CN107142275A - Applications of the eary maturity of grape gene VvWRKY13 in regulation and control plant in Synthesis pathway - Google Patents

Applications of the eary maturity of grape gene VvWRKY13 in regulation and control plant in Synthesis pathway Download PDF

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CN107142275A
CN107142275A CN201710311119.4A CN201710311119A CN107142275A CN 107142275 A CN107142275 A CN 107142275A CN 201710311119 A CN201710311119 A CN 201710311119A CN 107142275 A CN107142275 A CN 107142275A
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vvwrky13
fruit
tomato
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grape
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马倩
刘新
侯丽霞
张岁芳
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Qingdao Agricultural University
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    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life

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Abstract

The invention provides eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway.The present invention will be loaded with the EHA105 agrobacterium strains of recombinant plasmid, be obtained by leaf dish infestation methodVvWRKY13Overexpression tomato material, and selfing obtains the pure and mild transgenic line of at least three independence repeatedly.Detection transgene tomato strain difference is grown the correlation merit index of the ethylene emanation in period, the expression quantity of ethylene synthase key gene, ethene associated phenotypic traits, the length of transgenosis fruit different growth periods, and ripening fruits.The present invention passes through the results showVvWRKY13By the expression for regulating and controlling ethylene synthase key gene, participate in the biosynthesis of ethene, so as to shorten the young fruit expanding stage of transgene tomato, promote fruit early-maturing but do not change the function of ripe fruit quality, the Mature breeding for the berries fruit such as tomato provides theoretical foundation and experiment basis.

Description

Eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway
Technical field
Excavated the invention belongs to eary maturity of grape beneficial gene and Function Identification technical field, and in particular to eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway.
Background technology
Grape is as a kind of world's eurytopicity fruit, with its fashionable whole world of unique brewing character.With economic growth, in State's grape wine consumption is in rising trend, and grape -growing areas increases therewith.According to " international grape and grape wine tissue " The statistics of (international organization of vine and wine, OIV) issue is shown, in 2014 National expenditures is 79.9 ten thousand hectares in the grape -growing areas made grape wine, the 11% of global grape -growing areas is accounted for, more than 79.2 ten thousand The France of hectare leaps to the second in the world.At present, grape industry has become an important industry of agricultural industrialization.Cause This, verifies the molecular regulation mechanism that grape fruit grows, and to promoting grape research, improves the yield and product of China's grape Matter, increase China grape has particularly important realistic meaning with the competitiveness of international market at home.
Growing for grape fruit shows a kind of typical double " S " type curves, is generally divided into 3 growth phases： First growth cycle (young fruit expanding stage), deadtime (stone phase) and the second growth cycle.First growth cycle of grape fruit (young fruit expanding stage) continues nearly 60 days from florescence.At the end of this growth cycle, berry and embryo are basically formed, berry It is universal oval and grow into the 70% of ripening fruits volume.Second period is called deadtime (stone phase).This period holds The length of continuous time is relevant with grape variety characteristic, and this period of currant is more relatively short, has core grape to be substantially carried out embryo Development, plant skin hardening.This phase also carries out flavor substance and the synthesis of aromatic compound and the shape of some precursor substances Into.The mark that deadtime (stone phase) terminates is that change takes place in the color of grape pomace, therefore this turning point is also named and turned The color phase.It is generally acknowledged that the deadtime of grape fruit largely determines quality when berry is ripe.Afterwards, grape fruit Development enter last period --- the second growth cycle, Berry volume quickly increases again, and sugar content steeply rises, and contains Acid amount is rapid to be declined, and tannin content is significantly reduced, and anthocyanin and aromatic compound are largely synthesized, so that fruit shows spy Some colors, reach optimal edible state.At present, the research to fruit development is concentrated mainly on the second growth week Phase is the stage from fruit annesl to maturation, and the concern to the first two period is very limited.
The content of the invention
The present invention relates to eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway, while not The function and application of quality when changing fruit maturation, can excavate for eary maturity of grape beneficial gene and contribute, so as to solve contracting When short fruit growth period, it is difficult to keep the production problem of fruit quality.
To reach the purpose for solving the problems, such as above-mentioned production, the present invention is achieved using following technical scheme：
The invention provides eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway.
Further：The plant is tomato.
Further：Eary maturity of grape gene will be containedVvWRKY13Agrobacterium strains infect tomato, co-cultured；Will Explant after co-cultivation, which is placed in bud inducement cultivation base, carries out squamous subculture；Bud elongation medium is transferred to after explant germinates Culture；Bud is transferred to culture in root media afterwards and extremely obtains transgenosisVvWRKY13Tomato plant.
Further：7 days and veraison after spending, ethene is in transgenosisVvWRKY13Rate of release in tomato strain shows Write and be higher than wild-type tomatoes,VvWRKY13Influence the growing amount of Tomato Fruit Development early stage ethene.
Further：Ethylene synthase key gene SlACS1b、SlACO1WithSlACO4In transgenosisVvWRKY13Kind Expression quantity in solanberry reality is significantly raised,VvWRKY13By influenceing ethylene synthase related gene during Tomato Fruit Development Express to regulate and control the biosynthesis of ethene in tamato fruit.
Further：TransgenosisVvWRKY13Tomato plant seed has typical composing type triple response phenotype.
Further：TransgenosisVvWRKY13In tomato plantVvWRKY13The relative expression quantity and wild-type tomatoes of gene Compared to dramatically increasing,VvWKRY13It is overexpressed the excessive generation for promoting ethene.
Further：GeneVvWRKY13Overexpression shorten expanding stage in Fruit Development Process.
Further：GeneVvWRKY13Tomato strain fruit is overexpressed 2-3 days more early than wild type from blooming annesl,VvWRKY13Shorten the time of fruit young fruit development.
Further：GeneVvWRKY13It is overexpressed lycopene, soluble sugar and titratable in tomato strain fruit The content of acid is unchanged compared with wild type.
Compared with prior art, advantages of the present invention and advantageous effects are：
1st, the present invention is experimentally confirmed describedVvWRKY13Gene fruit young fruit expanding stage great expression, in deadtime and Second growth period expression quantity is reduced rapidly；And only worked through the checking of transgene tomato phenotypic character in young fruit expanding stage, it is right Deadtime and the second growth period have little to no effect.
2nd, it is of the present inventionVvWRKY13Gene is worked by regulating and controlling the growing amount of endogenous ethylene, the life without novel substance Into safe and reliable.
3rd, it is of the present inventionVvWRKY13Gene shortens young fruit and expanded by promoting the generation of young fruit expanding stage endogenous ethylene Phase, so as to reach shortening fruit development period, promote the effect of fruit early-maturing.
4th, it is of the present inventionVvWRKY13Important stage-deadtime and second growth of the gene in influence fruit quality Phase -- expression quantity is very low, has little influence on growing for the two period fruits, so as to not have completely to fruit quality finally Negative effect.
5th, the content of the index of quality total Soluble Sugar of fruit, titratable acid content and lycopene nothing is obvious after complete ripeness Change.
The present invention is experiment material from mode crop-tomato that berry is studied.By rightVvWRKY13Transgene tomato Strain is grown the research of associated phenotypic traits, it was demonstrated thatVvWRKY13By the generation of regulating fruit young fruit expanding stage ethene, Shorten the young fruit expanding stage of fruit, so that fruit early-maturing but not changing the quality of fruit.
Brief description of the drawings
Fig. 1 is the process that tomato variety ' Micro-Tom ' is viewed and admired in leaf disk method conversion in the present invention.5 width figures from left to right Piece is the tomato seedling sprouted respectively, the tomato cotyledon of preculture, the callus of germination, culture of rootage and is transferred in soil Transgenic Tomato Plants.
Fig. 2 is viewed and admired in tomato variety ' Micro-Tom ' transgenic line in the present inventionVvWRKY13Relative expression Amount.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are the transgenic line of 3 screened independent homozygosis respectively.
During Fig. 3 is the present inventionVvWKRY13It is overexpressed the ethylene emanation of tomato strain different times fruit.Wherein WT generations Table wild-type tomatoes, L3, L9, L14 are the transgenic line of 3 screened independent homozygosis respectively.
Fig. 4 is ethylene synthase key gene in the present inventionSlACS1a VvWKRY13When being overexpressed tomato strain difference Expression quantity in phase fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are 3 screened independent homozygosis respectively Transgenic line.
Fig. 5 is ethylene synthase key gene in the present inventionSlACS1b VvWKRY13When being overexpressed tomato strain difference Expression quantity in phase fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are 3 screened independent homozygosis respectively Transgenic line.
Fig. 6 is ethylene synthase key gene in the present inventionSlACS2 VvWKRY13It is overexpressed tomato strain different times Expression quantity in fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are turning for 3 screened independent homozygosis respectively Gene strain.
Fig. 7 is ethylene synthase key gene in the present inventionSlACS4 VvWKRY13It is overexpressed tomato strain different times Expression quantity in fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are turning for 3 screened independent homozygosis respectively Gene strain.
Fig. 8 is ethylene synthase key gene in the present inventionSlACS6 VvWKRY13It is overexpressed tomato strain different times Expression quantity in fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are turning for 3 screened independent homozygosis respectively Gene strain.
Fig. 9 is ethylene synthase key gene in the present inventionSlACO1 VvWKRY13It is overexpressed tomato strain different times Expression quantity in fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are turning for 3 screened independent homozygosis respectively Gene strain.
Figure 10 is ethylene synthase key gene in the present inventionSlACO3 VvWKRY13When being overexpressed tomato strain difference Expression quantity in phase fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are 3 screened independent homozygosis respectively Transgenic line.
Figure 11 is ethylene synthase key gene in the present inventionSlACO4 VvWKRY13When being overexpressed tomato strain difference Expression quantity in phase fruit.Wherein WT represents wild-type tomatoes, and L3, L9, L14 are 3 screened independent homozygosis respectively Transgenic line.
During Figure 12 is the present inventionVvWKRY13Be overexpressed tomato strain growing state, wherein A, B, C represent respectively 30 days, 45 days and the upgrowth situation of 60 days tomato plants.WT represents wild-type tomatoes, and L3, L9, L14 are 3 screened independences respectively Homozygosis transgenic line.
During Figure 13 is the present inventionVvWKRY13The fruit of tomato strain is overexpressed, wherein WT represents wild-type tomatoes, L3, L9, L14 are the transgenic line of 3 screened independent homozygosis respectively.
During Figure 14 is the present inventionVvWKRY13Tomato strain fruit sectional view is overexpressed, wherein WT represents wild-type tomatoes, L3, L9, L14 are the independent pure and mild transgenic line of screened 3 respectively.
Figure 15 is cultivated tomato kind ' Ailsa Craig ' in the present inventionVvWRKY13It is overexpressed the PCR identifications of strain. S1-S3：Transgenic sample；W：Wild type control；O：Negative control.
Figure 16 is cultivated tomato kind ' in Ailsa Craig ' transgenic line in the present inventionVvWRKY13Relative table Up to amount.Wherein WT represents wild-type tomatoes, and L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
During Figure 17 is the present inventionVvWRKY13It is overexpressed triple response of the tomato strain to ethene.Wherein WT represents wild type Tomato, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Figure 18 is ACC pairs in the present inventionVvWRKY13It is overexpressed the influence of tomato strain plumular axis length.Wherein WT represents open country Raw type tomato, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Figure 19 is AOA pairs in the present inventionVvWRKY13It is overexpressed the influence of tomato strain triple response.Wherein WT represents open country Raw type tomato, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Figure 20 is AOA pairs in the present inventionVvWRKY13It is overexpressed the influence of tomato strain triple response mesocotyl length.Its Middle WT represents wild-type tomatoes, and L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Figure 21 be transfer-gen plant different times in the present invention fruit in pectin enzyme geneSlPG1Relative expression quantity. Wherein WT represents wild-type tomatoes, and L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Figure 22 be transfer-gen plant different times in the present invention fruit in pectin enzyme geneSlPG3Relative expression quantity. Wherein WT represents wild-type tomatoes, and L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
During Figure 23 is the present inventionVvWKRY13It is overexpressed the content of lycopene in tomato strain complete ripeness fruit.Wherein WT generations Table wild-type tomatoes, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
During Figure 24 is the present inventionVvWKRY13It is overexpressed the content of total Soluble Sugar in tomato strain complete ripeness fruit.Wherein WT Wild-type tomatoes are represented, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
During Figure 25 is the present inventionVvWKRY13It is overexpressed the content of titratable acid in tomato strain complete ripeness fruit.Wherein WT generations Table wild-type tomatoes, L1, L2, L3 are the transgenic line of 3 screened independent homozygosis respectively.
Embodiment
Technical scheme is further described in detail below in conjunction with accompanying drawing, subordinate list and specific embodiment.
Embodiment 1
Illustrated by the present inventionVvWRKY13The proof of function and application of the gene in Synthesis pathway, is specifically included as follows Step：
1、VvWRKY13It is overexpressed the conversion and screening of tomato：
(1) a certain amount of commercially available seed for viewing and admiring tomato variety ' Micro-Tom ' is taken, with the 75 % s of ethanol soaking disinfection 30, Outwell rapidly.Again with the 4 % min of sodium hypochlorite soaking disinfection 15, with sterile water washing 7-8 times after sterilization, need to fully it wash every time Wash, thoroughly remove the disinfectant of residual.After seed washes clean, seed is broadcast in seed germination medium（1/2MS）.Dark training Support and treat within 3-4 days to move to 25 DEG C after germination, germination, the photoperiod is 16 h/8 h（Illumination/dark）Under conditions of cultivate.Treat Grow cotyledon, cotyledon just opens, do not grow true leaf before seedling be used to convert.
(2) willVvWRKY13CDS sequence (Genbank accession numbers：JQ692108) it is transferred to containing 35S promoter Super1300 plasmids.Afterwards, the plasmid is transferred in agrobacterium strains EHA105, and is stored in -80 DEG C of ultra low temperature freezer In, it is standby.- 80 DEG C will be stored in containVvWRKY13The agrobacterium strains of plasmid in YEB fluid nutrient mediums renewal cultivation and Expand culture.Wherein cultivating the YEB Liquid Culture based formulas used in Agrobacterium is：The g/L of tryptone 10, yeast extract 1 G/L, sucrose 5 g/L, MgSO4 7H2O 0.5 g/L, pH 7.0.121 DEG C of min of autoclaving 20, are cooled to room after packing The kanamycins and rifampin of 50 mg/L filtration sterilizations are added after temperature.
(3) take step (1) not grow the aseptic cotyledon of true leaf, cut off blade tip and petiole portion, remaining cotyledon is cut into 3 mm The square of × 3 mm size, is placed on the MS solid mediums of antibiotic-free, 28 DEG C of precultures 2 days.
(4) bacterium solution after step (2) expansion is shaken centrifuges 10 min in 5 000 r/min, and precipitation is diluted to sterilized water OD600=0.5-0.6 is as infecting liquid.Explant after step (3) preculture 2 days is infected into 5 min in liquid is infected.It is placed in Co-culture, co-cultured 2 days in 28 DEG C of darkrooms on the MS solid mediums of antibiotic-free.It is MS+2 to co-culture medium component The mg/L hygromycin of mg/L ZT+0.2 mg/L IAA+100 mg/L Ticarcillin/Clavulanate Acids+10.
(5) explant after co-culturing 2 days is taken out from darkroom, is all placed in bud inducement cultivation base, 7 are cultivated under light It is transferred to after it in new culture medium and carries out squamous subculture.After first time squamous subculture, subculture next time was typically carried out every 2 weeks and is trained Support, until explant germination is complete.Bud inducement cultivation based component is MS+2 mg/L ZT+0.2 mg/L IAA.
(6) after bud induction period, it is transferred in bud elongation medium, cultivates when explant germination bud length is about 2-3cm 3-4 weeks.Bud elongation medium composition is the mg/L of MS+0.5 mg/L ZT+0.2 mg/L IAA+100 mg/L Ticarcillin/Clavulanate Acids+10 tides Mycin.
(7) when bud is extended to 4-5 cm, cut after callus and bud is transferred in culture of rootage, cultivate 3-4 weeks. Wherein culture of rootage based component is the mg/L IBA of 1/2MS+10 mg/L hygromycin+2.
(8) it will take root vigorous, the seedling for growing into certain altitude is transferred to Tu Penli immediately.Leaf disk method converts the whole of tomato Individual process is as shown in Figure 1.
2、VvWRKY13The screening of transgene tomato and homozygosis
（1）The tomato leaf DNA of earth culture stage normal growth is extracted, enters performing PCR with the special primer of gene and identifies.It will detect To positive plant normally cultivated, and labeled as T1 generations.DetectionVvWRKY13PCR primer sequence used in gene is：
VvWRKY13-FP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；VvWRKY13-RP1：5’- GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；
（2）To obtain the transgenic line of homozygosis, T1 is for seed and sows for individual plant harvest, obtains T2 for transgene tomato seedling.
（3）T2 is extracted for transgene tomato blade DNA, removes and produces Gene Isolation plant, T2 is for seed for harvest.Plantation is received The seed obtained, obtains the transgene tomato seedling in T3 generations.
（4）PCR detects separation situations of the T3 for transgene tomato colony.The strain of Gene Isolation is occurred without in T3 generations i.e. For the transgenic line of homozygosis.DetectionVvWRKY13PCR primer sequence used in gene is consistent with (1).
（5）Using in qRT-PCR technology for detection transgenic linesVvWRKY13The relative expression quantity of gene.Testing result is such as Shown in Fig. 2.DetectionVvWRKY13QRT-PCR primer sequences used in gene expression amount are：
VvWRKY13-FP：5’-GGTTGCCAACAATCCCT-3’；
VvWRKY13-RP：5’-GTCATCTCCACCGATACTTC-3’；
（6）The seed of the transgene tomato strain of homozygosis is collected, for subsequent experimental.
3、VvWRKY13It is overexpressed the detection of tomato strain ethylene emanation, triple response and ethylene synthase gene expression amount
（1）With wild type andVvWRKY13Heterologous overexpression tomato plant fruit is material, and fruit of uniform size is chosen respectively 3-4, determines and spends latter 7 days, the Ethylene Production Rate of veraison and full ripe stage fruit.As a result as shown in figure 3, after spending 7 days and Veraison ethene existsVvWRKY13Rate of release in heterologous overexpression strain is significantly higher than wild type, and in full ripe stage difference not Substantially, showVvWRKY13It has impact on the ethylene emanation of tamato fruit young fruit.
In ethylene synthase approach, ACC synzyme and rate-limiting enzyme and key enzyme that acc oxidase is the approach.Key enzyme The expression of gene decides the growing amount of ethene, with wild type of the same period andVvWRKY13Heterologous overexpression tomato plant fruit For material, choose wild type of uniform size and be overexpressed tamato fruit 3-4 of plant, 7 d after measure is spent, veraison and complete The ethylene synthase approach related gene expression amount of ripe phase fruit.As a result as shown in Fig. 4-11,SlACS1b、SlACO1WithSlACO4 Relative expression quantity than wild type in transgenosis fruit is high；Remaining is then without larger difference.Comprehensive analysis shows,VvWRKY13It is main The expression of ethylene synthase key gene during tomato young fruit development is have impact on, and then influences the generation of tomato-ethylene.
DetectionSlACS1b、SlACO1WithSlACO4QRT-PCR primer sequences used in gene expression amount are：
SlACS1b-FP：5’-TGTTCTGAACCTGGTTGGT-3’；
SlACS1b-RP：5’-TAAAATCATCCAATCTTCGA-3’；
SlACO1-FP：5’-GTACCCGATCTTGACGA-3’；
SlACO1-RP：5’-GAAGTATGATGCCTCCTG-3’；
SlACO4-FP：5’-CTGTCATATTTCCAGCACC-3’；
SlACO4-RP：5’-AAGTTGACAGTAGTCTCCACAG-3’.
（2）The triple response of Plants To Ethylene is the specific reaction of plant, can be used for the bioassay of ethene.By second Plant seedlings after alkene processing show the expansion of the suppression of hypocotyl elongation growth, the horizontal overstriking of epicotyl, and summit hook Greatly.By wild type andVvWKRY13It is overexpressed after tomato seeds, aseptic process, dibbling is containing 10 μM of ACC and 10 μ On mol/L AOA MS culture mediums, after 28 DEG C of 12 h of normal culture, dark is carried out according to culture 7d, its growing state is observed.Knot Fruit shows：Relative to wild-type tomatoes,VvWRKY13Tomato plant is overexpressed under the processing of no exogenous ethylene, upper hypocotyl life Length is suppressed, shows the composing type triple response to ethene.On the culture medium containing 10 μM of ethylene synthesis inhibitors AOA,VvWKRY13The triple response difference for being overexpressed tomato strain and wild-type tomatoes diminishes.Result above is demonstrated in phenotypeVvWKRY13It is overexpressed the excessive generation for promoting ethene.
4、VvWRKY13It is overexpressed the detection of each growth period length of tomato strain fruit
（1）Observation simultaneously sowing wild-type tomatoes andVvWRKY13The developmental state of the tomato strain fruit of overexpression.As a result As shown in Figure 12, Tables 1 and 2,VvWRKY13It is overexpressed time of the strain tamato fruit from blooming annesl about in advance 2-3 My god, and annesl to the complete ripeness time be 10 days.ShowVvWRKY13Shorten the development time of tamato fruit young fruit.
Time statistics of the table 1 from blooming to fruit annesl
Strain Annesl time（dpa）
WT 42.67a±1.15
L1 36.33b±1.52
L2 39.33ab±4.0
L3 36.33b±1.52
The fruit of table 2 is from annesl to ripe time statistics
Strain Annesl to maturation（d）
WT 10a±1.15
L1 10a±1.52
L2 10a±4.04
L3 10a±1.52
（2）The pectin that polygalacturonase belongs in pectin enzyme, energy degradation of cell wall makes cell wall breakdown, participates in fruit Softening, its synthase gene isSlPG.Detect its synthetic geneSlPG1WithSlPG3Expression quantity, find its in wild type andVvWRKY13The relative expression quantity and indifference being overexpressed in tomato strain.VvWKRY13The fruit of tomato strain is overexpressed as schemed Shown in 13 and Figure 14.
DetectionSlPG1WithSlPG3Primer sequence used in gene expression amount is：
SlPG1-FP：5’-TGAGGACCAAATCGGAATC-3’；
SlPG1-RP：5’-TGTCGGACTAAGAAAGAATAACC-3’；
SlPG3-FP：5’-ATACAACAGTTTTCAGCAGTTCAAGT-3’；
SlPG3-RP：5’-GGTTTTCCACTTTCCCCTACTAA-3’.
5、VvWRKY13It is overexpressed the detection of tomato strain complete ripeness fruit quality
Lycopene belongs to carotenoid, is a kind of natural food coloring, is concentrated mainly on the red of various fruit and vegetable and fruit Among pulp or tissue, because of its former activity that is deficient in vitamin, there is the essence effect of going out to oxygen radical, therefore have pre- anti-cancer, support Face beauty, prevention cardiovascular and cerebrovascular disease, are also to weigh one of key index of Quality of Tomato Fruit.With full ripe stage wild type andVvWRKY13It is material to be overexpressed tomato strain fruit, determines the content of lycopene in full ripe stage fruit.As a result tomato is shown Red pigment in wild type andVvWRKY13Significant change does not occur for the content being overexpressed in tomato plant fruit.It can thus be appreciated thatVvWRKY13Overexpression not in tamato fruit lycopene content produce influence.
6th, tomato sugariness is the important component of Quality Characteristics in Tomato, and it depends primarily on soluble sugar in tamato fruit Content, therefore the quality of tomato can be evaluated with soluble sugar content.With full ripe stage wild type andVvWRKY13Overexpression kind The fruit of eggplant strain is material, determines soluble sugar content.As a result showVvWRKY13Transgenic line fruit and wild type fruit Obvious change does not occur for real sugared content, showsVvWRKY13Overexpression do not influence soluble sugar in tamato fruit to contain Amount.
7th, the acid in fruit is primarily referred to as the anion of total effectively organic acid, and main titratable acid is lemon in tomato Acid, is played an important role in the local flavor of tomato.We with full ripe stage wild type andVvWRKY13It is overexpressed tomato strain Fruit is the content of material tests titratable acid.As a result the two and no significant difference, explanation are shownVvWRKY13Do not influence tomato Middle titratable acid content.
To sum up show grapeVvWRKY13By promoting the generation of young fruit expanding stage endogenous ethylene, shorten young fruit expanding stage, So as to reach shortening fruit development period, promote the effect of fruit early-maturing, but fruit quality is had no adverse effect.
Embodiment 2
Illustrated by the present inventionVvWRKY13The proof of function and application in Synthesis pathway, specifically includes following step Suddenly：
1、VvWRKY13It is overexpressed the conversion and screening of tomato：
（1）Taking a certain amount of commercially available cultivated tomato kind, ' seed of Ailsa Craig (AC) ' is disappeared with 75 % ethanol immersion 30 s of poison, are outwelled rapidly.Again with the 4 % min of sodium hypochlorite soaking disinfection 15, with sterile water washing 7-8 times after sterilization, every time It need to fully wash, thoroughly remove the disinfectant of residual.After seed washes clean, seed is broadcast in seed germination medium（1/ 2MS）.Dark culturing treats to move to 25 DEG C for 3-4 days after germination, germination, and the photoperiod is 16 h/8 h（Illumination/dark）Bar Cultivated under part.Cotyledon to be grown, cotyledon just opens, do not grow true leaf before seedling be used to convert.
（2）By be stored in -80 DEG C the plasmid containing 35S promoter EHA105 agrobacterium strains in YEB fluid nutrient mediums Renewal cultivation and expansion are cultivated.Wherein cultivating the YEB Liquid Culture based formulas used in Agrobacterium is：The g/L of tryptone 10, ferment Female g/L of extract 1, sucrose 5 g/L, MgSO4 7H2O 0.5 g/L, pH 7.0.121 DEG C of min of autoclaving 20 after packing, It is cooled to the kanamycins and rifampin that 50 mg/L filtration sterilizations are added after room temperature.
（3）Take step (1) not go out the aseptic cotyledon of true leaf, cut off blade tip and petiole portion, remaining cotyledon is cut into 3 mm × 3 The square of mm size, is placed on the MS solid mediums of antibiotic-free, 28 DEG C of precultures 2 days.
（4）Bacterium solution after step (2) expansion is shaken centrifuges 10 min in 5 000 r/min, and precipitation is diluted to sterilized water OD600=0.5-0.6 is as infecting liquid.Explant after preculture 2 days is infected into 5 min in liquid is infected.It is placed in antibiotic-free MS solid mediums on co-culture, co-cultured 2 days in 28 DEG C of darkrooms.It is MS+2 mg/L ZT+ to co-culture medium component The mg/L hygromycin of 0.2 mg/L IAA+100 mg/L Ticarcillin/Clavulanate Acids+10.
（5）Explant after co-culturing 2 days is taken out from darkroom, bud inducement cultivation base is all placed in, cultivated 7 days under light It is transferred to afterwards in new culture medium and carries out squamous subculture.After first time squamous subculture, subculture next time was typically carried out every 2 weeks and is trained Support, until explant germination is complete.Bud inducement cultivation based component is MS+2 mg/L ZT+0.2 mg/L IAA.
（6）After bud induction period, it is transferred in bud elongation medium, cultivates when explant germination bud length is about 2-3cm 3-4 weeks.Bud elongation medium composition is the mg/L of MS+0.5 mg/L ZT+0.2 mg/L IAA+100 mg/L Ticarcillin/Clavulanate Acids+10 tides Mycin.
（7）When bud is extended to 4-5 cm, cut after callus and bud is transferred in root media, cultivate 3-4 weeks. Wherein culture of rootage based component is the mg/L IBA of 1/2MS+10 mg/L hygromycin+2.
（8）It will take root vigorous, the seedling for growing into certain altitude is transferred to Tu Penli immediately.
2、VvWRKY13The screening of transgene tomato and homozygosis
（1）The tomato leaf DNA of earth culture stage normal growth is extracted, enters performing PCR with the special primer of gene and identifies.As a result as schemed Shown in 15.It will detect that obtained positive plant is normally cultivated, and labeled as T1 generations.
DetectionVvWRKY13PCR primer sequence used in gene is：
VvWRKY13-FP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；
VvWRKY13-RP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’.
（2）To obtain the transgenic line of homozygosis, T1 is for seed and sows for individual plant harvest, obtains T2 for transgene tomato children Seedling.
（3）T2 is extracted for transgene tomato blade DNA, removes and produces Gene Isolation plant, T2 is for seed for harvest.Plantation is received The seed obtained, obtains the transgene tomato seedling in T3 generations.
（4）PCR detects separation situations of the T3 for transgene tomato colony.The strain of Gene Isolation is occurred without in T3 generations i.e. For the transgenic line of homozygosis.DetectionVvWRKY13PCR primer sequence used in gene is consistent with (1).
（5）Using in qRT-PCR technology for detection transgenic linesVvWRKY13The relative expression quantity of gene.Testing result is such as Shown in Figure 16.DetectionVvWRKY13QRT-PCR primer sequences used in gene expression amount are：
VvWRKY13-FP：5’-GGTTGCCAACAATCCCT-3’；
VvWRKY13-RP：5’-GTCATCTCCACCGATACTTC-3’；
（6）The seed of the transgene tomato strain of homozygosis is collected, for subsequent experimental.
3、VvWRKY13It is overexpressed the detection of tomato strain ethylene emanation, triple response and ethylene synthase gene expression amount
（1）With wild type andVvWRKY13Heterologous overexpression tomato plant fruit is material, and fruit of uniform size is chosen respectively 3-4, determines and spends latter 7 days, the Ethylene Production Rate of veraison and full ripe stage fruit.As a result show, 7 days and veraison after spending Ethene existsVvWRKY13Rate of release in heterologous overexpression strain is significantly higher than wild type, and unobvious in full ripe stage difference, ShowVvWRKY13It has impact on the ethylene emanation of tamato fruit young fruit.
In ethylene synthase approach, ACC synzyme and rate-limiting enzyme and key enzyme that acc oxidase is the approach.Key enzyme The expression of gene decides the growing amount of ethene, with wild type of the same period andVvWRKY13Heterologous overexpression tomato plant fruit For material, choose wild type of uniform size and be overexpressed tamato fruit 3-4 of plant, 7 d after measure is spent, veraison and complete The ethylene synthase approach related gene expression amount of ripe phase fruit.As a result show,SlACS1b、SlACO1WithSlACO4In transgenosis Relative expression quantity in fruit than wild type is high；Remaining is then without larger difference.Comprehensive analysis shows,VvWRKY13Mainly it have impact on The expression of ethylene synthase key gene during tomato young fruit development, and then influence the generation of tomato-ethylene.
DetectionSlACS1b、SlACO1WithSlACO4QRT-PCR primer sequences used in gene expression amount are：
SlACS1b-FP：5’-TGTTCTGAACCTGGTTGGT-3’；
SlACS1b-RP：5’-TAAAATCATCCAATCTTCGA-3’；
SlACO1-FP：5’-GTACCCGATCTTGACGA-3’；
SlACO1-RP：5’-GAAGTATGATGCCTCCTG-3’；
SlACO4-FP：5’-CTGTCATATTTCCAGCACC-3’；
SlACO4-RP：5’-AAGTTGACAGTAGTCTCCACAG-3’.
（2）The triple response of Plants To Ethylene is the specific reaction of plant, can be used for the bioassay of ethene.By second Plant seedlings after alkene processing show the expansion of the suppression of hypocotyl elongation growth, the horizontal overstriking of epicotyl, and summit hook Greatly.By wild type andVvWKRY13It is overexpressed after tomato seeds, aseptic process, dibbling is containing 10 μM of ACC and 10 μ On mol/L AOA MS culture mediums, after 28 DEG C of 12 h of normal culture, dark is carried out according to culture 7d, its growing state is observed.Knot Fruit is as shown in figures 17 to 20：Relative to wild-type tomatoes,VvWRKY13Tomato plant is overexpressed under the processing of no exogenous ethylene, Upper hypocotyl growth is suppressed, shows the composing type triple response to ethene.Containing 10 μM of ethylene synthesis inhibitors AOA On culture medium,VvWKRY13The triple response difference for being overexpressed tomato strain and wild-type tomatoes diminishes.Result above is in phenotype On demonstrateVvWKRY13It is overexpressed the excessive generation for promoting ethene.
4、VvWRKY13It is overexpressed the detection of each growth period length of tomato strain fruit
（1）Observation simultaneously sowing wild-type tomatoes andVvWRKY13The developmental state of the tomato strain fruit of overexpression.As a result It has been shown that,VvWRKY13Being overexpressed time of the strain tamato fruit from blooming annesl about shifts to an earlier date 2-3 days, and annesl to complete ripeness when Between be 10 days.ShowVvWRKY13Shorten the development time of tamato fruit young fruit.
（2）The pectin that polygalacturonase belongs in pectin enzyme, energy degradation of cell wall makes cell wall breakdown, participates in The softening of fruit, its synthase gene isSlPG.Detect its synthetic geneSlPG1WithSlPG3Expression quantity.As a result such as Figure 21 and Shown in Figure 22, its in wild type andVvWRKY13The relative expression quantity and indifference being overexpressed in tomato strain.DetectionSlPG1WithSlPG3Primer sequence used in gene expression amount is：
SlPG1-FP：5’-TGAGGACCAAATCGGAATC-3’；
SlPG1-RP：5’-TGTCGGACTAAGAAAGAATAACC-3’；
SlPG3-FP：5’-ATACAACAGTTTTCAGCA GTTCAAGT-3’；
SlPG3-RP：5’-GGTTTTCCACTTTCCCCTACTAA-3’.
5、VvWRKY13It is overexpressed the detection of tomato strain complete ripeness fruit quality
（1）Lycopene belongs to carotenoid, is a kind of natural food coloring, is concentrated mainly on various fruit and vegetable and fruit Among red pulp or tissue, because of its former activity that is deficient in vitamin, there is the essence effect of going out to oxygen radical, therefore have pre- anti-cancer Disease, skin maintenance, prevention cardiovascular and cerebrovascular disease, are also to weigh one of key index of Quality of Tomato Fruit.It is wild with full ripe stage Type andVvWRKY13It is material to be overexpressed tomato strain fruit, determines the content of lycopene in full ripe stage fruit.As a result as schemed Shown in 23, lycopene in wild type andVvWRKY13Significant change does not occur for the content being overexpressed in tomato plant fruit. It can thus be appreciated thatVvWRKY13Overexpression not in tamato fruit lycopene content produce influence.
（2）Tomato sugariness is the important component of Quality Characteristics in Tomato, and it depends primarily on soluble in tamato fruit Sugared content, therefore the quality of tomato can be evaluated with soluble sugar content.With full ripe stage wild type andVvWRKY13It is overexpressed The fruit of tomato strain is material, determines soluble sugar content.As a result it is as shown in figure 24VvWRKY13Transgenic line fruit with Obvious change does not occur for the sugared content of wild-type fruit, showsVvWRKY13Overexpression do not influence solvable in tamato fruit Property sugared content.
（3）Acid in fruit is primarily referred to as the anion of total effectively organic acid, and main titratable acid is lemon in tomato Lemon acid, is played an important role in the local flavor of tomato.We with full ripe stage wild type andVvWRKY13It is overexpressed tomato strain Fruit be material tests titratable acid content.As a result as shown in figure 25, the two and no significant difference, explanationVvWRKY13No Influence titratable acid content in tomato.
To sum up show grapeVvWRKY13By promoting the generation of young fruit expanding stage endogenous ethylene, shorten young fruit expanding stage, So as to reach shortening fruit development period, promote the effect of fruit early-maturing, but fruit quality is had no adverse effect.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality Apply example the present invention is described in detail, for the person of ordinary skill of the art, can still implement to foregoing Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.

Claims (10)

1. eary maturity of grape geneVvWRKY13Application in regulation and control plant in Synthesis pathway.
2. eary maturity of grape gene according to claim 1VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：The plant is tomato.
3. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：Eary maturity of grape gene will be containedVvWRKY13Agrobacterium strains infect tomato, co-cultured；Will be altogether Explant after culture, which is placed in bud inducement cultivation base, carries out squamous subculture；Trained after being transferred to bud elongation medium after explant germination Support；Bud is transferred to culture in root media afterwards and extremely obtains transgenosisVvWRKY13Tomato plant.
4. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：7 days and veraison after spending, ethene is in transgenosisVvWRKY13Rate of release in tomato strain is notable Higher than wild-type tomatoes,VvWRKY13Influence the growing amount of Tomato Fruit Development early stage ethene.
5. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：Ethylene synthase key gene SlACS1b、SlACO1WithSlACO4In transgenosisVvWRKY13Tomato fruit Expression quantity in reality is significantly raised,VvWRKY13By the expression for influenceing ethylene synthase related gene during Tomato Fruit Development To regulate and control the biosynthesis of ethene in tamato fruit.
6. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：TransgenosisVvWRKY13Tomato plant seed has typical composing type triple response phenotype.
7. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：TransgenosisVvWRKY13In tomato plantVvWRKY13The relative expression quantity of gene and wild-type tomatoes phase Than dramatically increasing,VvWKRY13It is overexpressed the excessive generation for promoting ethene.
8. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：GeneVvWRKY13Overexpression shorten expanding stage in Fruit Development Process.
9. eary maturity of grape gene according to claim 1VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：GeneVvWRKY13Tomato strain fruit is overexpressed 2-3 days more early than wild type from blooming annesl,VvWRKY13Shorten the time of fruit young fruit development.
10. eary maturity of grape gene according to claim 2VvWRKY13The answering in Synthesis pathway in regulation and control plant With, it is characterised in that：GeneVvWRKY13It is overexpressed lycopene in tomato strain fruit, soluble sugar and titratable acid Content is unchanged compared with wild type.
CN201710311119.4A 2017-05-05 2017-05-05 Application of grape early-maturing gene VvWRKY13 in regulation and control of ethylene biosynthesis in plants Expired - Fee Related CN107142275B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110066327A (en) * 2019-04-30 2019-07-30 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） Application of the protein TaWRKY13 in regulation stress resistance of plant
CN111118026A (en) * 2020-01-17 2020-05-08 浙江大学 Application of tomato LAT61 gene
CN111549041A (en) * 2020-04-22 2020-08-18 青岛农业大学 Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110066327A (en) * 2019-04-30 2019-07-30 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） Application of the protein TaWRKY13 in regulation stress resistance of plant
CN111118026A (en) * 2020-01-17 2020-05-08 浙江大学 Application of tomato LAT61 gene
CN111118026B (en) * 2020-01-17 2021-12-07 浙江大学 Application of tomato LAT61 gene
CN111549041A (en) * 2020-04-22 2020-08-18 青岛农业大学 Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof

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