Application of WOX gene in preparation of plants with increased yield of flavonoid compounds

Abstract

The invention belongs to the field of bioengineering, and particularly relates to application of a WOX gene in preparation of plants with increased yield of flavonoid compounds. More specifically, the invention relates to application of a WOX gene in preparation of lotus with increased yield of flavonoid compounds. The plant prepared by the method disclosed by the invention has the advantages that the yield of flavonoid compounds is increased, the plant can be better used as a raw material of cosmetics, and the effects of moisturizing, anti-aging and the like are improved.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. Specifically, it relates to the use of the WOX gene in the preparation of plants with increased flavonoid production; more specifically, it relates to the use of the WOX gene in the preparation of lotus plants with increased flavonoid production. Background Art

[0002] Flavonoid compounds are a class of polyphenolic secondary metabolites widely present in plants, with a C6-C3-C6 benzene ring backbone structure, formed by connecting two aromatic rings (ring A and ring B) through a three-carbon chain. They are important defense molecules for plants to cope with biotic and abiotic stresses (such as ultraviolet rays, pathogens).

[0003] Lotus, scientific name: Nelumbo nucifera, also known as lotus flower, is a perennial aquatic herb of the genus Nelumbo in the family Nelumbonaceae. Because of its flavonoid compounds, especially the flavonoids in the twin lotus, it is used in cosmetics (moisturizing, anti-aging). However, in existing lotus varieties, especially the twin lotus, the content of flavonoid compounds is relatively low, which is not conducive to being used as a raw material for cosmetics.

[0004] Therefore, there is a need in the art for a method for preparing plants with increased flavonoid production, which have increased flavonoid compounds. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing plants with increased flavonoid production, which have increased flavonoid compounds. The applicant has creatively found that the WOX gene used to regulate plant growth and development has the effect of increasing the flavonoid production of plants, especially the effect of increasing the flavonoid production in lotus.

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

[0007] The plants prepared by the method of the present invention have increased flavonoid production, and can be better used as raw materials for cosmetics, increasing effects such as moisturizing and anti-aging.

[0008] Further, the WOX gene is the WOX gene from lotus.

[0009] Further, the plant is lotus, and more specifically, the plant is twin 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 shown in SEQ ID NO.1 or 2. Exemplarily, a nucleotide sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence 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 shown in SEQ ID NO.3 or 4. Exemplarily, a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the protein sequence shown in SEQ ID NO.3 or 4.

[0012] In some specific embodiments, the primers for amplifying the WOX gene are as shown in SEQ ID NO.5 - 6, or SEQ ID NO.7 - 8.

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

[0014] Further, in the expression cassette, any common promoter in the art can be used as the promoter. In a specific embodiment, the promoter is the S35 promoter.

[0015] Further, in the expression cassette, any common terminator in the art can be used as the terminator. 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 yield of flavonoid compounds.

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

[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 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 a transgenic plant, characterized in that the above expression cassette, or the above expression vector, or the above 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 may be lotus. Further, the tissue may be a sterile seedling of lotus.

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

[0025] In some specific embodiments, the present invention provides a method for producing a transgenic plant, comprising: Obtaining the WOX gene of lotus; Preparing an expression vector containing the WOX gene of lotus; Transforming the expression vector into Agrobacterium rhizogenes; Using Agrobacterium rhizogenes with the expression vector to infect the tissue of lotus; and Continuing to culture to obtain a transgenic plant.

[0026] Further, the WOX gene of lotus has a nucleotide sequence as shown in SEQ ID NO.1 or 2.

[0027] Further, the obtaining of the WOX gene of lotus is obtained by amplification with primers as shown in SEQ ID NO.5 - 6, or SEQ ID NO.7 - 8.

[0028] Further, the expression vector is pCAMBIA1300.

[0029] Further, the Agrobacterium rhizogenes is Agrobacterium rhizogenes A4.

[0030] The present invention also provides the application of the above expression cassette, expression vector, host cell and method for producing a transgenic plant in producing a plant with an increased yield of flavonoids.

[0031] Further, the plant with an increased yield of flavonoids is lotus.

[0032] Even further, the plant with an increased yield of flavonoids is a twin lotus.

[0033] The present invention unexpectedly discovers that an expression cassette containing a nucleotide sequence as shown in SEQ ID NO.1 or 2 is constructed, and lotus is transformed by the Agrobacterium-mediated method to obtain a lotus transformation event with an increased yield of flavonoids.

[0034] Furthermore, the present invention also provides primer pairs for detecting the transformation event or the expression cassette, and the primer pairs are: The nucleotide sequences shown in SEQ ID NO.9 - 10; or The nucleotide sequences shown in SEQ ID NO.11 - 12. Description of the Drawings

[0035] Figure 1 For the homology analysis of the WOX genes of lotus and Arabidopsis thaliana; Figure 2 For the heat map of the flavonoid yield of transgenic plants. Detailed Embodiments

[0036] The present invention will be specifically described below in combination with specific implementation schemes and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation schemes and examples are used to illustrate the present invention, rather than limiting the present invention.

[0037] If there is no special instruction, all implementation manners and optional implementation manners of the present application can be combined with each other to form a new technical solution. If there is no special instruction, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0038] If there is no special instruction, all steps of the present application can be carried out in sequence or randomly, and preferably in sequence. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out in sequence, or can also include steps (b) and (a) carried out in sequence. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c), or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0039] The term "WOX gene" refers to the nucleotide sequence encoding the WOX protein. The WOX (WUSCHEL-related homeobox) protein is a plant-specific transcription factor family and belongs to the homeobox gene superfamily. Its name is derived from the first discovered member, WUSCHEL (WUS), which plays a key role in the maintenance of stem cells in the shoot apical meristem. Members of the WOX family are involved in multiple key processes of plant development by regulating the expression of downstream genes.

[0040] The term "identity" is the sequence similarity to a natural amino acid or nucleotide sequence. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0041] The term "transformation event" refers to the process of introducing exogenous DNA (such as plasmids, linear DNA fragments, etc.) into a host cell and enabling its expression or integration. Transformation can be divided into two types: Stable Transformation: The exogenous DNA integrates into the host genome and is inherited by offspring with the division of the host cell; and Transient Transformation: The exogenous DNA does not integrate into the genome and is only expressed in the host cell for a short period (usually lasting from several days to several weeks).

[0042] Hereinafter, examples of the present application will be described. The examples described below are exemplary and are only used to explain the present application and should not be construed as a limitation of the present application. For those not specified in the examples regarding specific techniques or conditions, the techniques or conditions described in the literature in the relevant field or according to the product specifications shall be followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0043] Example 1. Preparation of the expression cassette of the present invention Using the Arabidopsis thaliana AtWOX13 (AT4G35550) protein sequence as a bait, by retrieving the homologous protein in lotus and performing a homology analysis through MEGA, the genes NnWOX13-1 (Nn6g31691) and NnWOX13-2 (Nn5g30993) with the highest homology in lotus were obtained. The homology analysis is as Figure 1 shown.

[0044] The full length of the CDS of the expression 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 O 440 S16 , the total number of atoms is 4393, and the isoelectric point (PI) is 5.65. The nucleotide sequence of NnWOX13-1 is shown in SEQ ID NO.1: ATGGGTACAATACGAAATGGTGGAGAGACAAAAGAGGAGATGAAGATGATGGAATGGGAGAAGCAAGAGCAGCAGCAGCAGCAAAATGGTGTGATGATGTACGTGAAGGTTATGACAGATGAGCAGATGGAGCTTCTTCGGAAACAAATTTCTATTTATGCCACCATTTGTGAGGAGCTCGTCGAGATGCACAAAGCCATTACTGCTCAACGGGATCTCGCTGGAATGAGGCTGGGAAATCTGTACTTTGATCCATTAATGACTTCATCTGGTCACAAGATAACTGCAAGGCAGCGGTGGACCCCTGCACCCATTCAGCTCCAAATTCTTGAACGTATTTTTGATCAAGGCAATGGGACTCCTAGCAAGCAGAAGATCAAAGAGATAACTTCTGAACTGGCACAACATGGCCAAATTTCTGAAACAAATGTTTATAACTGGTTCCAGAACAGGAGGGCTCGATCAAAGAGGAAGCAACAAGTTGCAGTGCCAAACAATGCAGAATCAGAAATAGAGATAGAAGTTGAGTCCCCAGATGAAAAGAAGGCAAAGCCAGAGATCATTCACTTGCATGAGAATCAAGCTCCAAGTGCTGAGGACATGTGCTACCAAAGTCCTGGGATAATCTCTGCACTGCATTCCGTGGATCCACAGCATAATAAAGCAGAATCCATATTCTCATCAGATAATACTTCAAAATCTTCTGTCAGCTTGAGTCACTTGTCTTTCTATGAGAGTGTACTATCAAATTCAGGGTATGATCATTTGATTGGAAAAATGGAAGGCCCAGGGAGCTTTAATCCTTACCGGCAGGGGGAGGGCTATGACATGATCGGATGA (SEQ ID NO.1); The amino acid sequence is shown in SEQ ID NO.3: MGTIRNGGETKEEMKMMEWEKQEQQQQQNGVMMYVKVMTDEQMELLRKQISIYATICEELVEMHKAITAQRDLAGMRLGNLYFDPLMTSSGHKITARQRWTPAPIQLQILERIFDQGNGTPSKQKIKEITSELAQHGQISETNVYNWFQNRRARSKRKQQVAVPNNAESEIEIEVESPDEKKAKPEIIHLHENQAPSAEDMCYQSPGIISALHSVDPQHNKAESIFSSDNTSKSSVSLSHLSFYESVLSNSGYDHLIGKMEGPGSFNPYRQGEGYDMIG (SEQ ID NO.3).

[0045] The full-length CDS of the expression sequence of NnWOX13-2 is 807 bp, encoding 268 amino acids. The relative molecular mass of the protein is 30902.74 Da, and the molecular formula is C 1340 H 2120 N 386 O 426 S 14 , with a total of 4286 atoms and an isoelectric point (PI) of 6.08. The nucleotide sequence of NnWOX13-2 is shown in SEQ ID NO.2: ATGGGTACGGTAAGAAATGCTGTAGAAGGGGAAGAGAAGGTAAGGATAATGGAGTGGGAGAAACAAGAGCAGCAGCAGCAGCAGCCGCAGCAACAGCAAAATGCTGGGGTGATGTACGTGAAAGTTATGACAGATGAGCAGATGGAGCTGCTTCGGAAACAAATCTCTATTTACGCTACCATTTGTGAGCAGCTCGTCGAGATGCACAAAGCCATTACTGCTCAGCAGGATCTCGCTGGAATGAGGCTGGGAAATCTATATTGTGACCCACTGATGGCATCATCTGGACACAAAATAACTGCAAGGCAGCGATGGACTCCTACACCCATCCAGCTCCAAATTCTTGAACGTATCTTTGATCAAGGCAATGGGACTCCCAGCAAGCAGAAGATCAAAGAGATAACTTCTGAGTTGACACAACATGGACAGATTTCTGAAACAAATGTTTATAACTGGTTCCAGAACAGGAGGGCTAGATCAAAAAGGAAGCAACAGGTTTCGATACCAAATAATGCGGAATCGGAAGTGGAGACAGAGGTAGAGTCCCCAAAGGAAAAGAAGACAAAGCCAGAGAACATTCACTTTCATGAGAATCAAGCTCAAAAGGCTGAGGACCTATGCTTCCAAAGTGCTGAGATAAGCTCTGAACTGCACTCCTTAGATCCACATCCAACTAAAATGGAGGCCACGTTCCCATTGGATGGTACTTCAAAATCTTCTGGCAGTTTGACCCACATGTCTTTCTATGAGAGTGTGCTATCTAACCCAATTATGTATTGGCATAACTTTCATTCCAAAGAACAATGA (SEQ ID NO.2); The amino acid sequence is shown in SEQ ID NO.4: MGTVRNAVEGEEKVRIMEWEKQEQQQQQPQQQQNAGVMYVKVMTDEQMELLRKQISIYATICEQLVEMHKAITAQQDLAGMRLGNLYCDPLMASSGHKITARQRWTPTPIQLQILERIFDQGNGTPSKQKIKEITSELTQHGQISETNVYNWFQNRRARSKRKQQVSIPNNAESEVETEVESPKEKKTKPENIHFHENQAQKAEDLCFQSAEISSELHSLDPHPTKMEATFPLDGTSKSSGSLTHMSFYESVLSNPIMYWHNFHSKEQ (SEQ ID NO.4).

[0046] Using the cDNA reverse transcribed from the total RNA of lotus as a template, the following primers were selected to perform PCR reactions on the conserved sequences to obtain NnWOX13-1 (A) and NnWOX13-2 (B); F1: gaattcATGGGTACGGTAAGAAATGCTGTAGAAG (SEQ ID NO.5); R1: aagcttTCATTGTTCTTTGGAATGAAAGTTATGCCAA (SEQ ID NO.6); F2: gaattcATGGGTACAATACGAAATGGTGGAGAG (SEQ ID NO.7); R2: aagcttTCATCCGATCATGTCATAGCCCT (SEQ ID NO.8).

[0047] By cutting and recovering the gene DNA fragments A and B, the DNA fragments A and B and the pCAMBIA1300 vector were digested with EcoR I and Hind III restriction enzymes. Finally, the digested products of A and B were purified and ligated into the pCAMBIA1300 vector driven by the 35S promoter through the EcoR I and Hind III sites to form the NnWOX13-1::pCAMBIA1300 and NnWOX13-2::pCAMBIA1300 recombinant vectors (NnWOX13-1 recombinant vector and NnWOX13-2 recombinant vector). After transformation of DH5a Escherichia coli competent cells, positive clone detection was performed using the F1 / R1 and F2 / R2 primer pairs respectively, and the positive clones were sent to a sequencing company for sequence determination to confirm the correctness of the NnWOX13-1 and NnWOX13-2 expression clone sequences. It was shown that the expression cassette of the present invention was successfully prepared.

[0048] Example 2. Preparation of Lotus Containing Transformation Events of the Present Invention The recombinant vectors NnWOX13-1 and NnWOX13-2 prepared in Example 1 above were separately transformed into Agrobacterium rhizogenes A4. After correct identification of PCR colonies with the primer pairs F1 / R1 and F2 / R2, the sterile seedlings formed in the sterile system of lotus were infected to induce the overexpression of NnWOX13-1 and NnWOX13-2 genes in lotus hairy roots (hairy roots). The Agrobacterium rhizogenes with an OD 600 of 0.6 was centrifuged and collected, resuspended in YEP liquid medium, and acetosyringone with a final concentration of 0.1 mM was added. The bacterial solution was incubated with the wounded sterile lotus stems for 2 h, and then transferred to MS solid medium containing cef at concentration gradients of 0.5, 0.3, and 0.1 mg / L to remove Agrobacterium rhizogenes. Generally, the cef concentration was decreased by one level every 10 days until there was no contamination of Agrobacterium rhizogenes. The lotus hairy roots obtained by transforming the empty vector and the Arabidopsis thaliana transformed with the WOX gene in Arabidopsis thaliana were used as control materials. Genomic DNA was extracted from all the transformed tissues for positive detection, and primers were used to identify whether the target genes were successfully introduced into the hairy roots respectively.

[0049] 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 significant differences in expression levels compared with the control were screened and reserved.

[0050] qPCR-F3: TTATGACAGATGAGCAGATGGAGCT (SEQ ID NO.9); qPCR-R3: TCATTCCAGCGAGATCCTGCT (SEQ ID NO.10); qPCR-F4: CTTGAACGTATTTTTGATCAAGGCAATGG (SEQ ID NO.11); qPCR-R4: CTGGAACCAGTTATAAACATTTGTTTCAGAAATTTG (SEQ ID NO.12).

[0051] Example 3. Measurement of Flavonoids The total flavonoid content of the lotus seed core extract was determined by the Al(NO3)3-NaNO2 method. Accurately measure 0, 25, 50, 100, 150, and 200 μL of rutin standard solution with a mass concentration of 1 mg / mL into 2 mL centrifuge tubes. Sequentially add 50 μL of 5% NaNO2 solution and 50 μL of 10% Al(NO3)3 solution. After adding, mix well and let stand for 6 min; finally, add 500 μL of 5% NaOH solution, mix well and let stand for 15 min; use 75% ethanol solution to make the final volume of the reaction system up to 1 mL. Measure the absorbance value of the solution at a wavelength of 510 nm. Take the rutin mass concentration (mg / L) as the abscissa (X) and the absorbance value as the ordinate (Y) to draw a standard curve. And take 400 μL of the plant tissue extracts of the examples and comparative examples, and measure the absorbance value of the solution at a wavelength of 510 nm according to the above steps.

[0052] The linear regression equation of the standard curve is: Y = 0.0076X + 0.0064 (R2 = 0.9997), and the linear relationship is good in the range of rutin mass concentration from 25 to 200 mg / L. Calculate the total flavonoid content in the plant tissue extracts of the examples and comparative examples according to the regression equation. All results were repeated in 3 biological experiments.

[0053] The results are shown in Table 1. The heat map of its product is as Figure 2 shown.

[0054] Table 1

[0055] Nn1: Arabidopsis thaliana tissue extract (control group 1); Nn2: Lotus tissue extract prepared by the present invention (the present invention group); Nn3: Common lotus tissue extract (control group 2).

[0056] As can be seen from Table 1: Advantage of catechin increment: Compared with the Arabidopsis thaliana extract (Nn1) with this transformation event, the lotus tissue extract (Nn2) prepared by the present invention increased the content of 12 catechins and their derivatives by 1.8 - 5.6 times. Among them, the contents of 3 core components such as gallocatechin, gallate (EGCG), and catechin trimer all reached more than 3.2 times that of the Nn3 group.

[0057] Synergistic effect: The hyperoside-quercetin complex showed synchronous enrichment of 26 homologues in Nn2, and its total content increased by 217% compared with Nn3 (p < 0.01), and 4 new methylated modification products were detected.

[0058] Specific metabolites: Five characteristic derivatives such as ferulic acid were only detected with significant expression in the Nn2 group (detection limit > 10 μg / g). Specifically, as shown in Table 2.

[0059] Table 2

[0060] Therefore, it can be seen that the tissue extract of lotus prepared by the present invention has an increased yield of flavonoids.

[0061] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same composition and the same function and effect as the technical idea within the scope of the technical solution of this application are all included in the technical scope of this application. In addition, within the scope of not departing from the gist of this application, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways constructed by combining some constituent elements in the embodiments are also included in the scope of this application.

Claims

1. Use of the WOX gene in preparing plants with increased flavonoid production.

2. The use according to claim 1, characterized in that, The WOX gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO. 3 or 4.

3. The use according to claim 1, characterized in that, The plant is lotus.

4. The use according to any one of claims 1 to 3, characterized in that, The primers for amplifying the WOX gene are as shown in SEQ ID NO. 5 - 6 or SEQ ID NO. 7 - 8.

5. An expression cassette for increasing the production of flavonoid compounds, the expression cassette being sequentially connected by a promoter, a WOX gene, and a terminator, wherein, The nucleotide sequence of the WOX gene is as shown in SEQ ID NO. 1 or 2.

6. A primer pair for detecting the expression cassette as claimed in claim 5, the primer pair being: The nucleotide sequence shown in SEQ ID NO. 9 - 10; or The nucleotide sequence shown in SEQ ID NO. 11 - 12.

7. An expression vector, characterized in that, The expression vector contains the expression cassette as claimed in claim 5 or 6.

8. A host cell, characterized in that, The host cell contains the expression cassette as claimed in claim 5 or 6 or the expression vector as claimed in claim 7.

9. The host cell according to claim 8, wherein The host cell is Escherichia coli or Agrobacterium cell.

10. A method for producing a transgenic plant, characterized in that, Transforming a plant with the expression cassette as claimed in claim 5 or 6, or the expression vector as claimed in claim 7, or the host cell as claimed in claim 8 or 9 to obtain a transformed plant cell or tissue, and then culturing the transformed plant cell or tissue into a transgenic plant.

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