CN113563441A

CN113563441A - Strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof

Info

Publication number: CN113563441A
Application number: CN202110947261.4A
Authority: CN
Inventors: 张俊祥; 雷莹莹; 代红艳; 张志宏
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-10-29

Abstract

The invention relates to a strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof, belongs to the field of genetic engineering in molecular biology, and researches show that the strawberry FvMYB17 transcription factor has a function of promoting synthesis of strawberry polyphenol and triterpene substances. The invention provides a basis and a technical means for improving polyphenol and triterpene substances in plants by utilizing a genetic engineering technology, and has great application value.

Description

Strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof.

Background

The polyphenol compounds are complex secondary metabolites with a plurality of phenolic hydroxyl groups, and more than 6500 polyphenol compounds and derivatives thereof are contained in the whole plant kingdom and are secondary byproducts in the plant metabolic process. According to the number of aromatic rings and hydroxyl structures connected with the aromatic rings in the structure of the substance, the substance is divided into flavonoid compounds, phenolic acid compounds, stilbene compounds, lignans, polymeric lignins and the like. Flavonoids account for about two thirds of the total polyphenols ingested by humans, and are mostly present in the structure of derivatives and mostly in the form of glycosides. They are mainly classified into anthocyanins (anthocyanins), flavonoids (flavanones), flavanoids (flavanes), flavonols (flavanols), and flavanols (flavanols), and isoflavones can also be considered as a flavonoid. The polyphenol exists in various fruits, vegetables and various plant drinks including tea and coffee, has good antioxidant function, and also has the effects of strengthening vascular walls, promoting gastrointestinal digestion, reducing blood fat, enhancing human immunity, preventing arteriosclerosis, reducing blood pressure, inhibiting bacteria and cancer cell growth and the like.

Terpenes are one of three major secondary metabolites, and are classified into monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes and polyterpenes according to the number of isoprene units contained in the compound. Triterpenoids are largely classified into chain triterpenes, tetracyclic triterpenes and pentacyclic triterpenes. Pentacyclic triterpenes are common in medicinal plants, and the main structural types include ursane type, oleanane type, lupane type, friedelane type and the like. Triterpenes are contained in plants of Caryophyllaceae, Araliaceae, Leguminosae, Hippocastanaceae, Polygalaceae, Campanulaceae, Scrophulariaceae, etc., and have been reported to be less in Rosaceae. The compound has the physiological functions of removing free radicals, resisting cancer, resisting aging, resisting virus, reducing cholesterol and the like and has wide attention to medicinal value.

The previous researches on polyphenol and terpenoid substances mainly focus on the aspects of extraction detection and medicinal activity, and the research on transcription factors for regulating and controlling the synthesis of the metabolites is always a hot point of research in the scientific community. MYB transcription factors play an extremely important role in secondary metabolism of plants, and at the early stage, the strawberry FvMYB17 transcription factors are heterogeneously transformed into Arabidopsis thaliana, and FvMYB17 is found to promote anthocyanin synthesis. However, arabidopsis thaliana, also called arabian grass, belongs to arabidopsis plants in cruciferae, and has no fruit, while strawberry belongs to rosaceous strawberry plants, the fruit has strong fragrance and rich nutrition, and the arabidopsis thaliana belong to different families and have great phenotype difference. Therefore, it is difficult to predict and analyze the function of FvMYB17 in strawberry based on the results of heterologous transformation of FvMYB17 into arabidopsis thaliana. Whether the FvMYB17 transcription factor can promote anthocyanin synthesis in strawberries or whether the transcription factor can promote synthesis of other secondary metabolites is not clear at present. Therefore, only by homologous transformation of FvMYB17 into strawberry plants, the obtained FvMYB17 transgenic strawberry plants can be analyzed for functions. By comparing the content difference of the secondary metabolites of the FvMYB17 strawberry transgenic plant and the control plant, the result shows that FvMYB17 can not only promote the synthesis of anthocyanin, but also promote the synthesis of polyphenols such as tannin, flavonol, flavone, phenolic acids, flavanone, chalcone and the like, and even promote the synthesis of triterpenoids with completely different structures from anthocyanin. The results greatly enrich the function of regulating and controlling the plant secondary metabolite by FvMYB17, and lay an important foundation for subsequently utilizing FvMYB17 to improve the plant metabolite.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a strawberry transcription factor for promoting the synthesis of polyphenol and triterpene substances and application thereof. The technical scheme of the invention is that a plant expression vector of the constructed FvMYB17 is transformed into diploid forest strawberry 'Ruegen' by an agrobacterium-mediated method so as to realize functional analysis of the FvMYB17 gene; the regulation and control analysis of the FvMYB17 on polyphenol compounds and triterpene substances is realized by detecting secondary metabolites of fruits of the transgenic plants and the control plants.

The invention provides a strawberry transcription factor FvMYB17 for promoting synthesis of polyphenol and triterpene substances, wherein the amino acid sequence of the transcription factor is shown as a sequence 1; the nucleotide sequence of a strawberry transcription factor FvMYB17 for promoting the synthesis of polyphenol and triterpene substances is shown as a sequence 2;

the invention provides a plant expression vector pRI101-CaMV35S-FvMYB17 containing a transcription factor FvMYB17 for improving synthesis of polyphenol and triterpene substances, and the plant expression vector pRI101-CaMV 35-FvMYB 17 is transferred into diploid forest strawberry 'Ruegen' by an agrobacterium-mediated method.

The invention provides application of a strawberry FvMYB17 transcription factor in improving the content of plant polyphenol compounds.

The invention provides application of a strawberry FvMYB17 transcription factor in improving the content of plant triterpene substances.

The invention has the beneficial effects that: according to the invention, the function analysis reveals that the content of polyphenol and triterpenoid substances in strawberry fruits can be obviously improved by increasing the expression of strawberry FvMYB17, and a foundation is laid for improving the content of polyphenol and triterpenoid substances in other plants or creating a new strawberry germplasm with high polyphenol and triterpenoid substances through a gene process in the future.

Description of the figures

FIG. 1: FvMYB17 transgenic strawberry plant PCR identification electrophoretogram

Wherein, M: DL2000 Marker; FvMYB17 transgenic strawberry plants and control plants are shown.

FIG. 2: relative expression amount of FvMYB17 in FvMYB17 transgenic strawberry plants

FIG. 3: phenotypic analysis of FvMYB17 transgenic strawberry plants and wild-type control plants. Wherein CK is untransformed strawberry, OE-2/OE-5 is two independent strawberry transgenic strains of FvMYB17

I Whole plant II incomplete unfolded new leaf III completely unfolded leaf IV petiole V red fruit

The specific implementation mode is as follows:

in order to further clarify the present invention and not to limit the same, the following is described with reference to examples. The experimental procedures described in the following examples are conventional unless otherwise specified. The experimental drug materials are commercially available without specific reference.

Example 1 acquisition and phenotypic analysis of FvMYB17 transgenic strawberry plants

1.1 diploid forest strawberry 'Ruegen' transformation

(1) And (5) shaking the bacteria. Positive colonies detected as correct were picked and shaken overnight in a YEP liquid medium containing 25mg/L rifampicin and 100mg/L kanamycin at 28 ℃ in a shaking incubator overnight for 12-16h to orange yellow. The next dayIn the morning, 1 mL of the bacterial liquid is sucked and transferred into a new 50mL of YEP liquid culture medium, and the culture is continued until OD is reached₆₀₀The value is between 0.5 and 0.6.

(2) And (4) selecting materials. Selecting diploid Ruegen tissue culture strawberry seedlings with good growth potential, quickly cutting off the periphery of leaves, reserving a part with the width of 2-4mm, flattening the cut as much as possible, and wetting and soaking the cut part by using a co-culture liquid culture medium for later use.

(3) And (4) infection. Transferring the cut explants to a bacterial solution, beginning to infect for 8 min. Explants were plated on co-culture solid medium and co-cultured in the dark for 3 days.

(4) After co-culture, the explants were transferred to a delay medium (containing 200mg/L Cef and 200mg/L Tim), cultured for 4 days, transferred to a regeneration medium (containing 20mg/L Kan, 200mg/L Cef and 200mg/L Tim), and the specific time from the end of dark culture to light culture was determined according to the callus appearance of the explants. After the resistant bud appears, the explant is transferred to a differentiation medium (containing 30mg/LKan, 200mg/L Cef and 200mg/L Tim) for continuous culture.

1.2 identification of strawberry transgenic Positive lines

(1) When resistant seedlings grow to 7-8 leaves, DNA (deoxyribonucleic acid) of young leaves of strawberry transgenic plants is extracted (CTAB method), and the result is shown in figure 1 through PCR amplification and agarose gel electrophoresis detection, 768bp fragments appear in the transgenic plants, but the control plants do not exist, which indicates that the strawberry FvMYB17 overexpression vector is successfully introduced into the strawberries.

(2) When the resistant seedlings grow to 7-8 leaves, extracting RNA of young leaves of the strawberry transgenic plants, and carrying out RT-qPCR quantitative detection to determine whether the target genes are transferred.

The upstream primer is 5'-GATCAAAATTGCGAGCAGGT-3', and the upstream primer is,

the downstream primer is 5'-CTTTGTCCCAACCGACATTT-3', and the primer is,

the result is shown in fig. 2, and it can be seen from fig. 2 that the expression level of FvMYB17 in the transgenic plant is obviously increased compared with that in the control plant, which indicates that the strawberry FvMYB17 overexpression vector is successfully introduced into the strawberry.

1.3 phenotypic Observation of transgenic strawberry

And (3) placing the positive overexpression strain and the wild type strain in a sunlight greenhouse to culture in the same environment, and observing the phenotype change of different parts. As shown in FIG. 3, it was found that the newly grown leaves of the transgenic plants had significant pigment accumulation, especially the veins, and the color became lighter with the gradual unfolding of the leaves, but the veins were still darker than the control plants. The petiole part of the transgenic plants also had significant pigment accumulation and this phenotype was accompanied by this developmental process of the plants. The transgenic plants had red fruits that were not significantly different in color from the control.

Example 2 detection of secondary metabolites in fruits of FvMYB17 transgenic strawberry plants

Two strains of transgenic plants and the red fruit of wild plants are sampled simultaneously (each strain ensures at least 6 seedlings), mixed and ground respectively, and sent to a company for secondary metabolome detection. Differential analysis of metabolites was performed by setting the fold change.gtoreq.1.5, and the results are shown in Table 1. The content of main polyphenol compounds in two FvMYB17 overexpression strawberry transgenic lines is generally obviously higher than that of wild plants, and meanwhile, the content of partial triterpene substances is found to be obviously higher than that of the wild plants. The FvMYB17 can improve the content of polyphenol and triterpene substances in strawberry fruits.

TABLE 1 FvMYB17 transgenic strawberry plants and wild-type control plants differing in the content of polyphenols and triterpenoids in the fruits

Sequence listing

<110> Shenyang agriculture university

<120> strawberry transcription factor for promoting synthesis of polyphenol and triterpene substances and application thereof

<130> 2

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 256

<212> PRT

<213> forest strawberry (Fragaria visco)

<400> 1

Met Arg Lys Pro Cys Cys Asp Lys Gln Asp Thr Asn Lys Gly Ala Trp

1 5 10 15

Ser Lys Gln Glu Asp Gln Lys Leu Ile Asp Tyr Ile Arg Lys His Gly

20 25 30

Glu Gly Cys Trp Arg Thr Leu Pro Gln Ala Ala Gly Leu Leu Arg Cys

35 40 45

Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp Leu

50 55 60

Lys Arg Gly Lys Phe Ala Glu Asp Glu Glu Asp Leu Ile Ile Lys Leu

65 70 75 80

His Ala Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro

85 90 95

Gly Arg Thr Asp Asn Glu Val Lys Asn Tyr Trp Asn Ser His Leu Arg

100 105 110

Arg Lys Leu Ile Thr Met Gly Ile Asp Pro Asn Asn His Arg Pro Thr

115 120 125

Ser Thr Leu Phe Pro Arg Pro His Asn His His His Gln Asn Pro Pro

130 135 140

Gln Thr Leu Lys Ser Pro Ala Gly Ser Ala Ile Asn Asn Phe Asn His

145 150 155 160

Glu Pro Val Val Phe Glu Ser Lys Thr Pro Arg Gly Asp Asp Gln Asn

165 170 175

Cys Glu Gln Val Ser Asp Gly Arg Ser Cys Leu Glu Asp Asp Ser Ser

180 185 190

Cys Gly Gly His His Leu Pro Asp Leu Asn Leu Asp Leu Thr Val His

195 200 205

Leu Arg Val Ser Asn Asp Asp His Gln Tyr Leu Ser Lys Glu Leu Asn

210 215 220

His Leu His Leu Arg Ile Ser Asp Pro His Glu Met Ser Val Gly Thr

225 230 235 240

Lys Thr Asp Ile Phe Ala Ser Ser Thr Thr Leu Pro Leu Phe Arg Glx

245 250 255

<210> 2

<211> 768

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

atgaggaaac cctgctgcga caagcaagac acgaacaaag gagcttggtc gaagcaagaa 60

gatcagaagc tcatcgatta cattcgcaaa catggcgagg gttgttggcg tacccttcct 120

caagccgcag gccttcttcg atgcggtaaa agttgcagac ttcggtggat aaactatcta 180

cggccggacc ttaaaagggg caagtttgct gaagatgaag aagatctcat cattaagctt 240

catgcactcc taggcaatcg gtggtcgctg attgccggaa gattgccggg acgtacagac 300

aatgaagtga agaactattg gaactctcat ttgagacgaa agcttataac catgggtata 360

gatccaaaca atcatcgacc caccagcact ctcttccctc ggcctcataa tcatcatcat 420

caaaacccac cacagacact aaaatctcca gctggttctg ctatcaataa ttttaaccat 480

gagccagtag tgttcgagtc caaaactccg cgtggtgatg atcaaaattg cgagcaggtc 540

tcggatggca gaagttgctt agaggatgat tcttcttgtg gtggtcatca cctgcctgat 600

ttaaaccttg atctcactgt ccatctcagg gtttctaatg atgatcacca atatctcagt 660

aaggagctca atcatcttca tctcagaatt agtgaccctc atgaaatgtc ggttgggaca 720

aagactgata tatttgcctc atctaccacg cttcctctct tcagataa 768

Claims

1. A protein which is (a) or (b) or (c) below:

(a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(b) a protein consisting of amino acid sequences shown from 1 st to 255 th sites of the N end of a sequence 1 in a sequence table;

(c) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a) or (b) and is related to plant polyphenol and triterpene substances.

2. A gene encoding the protein of claim 1, wherein: the gene is a DNA molecule as described in any one of the following (1) to (4):

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) DNA molecules shown in 1 st to 768 th sites of a 5' end of a sequence 2 in a sequence table;

(3) DNA molecules which hybridize with the DNA sequences defined in (1) or (2) under stringent conditions and encode proteins associated with plant polyphenols and triterpene substances;

(4) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and encode proteins related to plant polyphenol and triterpene substances.

3. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 2 or 3.

4. Use of a protein according to claim 1, or a gene according to claim 2 or 3, for modulating plant polyphenol and triterpene substances.

5. A method for producing a transgenic plant, comprising introducing the gene of claim 2 or 3 into a plant of interest to obtain a transgenic plant; the content of polyphenol and triterpene substances of the transgenic plant is higher than that of the target plant.

6. A method for increasing the content of polyphenol and triterpene substances in plants, which is to increase the activity and/or expression level of the protein as claimed in claim 1 in the target plants and increase the content of polyphenol and triterpene substances in the plants.

7. Use of a protein according to claim 1, or a gene according to claim 2 or 3, or a method according to claim 6 or 7, in plant breeding.

8. The use of claim 7, wherein: the breeding aims to breed plants with high content of polyphenol and triterpene substances.