A kind of tangerine peel polymethoxyflavonoids biosynthesis gene and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a tangerine peel polymethoxyflavonoid biosynthesis gene and application. The present invention separates polyoxymethyltransferase CsOMT1 from sweet orange for the first time, verifies and analyzes that the polyoxymethyltransferase CsOMT1 can effectively participate in the biosynthesis of polymethoxyflavonoids, and can control the biosynthesis of polymethoxyflavonoids in sweet orange peel by regulating the expression amount of the polyoxymethyltransferase CsOMT1 gene, providing effective help for the subsequent production of fine tangerine peel.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a tangerine peel polymethoxyflavonoid biosynthesis gene and application thereof. Background Art

[0002] Polymethoxyflavonoids (PMFs) have multiple biological activities in plants, such as antioxidant, anti-inflammatory, and anti-cancer. A large number of research reports also show that PMFs play an important role in the treatment of diabetes, cardiovascular disease, colon cancer, obesity, dementia, etc., and are of great significance to human health.

[0003] Tangerine peel is a medicine for regulating qi. It is the dried and mature peel of the citrus plant and its cultivated varieties in the Rutaceae family. Tangerine peel has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. It is often taken as a decoction, or it can be soaked in water, cooked into porridge or stewed in soup. But no matter which method is used, it needs to be taken according to the doctor's instructions. At present, more than 40 polymethoxylated flavonoid compounds have been isolated and identified from citrus plants, with higher contents in citrus (Citrus reticulata Blanco) and sweet orange [Citrus sinensis (Linn.) Osbeck]. Tangeretin and nobiletin are representative compounds of polymethoxylated flavonoids. However, the mechanism of polymethoxylated flavonoid biosynthesis is still unknown, and no key genes and pathways that can be used to regulate the accumulation of polymethoxylated flavonoids in sweet orange have been invented. Summary of the invention

[0004] The present invention aims at the technical problems existing in the prior art, and provides a tangerine peel polymethoxyflavonoid biosynthesis gene and application. The present invention isolates polyoxymethyltransferase CsOMT1 from sweet orange for the first time, and verifies and analyzes that the polyoxymethyltransferase CsOMT1 can effectively participate in the biosynthesis of polymethoxyflavonoids. By regulating the expression of the polyoxymethyltransferase CsOMT1 gene, the biosynthesis of polymethoxyflavonoids in sweet orange peel can be controlled, providing effective help for the subsequent production of fine tangerine peel.

[0005] One of the purposes of the present invention is to provide a polyoxymethyltransferase CsOMT1 for the biosynthesis of tangerine peel polymethoxyflavonoids, the amino acid sequence of the polyoxymethyltransferase CsOMT1 is shown in SEQ ID NO.1.

[0006] On the other hand, the present invention also provides a tangerine peel polymethoxyflavonoids biosynthesis gene CsOMT1, and the nucleotide sequence of the gene CsOMT1 is shown in SEQ ID NO.2.

[0007] On the other hand, the present invention also provides a method for obtaining the tangerine peel polymethoxyflavonoid biosynthesis gene CsOMT1, comprising the following steps: using Arabidopsis thaliana OMT protein (NP_200227.1) as a reference, and performing BLAST analysis with the sweet orange genome database (http: / / www.citrusgenomedb.org / ) to obtain the sweet orange CsOMT1 protein.

[0008] Preferably, the method further comprises the following step: codon optimization of the obtained CsOMT1 protein to obtain its gene expression sequence.

[0009] On the other hand, the present invention also provides a method for constructing a recombinant expression system of tangerine peel polymethoxyflavonoids biosynthesis gene CsOMT1, the method comprising the following steps: recombinantly connecting the CsOMT1 gene sequence with an expression vector to obtain a recombinant expression vector.

[0010] Preferably, the recombinant expression vector is pKSE402.

[0011] Preferably, the construction method further comprises the following step: transforming the recombinant expression vector into Agrobacterium using electrotransformation to obtain recombinant Agrobacterium.

[0012] Preferably, the Agrobacterium is EHA105.

[0013] On the other hand, the present invention also provides the use of the polyoxymethyltransferase CsOMT1 in catalyzing the biosynthesis of polymethoxyflavonoids.

[0014] Preferably, the catalysis occurs in sweet orange.

[0015] Preferably, the catalysis is achieved by overexpressing the polyoxymethyltransferase CsOMT1.

[0016] Further preferably, the polymethoxyflavonoid biosynthesis includes nobiletin, heptamethoxyflavone and tangeretin.

[0017] The beneficial effects of the present invention are as follows: the present invention isolates polyoxymethyltransferase CsOMT1 from sweet orange for the first time, and verifies and analyzes that the polyoxymethyltransferase CsOMT1 can effectively participate in the biosynthesis of polymethoxyflavonoids, especially the accumulation of nobiletin, heptamethoxyflavonoids, and tangeretin is closely related to their gene expression. By regulating the gene expression of polyoxymethyltransferase CsOMT1, the biosynthesis of polymethoxyflavonoids in sweet orange peel can be controlled, providing effective help for the subsequent production of fine tangerine peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 RT-PCR analysis of CsOMT1 gene expression;

[0019] Figure 2 To analyze the effect of CsOMT1 gene on the biosynthesis of polymethoxyflavonoids. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein belong to the meanings commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0021] Example 1

[0022] A polyoxymethyltransferase CsOMT1 for the biosynthesis of tangerine peel polymethoxyflavonoids, the amino acid sequence of which is shown in SEQ ID NO.1, and the gene coding sequence of which is shown in SEQ ID NO.2.

[0023] Example 2

[0024] A method for obtaining a tangerine peel polymethoxyflavonoid biosynthesis gene CsOMT1 comprises the following steps:

[0025] Using the reported OMT protein in Arabidopsis thaliana (NP_200227.1) as a reference, BLAST analysis was performed with the sweet orange genome database (http: / / www.citrusgenomedb.org / ) to obtain the CsOMT1 protein, with the amino acid sequence as SEQ ID NO.1;

[0026] The CsOMT1 protein obtained in step 1) was codon optimized to obtain its gene expression sequence as SEQ ID NO.2.

[0027] Example 3

[0028] A recombinant expression system of tangerine peel polymethoxyflavonoid biosynthesis gene CsOMT1 is constructed, comprising the following steps:

[0029] The CsOMT1 gene sequence obtained in Example 2 was recombined with the pKSE402 expression vector to obtain a recombinant expression vector CsOMT1-pKSE402;

[0030] The cell cultured in step 1) was transformed into Agrobacterium EHA105 by electroporation to obtain recombinant Agrobacterium.

[0031] Example 4

[0032] Functional analysis of polyoxymethyltransferase CsOMT1 in the biosynthesis of polymethoxyflavonoids from tangerine peel: Taking sweet orange fruit as an example, the treatment group was injected with the recombinant Agrobacterium suspension described in Example 3 on the equatorial surface of the yellow peel layer of the fruit, and the control group was injected with the same amount of Agrobacterium suspension containing an empty vector. One week after infection, the peel of the injected area was sampled and analyzed for its gene expression and the changes in the content of nobiletin, heptamethoxyflavonoids, and tangeretin were detected.

[0033] The results are as follows Figure 1 The results showed that the CsOMT1 gene was transiently expressed in sweet orange fruit, and the expression level was significantly increased compared with the control group. In addition, nobiletin, heptamethoxyflavonoids, and tangeretin all showed significant increases ( Figure 2 ), further confirmed that the high expression of the polyoxymethyltransferase CsOMT1 of the present invention would participate in the biosynthesis of polymethoxyflavonoids, effectively increase the accumulation of polymethoxyflavonoids in sweet orange, and provide benefits for the later production of tangerine peel.

[0034] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polyoxymethyltransferase CsOMT1 for the biosynthesis of tangerine peel polymethoxyflavonoids, characterized in that: The amino acid sequence of the polyoxymethyltransferase CsOMT1 is shown in SEQ ID NO.

1.

2. A tangerine peel polymethoxyflavonoid biosynthesis gene CsOMT1, characterized in that: The nucleotide sequence of the tangerine peel polymethoxyflavonoids biosynthesis gene CsOMT1 is shown in SEQ ID NO.

2.

3. A method for obtaining the tangerine peel polymethoxyflavonoids biosynthesis gene CsOMT1 as claimed in claim 2, comprising the following steps: taking Arabidopsis thaliana OMT protein as a reference, performing BLAST analysis with the sweet orange genome database to obtain the sweet orange CsOMT1 protein, wherein the GenBank accession number of the Arabidopsis thaliana OMT protein is NP_200227.

1.

4. The method according to claim 3, characterized in that The method further comprises the following steps: codon optimization is performed on the obtained CsOMT1 protein to obtain its gene expression sequence.

5. A method for constructing a recombinant expression system of a tangerine peel polymethoxyflavonoid biosynthesis gene CsOMT1, characterized in that: The method comprises the following steps: recombinantly connecting the tangerine peel polymethoxyflavonoids biosynthesis gene CsOMT1 described in claim 2 with an expression vector to obtain a recombinant expression vector.

6. The construction method according to claim 5, characterized in that: The recombinant expression vector is pKSE402.

7. The construction method according to claim 5, characterized in that: The construction method further comprises the following steps: using the electrotransformation method to transform the recombinant expression vector into Agrobacterium to obtain recombinant Agrobacterium.

8. The construction method according to claim 7, characterized in that: The Agrobacterium is EHA105.

9. A use of the polyoxymethyltransferase CsOMT1 as claimed in claim 1 in catalyzing the biosynthesis of polymethoxyflavonoids, characterized in that: The catalysis occurs in sweet orange and is achieved by overexpressing the polyoxymethyltransferase CsOMT1.

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