Vietnamese sophora root phytoene dehydrogenase gene StPDS and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and relates to a reporter gene of subprostrate sophora, in particular to a phytoene dehydrogenase gene StPDS of subprostrate sophora and application of the phytoene dehydrogenase gene StPDS. The nucleotide sequence of the gene StPDS is as shown in SEQ ID No. 1. According to the invention, cloning and subcellular localization analysis are carried out on the StPDS gene in the chloroplast of subprostrate sophora for the first time; a subprostrate sophora StPDS gene silencing system is constructed, and the system has the characteristics of rapidness, high throughput, good effect and easiness in operation; according to the StPDS gene and the silence system, the expression level of the subprostrate sophora StPDS gene is effectively reduced, yellowing phenotype is caused, and the subprostrate sophora StPDS gene can be used as a reporter gene of a VIGS silence system in subprostrate sophora application. A solid theoretical and practical foundation is laid for application of the VIGS technology in the subprostrate sophora, and a basis is provided for verification and identification of other functional genes of the subprostrate sophora.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and relates to a reporter gene of Sophora flavescens, and in particular to a Sophora flavescens phytoene dehydrogenase gene. StPDS and its applications. Background Art

[0002] Sophora flavescens is a plant of the Leguminosae family. Sophora tonkinensis Gapnep.) (Chinese Pharmacopoeia Committee, 2020), first recorded in Kaibao Materia Medica, and included in classic documents such as Illustrated Materia Medica and the Pharmacopoeia of the People's Republic of China since 1963, are authentic medicinal materials and commonly used Chinese medicines in Guangxi's "Ten Flavors of Gui". The main material basis of Sophora flavescens is alkaloids and flavonoids. The quality evaluation component of its medicinal materials is clearly the total content of matrine and oxymatrine. It has multiple pharmacological effects such as anti-tumor, anti-arrhythmia, anti-hepatitis B virus, antioxidant, anti-inflammatory, and immune enhancement. It is one of the important raw materials for the production of nearly 30 Chinese patent medicines for the treatment of hepatitis, sore throat, anti-tumor, hemorrhoids, acne, etc., such as "Ganyanling Injection", "Guilin Watermelon Frost", "Anti-Virus Granules", "Nasopharyngeal Ling Tablets", "Kaihoujian Spray" and "Shenlian Capsules", etc. It is also used as a decoction piece in many prescriptions. Wild Sophora flavescens is mainly scattered in some limestone areas in Guangxi, Guizhou and Yunnan. It is a typical species in karst limestone areas. Due to the small amount of wild Sophora flavescens resources, cultivated Sophora flavescens has become the main source of Sophora flavescens medicinal materials. In the process of artificial cultivation, due to the long growth cycle of Sophora flavescens (3-4 years), small reproduction coefficient, large seed consumption, and serious diseases and insect pests, the production cost increases and the industry develops slowly. Therefore, in order to ensure the sustainable development of the Sophora flavescens industry, it is urgent to strengthen the innovation research of Sophora flavescens germplasm resources and cultivate new varieties of Sophora flavescens with high quality and high yield.

[0003] Virus-induced gene silencing (VIGS) technology is a fast and effective method for studying plant gene functions. It does not require genetic transformation and has the advantages of short cycle, low cost, simple operation and high silencing efficiency. It is widely used in the functional identification of genes related to plant growth and development, disease resistance and stress resistance, metabolic regulation, etc. It can be seen that the use of genetic engineering methods is an effective way to obtain new germplasm of Sophora flavescens, and currently VIGS technology has been successfully used in tobacco (publication number CN107815454A), Arabidopsis thaliana (publication number CN109206496A), wheat (publication number CN102124110A), rice (publication number CN113046364A), corn (publication number CN116855530A), tomato (publication number CN112501196A), peony (publication number CN118222608A), kenaf (publication number CN114480447A) and other plant research. However, due to the lack of effective genetic transformation and regeneration system methods, the in-depth research and application of endogenous functional genes of Sophora flavescens has been greatly restricted, thus affecting the innovative research on Sophora flavescens germplasm resources and even the sustainable development of the entire Sophora flavescens industry. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a gene encoding phytoene dehydrogenase from Radix Sophorae Tonkinensis StPDS and its applications.

[0005] The technical solution of the present invention is achieved in this way: Sophora flavescens phytoene dehydrogenase gene StPDS , whose nucleotide sequence is shown in SEQ ID No.1.

[0006] Sophora flavescens phytoene dehydrogenase gene StPDS The amino acid sequence of the expressed protein is shown in SEQ ID No.2.

[0007] Sophora flavescens phytoene dehydrogenase gene StPDS Application as a reporter gene of Sophora flavescens.

[0008] The above applications are achieved by constructing genes StPDS This is achieved by infecting Sophora flavescens plants with the recombinant vector.

[0009] Furthermore, the above-mentioned recombinant vector is a viral vector.

[0010] Preferably, the above-mentioned viral vector is tobacco rattle virus.

[0011] For the above application, the steps are: (1) Cloning Sophora flavescens PDSThe full length of the gene was constructed to construct the recombinant plasmid pEASY-Blunt- StPDS ; (2) Use the recombinant plasmid pEASY-Blunt- StPDS As a template, the primer pair of the specific fragment was used as a primer for amplification, the target fragment was recovered, and the target fragment was connected to the pTRV2 vector to construct pTRV2- StPDS Recombinant plasmid; (3) Place pTRV2- StPDS The bacterial solution of the recombinant plasmid and the bacterial solution of the pTRV1 auxiliary vector were mixed in equal proportions and then injected into the leaves of Sophora flavescens. After dark cultivation, yellow phenotype plants were obtained by normal cultivation.

[0012] The sequence of the specific fragment in the above step (2) is shown as SEQ ID No.3.

[0013] In the above step (3), the infiltration area of ​​the kenaf leaf injected is greater than 75% of the area of ​​the entire leaf.

[0014] The dark culture time in the above step (3) is 48 hours.

[0015] The present invention has the following beneficial effects: The present invention is the first to clone and analyze the subcellular localization of the phytoene desaturase (PDS) gene in the chloroplasts of Sophora flavescens; StPDS The gene silencing system has the characteristics of rapidity, high throughput, good effect and easy operation; the present invention StPDS The gene and silencing system effectively reduced the StPDS The gene expression level and chlorophyll synthesis are affected, resulting in a yellow phenotype, which can be used as a reporter gene in the application of the VIGS silencing system in Sophora flavescens. This has laid a solid theoretical and practical foundation for the application of VIGS technology in Sophora flavescens, provided a basis for the verification and identification of other functional genes in Sophora flavescens, and provided a reference for the application of phytoene dehydrogenase PDS and VIGS technology system in other crops. 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 Sophora flavescens StPDSAgarose gel electrophoresis of gene PCR amplification products; M: DL2000 DNAMarker; Lanes 1, 2, and 3: StPDS Gene.

[0018] Figure 2 Sophora flavescens StPDS Subcellular localization of genes.

[0019] Figure 3 To construct the recombinant viral vector pTRV2- StPDS Agarose gel electrophoresis of Agrobacterium colony PCR detection of empty vector pTRV2 and auxiliary vector pTRV1; M: DL2000 DNA Marker; Lanes 1-3: recombinant viral vector pTRV2- StPDS Lane 4: Agrobacterium monoclonal clone; Lane 4: empty vector pTRV2 Agrobacterium monoclonal clone; Lane 5: auxiliary vector pTRV1 Agrobacterium monoclonal clone.

[0020] Figure 4 This is a schematic diagram of the VIGS infection process using the back-of-leaf injection method and the yellowing phenotype of new leaves of Sophora flavescens.

[0021] Figure 5 Sophora flavescens Radix StPDS Gene silencing effect; pTRV2: empty vector pTRV2 control plant; 1-2: recombinant virus vector pTRV2- StPDS Silence plants. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0024] The primer sequences used in this application are as follows: The present application discloses a phytoene dehydrogenase gene from Sophora flavescens StPDS , whose nucleotide sequence is shown in SEQ ID No.1.

[0025] Sophora flavescens phytoene dehydrogenase gene StPDSThe amino acid sequence of the expressed protein is shown in SEQ ID No.2.

[0026] Sophora flavescens phytoene dehydrogenase gene StPDS Application as a reporter gene of Sophora flavescens.

[0027] The above applications are achieved by constructing genes StPDS This is achieved by infecting Sophora flavescens plants with the recombinant vector.

[0028] The specific examples are further described below: Example

[0029] 1. Mountain bean root PDS Gene cloning Total RNA was extracted from the roots of Sophora flavescens seedlings, and then the first-strand cDNA was synthesized according to the reverse transcription kit operation steps and stored at -20 °C for later use. StPDS The primers StPDS-F and StPDS-R of the gene sequence were used, and the high-fidelity enzyme Pfu DNA polymerase was used to amplify the gene sequence of Sophora flavescens with the primers StPDS-F and StPDS-R as the primer pair and cDNA as the template. StPDS Gene sequence.

[0030] The amplification system was: cDNA 1 μL, StPDS-F and StPDS-R 1 μL each (10 μM), 10 × Pfu buffer 2.5 μL, dNTP Mix 2 μL (2.5 mM), Pfu DNA polymerase 0.5 μL (2.5 U), and ddH2O was added to make up to 25 μL.

[0031] The amplification program was as follows: pre-denaturation at 95 °C for 5 min; 95 °C, 30 s; 56 °C, 30 s; 72 °C, 60 s; 30 cycles; and extension at 72 °C for 5 min.

[0032] The electrophoresis of the amplified product is shown in Figure 1 shown.

[0033] The PCR product was purified by gel purification recovery kit after electrophoresis and connected to the pEASY-Blunt cloning vector, transformed into Escherichia coli DH 5α competent cells, and then spread on LB plates containing Kan (100 mg / mL), inverted and cultured at 37°C for 8-12 h, and positive clones were selected for sequencing to obtain phytoene dehydrogenase-containing StPDS The recombinant bacteria pEASY-Blunt-StPDS with gene sequence.

[0034] 2. Mountain bean root PDS Subcellular localization (1) pBI121- StPDS Construction of transient expression vector The recombinant pEASY-Blunt-StPDS bacterial solution was used as a template, and the multiple cloning site sequence information of the transient expression vector pBI121 was combined with ClonExpressII technology to design amplification StPDS Primer pBI- StPDS -F and pBI- StPDS -R.

[0035] Amplification system: pEASY-Blunt- StPDS 1 μL of bacterial solution, 1 μL each of pBI-StPDS-F and pBI-StPDS-R (10 μM), 2.5 μL of 10×Pfu buffer, 2 μL of dNTP Mix (2.5 mM), 0.5 μL of Pfu DNA polymerase (2.5 U), and ddH2O to make up to 25 μL.

[0036] The amplification program was as follows: 95°C pre-denaturation for 5 min; 95°C, 30 s; 56°C, 30 s; 72°C, 60 s; 30 cycles; 72°C extension for 5 min. The PCR amplification product was purified by gel purification kit after electrophoresis and then connected to the Spe I linearized pBI121 vector.

[0037] The ligation system was: 280 ng of pBI121 vector linearized plasmid, 42 ng of insert fragment, 2 μL of 5×CEII Buffer, 1 μL of Exnase II, and ddH2O to make up to 10 μL.

[0038] The connection conditions were as follows: react at 37 °C for 10 min, cool to 4 °C and immediately cool on ice.

[0039] The ligation product was transformed into Escherichia coli DH 5α competent cells, then spread on LB plates containing Kan (100 mg / mL), cultured inverted at 37°C for 8-12 h, and the positive clones were selected for sequencing to obtain the recombinant bacteria pEASY-pBI121-StPDS.

[0040] (2) Instantaneous transformation of tobacco leaves Sow a number of tobacco seeds in a matrix containing vermiculite, and after one month of cultivation, wait for 6 to 8 leaves to grow before injection. Extract the plasmid of the recombinant bacteria pBI121-StPDS and transfer it into Agrobacterium EHA105 competent cells, then spread it on LB plates containing Kan (100 mg / mL), Rif (50 mg / mL) and Str (25 mg / mL), and invert and culture at 28 ℃ for 2 days. Using the Agrobacterium transformant colony as a template, PCR amplification of pBI-StPDS-F and pBI-StPDS-R was performed using gene-specific primers.

[0041] Pick the cells containing pBI121- StPDS A single colony of Agrobacterium carrying the plasmid was inoculated into 100 mL of LB liquid culture medium for expansion culture and cultured at 200 rpm until the OD was 0.6-0.8. The bacteria were collected by centrifugation at 4000 rpm for 4 minutes, and the bacteria were resuspended in LB liquid suspension containing 10 mM MgCl2 and 120 μM AS, and the OD was adjusted to 0.6. Select tobacco leaves with good growth conditions, inject from the lower epidermis of the tobacco leaves with a 1 mL syringe without a needle, and mark the injection area. After the injected tobacco plants were cultured in the dark for 24 hours, the leaves at the marked area were taken to make slides, and observed and photographed under a laser confocal microscope. The results are as follows Figure 2 As shown by Figure 2 Knowable StPDS The protein expressed by the gene is localized in chloroplasts.

[0042] VIGS vector construction and infection (1) Carry StPDS Construction of TRV recombinant vector with 1444 sequences The recombinant pEASY-Blunt-StPDS bacterial solution was used as a template, and according to the multiple cloning site sequence information of the pTRV2 vector and the ClonExpressII technical requirements, the SGN VIGS Tool online software (https: / / vigs.solgenomics.net / ) was used to design the amplification StPDS The primer pair pTRV2-StPDS-F and pTRV2-StPDS-R for the 300 bp specific fragment on the CDS domain of the gene (as shown in SEQ ID No. 3).

[0043] The amplification system was: 1 μL of pEASY-Blunt-StPDS bacterial solution, 1 μL each of pTRV2-StPDS-F and pTRV2-StPDS-R (10 μM), 2.5 μL of 10×Pfu buffer, 2 μL of dNTP Mix (2.5 mM), 0.5 μL of Pfu DNA polymerase (2.5 U), and ddH2O was added to make up to 25 μL.

[0044] The amplification program was as follows: pre-denaturation at 95 °C for 5 min; 95 °C, 30 s; 56 °C, 30 s; 72 °C, 30 s; 30 cycles; and extension at 72 °C for 5 min.

[0045] The PCR amplification product was purified by gel purification recovery kit after electrophoresis and then connected to the pTRV2 vector linearized by EcoRI and XhoI.

[0046] The ligation system was: 220 ng of pTRV2 vector linearized plasmid, 14 ng of insert fragment, 2 μL of 5×CEII Buffer, 1 μL of Exnase II, and ddH2O to make up to 10 μL.

[0047] The connection conditions were as follows: react at 37 °C for 10 min, cool to 4 °C and immediately cool on ice.

[0048] The ligation product was transformed into E. coli DH 5α competent cells, then spread on LB plates containing Kan (100 mg / mL), inverted and cultured at 37°C for 8-12 h, and positive clones were selected for sequencing to obtain the recombinant bacteria pTRV2-StPDS. Figure 3 As shown, it is proved that the present application constructed the recombinant viral vector pTRV2- StPDS .

[0049] (2) Cultivation of Sophora flavescens plants The seeds of Sophora flavescens with full grains were selected and sterilized with 3% H2O2 solution for 10 min, then rinsed with sterile distilled water for 4-5 times, soaked in sterile water at room temperature for 1 h, and then evenly placed in a seedling tray covered with vermiculite and placed in a light incubator (light / dark cycle of 14 / 10 h, day / night temperature of 28 / 26°C, relative humidity of 60 / 100, light intensity of 300 μM / (m 2 ·s)) After 21 days of cultivation, select seedlings with uniform growth and transplant them into the seedling medium. They can be injected only when they grow 6-9 leaves.

[0050] (3) VIGS infection Respectively extract the recombinant bacteria pTRV2- StPDSThe plasmids of pTRV2, pTRV1 and pTRV2 were transformed into Agrobacterium GV301 competent cells, then spread on LB plates containing Kan (100 mg / mL), Rif (50 mg / mL) and Str (25 mg / mL) and cultured inverted at 28 °C for 2 days.

[0051] Using Agrobacterium transformant colonies as templates, PCR amplification detection was performed using amplification primer pairs pTRV2-StPDS-F and pTRV2-StPDS-R, pTRV2-F and pTRV2-R, and pTRV1-F and pTRV1-R, respectively.

[0052] Pick a single colony of Agrobacterium containing pTRV2-StPDS, pTRV2 and pTRV1 plasmids and inoculate them into 100 mL LB liquid medium for expansion culture. Cultivate at 200 rpm until the OD is 0.8-1.0. Centrifuge at 4000 rpm for 10 min to collect the bacteria, resuspend the bacteria in sterile ddH2O2 suspension containing 10 mM MgCl2, 200 μM AS and 10 mM MES, and adjust the OD to 1.0; The bacterial suspension carrying the pTRV1 vector was mixed with the bacterial suspension carrying the pTRV2 vector and the bacterial suspension carrying the recombinant plasmid pTRV2-StPDS at a volume ratio of 1:1 to prepare two mixed bacterial solutions. The mixed bacterial solutions were incubated at room temperature in the dark for 4 hours and then used to inject Sophora flavescens leaves. A 1 mL syringe without a needle was used to inject from the lower epidermis of the young leaves of Sophora flavescens. The infiltration area should account for more than 75% of the entire leaf area. The injected Sophora flavescens plants were cultured in the dark for 48 hours and then cultured under normal conditions. The culture results are shown in Figure 2. Figure 4 As shown by Figure 4 It can be seen that the leaves of the Sophora flavescens seedlings injected with pTRV1 and pTRV2-StPDS bacterial solutions showed yellowing phenotype, while the leaves of the Sophora flavescens seedlings injected with pTRV1 and pTRV2 bacterial solutions did not show this phenotype. (4) After immersion StPDS Gene expression detection When the leaves showed yellowing phenotype (usually about 14 days after injection), total RNA was extracted from the leaves of Sophora tonkinensis. Actin is the internal reference gene, the internal reference gene primers are Actin-F and Actin-R, and the StPDS gene primers are qPCR-StPDS-F and qPCR-StPDS-R.

[0053] Real-time fluorescence quantitative PCR (qRT-PCR) StPDS The gene was expressed in the control group (injected with pTRV1 and pTRV2 empty vector bacteria) and silenced group (injected with pTRV1 and recombinant viral vector pTRV2- StPDSThe results are shown in Table 1. Figure 5 As shown by Figure 5 It can be seen that compared with the control, StPDS The expression level of the gene was significantly decreased in both silenced plants 1 and 2.

[0054] In summary: the present invention cloned a phytoene dehydrogenase (PDS) gene from Sophora flavescens, located in the chloroplast, constructed a tobacco rattle virus (TRV) recombinant vector carrying the StPDS sequence, and induced the silencing of the StPDS gene after infecting Sophora flavescens by leaf injection. After silencing, the expression level of the StPDS gene in the plant decreased significantly, and a typical yellowing phenotype appeared. The TRV recombinant vector carrying the StPDS sequence constructed by the present invention can be used as a positive control in the Sophora flavescens VIGS technology system, and explores and optimizes the Sophora flavescens VIGS technology system, laying a solid theoretical and practical foundation for the application of VIGS technology in Sophora flavescens, providing a basis for the verification and identification of other functional genes of Sophora flavescens, and also providing a reference for the application of phytoene dehydrogenase PDS and VIGS technology system in other crops.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Sophora flavescens phytoene dehydrogenase gene StPDS , whose nucleotide sequence is shown in SEQ ID No.

1.

2. The Sophora flavescens phytoene dehydrogenase gene of claim 1 StPDS The amino acid sequence of the expressed protein is shown in SEQ ID No.

2.

3. The Sophora flavescens phytoene dehydrogenase gene of claim 1 StPDS Application as a reporter gene of Sophora flavescens.

4. The use according to claim 3, Features: The application is to construct a gene StPDS This is achieved by infecting Sophora flavescens plants with the recombinant vector.

5. The use according to claim 4, Features: The recombinant vector is a viral vector.

6. The use according to claim 5, Features: The viral vector is tobacco rattle virus.

7. The use according to any one of claims 3 to 6, It is characterized in that The steps are: (1) Cloning Sophora flavescens PDS The full length of the gene was constructed to construct the recombinant plasmid pEASY-Blunt- StPDS ; (2) Use the recombinant plasmid pEASY-Blunt- StPDS As a template, the primer pair of the specific fragment was used as a primer for amplification, the target fragment was recovered, and the target fragment was connected to the pTRV2 vector to construct pTRV2- StPDS Recombinant plasmid; (3) Place pTRV2- StPDS The bacterial solution of the recombinant plasmid and the bacterial solution of the pTRV1 auxiliary vector were mixed in equal proportions and then injected into the leaves of Sophora flavescens. After dark cultivation, yellow phenotype plants were obtained by normal cultivation.

8. The use according to claim 7, Features: The sequence of the specific fragment in step (2) is shown as SEQ ID No.

3.

9. The use according to claim 7, Features: The infiltration area of ​​the kenaf leaf injected in step (3) is greater than 75% of the area of ​​the entire leaf.

10. The use according to claim 7, Features: The dark culture time in step (3) is 48 hours.

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