CN112094333B - Application of FaLBD39 in increasing anthocyanin accumulation in strawberry fruits and method for increasing anthocyanin accumulation in strawberry fruits - Google Patents
Application of FaLBD39 in increasing anthocyanin accumulation in strawberry fruits and method for increasing anthocyanin accumulation in strawberry fruits Download PDFInfo
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- CN112094333B CN112094333B CN202011070503.8A CN202011070503A CN112094333B CN 112094333 B CN112094333 B CN 112094333B CN 202011070503 A CN202011070503 A CN 202011070503A CN 112094333 B CN112094333 B CN 112094333B
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Abstract
The invention provides an application of FaLBD39 in increasing anthocyanin accumulation in strawberry fruits and a method for increasing anthocyanin accumulation in strawberry fruits, and relates to the technical field of biology, wherein the expression level of FaLBD39 gene has an important influence on anthocyanin accumulation in strawberry fruits. Therefore, by regulating the expression of the FaLBD39 gene in strawberry fruits, the anthocyanin content in strawberry fruits can be influenced. The expression of the FaLBD39 gene in strawberry fruits is reduced, so that the accumulation of anthocyanin in the strawberry fruits can be increased, and the strawberry fruits have higher nutritional value and market value.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to an application of FaLBD39 in increasing anthocyanin accumulation in strawberry fruits and a method for increasing anthocyanin accumulation in strawberry fruits.
Background
The strawberry is one of seven big fruits in the world, is beautiful in color, fragrant and delicious, and is rich in anthocyanin and other polyphenol substances. Anthocyanin, a natural plant pigment. The plant color-keeping agent can endow plants with colorful colors, and plays an important role in the growth and development of the plants and the fruit quality: the accumulation of anthocyanin can improve the disease resistance, oxidation resistance and ultraviolet resistance of plants; anthocyanin is also an important index for evaluating the maturity and quality of fruits. Anthocyanin has rich nutritive value and medicinal value, and its oxidation resistance makes it a natural powerful free radical scavenger, and has multiple health functions of resisting oxidation, resisting mutation, preventing cardiovascular and cerebrovascular diseases, protecting liver, inhibiting tumor cell generation, etc. Therefore, in modern society with frequent cardiovascular diseases and cancers, strawberries rich in anthocyanin are favored by people.
LBD is a class of transcription factors specific to higher plants, and members of this family usually contain a conserved LOB domain at the N-terminus, presumably to function in conjunction with a promoter. LBD is found in model plant Arabidopsis thaliana and is involved in regulating plant root, leaf, inflorescence and embryo development, and also in the synthesis of plant anthocyanin and the regulation of nitrogen metabolism. However, the application of the gene in the strawberry, especially in the strawberry fruit, is not reported yet.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
A first object of the present invention is to provide the use of the FaLBD39 gene for increasing the accumulation of anthocyanin in strawberry fruits, to alleviate at least one of the technical problems of the prior art.
The second purpose of the invention is to provide a method for increasing anthocyanin accumulation in strawberry fruits, which comprises the step of regulating and controlling the anthocyanin accumulation in the strawberry fruits by regulating and controlling the expression level of FaLBD39 gene in the strawberry fruits.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, there is provided the use of a FaLBD39 gene in increasing anthocyanin accumulation in strawberry fruits, the FaLBD39 gene including the gene expressing (a1) or (a 2):
(a1) the method comprises the following steps A protein comprising an amino acid sequence shown as SEQ ID No. 1;
(a2) the method comprises the following steps And (b) a protein having the same function as the protein having an amino acid sequence 70% or more identical to the amino acid sequence described in (a 1).
Further, the FaLBD39 gene is a gene expressing a protein having an amino acid sequence shown in SEQ ID NO. 1.
Further, the nucleotide sequence of the FaLBD39 gene includes (b1) or (b 2):
(b1) the method comprises the following steps Contains a nucleotide sequence shown as SEQ ID NO. 2;
(b2) the method comprises the following steps A nucleotide sequence having 70% or more identity to the nucleotide sequence of (b1) and having the same function.
Further, the nucleotide sequence of the FaLBD39 gene is a nucleotide sequence containing a nucleotide sequence shown as SEQ ID NO. 2.
Further, the expression of said FaLBD39 gene in strawberry fruits was down-regulated to increase the accumulation of anthocyanin in strawberry fruits.
In addition, the invention also provides a method for increasing anthocyanin accumulation in strawberry fruits, which comprises the step of regulating and controlling the expression level of FaLBD39 genes in the strawberry fruits.
Further, the expression of the FaLBD39 gene was down-regulated to increase the accumulation of anthocyanin in strawberry fruits.
Further, the FaLBD39 gene is connected with a pTRV vector to obtain a recombinant plasmid pTRV-FaLBD39, and the recombinant plasmid pTRV-FaLBD39 is expressed in strawberry fruits to increase the accumulation of anthocyanin in the strawberry fruits.
Further, the expression of the recombinant plasmid pTRV-FaLBD39 in strawberry fruits was achieved using Agrobacterium mediation.
Further, the method comprises the steps of:
(a) providing a linearized pTRV (pYL156) vector and a FaLBD39 gene with a pTRV (pYL156) vector recombinant fragment;
(b) after the linearized pTRV (pYL156) vector and the FaLBD39 gene with the recombinant fragment of the pTRV (pYL156) vector are incubated with recombinase, competent cells are transformed to obtain pTRV-FaLBD39 plasmid;
(c) the pTRV-FaLBD39 plasmid is transformed into Agrobacterium GV3101, and the FaLBD39 gene is transferred into strawberry fruits through Agrobacterium mediation.
Compared with the prior art, the invention has the following beneficial effects:
the inventor of the invention finds that the expression level of the FaLBD39 gene has an important influence on the accumulation of anthocyanin in strawberry fruits through research. Therefore, by regulating the expression of the FaLBD39 gene in strawberry fruits, the anthocyanin content in strawberry fruits can be influenced. The expression of the FaLBD39 gene in strawberry fruits is reduced, so that the accumulation of anthocyanin in the strawberry fruits can be increased, and the strawberry fruits have higher nutritional value and market value.
The invention also provides a method for increasing anthocyanin accumulation in strawberry fruits by regulating and controlling the expression level of the FaLBD39 gene in the strawberry fruits, the method is simple to operate and low in cost, and compared with the traditional method of applying exogenous substances such as auxin, cytokinin, abscisic acid and the like to plants, the method for directly regulating and controlling the gene of the strawberry fruits can regulate and control anthocyanin accumulation in the strawberry fruits more accurately, and the regulation and control efficiency is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a result graph of the states of strawberry fruits under different treatment modes provided in example 4 of the present invention.
Fig. 2 is a result graph of detecting the total anthocyanin content in strawberry fruits by applying the HPLC-DAD technique of the high performance liquid chromatography provided in embodiment 4 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
According to an aspect of the present invention, there is provided a use of a FaLBD39 gene for increasing the accumulation of anthocyanin in strawberry fruits, the FaLBD39 gene including a gene expressing (a1) or (a 2):
(a1) the method comprises the following steps A protein comprising an amino acid sequence shown as SEQ ID No. 1;
(a2) the method comprises the following steps And (b) a protein having the same function as the protein having an amino acid sequence 70% or more identical to the amino acid sequence described in (a 1).
The protein containing the amino acid sequence shown in SEQ ID No.1 means that the amino acid sequence of the protein can be the whole amino acid sequence shown in SEQ ID No.1, and also can be composed of the amino acid sequence shown in SEQ ID No.1 and other amino acids, and the functions of the other amino acids in the protein include, but are not limited to, the combination sites of a label for protein purification, a fluorescent protein marker and DNA, and the like, and in some specific embodiments, the protein can be, but is not limited to, HIS, GST, MyC, FLAG, HSV, V5, HA, GFP, RFP, BFP, CAT, DHFR, MBP, T7, thioredoxin, and the like.
Wherein "identity" refers to similarity to the amino acid sequence shown in SEQ ID NO. 1. The difference from the amino acid sequence shown in SEQ ID No.1 may be caused by one or several amino acid changes, deletions or insertions, which change(s) the protein molecule does not completely correspond to the amino acid sequence shown in SEQ ID No.1, but at least 70% or more, for example, but not limited to, 70%, 75%, 80%, 85%, 90% or 95% identity. Meanwhile, the protein molecules with more than 70 percent of identity with the amino acid sequence shown in SEQ ID NO.1 and the protein molecules containing the amino acid sequence shown in SEQ ID NO.1 have the same functions. Identity can be assessed visually or by computer software, for example, using blast software as is conventional in the art.
When the FaLBD39 gene is used for expressing a gene of a protein containing an amino acid sequence shown in SEQ ID NO.1, the effect of increasing anthocyanin accumulation in strawberry fruits is better.
In some alternative embodiments, the nucleotide sequence of the FaLBD39 gene includes (b1) or (b 2):
(b1) the method comprises the following steps Contains a nucleotide sequence shown as SEQ ID NO. 2;
(b2) the method comprises the following steps A nucleotide sequence having 70% or more identity to the nucleotide sequence of (b1) and having the same function.
The expression "comprising" means that the nucleotide sequence of the FaLBD39 gene may only comprise the nucleotide sequence shown in SEQ ID No.2, or may consist of the nucleotide sequence shown in SEQ ID No.2 and other nucleotide sequences, such as nucleotide sequences encoding functional units for protein purification, fluorescent protein markers, and binding sites for DNA, or encoding elements having a regulatory effect on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers, or transcription factor binding sites, etc.; the "containing" may also mean that the nucleotide sequence shown as SEQ ID NO.2 is discontinuous in the FaLBD39 gene, but a cDNA of the nucleotide sequence shown as SEQ ID NO.2 may be generated.
Wherein "identity" refers to similarity to the nucleotide sequence shown in SEQ ID NO. 2. Differences from the nucleotide sequence shown in SEQ ID NO.2 may be caused by changes or deletions or insertions of one or several nucleotides, as well as by the degeneracy of the codons, which change the sequence of the gene not completely identical to the nucleotide sequence shown in SEQ ID NO.2, but which have at least more than 70%, for example but not limited to 70%, 75%, 80%, 85%, 90% or 95% identity. Meanwhile, the genes with more than 70 percent of identity with the nucleotide sequence shown in SEQ ID NO.2 and the genes containing the nucleotide sequence shown in SEQ ID NO.2 have the same functions. Identity can be assessed visually or by computer software, for example, using blast software as is conventional in the art.
When the FaLBD39 gene is a gene containing a nucleotide sequence shown in SEQ ID NO.2, the application of the FaLBD39 gene in increasing anthocyanin accumulation in strawberry fruits is better.
The expression level of the FaLBD39 gene has an important influence on the accumulation of anthocyanin in strawberry fruits. Therefore, by regulating the expression of the FaLBD39 gene in strawberry fruits, the anthocyanin content in strawberry fruits can be influenced. Through research, the inventor of the invention finds that the expression of FaLBD39 gene in strawberry fruits is reduced, and the accumulation of anthocyanin in the strawberry fruits can be increased, so that the strawberry fruits have higher nutritional value and market value.
According to a second aspect of the present invention, there is also provided a method of increasing anthocyanin accumulation in strawberry fruits, the method comprising regulating the expression level of the FaLBD39 gene in strawberry fruits.
Because the gene FaLBD39 is related to the accumulation of anthocyanin in strawberry fruits, the regulation and control of the expression quantity of the gene FaLBD39 in the strawberry fruits can regulate and control the content of anthocyanin in the strawberry fruits, and compared with the traditional method of applying exogenous substances such as auxin, cytokinin, abscisic acid and the like to plants, the direct regulation and control of the gene of the strawberry fruits can regulate and control the accumulation of anthocyanin in the strawberry fruits more accurately, and the regulation and control efficiency is high. In addition, the method provided by the invention has the advantages of simple operation, low cost and the like.
Because the anthocyanin accumulation in the strawberry fruit can be increased, the strawberry fruit has higher nutritional value and market value, and therefore, in some preferred embodiments, the expression level of the FaLBD39 gene in the strawberry fruit is regulated downwards to increase the anthocyanin accumulation in the strawberry fruit. The expression of the FaLBD39 gene can be regulated and controlled by constructing a gene recombinant targeting vector, an antisense oligonucleotide technology, a methylation oligonucleotide technology or a zinc finger nuclease technology and the like, but the invention is not limited to this, and all the modes capable of achieving the effect of down-regulating the gene expression are within the protection scope of the invention.
In some preferred embodiments, the FaLBD39 gene is ligated to pTRV vector to give recombinant plasmid pTRV-FaLBD39, which is expressed in strawberry fruits to increase anthocyanin accumulation in strawberry fruits.
The VIGS technology (Virus-induced gene silencing, VIGS) has the advantages of short period, low cost, no need of genetic transformation and the like, and the functions of genes can be identified by adopting the VIGS technology in the embodiment, so that theoretical guidance is provided for cultivating engineering improved plants with the FaLBD39 gene knocked out by adopting technologies such as CRISPR/Cas9 and the like in the later stage.
In some specific embodiments, the method comprises the steps of:
(a) providing a linearized pTRV (pYL156) vector and a FaLBD39 gene with a pTRV (pYL156) vector recombinant fragment;
(b) after the linearized pTRV (pYL156) vector and the FaLBD39 gene with the recombinant fragment of the pTRV (pYL156) vector are incubated with recombinase, competent cells are transformed to obtain pTRV-FaLBD39 plasmid;
(c) the pTRV-FaLBD39 plasmid is transformed into Agrobacterium GV3101, and the FaLBD39 gene is transferred into strawberry fruits through Agrobacterium mediation.
The invention clones related genes FaLBD39 of strawberry anthocyanin by using the existing plant genetic engineering technology, transfers the genes into strawberry fruits by an agrobacterium-mediated transformation method, and proves that the anthocyanin accumulation amount in the transgenic fruits is obviously improved by comparative analysis.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1 cloning of Gene FaLBD39 that regulates the accumulation of anthocyanin in strawberry fruit
(1) And using the octaploid strawberry 'Hongyan' as a test material.
(2) And RNA extraction: after total RNA in the material is extracted by adopting an RNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., Ltd.), reverse transcription is carried out by using a reverse transcription kit (purchased from Beijing Tiangen Biochemical technology Co., Ltd.) and taking the RNA as a template to obtain a cDNA first chain.
(3) Cloning of the gene: taking the first strand of the reverse transcription cDNA as a template, carrying out PCR amplification by using primers FaLBD39-F and FaLBD39-R, and recovering a PCR product to obtain a 765bp target fragment.
FaLBD39-F:5’-ATGAGCTGCAACGGTTGCC-3’(SEQ ID NO.3)；
FaLBD39-R:5’-TCAAACAAAAAGGTTCAAAAGCTT-3’(SEQ ID NO.4)。
This fragment was ligated to pGWC-T vector, E.coli DH5a was transformed, and positive clones were examined by gene sequencing, thereby obtaining pGWC-FaLBD39 plasmid.
Example 2 construction of strawberry fruit overexpression vector
(1) In this example, the plant overexpression vector p3301-CaMV35S is preferred, and the p3301-CaMV35S vector is digested with EcoR1 (available from TaKaRa) to linearize the vector.
(2) PCR was carried out using pGWC-FaLBD39 plasmid obtained in example 1 as a template, and using primers FaLBD39-F1 and FaLBD39-R1 to clone a fragment having a recombinant sequence of p3301-CaMV35S, and a PCR product of FaLBD39 gene having a recombinant fragment of p3301 vector was obtained by recovering the PCR product using the following primers:
FaLBD39-F1:5’-AGCAGGCTTTGACTTTATGAGCTGCAACGGTTGCCGA-3’(SEQ ID NO.5)；
FaLBD39-R1:5’-TTTTGAACCTTTTTGTTTGAAAAGTCTCTAGACCCA-3’(SEQ ID NO.6)。
(3) the linearized p3301-CaMV35S vector and the PCR product of FaLBD39 gene with the recombinant fragment of p3301 vector were mixed with recombinase (purchased from Beijing Tiangen Biotechnology Co., Ltd.) uniformly, incubated at 55 ℃ for 15min, and inactivated at 85 ℃ for 5 min. Transforming Escherichia coli competence DH5a (purchased from Beijing Quanjijin biotechnology limited), sequencing and identifying recombinants to obtain p3301-FaLBD39 plasmid, transforming Agrobacterium GV3101 with the plasmid to obtain Agrobacterium GV3101-p3301-FaLBD39 strain containing recombinant plasmid, wherein the strain can be used for transforming strawberry fruits.
Example 3 construction of strawberry fruit silencing vector
(1) In this example, the plant silencing vector pTRV-FaLBD39 is preferably linearized by digesting the pTRV (pYL156) vector with Xba I (available from TaKaRa).
(2) The pGWC-FaLBD39 plasmid obtained in example 1 was used as a template, PCR amplification was carried out using primers FaLBD39-F2 and FaLBD39-R2, a fragment carrying the pTRV (pYL156) recombinant sequence was cloned, and the PCR product was recovered to obtain a PCR product of FaLBD39 gene carrying the pTRV (pYL156) vector recombinant fragment, using the following primers:
FaLBD39-F2:5’-AAAGGTACCATGAGGAAACCCTGCTGCGA-3’
(SEQ ID NO.7)；
FaLBD39-R2:5’-AAAGAATTCTCTGAAGAGAGGAAGCGTGG-3’
(SEQ ID NO.8)。
(2) and uniformly mixing the linearized pTRV (pYL156) vector and the PCR product of the FaLBD39 gene with the pTRV (pYL156) vector recombinant fragment with a recombinase (purchased from Beijing Tiangen Biotechnology Co., Ltd.), incubating for 15min at 55 ℃, and inactivating for 5min at 85 ℃. Transforming Escherichia coli competence DH5a (purchased from Beijing Quanzijin biotechnology limited), sequencing and identifying recombinants to obtain pTRV-FaLBD39 plasmid, transforming agrobacterium GV3101 with the plasmid to obtain agrobacterium GV3101-pTRV-FaLBD39 strain containing recombinant plasmid, wherein the strain can be used for transforming strawberry fruits.
Example 4 acquisition and phenotypic analysis of transgenic strawberry fruits
In this example, 30 strawberry plants with consistent growth vigor were selected, and averagely divided into 3 groups (control group, overexpression group, and silencing group), and 1-2 fruits with similar size were selected for each plant to perform transformation test, specifically:
(1) falbd39 obtaining of over-expressed strawberry fruit
Agrobacterium strain GV3101-p3301-FaLBD39 was inoculated into 100mL YEP liquid medium containing Kan (kanamycin) 50mg/L and Rif (rifampicin) 25mg/L, and shake-cultured at 28 ℃ and 200rpm until OD 600 became 1.0. Centrifuging at 3000rpm for 5min, removing supernatant, adding 10mL MS salt solution to resuspend thallus, placing at 28 deg.C, culturing at 100rpm for 4h with shaking, and injecting into strawberry fruit.
The MS salt solution contains MgCl2MES, acetosyringone.
The strawberry fruits are strawberry fruits with consistent growth vigor in a small green fruit period.
(2) Obtaining of strawberry fruit with FaLBD39 silencing
Agrobacterium strain GV3101-pTRV-FaLBD39 was inoculated into 100mL YEP liquid medium containing Kan (kanamycin) 50mg/L and Rif (rifampicin) 25mg/L, and shake-cultured at 28 ℃ and 200rpm until OD 600 became 1.0. Centrifuging at 3000rpm for 5min, removing supernatant, adding 10mL MS salt solution to resuspend thallus, placing at 28 deg.C, culturing at 100rpm for 4h with shaking, and injecting into strawberry fruit.
The MS salt solution contains MgCl2MES, acetosyringone.
The strawberry fruits are strawberry fruits with consistent growth vigor in a small green fruit period.
(3) And the results obtained
The fruits of the FALBD39 over-expression strawberries and the fruits of the FALBD39 silent strawberries are cultured to a red ripening stage, picked randomly and observed in a comparison mode, and the result is shown in figure 1, wherein a is the fruits of the strawberries in a control group, b is the fruits of the strawberries in an over-expression group, and c is the fruits of the strawberries in a silent group. Meanwhile, quantitative analysis is carried out by applying a high performance liquid chromatography HPLC-DAD technology, and the content of the total anthocyanin in the strawberry fruits is detected, wherein the result is shown in figure 2.
As can be seen from fig. 1 and fig. 2, the expression of FaLBD39 is limited by means of silencing or knockout, etc., so that the accumulation of anthocyanin in strawberry fruits can be effectively increased, and the nutritional value of the strawberry fruits is improved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> institute of plant of Chinese academy of sciences of Jiangsu province
Application of FaLBD39 in increasing anthocyanin accumulation in strawberry fruits and method for increasing anthocyanin accumulation in strawberry fruits
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Cys Val Leu Arg Ser Cys Leu Arg Trp Ile Asp Ser Pro Glu Ser Gln
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Gly Tyr Ala Thr Leu Phe Leu Ala Lys Phe Phe Gly Arg Ser Asp Leu
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Leu Ser Phe Ile Ser Ala Val Pro Glu Asn Gln Arg Pro Ala Leu Phe
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Gln Ser Leu Leu Phe Glu Ala Cys Gly Arg Thr Val Asn Pro Val Ser
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Gly Ala Val Gly Ile Leu Ser Ser Gly Asn Trp His Val Cys His Ala
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Ala Val Gln Thr Val Leu Ser Gly Gly Val Leu Arg Pro Leu Pro Ala
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Met Leu Thr Pro Asn Leu Asp Glu Ser Ser Gln Val Thr Ile Asn Arg
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Gly Ser Cys Gly Ile Gln Asn Pro Arg Tyr Thr Ser Val Pro Ser Met
130 135 140
Phe Asp Asp Asp Val Glu Pro Leu Asp Val Gly Leu Ser Leu Lys Pro
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Asn Leu Ala Ala Ala Gly Gly Arg Gly Arg Ala Arg Gly Trp Ser Cys
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Ala Val Asp Arg Met Arg Asp Thr Val Ser Phe Ser Ser Glu Asp Ser
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Leu Leu Asn Leu Phe Val
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aagtttttcg gccgtagcga cctcttgtcc ttcatctccg ccgtgcctga aaaccaacgc 180
cctgctcttt ttcagtctct gctttttgaa gcgtgcgggc gtacggtgaa cccggtgagc 240
ggagcggtgg ggattctctc gagcgggaac tggcacgtgt gtcacgcggc ggtccagacg 300
gtgttatccg gcggtgtttt gaggccgtta ccggcgatgc tgacgccaaa cctggacgaa 360
tcatcccaag tcactatcaa cagaggctca tgcggaattc aaaacccccg ctacacatcc 420
gtaccgtcca tgttcgacga cgacgtggag ccgttggatg taggactctc gctgaagccg 480
aatctcgcgg cggccggagg aagaggacga gcgaggggat ggagctgtgc agttgatcga 540
atgagagaca cggtgtcgtt tagctccgaa gattcggtca tgactactag cggtggcggc 600
ggcggttcgg acggtggccg caagcttttg aacctttttg tttga 645
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Claims (8)
- Use of the FaLBD39 gene for increasing the accumulation of anthocyanin in strawberry fruits, said FaLBD39 gene being a gene expressing a protein of the amino acid sequence shown in SEQ ID No. 1.
- 2. The use of claim 1, wherein the nucleotide sequence of the FaLBD39 gene is the nucleotide sequence shown in SEQ ID No. 2.
- 3. Use according to claim 1 or 2, wherein the expression of the FaLBD39 gene in strawberry fruits is down-regulated to increase the accumulation of anthocyanin in strawberry fruits.
- 4. A method for increasing anthocyanin accumulation in strawberry fruits, which comprises regulating the expression level of a FaLBD39 gene in strawberry fruits, wherein the FaLBD39 gene is a gene that expresses a protein having an amino acid sequence shown as SEQ ID No. 1.
- 5. The method of claim 4, wherein the expression of the FaLBD39 gene is downregulated to increase the accumulation of anthocyanin in strawberry fruits.
- 6. The method of claim 5, wherein the FaLBD39 gene is ligated with a pTRV vector to obtain a recombinant plasmid pTRV-FaLBD39, and the recombinant plasmid pTRV-FaLBD39 is expressed in strawberry fruits to increase anthocyanin accumulation in the strawberry fruits.
- 7. The method according to claim 6, wherein the expression of the recombinant plasmid pTRV-FaLBD39 in strawberry fruits is achieved using Agrobacterium mediation.
- 8. A method according to any of claims 4-7, characterized in that the method comprises the steps of:(a) providing a linearized pTRV vector and a FaLBD39 gene with a pTRV vector recombinant fragment;(b) after the linearized pTRV vector and the FaLBD39 gene with the recombinant pTRV vector fragment are incubated with recombinase, competent cells are transformed, and a pTRV-FaLBD39 plasmid is obtained;(c) the pTRV-FaLBD39 plasmid is transformed into Agrobacterium GV3101, and the FaLBD39 gene is transferred into strawberry fruits through Agrobacterium mediation.
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| CN104629489A (en) * | 2015-01-30 | 2015-05-20 | 浙江省农业科学院 | Method for extracting anthocyanin in strawberry fruits |
| CN106883292A (en) * | 2017-04-17 | 2017-06-23 | 山东农业大学 | Available from the MsLBD13 albumen and its encoding gene of functional form apple and application |
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| CN104629489A (en) * | 2015-01-30 | 2015-05-20 | 浙江省农业科学院 | Method for extracting anthocyanin in strawberry fruits |
| CN106883292A (en) * | 2017-04-17 | 2017-06-23 | 山东农业大学 | Available from the MsLBD13 albumen and its encoding gene of functional form apple and application |
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