CN110628778B - Gene, protein, vector, recombinant gene engineering bacterium for accelerating fruit maturation and application thereof - Google Patents

Abstract

The present invention relates to a gene for accelerating fruit ripening. The gene is: the coding region is the DNA molecule shown in SEQ ID NO.1; or the coding region is the DNA molecule shown in SEQ ID NO. and DNA molecules with the above homology. For the first time, the present invention excavates a gene (FRA gene) that promotes the early ripening of fruits outside the ethylene biosynthesis pathway, and constructs a carrier, thereby realizing the innovative research and development of the new germplasm of early-maturing fruits by using the transgenic method. Expression of FRA gene can accelerate fruit ripening.

Description

Gene, protein, vector, recombinant gene engineering bacterium for accelerating fruit maturation and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a gene (FRA gene), protein, vector, recombinant genetic engineering bacteria for accelerating fruit ripening and application thereof.

Background

The phenomenon of fruit ripening is mainly controlled by a series of physiological and biochemical reactions initiated by ethylene. In the fruit ripening process, the respiration is enhanced, the starch is hydrolyzed into soluble saccharide, and the organic acid is decomposed, so that the fruit sweetness is increased, and the sour and astringent taste is reduced; the pectin hydrolyzes to thin the cell wall, reduce the hardness of the fruit and soften the fruit, the chlorophyll pigments are degraded, and the anthocyanin and the carotenoid pigments are synthesized in a large quantity, so that the color of the fruit peel is changed from green to red or yellow. EFE (ethylene forming enzyme) is a key enzyme in ethylene biosynthesis. Researchers achieve the aim of inhibiting fruit softening by controlling the activity of the ethylene forming enzyme. A typical example in this respect is transgenic, storage-tolerant tomato. From 1990, professor Bidens professor of university of agriculture in Huazhong began to develop a transgenic storage-resistant tomato and succeeded in developing 'Huanan No. one', wherein the transgenic tomato is characterized by high fruit hardness, difficult softening and difficult decay, thereby meeting the market demands of long-time storage and long-distance transportation of the tomato. In 1997, the first agricultural biological transgenic safety evaluation in China was obtained in Huanan I, and the product is named as 'whooping fresh'. "Huafan No. one" is transgenic tomato with antisense EFE gene, and its principle for inhibiting fruit from softening is that after the antisense EFE gene is transcribed, it is combined with mRNA of EFE gene by means of base pairing, so that the expression quantity of EFE gene is reduced, and the EFE protein is reduced.

The maturity of fruits has a great influence on economic efficiency, and varieties which are early-maturing and come into the market generally have higher market prices, thereby causing facility-forcing culture means to be widely applied. The artificial control gene is used for regulating and controlling the fruit ripening and promoting the fruit ripening in advance, and the method is a research and development approach with great application potential and good prospect. The core work for realizing the approach is to excavate and develop genes with the effect of accelerating fruit maturation.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a gene (FRA gene), protein, a carrier, a recombinant gene engineering bacterium for accelerating fruit ripening and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a gene that accelerates the ripening of fruit, said gene being: the coding region is a DNA molecule shown in SEQ ID NO. 1;

or the following steps: the coding region is a DNA molecule which has 90 percent of homology with the DNA molecule shown in SEQ ID NO.1 and above.

A protein for accelerating fruit ripening, said protein being: a protein consisting of an amino acid residue sequence shown in SEQ ID NO. 2;

or, it is: has a protein consisting of an amino acid residue sequence with 90 percent or more of homology with the amino acid sequence shown in SEQ ID NO. 2.

A vector containing the gene for accelerating fruit ripening as described above.

Recombinant gene engineering bacteria containing the vector.

Moreover, the steps are as follows:

constructing a recombinant vector containing the gene for accelerating fruit maturation, transforming the recombinant vector into an agrobacterium strain EHA105, carrying out induction culture on the obtained recombinant gene engineering bacteria, and separating and purifying a culture solution to obtain thallus cells of the recombinant gene engineering bacteria containing the gene for accelerating fruit maturation.

The method comprises the following specific steps:

the method comprises the steps of transferring genes for accelerating fruit maturation into escherichia coli competent cells, and coating the transformed competent cells on an LB solid culture medium containing 500mM IPTG 8 muL and 20mg/ml X-gal 40 muL for overnight culture; the next day, white colonies were picked, inoculated into LB liquid medium containing 50. mu.g/ml kanamycin, cultured overnight at 37 ℃ at 200 rpm; taking 2-4ml of overnight culture bacterial liquid the next day, extracting plasmids, carrying out PCR identification on the plasmids, carrying out electrophoresis detection by using 1% agarose gel, and obtaining recombinant plasmids after the plasmids are qualified;

carrying out double enzyme digestion on the recombinant plasmid and the pRI 101-AN vector obtained in the step. The enzyme cutting system is as follows: each 25.0. mu.L contained: plasmid 5.0 μ L, 10 XQuickCut Buffer 2.5 μ L, NdeI and SmaI each 1 μ L, sterilized water 15.5 μ L, adding SmaI first before 30 deg.C reaction for 1h, then adding NdeI at 37 deg.C reaction for 1 h; carrying out electrophoresis on the enzyme digestion product on 1% agarose gel, recovering a target band, connecting the target band with the enzyme digestion product of a pRI 101-AN vector by using T4 ligase at 16 ℃ overnight, constructing a plant expression vector, transforming the connection product into AN escherichia coli cell, screening positive clone by blue and white spots, carrying out PCR amplification and double enzyme digestion verification, sequencing, and obtaining AN expression vector plasmid after the product is qualified;

after extracting expression vector plasmids, transforming the ligation products into EHA105 competent agrobacterium by an electric shock method, inoculating the transformed agrobacterium to a YEB solid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin, culturing for 2-3 days, selecting a single colony, shaking the single colony in a YEB liquid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin at 28 ℃ and 200rpm for 48 hours, carrying out PCR (polymerase chain reaction) method verification on the bacterial liquid, and separating and purifying after the bacterial liquid is verified to be qualified to obtain the recombinant genetic engineering bacteria.

The application of the gene for accelerating fruit maturation in plant breeding or accelerating fruit maturation is disclosed.

The use of a protein for increasing fruit ripening as described above in plant breeding or for increasing fruit ripening.

The invention has the advantages and positive effects that:

1. the invention excavates a gene (FRA gene) for promoting fruit to mature in advance outside an ethylene biosynthesis way for the first time, constructs a carrier, realizes the application of the gene to the innovation and the research of new germplasm of early maturing fruits by using a transgenic method, and can accelerate fruit maturation by over-expressing the FRA gene. The FRA gene is applied to cultivating a new early-maturing fruit variety, and the fruit maturing time of a FRA gene transferred tomato plant is obviously earlier than that of a wild type material.

2. The invention overcomes the defects of the prior art that the average single fruit weight of the fruits produced by a new transgenic germplasm (such as tomato and the like) is reduced, the pericarp is thickened, the sugar content is reduced, the number of seeds is reduced, the internal quality is deteriorated, and the like, so that the commodity of the fruits is reduced. After the gene is applied, the appearance and the internal quality of the obtained transgenic germplasm fruit are not changed, and the fruit commodity is good.

3. The invention provides a general germplasm innovation method and a technical system for early maturing fruits, can be used for germplasm innovation and new variety research and development of various early maturing varieties of fruits, and has wide and good application prospect in the field of fruit tree biotechnology breeding.

4. The invention also constructs a plant expression vector by using the FRA gene to obtain the recombinant gene engineering bacteria containing the vector.

Drawings

FIG. 1 is a photograph of the regeneration, rooting and transplantation of transgenic tomatoes according to the present invention using the gene for accelerating fruit ripening; wherein, the picture a is a state diagram of the regeneration of the tomato resistant adventitious bud, the picture b is a state diagram of the young shoot rooting obtained by the growth of the resistant bud, and the picture c is a state diagram of the transplanting of the regenerated resistant plant into a nutrition pot;

FIG. 2 is a photograph comparing fruits of a transgenic tomato and a wild type tomato using the gene for accelerating fruit ripening according to the present invention; wherein, the upper fruit is wild type, and the lower fruit is transgenic tomato.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A gene that accelerates the ripening of fruit, said gene being: the coding region is a DNA molecule shown in SEQ ID NO. 1;

or the following steps: the coding region is a DNA molecule which has 90 percent of homology with the DNA molecule shown in SEQ ID NO.1 and above.

The invention aims to provide a gene (FRA gene) for accelerating Fruit maturation, and any variant of a polynucleotide shown in SEQ ID NO.1 belongs to the protection scope of the invention as long as the variant has 90% or more homology with the polynucleotide due to the specificity of a nucleotide sequence. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide may be naturally occurring mutator variants or non-naturally occurring variants, including substitution, deletion and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein encoded thereby.

A protein for accelerating fruit ripening, said protein being: a protein consisting of an amino acid residue sequence shown in SEQ ID NO. 2;

or, it is: has a protein consisting of an amino acid residue sequence with 90 percent or more of homology with the amino acid sequence shown in SEQ ID NO. 2.

Due to the specificity of the amino acid sequence, any fragment of the peptide protein containing the amino acid sequence shown in SEQ ID No.2 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, as long as the peptide protein fragment or peptide protein variant has 90% or more homology with the aforementioned amino acid sequence, is included in the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan.

A vector containing the gene for accelerating fruit ripening as described above.

Recombinant gene engineering bacteria containing the vector.

Preferably, the steps are as follows:

constructing a recombinant vector containing the gene for accelerating fruit maturation, transforming the recombinant vector into an agrobacterium strain EHA105, carrying out induction culture on the obtained recombinant gene engineering bacteria, and separating and purifying a culture solution to obtain thallus cells of the recombinant gene engineering bacteria containing the gene for accelerating fruit maturation.

Preferably, the specific steps are as follows:

the method comprises the steps of transferring genes for accelerating fruit maturation into escherichia coli competent cells, and coating the transformed competent cells on an LB solid culture medium containing 500mM IPTG 8 muL and 20mg/ml X-gal 40 muL for overnight culture; the next day, white colonies were picked, inoculated into LB liquid medium containing 50. mu.g/ml kanamycin, cultured overnight at 37 ℃ at 200 rpm; taking 2-4ml of overnight culture bacterial liquid the next day, extracting plasmids, carrying out PCR identification on the plasmids, carrying out electrophoresis detection by using 1% agarose gel, and obtaining recombinant plasmids after the plasmids are qualified;

carrying out double enzyme digestion on the recombinant plasmid and the pRI 101-AN vector obtained in the step. The enzyme cutting system is as follows: each 25.0. mu.L contained: plasmid 5.0 μ L, 10 XQuickCut Buffer 2.5 μ L, NdeI and SmaI each 1 μ L, sterilized water 15.5 μ L, adding SmaI first before 30 deg.C reaction for 1h, then adding NdeI at 37 deg.C reaction for 1 h; carrying out electrophoresis on the enzyme digestion product on 1% agarose gel, recovering a target band, connecting the target band with the enzyme digestion product of a pRI 101-AN vector by using T4 ligase at 16 ℃ overnight, constructing a plant expression vector, transforming the connection product into AN escherichia coli cell, screening positive clone by blue and white spots, carrying out PCR amplification and double enzyme digestion verification, sequencing, and obtaining AN expression vector plasmid after the product is qualified;

after extracting expression vector plasmids, transforming the ligation products into EHA105 competent agrobacterium by an electric shock method, inoculating the transformed agrobacterium to a YEB solid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin, culturing for 2-3 days, selecting a single colony, shaking the single colony in a YEB liquid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin at 28 ℃ and 200rpm for 48 hours, carrying out PCR (polymerase chain reaction) method verification on the bacterial liquid, and separating and purifying after the bacterial liquid is verified to be qualified to obtain the recombinant genetic engineering bacteria.

The gene for accelerating fruit ripening can be applied to plant breeding or accelerating fruit ripening.

The protein for accelerating fruit ripening can be applied to plant breeding or accelerating fruit ripening.

The preparation and detection of the invention are as follows:

one, accelerating the obtaining of fruit mature gene FRA

Extracting total RNA from common peach fruits by using a Kit EASYspin Plus, synthesizing cDNA by using a reverse-Aid First Strand cDNA Synthesis Kit, taking the cDNA as a template, and using a PCR sequence specific primer pair:

F：GGAATTCCATATGGCTCTCTTTCTTTCTCTC，

R：TAACCCGGGACTACTCGATTTCTCCAC，

PCR amplification was performed, and the reaction system (25.0. mu.L): cDNA template 1.0 μ L, 5 XPCR buffer 2.5 μ L, 2.0 μ L of 2.5mM dNTPs, 0.2 μ L of 5U/μ L rTaq enzyme, 1.0 μ L of each of 10 μmol/L forward and reverse primers, dd H₂O17.3. mu.L. And (3) amplification procedure: pre-denaturation at 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 57 ℃ for 30s and 72 ℃ for 1 min; finally, extension is carried out for 8min at 72 ℃, the product is stored at 4 ℃, and the PCR product is detected by 1% agarose gel electrophoresis. The target fragment was recovered using the San-Prep column type DNA gel recovery kit, 4. mu.L of the target fragment was ligated with pEASY-Blunt Cloning Vector 1.0. mu.L at 25 ℃ for 15 minutes, and then the ligated fragment was transferred to E.coli competent cells, which were plated on LB solid medium containing 500mM IPTG 8. mu.L and 20mg/ml X-gal 40. mu.L for overnight culture. The next day, a white colony was picked and inoculated to a medium containing 50. mu.lg/ml kanamycin in LB liquid medium, 37 degrees, 200rpm overnight culture. The next day, 2-4ml of overnight culture broth was taken, plasmids were extracted with Takara Mini BEST plasmid purification Kit Ver 4.0, identified by PCR, detected by electrophoresis on 1% agarose gel, and sent to Biotechnology engineering (Shanghai) GmbH for sequencing.

In the present invention, the FRA gene may be obtained by a gene synthesis pathway.

Secondly, preparation of recombinant genetic engineering bacteria containing gene FRA for accelerating fruit maturation

The recombinant plasmid obtained in step one and the pRI 101-AN vector were subjected to double digestion with Nde I and Sma I, respectively. Enzyme digestion system (25.0. mu.L): plasmid 5.0 u L, 10 x Quickcut Buffer 2.5 u L, NdeI and SmaI each 1 u L, sterile water 15.5 u L, adding SmaI first prior to 30 degrees C reaction for 1h, and then NdeI at 37 degrees C reaction for 1 h. And (3) carrying out electrophoresis on the enzyme digestion product on 1% agarose gel, recovering a target band, connecting the target band with the enzyme digestion product of a pRI 101-AN vector by using T4 ligase at 16 ℃ overnight, constructing a plant expression vector, transforming the connection product into AN escherichia coli cell, screening positive clone by using a blue-white spot, carrying out PCR amplification and double enzyme digestion verification, and sending a sample to a company Limited in the biological engineering (Shanghai) for sequencing.

After the expression vector plasmid is extracted, the ligation product is transformed into EHA105 competent agrobacterium by an electric shock method, the transformed agrobacterium is inoculated on a YEB solid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin for culturing for 2-3 days, a single colony is picked up and shaken for 48h at the temperature of 28 ℃ and the speed of 200rpm in a YEB liquid culture medium containing 50 mu g/ml kanamycin and 100 mu g/ml streptomycin, and the bacterial liquid is verified by a PCR method.

Application of gene FRA for accelerating fruit ripening in new germplasm innovation of early ripening fruits

Adopting an agrobacterium infection method to infect the sterile hypocotyl of the tomato variety 'Micro-Tom', killing agrobacterium with antibiotic after two days, regenerating resistant adventitious buds on a screening culture medium, transferring the buds to a rooting culture medium to induce rooting, hardening seedlings, and transplanting the buds to a nutrition pot for cultivation. The results are shown in FIG. 1, in which a shows the state in which the resistant adventitious bud of tomato is regenerated, b shows the state in which the young shoot obtained by the growth of the resistant bud is rooted, and c shows the state in which the regenerated resistant plant is transplanted into a nutrition pot. The introduction of the foreign gene was verified by PCR. After blooming and fructification, the fruit development period of the transformant is compared with that of a wild tomato, and the fruit development period of the transformant is shortened by 2 to 5 days, so that the ripening time is obviously advanced. The results are shown in fig. 2, and fig. 2 shows the comparison of the state of the transgenic tomato fruits at maturity with the wild type fruits in the same growth stage, wherein the upper fruit is the wild type tomato and the lower is the transgenic tomato, and it can be seen that the maturity of the transgenic tomato is significantly higher than the wild type.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

<110> Tianjin college of agriculture

<120> gene, protein, vector, recombinant gene engineering bacterium for accelerating fruit maturation and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1206

<212> DNA

<213> coding region of gene for accelerating fruit ripening (Unknown)

<400> 1

ggaattccat atggctctct ttctttctct ctctctgttt catcttctcc tctgaaattt 60

tctgaattca catttcaagc tctgaaattt ttatcagaaa aaatgggtgt gccagaaacc 120

gacccacttt ctcagctaag cttgccaccg gggtttcgat tctatcccac agacgaagag 180

cttctggttc agtatctgtg ccgcaaggtt gctgggtacc aattcagtct gcaaattata 240

gctgaaattg atctctacaa gttcgatcca tgggttttac caagcaaagc aatatttggt 300

gaaaaagaat ggtacttctt tagtccgagg gaccggaaat acccaaatgg gtcacgaccc 360

aacagggtag ccgggtctgg gtactggaaa gccaccggca ctgataagat catcaccact 420

gaaggtcgaa aagttggaat aaaaaaagct ctggttttct atgtcggcaa agcccccaaa 480

ggcaccaaga ccaattggat tatgcatgag tatcgtctaa tcgaaccttc acgcaaaaat 540

ggcagctcca agttggatga atgggttttg tgtcgtattt acaagaagag ctcgagctca 600

gctgcgcaga aacccatgac gacgagcgtt tcgagcaaag agcacagcaa cggctcgtcg 660

tcttcctgct cgtctcagct tgacgacgtg ctcgagtggc tcccggatat tgacgaccgc 720

tgcttcactc tgccgcgcat aaactcgctc aaaacgctgc agcagcagca ggaggacagc 780

aagctcgggt ttcagatggg ttctggaaat ttcgactggg cgagtcttgc cgggctcaac 840

gtggtgcctg aactatgtcc caataaccag ccccaacaaa gccaagggca aatgaatgtg 900

aactatagca acaatgacat gtatgtccct tcgatcccgc cgctctgcca cgtggaatcg 960

ccgccggaga ggctcgcgaa gacggtggac gaggaggtgc agagcgggtt cagaactcag 1020

cgggttgata actcggggtt cttccagaac tcgaacgtta tgactcagaa cttttgcaac 1080

ccgaccgacc cgtacgggta cagtaaccga ctcggccggt cgggtttggg gtttggcggt 1140

gcggaaaagt gaaagtagaa ggggagtgca ttcatttcgg tggagaaatc gagtagtccc 1200

gggtta 1206

<210> 2

<211> 349

<212> PRT

<213> protein for accelerating fruit ripening (Unknown)

<400> 2

Met Gly Val Pro Gly Thr Ala Pro Leu Ser Gly Leu Ser Leu Pro Pro

1 5 10 15

Gly Pro Ala Pro Thr Pro Thr Ala Gly Gly Leu Leu Val Gly Thr Leu

20 25 30

Cys Ala Leu Val Ala Gly Thr Gly Pro Ser Leu Gly Ile Ile Ala Gly

35 40 45

Ile Ala Leu Thr Leu Pro Ala Pro Thr Val Leu Pro Ser Leu Ala Ile

50 55 60

Pro Gly Gly Leu Gly Thr Thr Pro Pro Ser Pro Ala Ala Ala Leu Thr

65 70 75 80

Pro Ala Gly Ser Ala Pro Ala Ala Val Ala Gly Ser Gly Thr Thr Leu

85 90 95

Ala Thr Gly Thr Ala Leu Ile Ile Thr Thr Gly Gly Ala Leu Val Gly

100 105 110

Ile Leu Leu Ala Leu Val Pro Thr Val Gly Leu Ala Pro Leu Gly Thr

115 120 125

Leu Thr Ala Thr Ile Met His Gly Thr Ala Leu Ile Gly Pro Ser Ala

130 135 140

Leu Ala Gly Ser Ser Leu Leu Ala Gly Thr Val Leu Cys Ala Ile Thr

145 150 155 160

Leu Leu Ser Ser Ser Ser Ala Ala Gly Leu Pro Met Thr Thr Ser Val

165 170 175

Ser Ser Leu Gly His Ser Ala Gly Ser Ser Ser Ser Cys Ser Ser Gly

180 185 190

Leu Ala Ala Val Leu Gly Thr Leu Pro Ala Ile Ala Ala Ala Cys Pro

195 200 205

Thr Leu Pro Ala Ile Ala Ser Leu Leu Thr Leu Gly Gly Gly Gly Gly

210 215 220

Ala Ser Leu Leu Gly Pro Gly Met Gly Ser Gly Ala Pro Ala Thr Ala

225 230 235 240

Ser Leu Ala Gly Leu Ala Val Val Pro Gly Leu Cys Pro Ala Ala Gly

245 250 255

Pro Gly Gly Ser Gly Gly Gly Met Ala Val Ala Thr Ser Ala Ala Ala

260 265 270

Met Thr Val Pro Ser Ile Pro Pro Leu Cys His Val Gly Ser Pro Pro

275 280 285

Gly Ala Leu Ala Leu Thr Val Ala Gly Gly Val Gly Ser Gly Pro Ala

290 295 300

Thr Gly Ala Val Ala Ala Ser Gly Pro Pro Gly Ala Ser Ala Val Met

305 310 315 320

Thr Gly Ala Pro Cys Ala Pro Thr Ala Pro Thr Gly Thr Ser Ala Ala

325 330 335

Leu Gly Ala Ser Gly Leu Gly Pro Gly Gly Ala Gly Leu

340 345

<210> 3

<211> 31

<212> DNA

<213> primer F (Unknown)

<400> 3

ggaattccat atggctctct ttctttctct c 31

<210> 4

<211> 27

<212> DNA

<213> primer R (Unknown)

<400> 4

taacccggga ctactcgatt tctccac 27

Claims (1)

1. The application of the gene for accelerating fruit maturation in tomato breeding or tomato maturation is characterized in that the gene for accelerating fruit maturation is as follows: the coding region is a DNA molecule shown in SEQ ID NO. 1.

Images