Application of E8 promoter and RH1 gene in creation of anthocyanin-rich tomato strain

Abstract

The invention discloses application of an E8 promoter and an RH1 gene to creation of an anthocyanin-rich tomato strain, and belongs to the technical field of tomato strain cultivation. The nucleotide sequence of the E8 promoter is as shown in SEQ ID No.1, the nucleotide sequence of the RH1 gene is as shown in SEQ ID No.2, and the CDS nucleotide sequence of the RH1 gene is as shown in SEQ ID No.3. The E8 promoter and the CDS of the RH1 gene are combined to construct an expression vector pCAMBIA2305-E8-RH1-5xFLAG, the expression vector is introduced into cotyledons of tomato plants by utilizing an agrobacterium-mediated genetic transformation technology, the RH1 gene is driven by the E8 promoter to be highly expressed in tomato fruits, and the expression vector pCAMBIA2305-E8-RH1-5xFLAG is obtained. Inducing accumulation of anthocyanin in the fruits; tests show that the expression level of the RH1 gene in transgenic tomato fruits is remarkably improved, the anthocyanin content is close to 0.3 mg / gFW, and the anthocyanin-rich tomato strain is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of tomato strain breeding and provides an E8 promoter and RH1 Application of genes in creating anthocyanin-rich tomato lines. Background Art

[0002] Tomatoes are rich in vitamins (such as V C 、V A 、V K and V B group), minerals, carotenoids, especially lycopene and dietary fiber and other nutrients; compared with other Solanaceae plants, such as eggplant, the flavonoid pathway in tomato fruit is not fully activated. Cultivated tomato fruits contain flavanones such as quercetin, myricetin and kaempferol, but cannot accumulate anthocyanins.

[0003] Since the beginning of the 21st century, scientists have begun to use genetic engineering to cultivate tomato strains rich in anthocyanins; early attempts to synthesize anthocyanins in tomato fruits focused on SlCHI Genetically, SlCHI When expressed ectopically in fruit, it increased flavonols by 78-fold, but failed to synthesize anthocyanins (see Muir et al. 2001; Schijlen et al. 2006 ); also overexpressed in tomato MYB or ikB Transcription factors, such as tomato endogenous SlANT1 ( Anthocyanin1 ), Arabidopsis PAP1 Or the expression of corn R2R3-MYB transcription factors C1 , it is possible to obtain tomato lines with anthocyanin spots on fruits (see Bovy et al. 2002; Mathews et al. 2003; Zuluaga et al. 2008 ); and in tomatoes, the fruit-specific promoter E8 drives snapdragon ikB transcription factors Delila ( Del )and MYB transcription factors Rosea1 ( Ros1 ) is expressed at high levels in the fruit, resulting in tomato lines with anthocyanin-rich skin and flesh; Del and Ros1 It can not only activate the late structural genes of anthocyanin synthesis, but also activate CHI The early structural genes for anthocyanin synthesis such as α-glucanin and β-glucanin are produced in tomatoes in amounts comparable to those of blackberries and blueberries (>2 mg / g) (see Butelli et al. 2008 ).

[0004] It is not difficult to find that when using genetic engineering methods to cultivate tomato varieties rich in anthocyanins, it is difficult for a single transcription factor to achieve the goal of increasing the anthocyanin content. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for producing a novel Medicago truncatula by using the E8 promoter and Medicago truncatula RH1 A new method for genetically creating tomato lines with high anthocyanin content.

[0006] The object of the present invention is achieved in this way: the E8 promoter is RH1 The application of the gene in creating anthocyanin-rich tomato lines, the nucleotide sequence of the E8 promoter is shown in SEQ ID No. 1, RH1 The nucleotide sequence of the gene is shown in SEQ ID No. 2. RH1 The CDS nucleotide sequence of the gene is shown in SEQ ID No. 3. The E8 promoter is linked to RH1 The CDS of the gene was combined to construct the expression vector pCAMBIA2305-E8-RH1-5xFLAG, and the expression vector was introduced into the cotyledons of tomato plants using Agrobacterium-mediated genetic transformation technology, and the expression vector was driven by the E8 promoter. RH1 The gene is highly expressed in tomato fruits, inducing anthocyanin accumulation in the fruits.

[0007] The wild tomato variety Ailsa Craig is referred to as wild tomato AC. The present invention cloned the E8 promoter from wild tomato AC and cloned the Medicago truncatula from the Medicago truncatula A17 plant. RH1 Gene, using E8 promoter and RH1 The CDS of the gene was combined to construct the expression vector pCAMBIA2305-E8-RH1-5xFLAG, and the expression vector was transformed by Agrobacterium-mediated genetic transformation and driven by the E8 promoter. RH1 The gene is highly expressed in tomato fruits, thereby achieving the accumulation of anthocyanins in the tomato fruits, and then creating a purple tomato variety rich in anthocyanins; experiments have shown that the anthocyanin content in the obtained purple tomato variety can reach about 0.3mg / g FW. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The phenotypes of wild-type tomato AC (WT strain) and transgenic tomato 1#, 2#, 3# strains are shown in Figure 2. RH1 Genetic testing, where: A is the comparison of the fruit surface color of WT strain and 1#, 2#, 3# strains; B is the comparison of the fruit cross-section color of WT strain and 1#, 2#, 3# strains; C is the comparison of the fruit cross-section color of WT strain and 1#, 2#, 3# strains RH1 Gene semi-quantitative PCR test results; D is the difference between WT strain and 1#, 2#, and 3# strains RH1 Gene quantitative PCR test results; Figure 2 The anthocyanin detection in WT and 2# strains, where: A is the liquid chromatography absorption peak diagram of anthocyanin in WT and 2# strains at 530nm; B is the quantitative detection result of anthocyanin content in WT and 2# strains; Figure 3 The map of the expression vector pCAMBIA2305-E8-RH1-5xFLAG. DETAILED DESCRIPTION

[0009] The present invention is further described below with reference to the accompanying drawings and test examples, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0010] The E8 promoter and RH1 The application of the gene in creating anthocyanin-rich tomato lines, the nucleotide sequence of the E8 promoter is shown in SEQ ID No. 1, RH1 The nucleotide sequence of the gene is shown in SEQ ID No. 2, RH1 The CDS nucleotide sequence of the gene is shown in SEQ ID No. 3. The E8 promoter is linked to RH1 The CDS of the gene was combined to construct the expression vector pCAMBIA2305-E8-RH1-5xFLAG, and the expression vector was introduced into the cotyledons of tomato plants using Agrobacterium-mediated genetic transformation technology, and the expression vector was driven by the E8 promoter. RH1 The gene is highly expressed in tomato fruits, inducing anthocyanin accumulation in the fruits.

[0011] The expression vector pCAMBIA2305-E8-RH1-5xFLAG was constructed as follows: 1) Extract DNA from young leaves of wild-type tomato AC plants; 2) Using wild-type tomato AC DNA as a template, PCR amplification of the tomato E8 promoter was performed using primer pair 1, yielding a 2200 bp amplification product. Primer pair 1 is as follows: 2305-pE8-HindⅢ-F: 5'-caggcatgcaagcttTTTGACATCCCTAATGATAT-3'; 2305-pE8-NcoⅠ-R: 5'-tcagatctaccatggCTTCTTTTGCACTGTGAATG-3'; 3) The PCR product was cloned into the linearized vector pCAMBIA2305 after double digestion with HindIII and NcoI using a one-step cloning reaction. A vector containing the E8 promoter was constructed and transformed into E. coli Top10. Positive recombinant plasmids were screened by PCR amplification and sequenced for identification. Sequencing results indicated that the recombinant plasmid E8::5xFLAG contained the E8 promoter sequence shown in SEQ ID No. 1. 4) Total RNA was extracted from the newly expanded mature compound leaves of Medicago truncatula A17 plants, and then reverse transcribed to obtain cDNA of Medicago truncatula A17; 5) Using the cDNA of Medicago truncatula A17 as a template, PCR amplification was performed using primer pair II to obtain a 732 bp PCR amplification product. Primer pair II is as follows:

[0012] 6) The PCR product was cloned into the linearized vector pCAMBIA2305-E8 after digestion with NcoI by One Step Cloning reaction to construct a vector containing RH1 The CDS sequence of the gene was expressed in the vector, which was transformed into Escherichia coli Top10. Positive recombinant plasmids were screened by PCR amplification and sequenced for identification. The sequencing results showed that the constructed vector contained the nucleotide sequence shown in SEQ ID No. 3, that is, the construction of pCAMBIA2305-E8-RH1-5xFLAG was completed.

[0013] The present invention is based on wild-type tomato AC and Medicago truncatula A17 plants. The wild-type tomato AC, Medicago truncatula A17, pCAMBIA2305 plasmid, and Agrobacterium tumefaciens EHA105 used in the research were provided by the Xishuangbanna Tropical Botanical Garden of the Chinese Academy of Sciences and are available to the public. The relevant experiments are as follows: Experimental Example 1 Construction of expression vector pCAMBIA2305-E8-RH1-5xFLAG 1. Extract DNA from young leaves of wild-type tomato AC plants; 2. Using wild-type tomato AC DNA as a template, PCR amplification of the tomato E8 promoter was performed using primer pair 1 to obtain an amplified product of approximately 2200 bp. Primer pair 1 is as follows: 2305-pE8-HindⅢ-F: 5'-caggcatgcaagcttTTTGACATCCCTAATGATAT-3'; 2305-pE8-NcoⅠ-R: 5'-tcagatctaccatggCTTCTTTTGCACTGTGAATG-3'; 3. The PCR product was cloned into the linearized vector pCAMBIA2305 after double digestion with HindIII and NcoI using a one-step cloning reaction. A vector containing the E8 promoter was constructed and transformed into E. coli Top10. Positive recombinant plasmids were screened by PCR amplification and sequenced for identification. Sequencing results showed that the recombinant plasmid E8::5xFLAG contained the E8 promoter with the nucleotide sequence shown in SEQ ID No. 1. 4. Extracting total RNA from the newly unfolded mature compound leaves of Medicago truncatula A17 plants, and then reverse-transcription to obtain cDNA of Medicago truncatula A17; 5. Using the cDNA of Medicago truncatula A17 as a template, PCR amplification was performed using primer pair II to obtain a PCR amplification product of approximately 732 bp. Primer pair II is as follows:

[0014] 6. The PCR product was cloned into the linearized vector pCAMBIA2305-E8 after digestion with NcoI by One Step Cloning reaction to construct a vector containing RH1 The CDS sequence of the gene was expressed in the vector, and E. coli Top10 was transformed. The positive recombinant plasmids were screened by PCR amplification and sequenced for identification. The sequencing results showed that the constructed vector contained the nucleotide sequence shown in SEQ ID No. 2 (i.e. RH1 The CDS sequence of the gene was obtained), and the expression vector pCAMBIA2305-E8-RH1-5xFLAG was constructed (see Figure 3 ).

[0015] Experimental Example 2 Genetic transformation of expression vector pCAMBIA2305-E8-RH1-5xFLAG in tomato 1. Material preparation 1-1. Preparation of culture medium or culture medium YEP liquid medium: Dissolve 10 g of peptone, 10 g of yeast extract, and 5 g of sodium chloride in an appropriate amount of distilled water, then dilute to 1 L with distilled water and sterilize by autoclaving at 121°C for 15 min. The pH of the following culture media or medium was adjusted to 5.8 and autoclaved at 121°C for 15 min: MS co-culture medium 1L: MS519 4.4g, sucrose 30g, sterilized, plus 2,4-D 200μL and KT 100μL; MS co-culture medium 1L: MS519 4.4g, sucrose 30g, agar 7g, sterilized with 2,4-D 200μL and KT 100μL; MS screening / germination medium 1L: MS519 4.4g, sucrose 30g, agar 7g, sterilized, add Kan 1mL (stock concentration 50mg / mL), cephalosporin 1mL, carbobenzyl 1mL (stock concentration 250mg / mL), ZT 1mL, IAA 30μL; MS bud differentiation medium 1 L: MS519 4.4 g, sucrose 30 g, agar 7 g, sterilized and added with Kan 1 mL, cephalosporin 1 mL, carbobenzyl 1 mL, ZT 0.2 mL, IAA 100 μL; 1 / 2 MS rooting medium 1 L: MS519 2.2 g, sucrose 15 g, agar 7 g, sterilized and added with Kan 1 mL, cephalosporin 1 mL, carbobenzyl 1 mL, and IAA 100 μL; The stock solution concentrations involved are: 2,4-D 1 mg / mL, KT 1 mg / mL, Kan 100 mg / mL, cephalosporin 200 mg / mL, carbenicillin 200 mg / mL, ZT 2 mg / mL, IAA 10 mg / mL; 1-2. Use wild-type tomato AC as transgenic material and set up a wild-type tomato AC control group ( Figure 1 、 2 (marked as WT); 2. Cultivate Agrobacterium containing the plant expression vector pCAMBIA2305-E8-RH1-5xFLAG in YEB liquid medium to OD600 = 1.2, collect the bacteria, and resuspend them in MS co-culture medium to OD600 = 0.4-0.6 to obtain a resuspended bacterial solution; 3. Excise the base and apex of wild-type tomato AC cotyledons to form neat wounds (total operation time ≤ 1 h), infect them with the resuspended bacterial solution obtained in step 3 for 10 min, aspirate dry, and place them with the adaxial side facing down in MS co-cultivation medium (with sterile filter paper) and culture in the dark for 2 days. 4. Transfer the cotyledon explants to MS screening / bud induction medium and culture under light for 3-6 weeks, changing the medium every 3 weeks. After buds appear on the callus tissue, transfer it to MS bud differentiation medium for bud differentiation until the main stem is formed. Cut a stem segment ≥ 2 cm and transfer it to 1 / 2 MS rooting medium to induce root development for 2-4 weeks. Wash the agar and transplant it to sterilized nutrient soil. Cover with film to keep it moist for 3-5 days, then gradually ventilate and grow the seedlings in the greenhouse. 6. Select three pCambia2305-E8-RH1-5XFLAG transgenic tomatoes (plants 1#, 2#, and 3#) and one WT plant. When the plants grow to the red fruit stage, perform phenotypic statistics. Figure 1 As shown: The skin color and cross-section color of the tomato fruit of the WT strain are both normal tomato red. Figure 1 Middle A, B; The tomato skins of plants 1#, 2#, and 3# show different shades of purple. The order of purple is from dark to light: 2#>1#>3#. Plant 3# shows yellow as the base color with a hint of purple. Figure 1 Middle A; After the tomato fruits of plants 1#, 2#, and 3# were cut cross-sectionally, the outer skin of the fruit of plant 2 was dark purple, and the surface of the section extending from the outer skin to the placenta was also dark purple; in contrast, the purple depth of the fruit of plant 1 decreased from the outer skin to the placenta, while the fruit of plant 3 was yellow from the outer skin to the placenta with some scattered purple spots. Figure 1 Middle B.

[0016] Test Example 3 RH1 Gene expression level detection Total RNA was extracted from strains 1#, 2#, 3# and WT in Experiment 2, and the cDNA obtained by reverse transcription was used as a template. RH1 Gene-specific quantitative primer pair III amplification RH1 For gene transcription products, primer pair III is as follows: RH1-RT-F: 5'-GCTTCCAGGTAGAACAGCTAAT-3'; RH1-RT-R: 5'- CACCAATTTGACTTGGAACCAT-3'; RT-PCR (Reverse Transcription Polymerase Chain Reaction) was performed using primer pair IV to amplify the tomato internal reference gene SlActin. Primer pair IV is as follows: SlActin-qF: 5'-GGATCTTGCTGGTCGTGATTT-3'; SlActin-qR: 5'-AGTCAAGAGCCACATAGGCA-3'.

[0017] The reaction system is: Template 1µL, 10× EasyTag® Buffer 1.5µL, dNTPs 1.2µL, 10µM Forward Primer 0.3µL, 10µM Forward Primer 0.3µL, EasyTag® DNA Polymerase 0.2µL, ddH2O 10.5µL; EasyTaq DNA Polymerase (EasyTaq® DNA Polymerase, AP111) is a product of Beijing Quanshijin Biotechnology.

[0018] The reaction procedure is:

[0019] The test results are as follows Figure 1 As shown in C, the WT tomato fruit did not appear RH1 Gene bands, and the tomato fruits of 1#, 2#, and 3# all showed RH1 The gene bands of strain 2# are the most obvious, while those of strains 1# and 3# are not obvious.

[0020] Further, using the cDNA obtained by reverse transcription as a template, RH1 Gene-specific quantitative primer pair V was used to amplify the RH1 gene transcript. Primer pair V is as follows: RH1-qRT-F: 5'-ACGAAAGAAGGTTGAATCAGAAGC-3'; RH1-qRT-R: 5'-CATGAACAAAATGTGAACGAGATGA-3'; The tomato internal reference gene SlActin was amplified using primer pair VI for qRT-PCR (Quantitative Real-time PCR). Primer pair VI is as follows: SlActin-qF: 5'-GGATCTTGCTGGTCGTGATTT-3'; SlActin-qR: 5'-AGTCAAGAGCCACATAGGCA-3'.

[0021] The reaction system was as follows: 2×qPCR MIX 7.5µL, 10µM Forward Primer 0.3µL, 10µM Reverse Primer 0.3µL, cDNA Template 1.5µL / 50ng, and RNase-free H2O to a total volume of 15µL. QuantFastSYBR Green qPCR SuperMix was a product of Hangzhou Maibo Biotechnology Co., Ltd.

[0022] The reaction procedure is:

[0023] The test results are as follows Figure 1 As shown in D, no RH1 Gene expression was detected in tomato fruits of 1#, 2# and 3# plants. RH1 Gene expression, of which strain 3# had a small amount RH1 Gene expression, strain 1 RH1 The gene expression level was significantly higher than that of strain 3#, and strain 2# had the highest expression level, which was nearly 6 times that of strain 1#. Figure 1 A, B, C, and D can be confirmed that the WT strain does not show purple and RH1 The purple depth of strains 1#, 2#, and 3# is related to gene expression. RH1 The gene expression levels showed a positive correlation.

[0024] Test Example 4 Anthocyanin content detection 1. Use the 2# tomato plant in Experiment 2 as the test sample and the WT plant as the control test sample; wash away impurities with distilled water immediately after collection, and absorb the moisture on the sample surface with filter paper; 2. Weigh 50-100 mg of sample respectively, place it into a 10 mL brown centrifuge tube, add steel balls, and grind it in a tissue grinder at 60 Hz for 2 minutes to obtain the test sample; 3. Ultrasonic-assisted acidification and methanol extraction: add 2 mL of hydrochloric acid / methanol (v / v = 0.1 / 99.9) to the ground test sample, ultrasonicate in a 4°C ice-water bath for 15 minutes to obtain anthocyanin extract, and place in a -20°C refrigerator in the dark overnight; 4. After centrifugation of the anthocyanin extract, the supernatant was collected for HPLC detection. The detection parameters were as follows: Waters Alliance series e2695-2998 liquid chromatography, Agilent TC-C18 column; 0.1% HCOOH (mobile phase A) and acetonitrile (mobile phase B) gradient elution; flow rate 0.8 mL / min, pressure 1000-1400 psi; elution program as shown in Table 1, quantitative loop volume 100 μL, injection volume 10-30 μL, column and detection cell temperature 35°C, diode array detector (DAD) used, wavelength 530 nm; Table 1 Elution procedure

[0025] 6. Hydrochloric acid hydrolyzes the anthocyanin extract of the sample. Rotary evaporation is stopped until the bottom of the bottle is reached. 3-4 mL of 2.5 M hydrochloric acid-methanol solution is added to the rotary evaporation bottle and the residue is quickly dissolved. The residue is completely transferred to a 10 mL brown screw-capped pressure-resistant glass bottle. A suitable magnetic stirrer is added and the polytetrafluoroethylene cap is tightened. The anthocyanin extract is steamed in a water bath at 90-92°C in the dark under magnetic stirring for 45-60 minutes and then cooled. The obtained anthocyanin sample is ready for testing. 7. Quantitative parameters: Weigh 1 mg of chromatographic standard and add 1 mL of methanol to prepare an initial standard of 1 mg / mL. Dilute the initial standard to 30 mg / L and 0.9 mg / L mixed standards A and B, respectively. Inject different volumes from 5 to 60 μL and plot a standard curve with 4 to 5 points (including zero) covering the concentration range of 0.3 to 3 mg / L in the quantitative loop. 8. Perform anthocyanin quantitative detection on the anthocyanin sample and anthocyanin standard sample according to the detection procedure in step 4; 9. Test results: After detection in step 4, the 2# tomato fruit material had obvious signal intensity at 530nm wavelength, while the WT tomato fruit material had no signal at 530nm wavelength. Figure 2 Middle A; After testing in step 8, no anthocyanins were detected in the WT tomato fruit material, while three types of anthocyanins were detected in the 2# tomato fruit material, with contents of delphinidin (Del) 0.101 mg / g FW, petunidin (Pet) 0.169 mg / g FW, and pelargonidin (Pel) 0.022 mg / g FW, totaling 0.292 mg / g FW, see Figure 2 Middle B; The results showed that the E8 promoter and RH1 Gene expression vector pCAMBIA2305-E8-RH1-5xFLAG was constructed to induce RH1 High gene expression increases the anthocyanin content in the fruit (including the peel and flesh), and can be used to obtain anthocyanin-rich tomato lines, such as strain 2#.

Claims

1. An E8 promoter and RH1 The application of the gene in creating anthocyanin-rich tomato strains is characterized by: The nucleotide sequence of the E8 promoter is shown in SEQ ID No. 1, RH1 The nucleotide sequence of the gene is shown in SEQ ID No. 2, RH1 The CDS nucleotide sequence of the gene is shown in SEQ ID No.

3. The E8 promoter is linked to RH1 The CDS of the gene was combined to construct the expression vector pCAMBIA2305-E8-RH1-5xFLAG, and the expression vector was introduced into the cotyledons of tomato plants using Agrobacterium-mediated genetic transformation technology, and the expression vector was driven by the E8 promoter. RH1 The gene is highly expressed in tomato fruits, inducing anthocyanin accumulation in the fruits.

2. The use according to claim 1, characterized in that The expression vector pCAMBIA2305-E8-RH1-5xFLAG was constructed as follows: 1) Extract DNA from young leaves of wild-type tomato AC plants; 2) Using wild-type tomato AC DNA as a template, PCR amplification of the tomato E8 promoter was performed using primer pair 1, yielding a 2200 bp amplification product. Primer pair 1 is as follows: 2305-pE8-HindⅢ-F: 5'-caggcatgcaagcttTTTGACATCCCTAATGATAT-3'; 2305-pE8-NcoⅠ-R: 5'-tcagatctaccatggCTTCTTTTGCACTGTGAATG-3'; 3) The PCR product was cloned into the linearized vector pCAMBIA2305 after double digestion with HindIII and NcoI using a one-step cloning reaction. A vector containing the E8 promoter was constructed and transformed into E. coli Top10. Positive recombinant plasmids were screened by PCR amplification and sequenced for identification. Sequencing results indicated that the recombinant plasmid E8::5xFLAG contained the E8 promoter sequence shown in SEQ ID No.

1. 4) Total RNA was extracted from the newly expanded mature compound leaves of Medicago truncatula A17 plants, and then reverse transcribed to obtain cDNA of Medicago truncatula A17; 5) Using the cDNA of Medicago truncatula A17 as a template, PCR amplification was performed using primer pair II to obtain a 732 bp PCR amplification product. Primer pair II is as follows: 2305-RH1CDS-5XFLAG NcoI F: 5'-gaggcgcgccggatcATGGCGAATACAAGCGGCGT-3'; 2305-RH1CDS-5XFLAG NcoI R:5'-catcatctttgtagtcAAGATCTCGAAGAAAT-3'; 6) The PCR product was cloned into the linearized vector pCAMBIA2305-E8 after digestion with NcoI by One Step Cloning reaction to construct a vector containing RH1 The CDS sequence of the gene was expressed in the vector, which was transformed into Escherichia coli Top10. Positive recombinant plasmids were screened by PCR amplification and sequenced for identification. The sequencing results showed that the constructed vector contained the nucleotide sequence shown in SEQ ID No. 3, that is, the construction of pCAMBIA2305-E8-RH1-5xFLAG was completed.

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