for pre-culture, and dark culture was performed at 25°C for 2 days.

[0037] (2.2) Co-culture of Agrobacterium and explants The O. cummingsii stem segment explants were transferred into the Agrobacterium containing the activated OpJRB1 ​The Agrobacterium rhizogenes B5 medium suspension (OD value is about 0.3) of the gene knockout vector or the overexpression vector was soaked for 10 minutes, the explants were fully contacted with the bacterial solution by gently shaking, and after the infection was completed, the stem surface was wiped dry and placed on the B5 medium surface and cultured in the dark at 28°C for 2 days.

[0038] (2.3) Induction and subculture of hairy roots After the above material was cultured in the dark for 2 days, it was transferred to the first sterilized solid medium (B5 + Cb 300 mg / L) and cultured in the dark at 25°C, and the medium was replaced every 2 weeks or so. After about 14-20 days, small calli appeared at the wound of the above explants, and then yellowish hairy roots grew successively at the calli. At this time, the stem segments with hairy roots were transferred and divided into batches on the second sterilized solid medium (B5 + Cb 200 mg / L), and the hairy roots grew faster on this medium. After 2-3 weeks, the hairy roots grew to 3-4 cm and began to branch, at which time the hairy roots with good growth and branches were cut from the calli and transferred to the third sterilized solid medium (B5 + Cb 100 mg / L) and cultured in the dark at 25°C. After a period of culture, the clonal lines with good growth and no overflow were selected from the third sterilized solid medium, and the newly grown tips of the hairy roots about 2 cm long were cut from these clonal lines and transferred to the B5 solid medium for further culture, with the culture conditions being 25°C in the dark. After about 2 weeks of culture, the lines without overflow were selected and independently numbered.

[0039] (3) PCR detection of transgenic O. basilicum hairy roots As shown in Table 4, the RolB gene was detected by PCR using the upstream and downstream primers (SEQ ID NO. 9-10) of the RolB gene expressed in the hairy roots. The target sequence was detected by PCR using M13F (SEQ ID NO. 11) and OpJRB1sgRNA-R (SEQ ID NO. 8) in pMD18T-CRISPR / Cas9, and the positive lines obtained were further verified by sequencing. According to the 35S promoter region upstream of the expression cassette of the target gene and OpJRB1 The forward primer (SEQ ID NO. 12) and reverse primer (SEQ ID NO. 13) were designed for PCR detection of the O. basilicum overexpression transgenic lines.

[0040] Table 4 OpJRB1 PCR primers for identification of transgenic hairy root lines

[0041] The plant expression vector is transformed into Agrobacterium rhizogenes to obtain a strain containing OpJRB1 The Agrobacterium rhizogenes strain of the gene plant knockout expression vector and the overexpression vector is used to infect the short snake root stem segment, and the transgenic short snake root hairy root is obtained by PCR detection and sequencing verification, and the knockout strain (GUS-1, GUS-2, GUS-3 and GUS-4) and the overexpression transgenic strain (OEP-1, OEP-2, OEP-3 and OEP-4) are obtained. OpJRB1-KO1 、 OpJRB1-KO23 、 OpJRB1-KO36 )and overexpression transgenic strain (OEP-1, OEP-2, OEP-3 and OEP-4) are obtained. OpJRB1-OE3 、 OpJRB1-OE6 、 OpJRB1-OE41 The obtaining of the transgenic short snake root hairy root provides direct materials for the verification of gene function and the screening of short snake root hairy root with higher camptothecin content.

[0042] 4. Determination of the content of camptothecin in the transgenic short snake root hairy root strain

[0043] (1) Hairy root liquid culture The well-grown hairy roots in Example 3 are selected, the middle and tip ends are cut, and are inoculated into 100 mL of B5 liquid culture medium, and then are expanded and cultured by using a shaking bed under dark conditions at 25°C and 100 rpm. The hairy roots are taken out of the culture container, and the surface medium is absorbed by using an absorbent paper. First, 3 parts of the hairy roots are cut, each weighing about 0.2-0.6 g, are wrapped with tin foil paper and are labeled, are quickly frozen in liquid nitrogen, and then are placed in a-80°C constant temperature box for storage, for subsequent RNA extraction. The remaining hairy roots are wrapped with an absorbent paper and are labeled, and then are placed in a 50°C constant temperature box for drying. After the weight is unchanged, the hairy roots are taken out, and are used for subsequent camptothecin extraction and content determination.

[0044] (2) Extraction of camptothecin in the hairy roots The above dried hairy roots to a constant weight are taken out, are ground into powder, 50 mg of the powder is weighed, is loaded into a 2 mL centrifuge tube, about 1 mL of chromatographically pure methanol is added, the centrifuge tube cap is tightly covered, and is sealed with a sealing film. After being vortexed and shaken for 3 min, the sample is placed in an ultrasonic instrument ultrasonic water bath for extraction for 1 hour. Then, the sample is centrifuged in a centrifuge at a speed of 12,000 rpm for 10 min. After centrifugation, the supernatant is taken, is filtered by using a 0.22 μm organic phase filter membrane, and the camptothecin crude extract after filtration is stored at-20°C for standby use.

[0045] (3) HPLC determination of the content of camptothecin in the hairy roots Firstly, camptothecin standard sample was configured to make standard curve. The configuration method of camptothecin standard sample was as follows: 1 mg camptothecin standard sample was weighed by an analytical balance and placed in a small beaker, 10 mL of methanol was added into the beaker, then the small beaker was ultrasonically treated in a water bath of an ultrasonic instrument for 10-20 min, and after complete dissolution, the standard sample solution with a final concentration of 100 μg / mL was obtained. When the standard sample was measured, it was diluted into multiple different concentration gradients, so as to make a standard curve.

[0046] 10 μL of each of the camptothecin crude extracts was injected into a high performance liquid chromatograph. The used chromatographic column was a C-18 reversed phase silica gel column (Symmetry Shield TM C18, 5 μm, 250x4.6 mm, Waters); the chromatographic conditions were as follows: mobile phase acetonitrile: 1 ‰ formic acid water = 30:70; column temperature 30 ℃; flow rate was set to 1 mL / min; detection wavelength was set to 254 nm. After detection, the peak area of camptothecin component of each sample was recorded in the HPLC, and the peak area was substituted into the above-mentioned standard curve, and the calculated camptothecin content in the sample was obtained. The results showed that the camptothecin content in the knockout strain (Example 1) OpJRB1-KO1 、 OpJRB1-KO23 、 OpJRB1- KO36 ) was significantly lower than that in the control (Example 1) 2300-EV ), while the camptothecin content in the overexpression transgenic strain (Example 2) OpJRB1-OE3 、 OpJRB1-OE6 、 OpJRB1-OE41 ) was significantly higher than that in the control (Example 1) 2306-EV ) (see Figure 4 and Figure 5 ). Note: * represents P<0.05 compared with the control group, and ** represents P<0.01 compared with the control group.

[0047] Example 4: OpJRB1 Mechanism of action of transcription factor Dual-LUC detection OpJRB1 Activation ability of camptothecin biosynthetic gene OpTDC promoter 1. Vector construction and Agrobacterium transformation The promoters of OpTDC1 and OpLAMT1 genes were constructed into pGreenII0800-fLUC expression vector, the OpJRB1 gene was constructed into pHB-YFP expression vector, and the constructed plasmids were transformed into Agrobacterium GV3101 strain, and positive clone strains were obtained by bacterial liquid PCR detection.

[0048] 2. Dual luciferase reporter gene detection (Dual-LUC) The positive clone strains containing the promoters of OpTDC1 and OpLAMT1 genes were mixed with the Agrobacterium GV3101 strain containing the OpJRB1 gene, and the mixed bacterial solution was used for plant transformation.pHB-YFP and pHB-OpJRB1-YFP GV3101 positive strain of plasmid and pTDC1-fLUC plasmid and pLAMT1-fLUC GV3101 positive strain of plasmid was expanded at 28℃, centrifuged to remove supernatant, and resuspended in MS liquid medium to OD 600 0.6, 2 μL and 20 μL of 0.1 M acetosyringone and 0.5 M MES solution (pH: 5.7) were added per mL, mixed, and left to stand at room temperature for 3 h, and GV3101 bacterial liquid containing pHB-YFP or pHB-OpJRB1-YFP plasmid was mixed with GV3101 bacterial liquid containing pTDC1-fLUC and pLAMT1-fLUC plasmid at 1:1, and transiently injected into tobacco. Two days later, tobacco leaves were punched with a puncher, and then quickly frozen in liquid nitrogen, and the LUC / REN ratio was determined according to the fluorescence detection kit and instrument of Promega. The results showed that the short snake root OpJRB1 transcription factor can activate the expression of camptothecine synthesis genes OpTDC1 and OpLAMT1 (see Figure 6 , Figure 7 ). Note: ** represents P<0.01 compared with the control group.

[0049] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations to the present application without creative work based on the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art within the concept of the present application should be within the protection scope determined by the present 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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