CN114438102A - Strawberry ethylene response FaERF13 gene and application thereof in changing strawberry fruit mature period - Google Patents
Strawberry ethylene response FaERF13 gene and application thereof in changing strawberry fruit mature period Download PDFInfo
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Abstract
The invention discloses strawberry ethylene regulation and controlFaERF13Genes and the application thereof,FaERF13the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the protein amino acid sequence is shown as SEQ ID NO. 2. The invention is realized by mixing strawberryFaERF13After gene separation, VIGS vector is constructed to transform strawberry fruits, and the maturation period of the fruits with gene silencing is obviously earlier than that of negative control, which shows thatFaERF13The gene is a negative regulatory factor for strawberry fruit ripening,FaERF13the gene has important application value in cultivating new strawberry varieties with early or delayed mature fruits (storage resistance)。
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a strawberry fruit maturity regulating gene FaERF13 and application thereof.
Background
Strawberry (Fragaria × ananassa Duch.) is a perennial herb of the family rosaceae, and is popular among consumers because of its characteristics such as special aroma, beautiful fruit type, and rich in vitamin C. The facility strawberries are consistent in growth, development and mature period and are easy to form and come into the market collectively. Therefore, the research on how to change the mature period of the strawberries, to bring the strawberries into the market or prolong the fruit period can obtain higher economic value. Fruits are classified into climacteric fruits and non-climacteric fruits according to whether or not a peak of respiration occurs before the fruit ripens. The climacteric fruit respiration transition is accompanied with ethylene transition, while the non-climacteric fruit has no ethylene transition phenomenon before and after the fruit is mature. Currently, studies on ethylene regulation of fruit ripening are mainly focused on climacteric fruits, and the functions and mechanisms in regulation of ripening of non-climacteric fruits such as strawberries are not clear. Strawberry fruit is capable of producing ethylene and expressing ethylene receptor genes. Exogenous ethephon promotes the coloring and softening of strawberry fruits and partially restores the coloring of fruits of RNAi strains.
ERF transcription factors are ethylene response factors involved in the ethylene signal transduction pathway, and are one of the AP2/ERF transcription factor superfamily. The proteins encoded by the AP2/ERF superfamily genes all comprise a conserved AP2/ERF structural domain, a DNA-binding domain AP2 structural domain consisting of 60-70 amino acids and consisting of 3 beta folds and 1 alpha helix. The AP2 domain was originally identified in the APETAL2 protein that regulates peanut growth in Arabidopsis. One protein module that binds the GCC cassette, the ERF domain, was then found in tobacco ethylene response proteins ERF1, ERF2, ERF3 and ERF4, regulating the level of transcription of ethylene response genes. The ERF gene family is the most important large family in the plant AP2/ERF transcription factor superfamily, and plays an important role in the processes of plant growth and development and stress response. Research shows that FaERF9 can be combined with FaMYB98 to induce the transcription level of a key synthetase encoding gene FaQR of strawberry important flavor substances 4-hydroxy-2, 5-dimethyl-3 (2H) furanone, and the AP2/ERF family can also regulate the biosynthesis of plant hormones, including ethylene, cytokinin, gibberellin and abscisic acid. AP2/ERF is also involved in the response of auxin, cytokinin, abscisic acid and jasmonic acid signals and is a key factor for linking phytohormone signals.
The current research on whether ethylene regulates and controls the ripening of strawberry fruits and the mechanism problem in the fruit needs to be solved urgently, the research on the function of the ERF gene in the ripening regulation of the strawberry fruits can provide a gene breeding tool for regulating and controlling the ripening period of the strawberries. On one hand, the clarification and application of the regulation and maturation function of the related genes can promote early maturation of strawberries and achieve the application value of improving the economic added value in the market in advance, on the other hand, the fresh strawberries are not storage-resistant and easy to deteriorate, the application of the related genes can delay the maturation period and achieve the effect of reducing logistics and storage cost due to peak staggering in the fruit period.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a strawberry ethylene response FaERF13 gene and application thereof in changing the fruit ripening period of strawberries, one aim is to provide a strawberry ethylene response gene FaERF13 which meets the use requirement, and the other aim is to provide application of the strawberry ethylene response gene FaERF13.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a strawberry ethylene response gene FaERF13 has a nucleotide sequence shown in SEQ ID NO. 1.
The amino acid sequence of the expression protein of the strawberry ethylene response gene FaERF13 is shown in SEQ ID NO. 2.
A pNC-TRV2-VIGS vector containing the strawberry ethylene response gene FaERF13.
The pNC-Cam1304-35S overexpression vector containing the strawberry ethylene response gene FaERF13.
The pNC-Cam1304-RNAi vector containing the strawberry ethylene response gene FaERF13.
The effect of the strawberry ethylene response gene FaERF13 in changing the fruit ripening of strawberries.
Has the advantages that: the FaERF13 gene is cloned by taking strawberry variety 'peach fumigation' as a material. The expression modes of the FaERF13 gene in different maturation stages are detected by a real-time fluorescent quantitative detection technology, and the fact that the VIGS vector is constructed to instantaneously transform strawberry fruits is proved to be a negative regulatory factor for strawberry fruit maturation, and the vector for stably over-expressing or silencing the gene is constructed, so that the method has important application value in the breeding fields of improving the yield of strawberries, promoting the maturation of strawberries and the like.
Drawings
FIG. 1 is a diagram of spatiotemporal expression patterns of the FaERF13 gene;
FIG. 2 is a plasmid map of plant VIGS vector PNC-TRV 2;
FIG. 3 is a graph comparing phenotypic changes in FaERF13 silencing versus negative groups;
FIG. 4 is a graph of the expression pattern of the FaERF13 gene silencing group versus the negative group;
FIG. 5 is a plasmid map of plant overexpression vector pNC-Cam 1304-35S;
FIG. 6 is a plasmid map of plant RNAi vector pNC-Cam 1304-RNA.
Detailed Description
The invention is further illustrated by the following examples and figures.
Example 1
Cloning of the FaERF13 Gene
The cDNA of 'peach fumigation' strawberry fruit is used as a template, a gene coding region segment is amplified, connected with a cloning vector and sequenced to obtain a gene nucleotide sequence.
(1) Primer design
Design of gene FaERF13 amplification primer based on octaploid strawberry genome
Primer F：5’-CTATCTAATCTTTGCCTTGG-3’
Primer R：5’-ATGGTTGTCTGTCCGTACAT-3’
(2) PCR amplification
Extracting RNA by taking a lean fruit receptacle removed in the mature period of the 'peach fumigation' strawberry as a template, carrying out reverse transcription to obtain a cDNA template, and carrying out PCR amplification on a CDS sequence of FaERF 13:
PCR amplification System (Total 50. mu.l):
reaction procedure:
(3) ligation of purified fragments to cloning vectors
Ligation vector pEASY-Blunt Cloning Kit (gold, Beijing, full complement)
Reaction (5 μ L):
PCR Product： 50ng
pEASY-Blunt Zero Cloning Vector 1μL
ddH2O up to 5μL
reaction conditions are as follows: 10min at 25 DEG C
E, transformation of escherichia coli: thawing freshly prepared or frozen E.coli Trans-T1 competent cells at-80 deg.C on ice; adding the ligation product into 50 microliter of competent cells, gently mixing, and performing ice bath for about 20 min; heating in 42 deg.C water bath for 60sec, and rapidly placing on ice for 2 min; adding 500 μ L LB liquid culture medium, shaking at 37 deg.C and 200rpm for 1 h; centrifuging at 4000rpm for 2min, discarding part of supernatant, retaining 100 μ L of bacterial liquid, and mixing; the bacterial liquid was smeared on LB screening plates containing kan and cultured overnight at 37 ℃ in an inverted state.
(4) Positive clone screening and sequencing analysis
Selecting a single colony from a screening culture plate, inoculating the single colony in an LB liquid culture medium, and shaking the bacteria at 37 ℃ and 250rmp for 6 h; bacteria liquid PCR detection is directly carried out by taking the bacteria liquid cultured for 6h as a template
Reaction system (20 μ L):
reaction procedure:
clone sent to Biotech company (Nanjing) for sequencing with PCR detection of bacterial liquid as positive
Example 2
FaERF13 gene space-time expression pattern analysis
The abundance of the FaERF13 gene in different fruit development stages is quantified through a fluorescent quantitative PCR technology, a fluorescent quantitative PCR primer forward primer 5'-GGCGTATGACCGAGCTGCTT-3' and a fluorescent quantitative PCR primer reverse primer 5'-TACCGGCTCAACGTTGTTGGA-3' are designed based on the ORF region of the FaERF13 gene, and a reference primer forward primer 5'-TTCACGAGACCACCTATAACTC-3' and a reference primer reverse primer 5'-GCTCATCCTATCAGCGATT-3' are designed based on the Actin gene. The reaction system refers to a Novoweb ChamQ SYBR Qpcr Master Mix kit, and the PCR reaction program is as follows: 95 ℃ for 30 sec; 95 ℃ 10sec, 60 ℃ 30sec, 40 cycles. Three biological replicates were performed for each experiment. The relative expression levels of the FaERF13 gene at different flowering stages and at different parts of the floral organ were calculated by performing quantitative fluorescent PCR using a quantitative fluorescent PCR instrument CFX96TM (Bio-RAD) with reference to the PCR system and procedure described in the manufacturer's instructions to obtain the number of cycles at which the fluorescence threshold was reached.
Selecting fruits of 'peach fumigation' strawberry G (green fruit), GW (green-to-ginkgo), W (ginkgo) and R (red fruit), selecting three receptacle with consistent size and color as biological repetition in each period, removing lean fruit to extract RNA, and performing expression analysis, wherein the expression level of FaERF13 gene is continuously reduced along with the development of the fruits as shown in figure 2, so that the expression of FaERF13 is proved to be negatively related to the mature period of the strawberry fruits.
Example 3FaERF13-VIGS vector construction and transient transformation of "peach Fumigation" fruits
(1) Vector construction
Design small fragment primer FaERF13.1_ F: 5 'CAGCGCTAAAACCCGAGGA-3'; FaERF13.1_ R: 5 '-CAAGCCACACTCTCGCTCCA 3'), using FaERF13cDNA as template to amplify the FaERF13.1 sequence, PCR procedure as in example 1, using Nimble cloning kit to insert the FaERF13 expression cassette into the VIGS vector PNC-TRV 2.
The system is as follows
PCR Product 50ng
pNC-Cam1304-35S 20-120ng
Nimble Mix 5μL
ddH2The volume of O was adjusted to 10. mu.L, and the cells were incubated at 25 ℃ for 10 min.
(2) Preparation of the invaded liquor
The vector plasmid is transformed into agrobacterium GV3101 competence by a liquid nitrogen freeze-thaw method: taking out the competent cells from-80 ℃, thawing at 4 ℃ and placing on ice; adding the plasmid to be transformed into 100 μ l of competent cells, stirring with hands to the tube bottom, mixing, standing on ice for 10min, liquid nitrogen for 5min, water bath at 37 deg.C for 5min, and ice bath for 5 min; adding 900 mul of YEB liquid culture medium without antibiotics, and performing shake culture at 28 ℃ for 2-3 hours; the cells were harvested by centrifugation at 6000rpm for one minute, and approximately 100. mu.l of the supernatant was left to gently blow and resuspend the pellet, spread on YEB plates containing Kan (50. mu.g/m) and Rif (50. mu.g/m), and placed upside down in an incubator at 28 ℃ for 2-3 days.
The PNC-TRV2-FaERF13 Agrobacterium positive strain was identified as in example 1. Inoculating into YEB liquid culture medium containing Kan (50 μ g/m) and Rif (50 μ g/m), culturing at 28 deg.C for 16h in shaking table, removing supernatant at 4000rpm for 10min, collecting thallus, and gradually adding resuspension (10mM MgCl. sub.10 mM)210mM MES, 200. mu.M acetosyringone), OD600 was measured using a spectrophotometer, the cells were finally resuspended to a final concentration of 2.0, the resuspended solution was left to stand away from the sun at room temperature for 3 hours, the PNC-TRV1 resuspension and PNC-TRV2-FaERF13.1 resuspension were mixed in equal volumes to prepare for injection
(3) Strawberry fruit transient transformation
Selecting GW and W stage strawberry peach fumigation fruits, injecting agrobacterium tumefaciens resuspension from a receptacle part, and infecting. The PNC-TRV1 heavy suspension was mixed with PNC-TRV2 empty heavy suspension in equal volume as a negative control. Phenotypic changes were observed by daily photographs after infestation. The fruits in the VIGS group begin to turn color in the next day, the fruits in the fourth day are completely mature, the fruits in the MOCK group do not change greatly in development and are still in the ginkgo stage, and the maturity is obviously lower than that in the VIGS group (figure 3).
(4) qRT-PCR detection and phenotype observation of VIGS silent fruit
In order to verify whether the change of premature ripening of strawberry fruits of the VIGS treatment group 'peach blooms' is related to the gene FaERF13 transfer, the invention adopts a common fluorescent quantitative PCR method to detect the expression quantity of the gene FaERF13 of the MOCK group and the VIGS treatment group, and the fluorescent quantitative PCR mode is the same as that of the example 2.
By taking an internal reference gene FaActin1 as a control, expression patterns of FaERF13 in different treatment groups are analyzed and found (figure 4), the expression level of the VIGS treatment group FaERF13 is about 50% lower than that of the CK group, so that the fact that the transcription factor of FaERF13 is silenced is proved to be effective, the fact that the fruit ripening of 'peach fumigation' strawberry is greatly advanced due to the silencing of the transcription factor of FaERF13 is proved, and the fact that the transcription factor of FaERF13 plays a role in negative regulation on the fruit ripening of the strawberry is proved.
Example 4
Construction of pNC-Cam1304-35S overexpression vector and RNA interference vector of FaERF13 and application prospect thereof
(1) Construction of pNC-Cam1304-35S FaERF13 overexpression vector and application prospect thereof
FaERF13-pNC-Cam1304-35S primers 5'-AGTGGTCTCTGTCCAGTCCTCTATCTAATCTTTGCCTTGG-3' and 5'-GGTCTCAGCAGACCACAAGTATGGTTGTCTGTCCGTACAT-3' with NC cloning joints are designed, FaERF13 obtained in example 1 is used as a template cDNA, a FaERF13 target insert fragment is obtained through amplification, and the PCR amplification and product recovery processes are the same as those in example 1.
The rapid Cloning of the insert and pNC-Cam1304-35S vector was performed by one-step method using a Nimble Cloning kit (NC Biotech), the system was the same as example 3, the E.coli transformation and positive clone screening method was the same as example 1, and the positive bacteria liquid was sequenced by Biochemical company (Nanjing) to obtain pNC-Cam1304-35S, FaERF13 overexpression vector.
The FaERF13-Cam1304-35S overexpression vector transforms strawberries by an agrobacterium-mediated method, a new strawberry variety with late ripening fruits is expected to be obtained, and the fruit production period of the strawberries can be prolonged (the commodity period is prolonged) by combining the early ripening variety and the genetically improved variety.
(2) pNC-Cam 1304-RNAi: FaERF13 vector construction and application prospect thereof
The primer design with NC joint, the acquisition of the FAERF13 insert and the NC cloning process are the same as those of the pNC-Cam1304-35S, the construction process of the FAERF13 vector, the pNC-Cam1304-RNAi vector has a positive NC cloning frame and a negative NC cloning frame, the FAERF13 insert is assembled in the positive direction and the vector is assembled in the reverse direction at the same time through one-step reaction, and the pNC-Cam1304-RNAi is obtained: ERF13 interferes with the vector. After the vector is transformed into plant cells such as strawberries and the like, the vector can be efficiently transcribed and can spontaneously form an RNAi hairpin structure, so that the FAERF13 gene can be rapidly and efficiently silenced.
pNC-Cam 1304-RNAi: the ERF13 interference vector can transform strawberry tissue culture seedlings by using an agrobacterium-mediated method or a gene gun method, and is expected to obtain new varieties with early fruit maturity, and particularly has great potential for application in aspects of advancing marketing time and enhancing commodity competitiveness of late-maturing strawberry varieties.
Sequence listing
<110> Yangzhou university
<120> strawberry ethylene response FaERF13 gene and application thereof in changing fruit maturation period of strawberries
<160> 12
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gacgacatca tccccttgtt tttccccagc tctgctactg cttcaacctg catgagtggg 180
ttttttacca cagagaactg gtgggaatca gcgctaaaac ccgaggactc tgaaaccgaa 240
gctgtaagtg ccccagggga cgacgtcgag gttaaactga gggagcaagc tcgtgttgcg 300
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gggaagtacg cggcggagat aagagatccg gcgaagaatg gagcgagagt gtggcttggg 420
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145 150 155 160
Gly Arg Lys Ala Lys Leu Asn Phe Pro His Leu Ile Gly Ser Asn Asn
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Tyr Ser Thr Ser Thr Ser Ser Ala Ser Glu Asp Ser Glu Ser Pro Lys
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Claims (7)
1. Strawberry ethylene response geneFaERF13The nucleotide sequence is shown in SEQ ID NO. 1.
2. The strawberry ethylene response gene of claim 1FaERF13The amino acid sequence of the expression protein is shown as SEQ ID NO. 2.
3. A strawberry ethylene response gene comprising the strawberry of claim 1FaERF13Plant overexpression vector PNC-Cam1304-35S:FaERF13。
4. a strawberry ethylene response gene comprising the strawberry of claim 1FaERF13Plant interference vector pNC-Cam 1304-RNAi:FaERF13。
5. the carrier of claim 3, wherein: the vector is assembled with an NC Cloning Frame (Nimble Cloning Frame), and gene fragments can be rapidly assembled by a one-step method without enzyme digestion connection; the vector is assembled with a 35S strong promoter, and can efficiently start gene expression.
6. The carrier of claim 4, wherein: the vector is assembled with an expression frame of 'NC cloning frame-intron-NC cloning frame' and an expression frame of kanamycin resistance gene, wherein chloramphenicol resistance gene is assembled in an intron, and the cloning positive rate can be close to 100% by screening of two antibiotics of kanamycin and chloramphenicol; two NC Cloning frames (Nimble Cloning frames) can insert gene fragments in a forward direction and insert gene fragments in a reverse direction simultaneously in a one-step reaction, and a silencing complex is automatically formed to efficiently interfere gene expression.
7. Use of the strawberry ethylene response gene FaERF13 of claim 1 for regulating strawberry fruit ripening.
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| CN115976067A (en) * | 2022-12-01 | 2023-04-18 | 四川农业大学 | Application of FaU-box E3 gene family in regulating and controlling strawberry fruit ripening |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576288A (en) * | 2018-12-05 | 2019-04-05 | 浙江五合生物科技有限公司 | A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its application |
| CN110894221A (en) * | 2019-12-12 | 2020-03-20 | 中国科学院植物研究所 | Strawberry maturation associated transcription factor gene FaNAC2 and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576288A (en) * | 2018-12-05 | 2019-04-05 | 浙江五合生物科技有限公司 | A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its application |
| CN110894221A (en) * | 2019-12-12 | 2020-03-20 | 中国科学院植物研究所 | Strawberry maturation associated transcription factor gene FaNAC2 and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115976067A (en) * | 2022-12-01 | 2023-04-18 | 四川农业大学 | Application of FaU-box E3 gene family in regulating and controlling strawberry fruit ripening |
| CN115976067B (en) * | 2022-12-01 | 2023-09-05 | 四川农业大学 | Application of FaU-box E3 gene family in regulation and control of strawberry fruit ripening |
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