CN104611345B - A kind of gene for improveing fruit quality and its coded product and application - Google Patents
A kind of gene for improveing fruit quality and its coded product and application Download PDFInfo
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Abstract
The invention discloses a kind of gene M aOFP1 of new improvement fruit quality, while being related to related application of the MaOFP1 genes in improvement fruit quality.At least one of 4) genetically modified plants method is cultivated the invention provides one kind, by MaOFP1 channel genes purpose plants, to obtain with following 1) genetically modified plants of feature：1) fruit weight of the genetically modified plants is more than the purpose plant；2) the pulp thickness of the genetically modified plants fruit is more than the purpose plant；3) peel thickness of the genetically modified plants fruit is more than the purpose plant；4) soluble solid content of the genetically modified plants fruit is more than the purpose plant；The nucleotides sequence of the MaOFP1 genes is classified as SEQ ID No.1 in SEQ ID No.1 or the sequence table in sequence table from the nucleotides of 5 ' end the 1st 435；The amino acid sequence of the coded product MaOFP1 albumen of the MaOFP1 genes is the SEQ ID No.2 in sequence table.The experiment proves that, the MaOFP1 genes of banana are imported in wild-type tomatoes, obtain the genetically modified plants of quality-improving, be that plant breeding is laid a good foundation by the present invention.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of gene and its coded product for improveing fruit quality
With application.
Background technology
OFP (ovate family protein, OFP) (avette family protein gene) is the class existed only in plant
Gene, the protein of such gene code has a nucleoid positioning signal and a unknown function being made up of 70 amino acid
(DUF623) domains (Liu etc., 2002) of conserved domain Domain ofUnknown Function 623, later this
Domain is renamed as OVATE domains (Hackbusch etc., 2005) again.OVATE genes are separated in tomato are accredited as at first
The important regulating and controlling factor of fruit shapes, is that terminator occurs and causes tamato fruit in advance in translation process by OVATE genes
By the circular transformation (Liu etc., 2002) to pyriform.Later, it have also discovered in arabidopsis with conservative OVATE domains
AtOFPs gene families, and show that it has effect (Hackbusch etc., 2005 of the cell membrane elongation of regulation Plant Secondary Materials；
Pagnussat etc., 2007；Wang etc. 2007；2011；Li etc. is 2011).Tsaballa etc. is cloned into an OVATE from capsicum
Gene C aOvate, it participates in the shape for determining fruit.However, the research at present for this genoid function is fairly limited,
In terms of some reports also only rest on its regulating and controlling effect to fruit shapes, for the research of its regulating and controlling effect to fruit quality
Have no report.
Banana (Musa acuminata Colla) is the world important cereal crops and industrial crops, is also to be only second to mandarin orange
The second-biggest-in-the-world fruit of tangerine, is one of maximum fruit of global fresh fruit consumption figure.With banana industry development and people's lives
The raising of level, for the requirement more and more higher of banana quality.The high-quality banana that fruit type is attractive in appearance, nutrition and health care, resistance to storage are fresh-keeping
As popular demand, quality-improving also just turn into banana industry can sustainable development Key Scientific And Technical Problems.But at present
Research to banana quality responses and regulatory mechanism is not goed deep into also very much.This research is using banana MuMADS1 as bait, using ferment
An ovate family protein has been fished out in the cDNA library that female two-hybrid techniques adopt latter 2 days from banana, has been named
For MaOFP1.Speculate that the gene may play an important roll during banana quality responses.
The content of the invention
It is an object of the present invention to provide MaOFP1 genes and its coded product.
The nucleotides sequence of the MaOFP1 is classified as the SEQ ID No.1 in sequence table；
The amino acid sequence of the MaOFP1 albumen is the SEQ ID No.2 in sequence table.
It is a further object to provide the new of MaOFP1 genes or MaOFP1 gene encoding production MaOFP1 albumen
Purposes.
The invention provides the coded product MaOFP1 albumen of MaOFP1 genes or MaOFP1 genes in improvement fruit
Application in quality；
The improvement fruit quality is embodied in the soluble solid content for improving fruit, and increase fruit is heavy, increase
Plus fruit pulp thickness；
The plant is specially dicotyledon or monocotyledon；The dicotyledon further be specially kind
Eggplant；
The nucleotides sequence of the MaOFP1 genes is classified as the SEQ ID in SEQ ID No.1 or the sequence table in sequence table
No.1 is from the nucleotides of 5 ' end 1-435；The amino acid sequence of the MaOFP1 albumen is the SEQ ID in sequence table
No.2。
Third object of the present invention is to provide a kind of cultivation genetically modified plants method.
The method that the present invention is provided, has following 1) -4 by MaOFP1 channel genes purpose plants, to obtain) at least one
Plant the genetically modified plants of feature：
1) fruit weight of the genetically modified plants is more than the purpose plant；
2) the pulp thickness of the genetically modified plants fruit is more than the purpose plant；
3) peel thickness of the genetically modified plants fruit is more than the purpose plant；
4) soluble solid content of the genetically modified plants fruit is more than the purpose plant；
In the above method, the nucleotides sequence of the MaOFP1 genes is classified as SEQ ID No.1 or sequence table in sequence table
In SEQ ID No.1 from the nucleotides of 5 ' end 1-435；The ammonia of the coded product MaOFP1 albumen of the MaOFP1 genes
Base acid sequence is the SEQ ID No.2 in sequence table.
In the above method, the MaOFP1 genes import the purpose plant by expression vector.
In the above method, the expression vector is to insert the MaOFP1 genes in pCAMBIA1304, obtained expression
The carrier of MaOFP1 albumen；In an embodiment of the present invention, expression vector is specially from 5 ' by the SEQ ID No.1 in sequence table
The carrier obtained between the nucleotides inserted pCAMBIA1304 of end 1-435 NcoI and SpeI restriction enzyme sites.
In the above method, the purpose plant is dicotyledon or monocotyledon；The dicotyledon is specific
For tomato.
Fourth object of the present invention is to provide a kind of expression vector.
The expression vector that the present invention is provided, for MaOFP1 genes are inserted in pCAMBIA1304, obtained expression MaOFP1
The carrier of albumen；In an embodiment of the present invention, expression vector is specially from 5 ' ends the by the SEQ ID No.1 in sequence table
The carrier obtained between 1-435 nucleotides inserted pCAMBIA1304 NcoI and SpeI restriction enzyme sites.
The nucleotides sequence of the MaOFP1 genes is classified as the SEQ ID in SEQ ID No.1 or the sequence table in sequence table
No.1 is from the nucleotides of 5 ' end 1-435；The amino acid sequence of the MaOFP1 albumen is the SEQ ID in sequence table
No.2。
The experiment proves that, MaOFP1 is imported wild-type tomatoes by the present invention, obtains turning for 35S promoter driving
MaOFP1 tomato strains, overexpression MaOFP1 adds fruit weight, pulp peel thickness and soluble solid and contained
Amount.These results show that MaOFP1 is played an important role in fruit quality forming process.
Brief description of the drawings
Fig. 1 is RT-PCR expression analysis of the MaOFP1 in banana different tissues
Fig. 2 detects for Southern traces
Fig. 3 detects to turn MaOFP1 Quality of Tomato Fruit.Wherein A is Red mature fruit vertical section；B examines for Red mature fruit weight
Survey；C is Red mature fruit pulp Thickness sensitivity；D Red mature fruits peel thickness is detected；E is Red mature fruit soluble solid content
Detection.
Fig. 4 is expression analysis of the MaOFP1 in transgenic line Red mature fruit
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Banana (Musa acuminateL.AAA group cv.Brazilian) fruit (Post flowering in following embodiments
100-120 days) bought from China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's biotechnology research institute Chengmai banana plantation.Pick up from green ripe stage
Quick-frozen latter -80 DEG C save backup in fruit, flower, ovary, root, stem and the Ye Yu liquid nitrogen of (100 days after spending).
Tomato (Lycopersivonesculenlum Mill.) is sheerly for AV6, hereinafter referred to as wild-type tomatoes, is purchased from
Chinese Academy of Tropical Agricultural Sciences quality resource research institute vegetables center.It is planted in the tropical biotechnology of Chinese Academy of Tropical Agricultural Sciences
- 80 DEG C save backup after research institute's Experimental Base, collection Transgenic tomato fruit, root, stem, flower and blade liquid nitrogen flash freezer.
Embodiment 1, the acquisition for turning MaOFP1 tomatoes
1st, gene M aOFP1 acquisition
Using the MATCHMAKER of Clontech companiesTMThe kits of GAL4Two-Hybrid System 3, with banana
MuMADS1 is bait, the cDNA library that banana is adopted latter 2 days is built, using co-transformation method transformed yeast AH109, in SD/-
Screened on Ade/-His-Leu/-Trp+x-a-gal, picking blue colonies, extract yeast plasmid DNA as template, use kit
In long range PCR primer (P1：5′-CTATTCGATGATGAAGATACCCCACCAAACCC-3′and P2：5′-
GTGAACTTGCGGGGTTTTTCAGTATCTACGATT-3 ' and the Advantage 2PCR Polymerase Mix
(Clontech, Cat.No.639201) is expanded, and obtained PCR primer is sequenced, and total length is redesigned after bioinformatic analysis
Primer, expands the SEQ ID No.1 that obtained nucleotides sequence is classified as in sequence table, and the unnamed gene shown in the nucleotide sequence is
MaOFP1, its albumen encoded is MaOFP1, and the amino acid sequence of the albumen is the SEQ ID No.2 in sequence table.
2nd, banana MaOFP1 expression analysis
Banana Root, stem, leaf, flower, ovary and banana progress Total RNAs extraction, the cDNA reverse transcriptions of adopting latter 0 day.
Primer, OVRTP1 are designed in non-conservative area according to MaOFP1 sequences：5′-GCCATTAACGGCCCACAGTTC-3′；
OVRTP2：5 '-TGGTGGAGAGGTGAATTCAAG-3 ' carry out real-time qRT-PCR analyses.Using ACTIN as internal reference, expand
The primer sequence for increasing internal reference is AP1：5 '-AGGCAGGATTTGCTGGTGA-3 ' and AP2：5’-ACCAGTGGTACGACCGCTA-
3 ', amplification length is 398bp.
As a result as shown in figure 1, MaOFP1 banana different tissues the in good time quantitative PCR expression analysis of fluorescence, it can be seen that
The gene is main to express in ovary and after adopting in 0 day fruit, is not expressed in other tissues or expression quantity is seldom.Speculate its
Play an important roll in fruit development and quality responses.
3rd, the amplification of expression vector
Leaf of banana DNA is extracted, with P1：5-CGCCATGGATGGAGTACCCATACGAC-3, P2：5-
CGACTAGTGTATTGCTCAGCCAAC-3 is primer, obtains 435bp PCR primer.
By NcoI the and SpeI digestions of obtained PCR primer, obtained digestion products are with passing through same digestion
PCAMBIA1304 carriers (public can obtain from China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute's biotechnology research institute) are connected, and are connected
Practice midwifery thing, connection product is converted into Escherichia coli, transformant is obtained.The plasmid of transformant is extracted, sequencing is sent to, is as a result the matter
Grain is from the nucleotides inserted pCAMBIA1304 of 5 ' end 1-435 NcoI and SpeI by the SEQ ID No.1 in sequence table
The carrier obtained between restriction enzyme site, is named as pCAMBIA1304-MaOFP1.
4th, the acquisition of recombinant bacterium
PCAMBIA1304-MaOFP1 is transferred into Agrobacterium tumefaciens LBA4404, and (public can be therefrom
Tropical agricultural science institute of state torrid zone biotechnology research institute is obtained) in, recombinant bacterium is obtained, the plasmid order-checking of recombinant bacterium is extracted, should
Plasmid is pCAMBIA1304-MaOFP1, and the recombinant bacterium containing the plasmid is named as into LBA4404/pCAMBIA1304-
MaOFP1。
5th, the acquisition and identification of MaOFP1 tomatoes are turned
1) acquisition of MaOFP1 tomatoes is turned
Above-mentioned LBA4404/pCAMBIA1304-MaOFP1 is infected to the blade of wild-type tomatoes, Agrobacterium has been infected about
500 leaf dishes.The bud that differentiates obtains 30 resistant budses by screening, last transplant survival 15 plants of resistant plants, conversion effect
Rate is 3%；15 plants of T0 are obtained for the tomato for turning MaOFP1.
2) identification of MaOFP1 tomatoes is turned
Extract T0 generation turn MaOFP1 tomato carry out Southern trace detections, using unconverted wild-type tomatoes as pair
According to probe primer is P1：5′-GCCATTAACGGCCCACAGTTC-3′；P2：5′-TGGTGGAGAGGTGAATTCAAG-3.Knot
Fruit is as shown in Fig. 2 L1, L2, L3, L4, L5, L6, L7 are the different strains for turning MaOFP1 tomatoes in T0 generations, and CK- is unconverted open country
Raw type tomato, is negative control, and CK+ is pCAMBIA1304-MaOFP1 plasmids, is positive control, it can be seen that obtain 4 plants of volumes
Number turn MaOFP1 tomato for L3, L5, L6, L7 positive T0 generations.
Empty carrier pCAMBIA1304 is transferred in wild-type tomatoes using same method, obtain T0 generation turn empty carrier kind
Eggplant, carries out Southern trace detections, is not detected by purpose fragment according to the method described above, illustrates that obtaining positive T0 generations turns empty carrier
Tomato.
Above-mentioned T0 is sowed for plant, sowing, until obtaining T2 for homozygous lines.
The functional study of embodiment 2, MaOFP1
1st, the phenotype of MaOFP1 tomatoes is turned
Sowing numbering is L3, L5, L6, L7 T2 generations to turn MaOFP1 tomatoes (MaOFP1), T2 generations and turn empty carrier tomato and not
Wild-type tomatoes (control) are converted, above strain tomato seeds are broadcast in pot for growing seedlings, under 25 DEG C, illumination 3000lux, 8h/d
Seedling is transferred to crop field in 2 months or so by culture, growth when plant strain growth stalwartness when plant height reaches 15-20cm, observes red ripe phase fruit
It is real.Each 20 plants of strain, is tested in triplicate, results averaged ± standard deviation.
As a result as shown in figure 3, wherein A is Red mature fruit vertical section；B is regulation and control of the MaOFP1 to Red mature fruit weight, C, D
Regulation and control for MaOFP1 to Red mature fruit fruit pulp thickness, E is tune of the MaOFP1 to Red mature fruit soluble solid content
Control.
Find out in Fig. 3 A, T2 generations turn MaOFP1 tomato plants fruit compared with wild-type tomatoes and become big, weight increase；Statistics
Fruit weight (fruit weight), it is as a result following (shown in Fig. 3 B)：Numbering is to turn the list of MaOFP1 tomatoes L3, L5, L6, L7 4 T2 generations
Fruit is respectively (unit again：Gram)：13.23 ± 0.26,13.85 ± 0.11,11.68 ± 0.28,12.68 ± 0.15, and wild type
Tomato fruit weighs 9.37 ± 0.399 grams.
In Fig. 3 C, T2 generation turn MaOFP1 tomato plants pulp with wild-type tomatoes compared with thicken count numbering be L3, L5,
In L6, L7 4 T2 generations, turn the pulp thickness of MaOFP1 tomatoes, as a result as follows：2.59±0.11、2.80±0.10、2.36±
0.02nd, 2.49 ± 0.10, and wild-type tomatoes pulp thickness is 2.18 ± 0.02.
In Fig. 3 D, T2 generation turn MaOFP1 tomato plants with wild-type tomatoes compared with pericarp increase it is thin, count numbering be L3, L5,
In L6, L7 4 T2 generations, turn the peel thickness of MaOFP1 tomatoes, as a result as follows：0.306±0.009、0.372±0.016、0.387
± 0.009,0.382 ± 0.003, and wild-type tomatoes peel thickness is 0.265 ± 0.013.
In Fig. 3 E, in T2 generations, turn MaOFP1 tomato plants red ripe phase fruit soluble solids content compared with wild-type tomatoes
Increase, statistics numbering is to turn the soluble solid content of MaOFP1 tomatoes L3, L5, L6, L7 4 T2 generations, as a result as follows：
4.55 ± 0.048,5.15 ± 0.085,5.75 ± 0.625,6.0 ± 0.42, and the red ripe phase fruit solubility of wild-type tomatoes is solid
Shape thing is 4.10 ± 0.17.
2nd, expression of the MaOFP1 in transgenic line
The T2 that numbering is L3, L5, L6, L7 is gathered respectively to invert for the ripe phase fruit of MaOFP1 tomato reds, extraction total serum IgE is turned
Record cDNA and utilize OVRTP 1：5′-GCCATTAACGGCCCACAGTTC-3′；OVRTP2：5′-
TGGTGGAGAGGTGAATTCAAG-3 ' carry out qRT-PCR detects MaOFP1 expression.Reference gene accession number is
(AK318637), primer sequence is actinP1：5′-CCAACAGAGAGAAGATGA-3′actinP2：5′-
ATGTCTCTTACAATTTCCCG-3′。
As a result as shown in figure 4, being wherein substantially not detectable the expression of the gene in the red ripe phase fruit of wild-type tomatoes, and
Expression quantity is all larger in L3, L5, L6 and L7 strain fruit, is respectively：9.196、7.948、6.886、8.411.Speculate that they are right
Quality responses have larger effect.
From the above, it can be seen that MaOFP1 shows significant regulating and controlling effect to Quality of Tomato Fruit.Fruit becomes big, fruit
Weight increase, fruit pulp thickness increase, fruit soluble solids content is dramatically increased, and the result of study of this research is disclosed
MaOFP1 genes are in the potential value influenceed on fruit quality, it is possible to the gene money of preciousness is provided for improvement fruit quality from now on
Source, while the research to fruit quality formation mechenism provides new point of penetration.
Claims (2)
- The application of 1.MaOFP1 genes or MaOFP1 gene encoding production MaOFP1 albumen in improvement fruit quality,The improvement fruit quality is embodied in the weight for improving fruit, fruit pulp thickness, fruit solubility solid Thing content；The plant is tomato；The nucleotides sequence of the MaOFP1 genes is classified as the SEQ ID No.1 in SEQ ID No.1 or the sequence table in sequence table From the nucleotides of 5 ' end 1-435；The amino acid sequence of the coded product MaOFP1 albumen of the MaOFP1 genes is sequence SEQ ID No.2 in table.
- 2. one kind cultivates genetically modified plants method, have following 1) -4 by MaOFP1 channel genes purpose plants, to obtain) in extremely A kind of few genetically modified plants of feature：1) fruit weight of the genetically modified plants is more than the purpose plant；2) it is described to turn base Because the pulp thickness of fruit is more than the purpose plant；3) peel thickness of the genetically modified plants fruit is more than the mesh Plant；4) soluble solid content of the genetically modified plants fruit is more than the purpose plant；The plant is tomato；The nucleotides sequence of the MaOFP1 genes is classified as the SEQ ID No.1 in SEQ ID No.1 or the sequence table in sequence table From the nucleotides of 5 ' end 1-435；The amino acid sequence of the coded product MaOFP1 albumen of the MaOFP1 genes is sequence SEQ ID No.2 in table.
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| CN112457380B (en) * | 2019-09-09 | 2022-07-05 | 中国科学院遗传与发育生物学研究所 | Protein for regulating and controlling content of plant fruit shape and/or fruit juice, related biological material and application thereof |
| CN110878324A (en) * | 2019-12-09 | 2020-03-13 | 新疆农业科学院园艺作物研究所 | Preparation method of tomato material with high fruit solid content |
| CN116814673A (en) * | 2022-04-27 | 2023-09-29 | 中国农业科学院农业基因组研究所 | Genomic structural variation for regulating and controlling content of soluble solids in tomato fruits, related products and application |
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