Use of WOX gene in preparing plants with increased flavonoid production

Abstract

This invention belongs to the field of bioengineering and, more specifically, relates to the use of WOX genes in producing plants with increased flavonoid production. More specifically, it relates to the use of WOX genes in producing lotus plants with increased flavonoid production. Plants produced using the methods of the present invention have increased flavonoid production and can be used as raw materials for cosmetics, enhancing benefits such as moisturizing and anti-aging effects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular, relates to the use of WOX genes in preparing plants with increased flavonoid production; more particularly, relates to the use of WOX genes in preparing lotus with increased flavonoid production. Background Art

[0002] Flavonoids are a class of polyphenolic secondary metabolites widely found in plants. They have a C6-C3-C6 benzene ring skeleton, consisting of two aromatic rings (A and B) connected by a three-carbon chain. They are important defense molecules for plants against biotic and abiotic stresses, such as ultraviolet light and pathogens.

[0003] Nelumbo nucifera, also known as lotus or lotus, is a perennial aquatic herb in the genus Nelumbo, family Nelumbonaceae. Its flavonoids, particularly those found in the lotus with two stems, are used in cosmetics for moisturizing and anti-aging effects. However, existing lotus varieties, particularly the lotus with two stems, have low flavonoid content, making them unsuitable for use as a cosmetic ingredient.

[0004] Therefore, there is a need in the art for a method of preparing a flavonoid-enhanced plant having increased flavonoid content. Summary of the Invention

[0005] In response to the shortcomings of the prior art, the present invention aims to provide a method for producing plants with increased flavonoid production. The present invention has creatively discovered that WOX genes, which are used to regulate plant growth and development, can increase plant flavonoid production, particularly in lotus plants.

[0006] In view of this, in a first aspect, the present invention provides use of a WOX gene in preparing a plant with increased flavonoid production.

[0007] The plant prepared by the method of the present invention has increased flavonoid compound production, and can be better used as a raw material for cosmetics to enhance effects such as moisturizing and anti-aging.

[0008] Furthermore, the WOX gene is a WOX gene from lotus.

[0009] Furthermore, the plant is a lotus, and further, the plant is a double lotus.

[0010] Furthermore, the WOX gene has a nucleotide sequence as shown in SEQ ID NO.1 or 2, or a nucleotide sequence having more than 80% identity with the nucleotide sequence as shown in SEQ ID NO.1 or 2, illustratively, a nucleotide sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence as shown in SEQ ID NO.1 or 2.

[0011] Furthermore, the WOX gene encodes a protein comprising an amino acid sequence as shown in SEQ ID NO. 3 or 4, or encodes a protein comprising an amino acid sequence having more than 80% identity with the amino acid sequence as shown in SEQ ID NO. 3 or 4, illustratively, a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the protein sequence as shown in SEQ ID NO. 3 or 4.

[0012] In some specific embodiments, the primers used to amplify the WOX gene are shown as SEQ ID NOs. 5-6, or SEQ ID NOs. 7-8.

[0013] In a second aspect, the present invention provides an expression cassette for increasing the yield of flavonoid compounds, wherein the expression cassette is composed of a promoter, a WOX gene and a terminator connected in sequence, wherein the nucleotide sequence of the WOX gene is shown in SEQ ID NO. 1 or 2.

[0014] Furthermore, in the expression cassette, the promoter may use any common promoter in the art. In a specific embodiment, the promoter is the S35 promoter.

[0015] Furthermore, in the expression cassette, the terminator may use any common promoter in the art. In a specific embodiment, the terminator is the E9 terminator.

[0016] In a third aspect, the present invention provides an expression vector, characterized in that the expression vector contains the above-mentioned expression cassette for increasing the production of flavonoid compounds.

[0017] In some specific embodiments, the expression vector can be pCAMBIA1300, pEAQ, or pCXSN vector.

[0018] In a fourth aspect, the present invention further provides a host cell, characterized in that the host cell contains the above-mentioned expression cassette or expression vector.

[0019] In some specific embodiments, the host cell is a prokaryotic cell.

[0020] In some specific embodiments, the host cell is an Escherichia coli or Agrobacterium cell.

[0021] In a specific embodiment, the host cell is Agrobacterium rhizogenes A4.

[0022] In a fifth aspect, the present invention also provides a method for producing transgenic plants, characterized in that the above-mentioned expression cassette, or the above-mentioned expression vector, or the above-mentioned host cell is transformed into a plant to obtain a transformed plant cell or tissue, and then the transformed plant cell or tissue is cultured into a transgenic plant.

[0023] In some specific embodiments, the plant of the present invention can be lotus. Further, the tissue can be a sterile seedling of lotus.

[0024] In some specific embodiments, the plant of the present invention may be a lotus with two stems.

[0025] In some specific embodiments, the present invention provides a method for producing a transgenic plant, comprising:

[0026] Obtain the WOX gene from lotus;

[0027] preparing an expression vector containing the lotus WOX gene;

[0028] transforming the expression vector into Agrobacterium rhizogenes;

[0029] Infecting lotus tissue with Agrobacterium rhizogenes carrying the expression vector; and

[0030] The transgenic plants were obtained by continuing the cultivation.

[0031] Furthermore, the WOX gene of the lotus has a nucleotide sequence as shown in SEQ ID NO. 1 or 2.

[0032] Furthermore, the WOX gene of lotus is obtained by amplification using primers shown in SEQ ID NOs. 5-6, or SEQ ID NOs. 7-8.

[0033] Furthermore, the expression vector is pCAMBIA1300.

[0034] Furthermore, the Agrobacterium rhizogenes is Agrobacterium rhizogenes A4.

[0035] The present invention also provides the use of the above-mentioned expression cassette, expression vector, host cell and method for producing transgenic plants in producing plants with increased flavonoid yield.

[0036] Furthermore, the plant with increased flavonoid production is lotus.

[0037] Furthermore, the plant with increased flavonoid production is lotus.

[0038] The present invention unexpectedly discovered that by constructing an expression cassette comprising a nucleotide sequence as shown in SEQ ID NO. 1 or 2 and transforming lotus using Agrobacterium-mediated method, a lotus transformation event with increased flavonoid production was obtained.

[0039] Furthermore, the present invention also provides a primer pair for detecting the transformation event or expression cassette, wherein the primer pair is:

[0040] The nucleotide sequence shown in SEQ ID NO.9-10; or

[0041] The nucleotide sequences shown in SEQ ID NOs. 11-12. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Homology analysis of WOX genes in Lotus and Arabidopsis;

[0043] Figure 2 This is a heat map of flavonoid production in transgenic plants. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0045] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution. Unless otherwise specified, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0046] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), indicating that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0047] The term "WOX gene" refers to the nucleotide sequence encoding a WOX protein. WOX (WUSCHEL-related homeobox) proteins are a family of plant-specific transcription factors belonging to the homeobox gene superfamily. Their name derives from the first member discovered, WUSCHEL (WUS), which plays a key role in maintaining stem cells in the shoot apical meristem. WOX family members participate in multiple key processes in plant development by regulating the expression of downstream genes.

[0048] The term "identity" refers to sequence similarity to natural amino acid or nucleotide sequences. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0049] The term "transformation event" refers to the process by which exogenous DNA (such as plasmids or linear DNA fragments) is introduced into host cells and expressed or integrated. Transformation can be divided into two types: stable transformation, in which the exogenous DNA integrates into the host genome and is inherited by progeny as the host cell divides; and transient transformation, in which the exogenous DNA is not integrated into the genome and is only expressed in the host cell for a short period of time (usually lasting several days to weeks).

[0050] Below, the embodiment of the present application is described. The embodiment described below is exemplary and is only used to explain the present application, and is not to be construed as limiting the present application. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0051] Example 1. Preparation of the expression cassette of the present invention

[0052] Using the Arabidopsis AtWOX13 (AT4G35550) protein sequence as bait, we searched for homologous proteins in lotus and performed homology analysis using MEGA. We obtained the most homologous genes NnWOX13-1 (Nn6g31691) and NnWOX13-2 (Nn5g30993) in lotus. The homology analysis showed that Figure 1 shown.

[0053] The full length of the CDS sequence of NnWOX13-1 is 840 bp, encoding 279 AA amino acids. The relative molecular mass of the protein is 31710.67 Da, and the molecular formula is C 1373 H 2173 N 391 O440 S 16 , with a total of 4393 atoms and an isoelectric point (PI) of 5.65. The nucleotide sequence of NnWOX13-1 is shown in SEQ ID NO.1:

[0054] ATGGGTACAATACGAAATGGTGGAGAGACAAAAGAGGAGATGAAGATGATGGAATGGGAGAAGCAAGAGCAGCAGCAGCAGCAAAATGGTGTGATGATGTACGTGAAGGTTATGACAGATGAGCAGATGGAGCTTCTTCGGAAACAAATTTCTATTTATGCCACCATTTGTGAGGAGCTCGTCGAGATGCACAAAGCCATTACTGCTCAACGGGATCTCGCTGGAATGAGGCTGGGAAATCTGTACTTTGATCCATTAATGACTTCATCTGGTCACAAGATAACTGCAAGGCAGCGGTGGACCCCTGCACCCATTCAGCTCCAAATTCTTGAACGTATTTTTGATCAAGGCAATGGGACTCCTAGCAAGCAGAAGATCAAAGAGATAACTTCTGAACTGGCACAACATGGCCAAATTTCTGAAACAAATGTTTATAACTGGTTCCAGAACAGGAGGGCTCGATCAAAGAGGAAGCAACAAGTTGCAGTGCCAAACAATGCAGAATCAGAAATAGAGATAGAAGTTGAGTCCCCAGATGAAAAGAAGGCAAAGCCAGAGATCATTCACTTGCATGAGAATCAAGCTCCAAGTGCTGAGGACATGTGCTACCAAAGTCCTGGGATAATCTCTGCACTGCATTCCGTGGATCCACAGCATAATAAAGCAGAATCCATATTCTCATCAGATAATACTTCAAAATCTTCTGTCAGCTTGAGTCACTTGTCTTTCTATGAGAGTGTACTATCAAATTCAGGGTATGATCATTTGATTGGAAAAATGGAAGGCCCAGGGAGCTTTAATCCTTACCGGCAGGGGGAGGGCTATGACATGATCGGATGA (SEQ ID NO.1);

[0055] The amino acid sequence is shown in SEQ ID NO.3:

[0056] MGTIRNGGETKEEMKMMEWEKQEQQQQQNGVMMYVKVMTDEQMELLRKQISIYATICEELVEMHKAITAQRDLAGMRLGNLYFDPLMTSSGHKITARQRWTPAPIQLQILERIFDQGNGTPSKQKIKEITSELAQHGQISE TNVYNWFQNRRARSKRKQQVAVPNNAESEIEIEVESPDEKKAKPEIIHLHENQAPSAEDMCYQSPGIISALHSVDPQHNKAESIFSSDNTSKSSVSLSHLSFYESVLSNSGYDHLIGKMEGPGSFNPYRQGEGYDMIG (SEQ ID NO.3).

[0057] The CDS of NnWOX13-2 is 807 bp in length, encoding 268 amino acids. The protein has a molecular weight of 30902.74 Da and a molecular formula of C 1340 H 2120 N 386 O 426 S 14 The total number of atoms is 4286 and the isoelectric point (PI) is 6.08. The nucleotide sequence of NnWOX13-2 is shown in SEQ ID NO.2:

[0058] ATGGGTACGGTAAGAAATGCTGTAGAAGGGGAAGAGAAGGTAAGGATAATGGAGTGGGAGAAACAAGAGCAGCAGCAGCAGCAGCCGCAGCAACAGCAAAATGCTGGGGTGATGTACGTGAAAGTTATGACAGATGAGCAGATGGAGCTGCTTCGGAAACAAATCTCTATTTACGCTACCATTTGTGAGCAGCTCGTCGAGATGCACAAAGCCATTACTGCTCAGCAGGATCTCGCTGGAATGAGGCTGGGAAATCTATATTGTGACCCACTGATGGCATCATCTGGACACAAAATAACTGCAAGGCAGCGATGGACTCCTACACCCATCCAGCTCCAAATTCTTGAACGTATCTTTGATCAAGGCAATGGGACTCCCAGCAAGCAGAAGATCAAAGAGATAACTTCTGAGTTGACACAACATGGACAGATTTCTGAAACAAATGTTTATAACTGGTTCCAGAACAGGAGGGCTAGATCAAAAAGGAAGCAACAGGTTTCGATACCAAATAATGCGGAATCGGAAGTGGAGACAGAGGTAGAGTCCCCAAAGGAAAAGAAGACAAAGCCAGAGAACATTCACTTTCATGAGAATCAAGCTCAAAAGGCTGAGGACCTATGCTTCCAAAGTGCTGAGATAAGCTCTGAACTGCACTCCTTAGATCCACATCCAACTAAAATGGAGGCCACGTTCCCATTGGATGGTACTTCAAAATCTTCTGGCAGTTTGACCCACATGTCTTTCTATGAGAGTGTGCTATCTAACCCAATTATGTATTGGCATAACTTTCATTCCAAAGAACAATGA (SEQ ID NO.2);

[0059] The amino acid sequence is shown in SEQ ID NO.4:

[0060] MGTVRNAVEGEEKVRIMEWEKQEQQQQQPQQQQNAGVMYVKVMTDEQMELLRKQISIYATICEQLVEMHKAITAQQDLAGMRLGNLYCDPLMASSGHKITARQRWTPTPIQLQILERIFDQGGNTPSKQKIKEITS ELTQHGQISETNVYNWFQNRRARSKRKQQVSIPNNAESEVETEVESPKEKKTKPENIHFHENQAQKAEDLCFQSAEISSELHSLDPHPTKMEATFPLDGTSKSSGSLTHMSFYESVLSNPIMYWHNFHSKEQ (SEQ ID NO.4).

[0061] Using cDNA reverse-transcribed from total RNA of lotus as template, the following primers were used for PCR reaction on the conserved sequence to obtain NnWOX13-1 (A) and NnWOX13-2 (B);

[0062] F1: gaattcATGGGTACGGTAAGAAATGCTGTAGAAG (SEQ ID NO.5);

[0063] R1: aagcttTCATTGTTCTTTGGAATGAAAGTTATGCCAA (SEQ ID NO. 6);

[0064] F2: gaattcATGGGTACAATACGAAATGGTGGAGAG (SEQ ID NO.7);

[0065] R2: aagcttTCATCCGATCATGTCATAGCCCT (SEQ ID NO. 8).

[0066] The gene DNA fragments A and B were recovered by gel cutting and DNA fragments A and B and pCAMBIA1300 vector were used EcoR I and Hind III Restriction enzyme digestion was performed, and finally the A and B digestion products were purified by EcoR I and HindThe two sites of the 35S promoter were ligated into the pCAMBIA1300 vector driven by the 35S promoter, forming the NnWOX13-1::pCAMBIA1300 and NnWOX13-2::pCAMBIA1300 recombinant vectors (NnWOX13-1 and NnWOX13-2 recombinant vectors). After transformation into competent DH5a E. coli, positive clones were detected using the F1 / R1 and F2 / R2 primer pairs, respectively. These clones were sent to a sequencing company for sequence analysis to confirm the correctness of the NnWOX13-1 and NnWOX13-2 expression clone sequences. This demonstrated the successful preparation of the expression cassettes of the present invention.

[0067] Example 2: Preparation of Lotus Containing Transformation Events of the Present Invention

[0068] The NnWOX13-1 and NnWOX13-2 recombinant vectors prepared in Example 1 were transformed into Agrobacterium rhizogenes A4, and after the colonies were identified by PCR with primers F1 / R1 and F2 / R2, they were used to infect the sterile seedlings formed in the lotus sterile system to induce the production of lotus hairy roots (hairy roots) with overexpression of NnWOX13-1 and NnWOX13-2 genes. 600 Agrobacterium rhizogenes were harvested by centrifugation at a concentration of 0.6, resuspended in YEP liquid medium, and supplemented with a final concentration of 0.1 mM acetoeugenol. The bacterial suspension was incubated with wounded, sterile lotus stems for 2 hours. The cells were then transferred to MS solid medium containing a gradient of CEF concentrations of 0.5, 0.3, and 0.1 mg / L to remove the Agrobacterium rhizogenes. The CEF concentration was generally reduced every 10 days until no Agrobacterium rhizogenes contamination was present. Lotus hairy roots transformed with an empty vector and Arabidopsis thaliana transformed with the WOX gene served as controls. Genomic DNA was extracted from all transformed tissues for positive results, and primers were used to verify successful introduction of the target gene into the hairy roots.

[0069] The expression levels of NnWOX13-1 and NnWOX13-2 in the induced hairy roots were detected by RT-qPCR using qPCR-F3 / R3 and qPCR-F4 / R4, respectively, and the hairy roots with expression levels significantly different from the control were screened for future use.

[0070] qPCR-F3: TTATGACAGATGAGCAGATGGAGCT (SEQ ID NO.9);

[0071] qPCR-R3:TCATTCCAGCGAGATCCTGCT (SEQ ID NO.10);

[0072] qPCR-F4:CTTGAACGTATTTTTGATCAAGGCAATGG (SEQ ID NO. 11);

[0073] qPCR-R4: CTGGAACCAGTTATAAACATTTGTTTCAGAAATTTG (SEQ ID NO. 12).

[0074] Example 3: Measurement of flavonoids

[0075] The total flavonoid content of lotus seed core extract was determined using the Al(NO3)3-NaNO2 method. Accurately measure 0, 25, 50, 100, 150, and 200 μL of a 1 mg / mL rutin standard solution into a 2 mL centrifuge tube. Add 50 μL each of 5% NaNO2 solution and 10% Al(NO3)3 solution, mix thoroughly, and let stand for 6 minutes. Finally, add 500 μL of 5% NaOH solution, mix thoroughly, and let stand for 15 minutes. The reaction volume was adjusted to 1 mL with 75% ethanol. The absorbance of the solution was measured at 510 nm. A standard curve was constructed with rutin concentration (mg / L) as the abscissa (X) and absorbance as the ordinate (Y). 400 μL of the plant tissue extracts from the examples and comparative examples were also measured at 510 nm using the same procedure.

[0076] The linear regression equation for the standard curve was: Y = 0.0076X + 0.0064 (R² = 0.9997), showing good linearity within the rutin concentration range of 25 to 200 mg / L. The total flavonoid content in the plant tissue extracts of the Examples and Comparative Examples was calculated based on the regression equation. All results were obtained by performing three biological replicates.

[0077] The results are shown in Table 1. The product thermodynamic diagram is shown in Figure 2 shown.

[0078] Table 1

[0079]

[0080] Nn1: Arabidopsis tissue extract (control group 1);

[0081] Nn2: lotus tissue extract prepared by the present invention (the present invention group);

[0082] Nn3: extract from common lotus tissue (control group 2).

[0083] As can be seen from Table 1:

[0084] Incremental advantage of tea catechins: Compared with the Arabidopsis extract (Nn1) with this transformation event, the lotus tissue extract (Nn2) prepared by the present invention increases the content of 12 catechins and their derivatives by 1.8-5.6 times, among which the three core components, including gallocatechin, gallate (EGCG), and catechin trimer, all reach more than 3.2 times that of the Nn3 group.

[0085] Synergistic enhancement phenomenon: The hyperoside-quercetin complex showed synchronous enrichment of 26 homologues in Nn2, and its total content was increased by 217% compared with Nn3 (p<0.01), and 4 new methylation modification products were detected.

[0086] Specific metabolites: Five characteristic derivatives such as ferulic acid were only detected in the Nn2 group with significant expression (detection limit > 10 μg / g), as shown in Table 2.

[0087] Table 2

[0088]

[0089] Therefore, it can be seen that the lotus tissue extract prepared in the present invention has an increased yield of flavonoids.

[0090] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. Use of a WOX gene, an expression cassette comprising a WOX gene, an expression vector, or a host cell in preparing a plant with increased flavonoid production, characterized in that: The WOX gene encodes a protein with an amino acid sequence as shown in SEQ ID NO. 3 or 4; wherein the plant is lotus.

2. The use according to claim 1, characterized in that The primers used to amplify the WOX gene are shown in SEQ ID NOs. 5-6, or SEQ ID NOs. 7-8.

3. The use according to claim 1, characterized in that The expression cassette comprising the WOX gene is formed by sequentially connecting a promoter, a WOX gene and a terminator, wherein the nucleotide sequence of the WOX gene is shown as SEQ ID NO. 1 or 2.

4. The use according to any one of claims 1 to 3, characterized in that The primer pairs for detecting the expression cassette are: The nucleotide sequence shown in SEQ ID NO.9-10; or The nucleotide sequences shown in SEQ ID NOs. 11-12.

5. The use according to any one of claims 1 to 3, characterized in that The host cell is Escherichia coli or Agrobacterium cell.

6. A method for producing a transgenic plant with increased flavonoid production, characterized in that: The WOX gene is transformed into lotus to obtain transformed lotus cells or tissues, and then the transformed lotus cells or tissues are cultured into transgenic lotus, wherein the WOX gene encodes a protein with an amino acid sequence as shown in SEQ ID NO. 3 or 4.

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