CN107142275B

CN107142275B - Application of grape early-maturing gene VvWRKY13 in regulation and control of ethylene biosynthesis in plants

Info

Publication number: CN107142275B
Application number: CN201710311119.4A
Authority: CN
Inventors: 马倩; 刘新; 侯丽霞; 张岁芳
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2017-05-05
Filing date: 2017-05-05
Publication date: 2020-03-31
Anticipated expiration: 2037-05-05
Also published as: CN107142275A

Abstract

The invention provides a grape prematurity geneVvWRKY13The application of the compound in regulating ethylene biosynthesis in plants. The invention obtains the EHA105 agrobacterium strain carrying the recombinant plasmid by a leaf disc infection methodVvWRKY13And repeatedly selfing to obtain at least 3 independent pure transgenic lines. Detecting the ethylene production amount of the transgenic tomato line in different growth and development periods, the expression amount of an ethylene synthesis key enzyme gene, ethylene-related phenotypic characters, the lengths of different growth periods of the transgenic fruits and related quality indexes of mature fruits. The invention proves through experimental resultsVvWRKY13Through regulating and controlling the expression of key enzyme genes for synthesizing ethylene and participating in the biosynthesis of ethylene, the young fruit expanding period of the transgenic tomato is shortened, the premature fruit is promoted without changing the quality of the mature fruit, and theoretical basis and experimental basis are provided for the premature breeding of berry fruits such as tomatoes and the like.

Description

Application of grape early-maturing gene VvWRKY13 in regulation and control of ethylene biosynthesis in plants

Technical Field

The invention belongs to the technical field of favorable gene mining and function identification of grape prematurity, and particularly relates to a grape prematurity geneVvWRKY13The application of the compound in regulating ethylene biosynthesis in plants.

Background

Grape is a world wide-adaptability fruit, and is popular in the world due to the unique brewing characteristics of grape. With the economic growth, the consumption of Chinese grape wine is on the rise, and the planting area of the grape is increased. According to the statistical data published by the international organization of grapes and wine (OIV), the grape planting area for brewing wine in the Chinese in 2014 is 79.9 ten thousand hectares, which accounts for 11 percent of the global grape planting area and is over the second of the world living in France of 79.2 ten thousand hectares. At present, the grape industry has become an important industry for agricultural industrialization in China. Therefore, the method has important practical significance for promoting grape research, improving the yield and quality of grapes in China and increasing the competitiveness of grapes in domestic and international markets by detecting the molecular regulation mechanism of grape fruit growth and development.

The growth and development of grape fruits presents a typical double "S" shaped curve, which is generally divided into 3 growth stages: a first growth cycle (young fruit expansion phase), a lag phase (hard core phase) and a second growth cycle. The first growth cycle (young fruit expansion phase) of the grape fruit lasts for nearly 60 days from the flowering phase. At the end of this growth cycle, berries and embryos essentially form, the berries generally ovalize and grow to 70% of the mature fruit volume. The second period is called the lag phase (hard nucleus phase). The duration of this time period is related to the variety characteristics of grapes, the time period of seedless grapes is relatively short, and nucleated grapes mainly develop embryo and harden seed coat. The synthesis of flavour and aroma compounds, as well as the formation of some precursor substances, also takes place at this stage. The end of the lag phase (sclerosing phase) is marked by the onset of a change in the color of the grape pericarp, and this turning point is therefore also called the hyperchromic phase. It is believed that the lag phase of the grape fruit largely determines the quality of the berry at maturity. And then, the grape fruit development enters the last period, namely the second growth cycle, the berry volume is rapidly increased again, the sugar content is rapidly increased, the acid content is rapidly reduced, the tannin content is remarkably reduced, and a large amount of anthocyanin and aromatic compounds are synthesized, so that the grape fruit presents unique color, fragrance and taste, and the best edible state is achieved. Currently, the research on the growth and development of fruits mainly focuses on the second growth cycle, i.e. the stage from fruit color change to ripening, and the attention to the first two stages is limited.

Disclosure of Invention

The invention relates to a grape prematurity geneVvWRKY13The application of the compound in regulating and controlling ethylene biosynthesis in plants, and simultaneously, the function and the application of the compound do not change the quality of mature fruits, can make a contribution to the gene mining for the early maturity of grapes, thereby solving the problem of shrinkageThe production problem that the fruit quality is difficult to maintain when the fruit growth period is short.

In order to solve the production problem, the invention adopts the following technical scheme:

the invention provides a grape prematurity geneVvWRKY13The application of the compound in regulating ethylene biosynthesis in plants.

Further: the plant is tomato.

Further: will contain grape early maturing geneVvWRKY13The agrobacterium strain of (a) infects tomatoes for co-culture; placing the explants after co-culture in a bud induction culture medium for subculture; transferring the explant into a bud elongation culture medium for culture after the explant germinates; then transferring the bud into rooting culture medium to culture so as to obtain transgeneVvWRKY13Tomato plants.

Further: ethylene is transgenic 7 days after flowering and at the time of color transitionVvWRKY13The release rate in tomato lines is significantly higher than in wild type tomatoes,VvWRKY13influences the generation amount of ethylene in the early development stage of tomato fruits.

Further: ethylene synthesis key enzyme geneSlACS1b、SlACO1AndSlACO4in transgenesisVvWRKY13The expression level in tomato fruits is obviously increased,VvWRKY13the biosynthesis of ethylene in tomato fruits is regulated and controlled by influencing the expression of ethylene synthesis related genes in the tomato fruit development process.

Further: transgenosisVvWRKY13Tomato plant seeds have a typical constitutive triple response phenotype.

Further: transgenosisVvWRKY13In tomato plantsVvWRKY13The relative expression quantity of the gene is obviously increased compared with that of the wild tomato,VvWKRY13overexpression promotes the overproduction of ethylene.

Further: geneVvWRKY13The overexpression of (a) shortens the expansion period during fruit development.

Further: geneVvWRKY13The fruit of the over-expression tomato line is 2 to 3 days earlier than the wild type from flowering to color change,VvWRKY13the development time of young fruits of the fruits is shortened.

Further: geneVvWRKY13The contents of lycopene, soluble sugars and titratable acid in the over-expressed tomato line fruits were all unchanged compared to the wild type.

Compared with the prior art, the invention has the advantages and beneficial technical effects that:

1. the invention proves thatVvWRKY13The gene is expressed in a large amount in the young fruit expanding period of the fruit, and the expression level is rapidly reduced in the lag phase and the second growth phase; and the transgenic tomato phenotypic character verifies that the tomato plant only plays a role in the young fruit expansion period, and almost has no influence on the lag phase and the second growth phase.

2. The invention is as describedVvWRKY13The gene plays a role in regulating the generation amount of endogenous ethylene, has no generation of new substances, and is safe and reliable.

3. The invention is as describedVvWRKY13The gene can shorten the expansion period of young fruits by promoting the generation of endogenous ethylene in the expansion period of young fruits, thereby achieving the effects of shortening the development period of fruits and promoting the precocity of fruits.

4. The invention is as describedVvWRKY13The gene has very low expression level in important stages affecting the fruit quality, namely a lag phase and a second growth phase, and hardly affects the growth and development of the fruit in the two stages, so that the fruit quality is not negatively affected at all finally.

5. The quality index of the fully ripe fruit has no obvious change in soluble total sugar, titratable acid content and lycopene content.

The invention selects tomato as experimental material. By pairsVvWRKY13The research of the phenotype characters related to the growth and development of the transgenic tomato strains proves thatVvWRKY13The young fruit expanding period of the fruit is shortened by regulating and controlling the generation of ethylene in the young fruit expanding period of the fruit, so that the fruit is premature without changing the quality of the fruit.

Drawings

FIG. 1 is a process of transforming ornamental tomato variety ` Micro-Tom ` by the leaf disc method of the present invention. The 5 pictures from left to right are germinated tomato seedlings, pre-cultured tomato cotyledons, germinated calli, rooting cultures and transgenic tomato plants transferred to soil, respectively.

FIG. 2 shows the transgenic lines of ornamental tomato variety ` Micro-Tom ` according to the present inventionVvWRKY13Relative expression amount of (3). Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 3 is a drawing of the present inventionVvWKRY13Over-expressing the ethylene production of the tomato lines at different stages of the fruit. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 4 shows the key enzyme genes for ethylene synthesis in the present inventionSlACS1aIn thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 5 shows the key enzyme genes for ethylene synthesis in the present inventionSlACS1bIn thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 6 shows the key enzyme genes for ethylene synthesis in the present inventionSlACS2In thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 7 shows the key enzyme genes for ethylene synthesis in the present inventionSlACS4In thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 8 shows the key enzyme genes for ethylene synthesis in the present inventionSlACS6In thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 9 shows the key enzyme genes for ethylene synthesis in the present inventionSlACO1In thatVvWKRY13Over-expression tomato plant line different-period fruitsThe expression level in (1). Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 10 shows the key enzyme genes for ethylene synthesis in the present inventionSlACO3In thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 11 shows the key enzyme genes for ethylene synthesis in the present inventionSlACO4In thatVvWKRY13Over-expressing the expression level in the fruit of tomato lines at different periods. Wherein WT represents wild type tomato, and L3, L9, and L14 are 3 independent homozygous transgenic lines screened respectively.

FIG. 12 is a drawing of the present inventionVvWKRY13The growth of the over-expressed tomato lines, wherein A, B, C represents the growth status of 30, 45 and 60 days tomato plants, respectively. WT stands for wild type tomato and L3, L9, L14 are 3 independent homozygous transgenic lines screened for, respectively.

FIG. 13 shows the present inventionVvWKRY13Fruits of over-expressed tomato lines, where WT represents wild type tomato, L3, L9, L14 are 3 independent homozygous transgenic lines screened, respectively.

FIG. 14 shows the present inventionVvWKRY13Fruit transection map of overexpression tomato lines, where WT represents wild type tomato and L3, L9, L14 are 3 independent homozygous transgenic lines screened, respectively.

FIG. 15 is a drawing of the tomato cultivar 'Ailsa Craig' of the present inventionVvWRKY13And (4) performing PCR identification on the overexpression strain. S1-S3: a transgenic sample; w: a wild-type control; o: and (5) negative control.

FIG. 16 is a transgenic line of the invention of the cultivated tomato variety' Ailsa CraigVvWRKY13Relative expression amount of (3). Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 17 is a drawing of the present inventionVvWRKY13Triple reaction of over-expressed tomato lines to ethylene. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent ones screened respectivelyHomozygous transgenic lines.

FIG. 18 shows an ACC pair in the present inventionVvWRKY13Influence of the length of the embryonic axis of the over-expressed tomato lines. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 19 shows AOA pairs in the present inventionVvWRKY13Influence of triple reactions of over-expressed tomato lines. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 20 shows AOA pairs in the present inventionVvWRKY13Influence of the length of the embryonic axis in triple reactions of over-expressed tomato lines. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 21 shows the pectase genes in fruits of different stages of the transgenic plants of the inventionSlPG1Relative expression amount of (3). Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 22 shows the pectase genes in fruits of different periods of the transgenic plants of the present inventionSlPG3Relative expression amount of (3). Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 23 is a drawing of the present inventionVvWKRY13Overexpresses the lycopene content in the fully ripe fruit of the tomato line. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 24 shows the present inventionVvWKRY13Over-expression of the soluble total sugar content in the fully ripe fruit of the tomato line. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

FIG. 25 shows the present inventionVvWKRY13Over-expression of titratable acid content in the fully ripe fruit of a tomato line. Wherein WT represents wild type tomato, and L1, L2, and L3 are 3 independent homozygous transgenic lines screened respectively.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the accompanying drawings, the attached tables and the specific embodiments.

Example 1

The invention is illustratedVvWRKY13The function and application of the gene in ethylene biosynthesis are proved, and the method specifically comprises the following steps:

1、VvWRKY13transformation and screening of over-expressed tomatoes:

(1) a certain amount of seeds of a commercial ornamental tomato variety 'Micro-Tom' are taken, soaked and disinfected in 75% ethanol for 30 s and quickly poured out. Soaking and sterilizing with 4% sodium hypochlorite for 15 min, washing with sterile water for 7-8 times, and thoroughly removing residual disinfectant. After the seeds were washed clean, the seeds were sown in seed germination medium (1/2 MS). Culturing in dark for 3-4 days until seed germinates, transferring to 25 deg.C, and culturing under light/dark condition with photoperiod of 16 h/8 h. When cotyledons grow out, seedlings which are just opened and before true leaves grow out are used for transformation.

(2) Will be provided withVvWRKY13The CDS sequence of (Genbank accession number: JQ692108) was transformed into the Super1300 plasmid containing the 35S promoter. Thereafter, the plasmid was transferred into Agrobacterium strain EHA105 and stored in an ultra-low temperature refrigerator at-80 ℃ for future use. Storing at-80 deg.CVvWRKY13The Agrobacterium strain of the plasmid was resume and expanded in YEB liquid medium. Wherein the YEB liquid culture medium used for culturing the agrobacterium is prepared from the following components in percentage by weight: 10 g/L tryptone, 1g/L yeast extract, 5 g/L sucrose, 0.5 g/L MgSO4.7H2O, pH 7.0. Subpackaging, autoclaving at 121 deg.C for 20 min, cooling to room temperature, and adding 50 mg/L filter-sterilized kanamycin and rifampicin.

(3) Taking the sterile cotyledon without true leaf in the step (1), cutting off the leaf apex and the leaf stalk, cutting the rest cotyledon into a square block with the size of 3mm multiplied by 3mm, placing the square block on an MS solid culture medium without antibiotics, and pre-culturing for 2 days at 28 ℃.

(4) And (3) centrifuging the bacterial liquid expanded and shaken in the step (2) for 10 min at 5000 r/min, and diluting the precipitate with sterile water until OD600=0.5-0.6 is used as an infection liquid. And (4) infecting the explants pre-cultured for 2 days in the step (3) in an infection solution for 5 min. Co-culture was performed on MS solid medium without antibiotics in a dark room at 28 ℃ for 2 days. The components of the co-culture medium are MS +2mg/L ZT +0.2 mg/L IAA +100 mg/L timentin +10 mg/L hygromycin.

(5) The explants after 2 days of co-culture were taken out of the dark, placed in the bud induction medium, cultured in the light for 7 days, and transferred to a new medium for subculture. After the first subculture, the next subculture is generally carried out every 2 weeks until the explants germinate completely. The bud induction medium comprises MS +2mg/L ZT +0.2 mg/L IAA.

(6) After the bud induction period, the explant is transferred into a bud elongation culture medium when the sprouting bud length of the explant is about 2-3cm, and is cultured for 3-4 weeks. The bud elongation culture medium comprises MS +0.5 mg/L ZT +0.2 mg/L IAA +100 mg/L timentin +10 mg/L hygromycin.

(7) When the bud is extended to 4-5 cm, the callus is cut off and the bud is transferred to rooting culture for 3-4 weeks. Wherein the rooting medium comprises 1/2MS +10 mg/L hygromycin +2mg/L IBA.

(8) And (4) transferring the plantlets which have vigorous rooting and grow to a certain height into a soil pot immediately. The whole process of leaf disc transformation of tomato is shown in figure 1.

2、VvWRKY13Screening and Homozygosis of transgenic tomato

(1) Extracting the DNA of tomato leaves growing normally in the soil culture stage, and carrying out PCR identification by using specific primers of genes. The positive plants obtained by detection are normally cultured and marked as T1 generation. Detection ofVvWRKY13The PCR primer sequence used by the gene is as follows:

VvWRKY13-FP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；VvWRKY13-RP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；

(2) to obtain homozygous transgenic lines, seeds of T1 generation are harvested and sowed to obtain T2 generation transgenic tomato seedlings.

(3) Extracting the T2 transgenic tomato leaf DNA, removing the generated gene separated plant, and harvesting the T2 seed. And planting the harvested seeds to obtain T3 generation transgenic tomato seedlings.

(4) The segregation of the T3 transgenic tomato population was tested by PCR. In the T3 generationThe plant line without gene segregation is the homozygous transgenic plant line. Detection ofVvWRKY13The PCR primer sequences used for the genes were identical to those in (1).

(5) Detection of transgenic strains by using qRT-PCR technologyVvWRKY13Relative expression level of gene. The results of the detection are shown in FIG. 2. Detection ofVvWRKY13The qRT-PCR primer sequence used for gene expression is as follows:

VvWRKY13-FP：5’-GGTTGCCAACAATCCCT-3’；

VvWRKY13-RP：5’-GTCATCTCCACCGATACTTC-3’；

(6) seeds of homozygous transgenic tomato lines were collected for subsequent experiments.

3、VvWRKY13Detection of over-expression tomato strain ethylene generation amount, triple reaction and ethylene synthesis gene expression amount

(1) In the wild type andVvWRKY13the heterogeneously over-expressed tomato plant fruits are taken as materials, 3-4 fruits with uniform sizes are respectively selected, and the ethylene release rate of the fruits in 7 days after flowering, the color transition period and the mature period is measured. The results are shown in FIG. 3, with ethylene at 7 days after anthesis and at the color transitionVvWRKY13The release rate in heterologous overexpression lines was significantly higher than that of the wild type, but the difference was not significant at the full stage, indicating thatVvWRKY13Influences the ethylene generation amount of young tomato fruits.

In the ethylene synthesis pathway, ACC synthase and ACC oxidase are the rate-limiting and key enzymes of the pathway. Expression of key enzyme gene determines the amount of ethylene produced, and the wild type at the same timeVvWRKY13Selecting 3-4 tomato fruits with uniform sizes of wild type and over-expression plants as materials, and measuring the expression quantity of ethylene synthesis pathway related genes of fruits at 7d after flowering, at the color transition stage and at the mature stage. The results are shown in figures 4-11,SlACS1b、SlACO1andSlACO4the relative expression amount in the transgenic fruit is higher than that in the wild type; the rest have no great difference. The comprehensive analysis shows that the content of the compound,VvWRKY13mainly influences the expression of key enzyme genes for ethylene synthesis in the development process of young tomato fruits, and further influences the generation of tomato ethylene.

Detection ofSlACS1b、SlACO1AndSlACO4the qRT-PCR primer sequence used for gene expression is as follows:

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 a response specific to plants and can be used for the bioassay of ethylene. The ethylene-treated plantlets exhibited inhibition of the elongation growth of the epicotyl, thickening of the epicotyl in the transverse direction, and enlargement of the apical hook. Mixing wild type andVvWKRY13tomato seeds are over-expressed, after aseptic treatment, the seeds are spotted on MS culture medium containing 10 mu M ACC and 10 mu mol/L AOA, after normal culture for 12 h at 28 ℃, dark culture is carried out for 7d, and the growth condition is observed. The results show that: in contrast to the wild type tomato plants,VvWRKY13the over-expression tomato plant has inhibited growth of upper and lower embryonic axes in the absence of exogenous ethylene treatment, and shows constitutive triple response to ethylene. On a medium containing 10. mu.M of AOA,VvWKRY13the triple response differences between over-expressed tomato lines and wild type tomatoes became smaller. The above results demonstrate phenotypicallyVvWKRY13Overexpression promotes the overproduction of ethylene.

4、VvWRKY13Detection of fruit growth period and fruit growth period of over-expressed tomato line

(1) Observing simultaneously sown wild type tomatoes andVvWRKY13development of fruits of over-expressed tomato lines. The results are shown in FIG. 12, Table 1 and Table 2,VvWRKY13the time from flowering to color conversion of the over-expression strain tomato fruit is about 2-3 days ahead, and the time from color conversion to maturity is 10 days. Show thatVvWRKY13The development time of young fruits of the tomato fruits is shortened.

TABLE 1 transition from flowering to fruitTemporal statistics of color

Line of plants	Color conversion time (dpa)
		WT	42.67a±1.15
L1	36.33b±1.52
		L2	39.33ab±4.0
L3	36.33b±1.52

TABLE 2 statistics of fruit time from color transition to ripening

Line of plants	Color change to maturity (d)
		WT	10a±1.15
L1	10a±1.52
		L2	10a±4.04
L3	10a±1.52

(2) Polygalacturonase belongs to pectinase, can degrade pectin in cell wall to decompose cell wall and participate in softening fruit, and its synthetase gene isSlPG. Detection of its synthetic geneSlPG1AndSlPG3the expression level of (A) was found to be in the wild type andVvWRKY13there was no difference in the relative expression levels in the over-expressed tomato lines.VvWKRY13Fruits of over-expressed tomato lines are shown in FIGS. 13 and 14.

Detection ofSlPG1AndSlPG3the primer sequence used for gene expression level is as follows:

SlPG1-FP：5’-TGAGGACCAAATCGGAATC-3’；

SlPG1-RP：5’-TGTCGGACTAAGAAAGAATAACC-3’；

SlPG3-FP：5’-ATACAACAGTTTTCAGCAGTTCAAGT-3’；

SlPG3-RP：5’-GGTTTTCCACTTTCCCCTACTAA-3’。

5、VvWRKY13detection of quality of over-expressed tomato plant line fully ripe fruit

Lycopene belongs to carotenoid, is a natural edible pigment, is mainly concentrated in red pulp or tissues of various fruits and vegetables and fruits, and has a quenching effect on oxygen free radicals due to lack of provitamin activity, so that the lycopene has the effects of preventing cancers, beautifying and beautifying, preventing cardiovascular and cerebrovascular diseases, and is one of key indexes for measuring the quality of tomato fruits. To complete the mature period of wild type andVvWRKY13over-expression tomato line fruit is used as material, and the content of lycopene in the fruit in the mature period is determined. The results show that lycopene is present in wild type andVvWRKY13the content in the fruit of the over-expressed tomato plant did not change significantly. Thus, it can be seen thatVvWRKY13The over-expression of (A) has no effect on the lycopene content in the tomato fruit.

6. Tomato sweetness is an important component of tomato quality traits and depends mainly on the soluble sugar content in tomato fruits, so the soluble sugar content can be used to evaluate tomato quality. To complete the mature period of wild type andVvWRKY13fruits of over-expressed tomato lines were used as material and the soluble sugar content was determined. Results displayVvWRKY13The sugar content of the transgenic line fruit and the wild fruit is not obviously changed, which shows thatVvWRKY13The overexpression of (a) does not affect the soluble sugar content in tomato fruits.

7. The acid in the fruit mainly refers to the anion of the total effective organic acid radical, and the titratable acid in the tomato is citric acid which plays an important role in the flavor of the tomato. We have completed the maturity stages of wild type andVvWRKY13fruits of over-expressed tomato lines were used as material to detect the content of titratable acids. The results show that there is no obvious difference between the two, which showsVvWRKY13Does not affect the titratable acid content in the tomato.

The conclusion shows that the grapesVvWRKY13By promoting the generation of endogenous ethylene in the enlargement period of young fruits, the young fruits are shortenedThe expanding period, thereby achieving the effects of shortening the fruit development period and promoting the fruit precocity without negative influence on the fruit quality.

Example 2

The invention is illustratedVvWRKY13The function and application of the method in ethylene biosynthesis are proved, and the method specifically comprises the following steps:

1、VvWRKY13transformation and screening of over-expressed tomatoes:

(1) a certain amount of seeds of commercial cultivated tomato variety 'Ailsa Craig (AC)' are taken, soaked and sterilized with 75% ethanol for 30 s, and quickly poured out. Soaking and sterilizing with 4% sodium hypochlorite for 15 min, washing with sterile water for 7-8 times, and thoroughly removing residual disinfectant. After the seeds were washed clean, the seeds were sown in seed germination medium (1/2 MS). Culturing in dark for 3-4 days until seed germinates, transferring to 25 deg.C, and culturing under light/dark condition with photoperiod of 16 h/8 h. When cotyledons grow out, seedlings which are just opened and before true leaves grow out are used for transformation.

(2) The EHA105 Agrobacterium strain containing the 35S promoter plasmid, stored at-80 ℃ was resume and expanded in YEB liquid medium. Wherein the YEB liquid culture medium used for culturing the agrobacterium is prepared from the following components in percentage by weight: 10 g/L tryptone, 1g/L yeast extract, 5 g/L sucrose, 0.5 g/L MgSO4.7H2O, pH 7.0. Subpackaging, autoclaving at 121 deg.C for 20 min, cooling to room temperature, and adding 50 mg/L filter-sterilized kanamycin and rifampicin.

(3) Taking the sterile cotyledon without true leaves in the step (1), cutting off the leaf apex and the leaf stalk, cutting the rest cotyledon into a square block with the size of 3mm multiplied by 3mm, placing the square block on an MS solid culture medium without antibiotics, and pre-culturing for 2 days at 28 ℃.

(4) And (3) centrifuging the bacterial liquid expanded and shaken in the step (2) for 10 min at 5000 r/min, and diluting the precipitate with sterile water until OD600=0.5-0.6 is used as an infection liquid. Explants after 2 days of pre-culture were infected in the infection fluid for 5 min. Co-culture was performed on MS solid medium without antibiotics in a dark room at 28 ℃ for 2 days. The components of the co-culture medium are MS +2mg/L ZT +0.2 mg/L IAA +100 mg/L timentin +10 mg/L hygromycin.

(7) When the bud is extended to 4-5 cm, the callus is cut off, and the bud is transferred to a rooting medium for 3-4 weeks. Wherein the rooting medium comprises 1/2MS +10 mg/L hygromycin +2mg/L IBA.

(8) And (4) transferring the plantlets which have vigorous rooting and grow to a certain height into a soil pot immediately.

2、VvWRKY13Screening and Homozygosis of transgenic tomato

(1) Extracting the DNA of tomato leaves growing normally in the soil culture stage, and carrying out PCR identification by using specific primers of genes. The results are shown in FIG. 15. The positive plants obtained by detection are normally cultured and marked as T1 generation.

Detection ofVvWRKY13The PCR primer sequence used by the gene is as follows:

VvWRKY13-FP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’；

VvWRKY13-RP1：5’-GCTCTAGAATGTCTACTACTTCTCAAGCC-3’。

(4) The segregation of the T3 transgenic tomato population was tested by PCR. The lines which did not show gene segregation in the T3 generation were homozygous transgenic lines. Detection ofVvWRKY13For geneThe PCR primer sequence of (1) was identical to that of (1).

(5) Detection of transgenic strains by using qRT-PCR technologyVvWRKY13Relative expression level of gene. The detection results are shown in fig. 16. Detection ofVvWRKY13The qRT-PCR primer sequence used for gene expression is as follows:

VvWRKY13-FP：5’-GGTTGCCAACAATCCCT-3’；

VvWRKY13-RP：5’-GTCATCTCCACCGATACTTC-3’；

(1) In the wild type andVvWRKY13the heterogeneously over-expressed tomato plant fruits are taken as materials, 3-4 fruits with uniform sizes are respectively selected, and the ethylene release rate of the fruits in 7 days after flowering, the color transition period and the mature period is measured. The results show that ethylene is present 7 days after the flower and at the color transitionVvWRKY13The release rate in heterologous overexpression lines was significantly higher than that of the wild type, but the difference was not significant at the full stage, indicating thatVvWRKY13Influences the ethylene generation amount of young tomato fruits.

In the ethylene synthesis pathway, ACC synthase and ACC oxidase are the rate-limiting and key enzymes of the pathway. Expression of key enzyme gene determines the amount of ethylene produced, and the wild type at the same timeVvWRKY13Selecting 3-4 tomato fruits with uniform sizes of wild type and over-expression plants as materials, and measuring the expression quantity of ethylene synthesis pathway related genes of fruits at 7d after flowering, at the color transition stage and at the mature stage. The results show that it is possible to display,SlACS1b、SlACO1andSlACO4the relative expression amount in the transgenic fruit is higher than that in the wild type; the rest have no great difference. The comprehensive analysis shows that the content of the compound,VvWRKY13mainly influences the expression of key enzyme genes for ethylene synthesis in the development process of young tomato fruits, and further influences the generation of tomato ethylene.

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 a response specific to plants and can be used for the bioassay of ethylene. The ethylene-treated plantlets exhibited inhibition of the elongation growth of the epicotyl, thickening of the epicotyl in the transverse direction, and enlargement of the apical hook. Mixing wild type andVvWKRY13tomato seeds are over-expressed, after aseptic treatment, the seeds are spotted on MS culture medium containing 10 mu M ACC and 10 mu mol/L AOA, after normal culture for 12 h at 28 ℃, dark culture is carried out for 7d, and the growth condition is observed. The results are shown in FIGS. 17-20: in contrast to the wild type tomato plants,VvWRKY13the over-expression tomato plant has inhibited growth of upper and lower embryonic axes in the absence of exogenous ethylene treatment, and shows constitutive triple response to ethylene. On a medium containing 10. mu.M of AOA,VvWKRY13the triple response differences between over-expressed tomato lines and wild type tomatoes became smaller. The above results demonstrate phenotypicallyVvWKRY13Overexpression promotes the overproduction of ethylene.

(1) Observing simultaneously sown wild type tomatoes andVvWRKY13development of fruits of over-expressed tomato lines. The results show that it is possible to display,VvWRKY13the time from flowering to color conversion of the over-expression strain tomato fruit is about 2-3 days ahead, and the time from color conversion to maturity is 10 days. Show thatVvWRKY13The development time of young fruits of the tomato fruits is shortened.

(2) Polygalacturonase belongs to pectinase, can degrade pectin in cell wall to decompose cell wall and participate in softening fruit, and its synthetase gene isSlPG. Detection of its synthetic geneSlPG1AndSlPG3the expression level of (3). The results are shown in FIGS. 21 and 22, which are in the wild type andVvWRKY13there was no difference in the relative expression levels in the over-expressed tomato lines. Detection ofSlPG1AndSlPG3the primer sequence used for gene expression level is as follows:

SlPG1-FP：5’-TGAGGACCAAATCGGAATC-3’；

SlPG1-RP：5’-TGTCGGACTAAGAAAGAATAACC-3’；

SlPG3-FP：5’-ATACAACAGTTTTCAGCA GTTCAAGT-3’；

SlPG3-RP：5’-GGTTTTCCACTTTCCCCTACTAA-3’。

(1) Lycopene belongs to carotenoid, is a natural edible pigment, is mainly concentrated in red pulp or tissues of various fruits and vegetables and fruits, and has a quenching effect on oxygen free radicals due to lack of provitamin activity, so that the lycopene has the effects of preventing cancers, beautifying and beautifying, preventing cardiovascular and cerebrovascular diseases, and is one of key indexes for measuring the quality of tomato fruits. To complete the mature period of wild type andVvWRKY13over-expression tomato line fruit is used as material, and the content of lycopene in the fruit in the mature period is determined. The results are shown in FIG. 23, with lycopene in the wild type and in the wild typeVvWRKY13The content in the fruit of the over-expressed tomato plant did not change significantly. Thus, it can be seen thatVvWRKY13The over-expression of (A) has no effect on the lycopene content in the tomato fruit.

(2) Tomato sweetness is an important component of tomato quality traits and depends mainly on the soluble sugar content in tomato fruits, so the soluble sugar content can be used to evaluate tomato quality. To complete the mature period of wild type andVvWRKY13fruits of over-expressed tomato lines were used as material and the soluble sugar content was determined. The results are shown in FIG. 24VvWRKY13The sugar content of the transgenic line fruit and the wild fruit is not obviously changed, which shows thatVvWRKY13The overexpression of (a) does not affect the soluble sugar content in tomato fruits.

(3) The acid in the fruit is mainlyThe active organic acid radical refers to anions of total effective organic acid radicals, and the main titratable acid in the tomato is citric acid, which plays an important role in the flavor of the tomato. We have completed the maturity stages of wild type andVvWRKY13fruits of over-expressed tomato lines were used as material to detect the content of titratable acids. The results are shown in FIG. 25, which shows that there is no significant difference between the two, indicating thatVvWRKY13Does not affect the titratable acid content in the tomato.

The conclusion shows that the grapesVvWRKY13The growth period of the fruits is shortened by promoting the generation of endogenous ethylene in the young fruit expansion period, so that the effects of shortening the development period of the fruits and promoting the precocity of the fruits are achieved, but no negative influence is caused on the quality of the fruits.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. Grape prematurity geneVvWRKY13The application of the compound in regulating and controlling the ethylene biosynthesis in plants is characterized in that: the grape prematurity geneVvWRKY13GenBank accession No. JQ692108 of CDS sequence of (a); the plant is tomato; ethylene is transgenic 7 days after flowering and at the time of color transitionVvWRKY13The release rate in tomato lines is higher than that of wild type tomatoes,VvWRKY13influence the generation amount of ethylene in the early development stage of the tomato fruit; geneVvWRKY13The over-expression of (a) shortens the expansion period in the fruit development process; geneVvWRKY13The fruit of the over-expression tomato line is 2 to 3 days earlier than the wild type from flowering to color change,VvWRKY13the development time of young fruits of the fruits is shortened.

2. The grape precocity gene of claim 1VvWRKY13The application of the compound in regulating and controlling the ethylene biosynthesis in plants is characterized in that: will contain grape early maturing geneVvWRKY13The agrobacterium strain of (a) infects tomatoes for co-culture; placing the explants after co-culture in a bud induction culture medium for subculture; transferring the explant into a bud elongation culture medium for culture after the explant germinates; then transferring the bud into rooting culture medium to culture so as to obtain transgeneVvWRKY13Tomato plants.

3. The grape precocity gene of claim 1VvWRKY13The application of the compound in regulating and controlling the ethylene biosynthesis in plants is characterized in that: ethylene synthesis key enzyme geneSlACS1b、SlACO1AndSlACO4in transgenesisVvWRKY13The expression level in the tomato fruit is increased,VvWRKY13by influencing the ethylene synthesis key enzyme gene in the tomato fruit development processSlACS1b、SlACO1AndSlACO4to regulate the biosynthesis of ethylene in tomato fruits.

4. The grape precocity gene of claim 1VvWRKY13The application of the compound in regulating and controlling the ethylene biosynthesis in plants is characterized in that: transgenosisVvWRKY13In tomato plantsVvWRKY13The relative expression quantity of the gene is increased compared with that of the wild tomato,VvWKRY13overexpression promotes the overproduction of ethylene.

5. The grape precocity gene of claim 1VvWRKY13The application of the compound in regulating and controlling the ethylene biosynthesis in plants is characterized in that: geneVvWRKY13The contents of lycopene, soluble sugars and titratable acid in the over-expressed tomato line fruits were all unchanged compared to the wild type.