CN112877338B

CN112877338B - Method for lengthening tomato fruit shape

Info

Publication number: CN112877338B
Application number: CN202110195608.4A
Authority: CN
Inventors: 张俊红; 艾国; 叶志彪; 张余洋; 王家发; 李国斌; 张得迪
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2022-06-10
Anticipated expiration: 2041-02-19
Also published as: CN112877338A

Abstract

The invention relates to a method for lengthening tomato fruit shape, which is used for lengthening tomato fruit shape by regulating and controlling overexpression of DWARF gene in tomato. Compared with the prior art, the invention increases the ratio of the longitudinal diameter to the transverse diameter of the tomato by regulating and controlling the overexpression of the DWARF gene in tomato plants, so that fruits can be lengthened.

Description

Method for lengthening tomato fruit shape

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a method for lengthening the shape of a tomato fruit.

Background

Tomatoes are one of the widest cultivated fruits and vegetables in the world. The shape of the tomato fruits is in important connection with the properties such as the size and the appearance quality of the fruits, the long-fruit-shaped tomatoes have inherent advantages in mechanical harvesting and subsequent transportation compared with round-fruit-shaped tomatoes, and meanwhile, the long-fruit-shaped tomatoes can meet the requirements of different consumers.

Brassinolide (BL), also called Brassinolide, is a natural sterol plant hormone with high physiological activity, and is the Brassinosterol (BRs) with the highest biological activity found at present. BR is widely present in organs such as pollen, seeds, stems and leaves of plants, and has been internationally praised as the sixth major plant hormone in addition to the existing 5 hormones (auxin, abscisic acid, gibberellin, cytokinin, ethylene). Brassinolide has very wide functions in the growth and development of plants and various stress resistance processes, and has obvious influence on the aspects of expansion of seed cells, shapes of plants and fruits and the like.

The influence of plum microcrystals on the growth of tomato reproductive organs and the absorption and utilization of light energy of photosystem II by hormones such as cytokinin is reported to be that the fruit enlargement is promoted by externally spraying tomato with BR, but the fruit shape is not substantially changed after the transverse diameter and the longitudinal diameter of the fruit are enlarged by the method. Gansu reports that the construction of an excess vector of the SLBRI1 in the research of the construction of a specific expression vector of a tomato BRs receptor gene SLBRI1 fruit and genetic transformation of a tomato causes the longitudinal and transverse diameters of a tomato fruit to be simultaneously enlarged, and the whole tomato expands without causing the whole change of the fruit shape.

The existing method for controlling the shape of the tomato fruit by using the BR regulating and controlling method only increases the transverse and longitudinal diameters of the tomato fruit at the same time, and does not obviously increase the difference between the two methods, so that the shape of the tomato fruit forms a long shape.

Disclosure of Invention

In order to solve the technical problem, the invention provides a method for lengthening the shape of tomato fruits.

The specific technical scheme is as follows:

a method for prolonging the shape of tomato fruits is characterized in that the overexpression of DWARF genes in tomatoes is regulated, so that the shape of the tomato fruits is prolonged, and the sequences of the DWARF genes are shown in SEQ.

Compared with the prior art, the invention increases the ratio of the longitudinal diameter to the transverse diameter of the tomato by regulating and controlling the overexpression of DWARF genes in tomato plants, so that fruits can be lengthened.

Further, the method comprises the following steps:

step S1, constructing an overexpression vector of the DWARF gene;

step S2, the tomato explants are dip-dyed after the overexpression vector is transferred into agrobacterium;

and S3, selecting the tomato explants soaked in the step S2, growing callus, and further culturing to obtain the tomato germplasm with longer fruits.

Further, in the step S3, after the callus grows into seedlings, plant tissues of the seedlings are subjected to DWARF gene expression level detection, and a plant with the sequence number of Solyc11g005330 gene as an internal reference gene is screened out, wherein the DWARF gene expression level of the plant is increased by 50-200 times, and the plant is further cultivated to obtain lengthened tomato fruits.

The technical effect of adopting the further technical scheme is as follows: the plant with 50-200 times of DWARF gene relative expression can increase the ratio of the longitudinal diameter to the transverse diameter of the fruit.

Further, a plant with DWARF gene expression quantity improved by 70-90 times is screened out.

The beneficial effect of adopting the further technical scheme is that: when the relative expression level of DWARF genes is 70-90, the aspect ratio of DWARF genes is further increased, and the fruits have more obvious elongation effect visually.

Further, in step S3, the method for measuring the relative expression level of DWARF gene includes: the method comprises the steps of treating tissues of tomato seedlings, extracting total RNA of the tissues, carrying out reverse transcription to obtain cDNA of the tissues, and detecting the expression quantity of the cDNA by using a detection primer, wherein the upstream sequence of the detection primer is shown as SEQ. NO.8, and the downstream sequence of the detection primer is shown as SEQ. NO. 9.

Further, in the step S1, the overexpression vector of DWARF gene includes a desired fragment containing the DWARF gene.

Further, the skeleton vector of the overexpression vector of the DWARF gene is pHellsgate8 vector.

Further, the tomato germplasm is Ailsa Craig.

An overexpression vector of DWARF gene, which is different from the overexpression vector of DWARF gene, and comprises a target fragment containing ORF of DWARF gene.

Compared with the prior art, the overexpression vector of the DWARF gene constructed by the invention can realize overexpression of the DWARF gene in tomato, so that the shape of tomato fruits is lengthened.

Further, the overexpression vector backbone vector of DWARF gene is pHellsgate8 vector.

The method for constructing the overexpression vector of the DWARF gene is different from the method for constructing the overexpression vector of the DWARF gene in that the method comprises the following steps:

a1, using cDNA reverse transcription of total RNA of tomato tissue as a template, and adopting an amplification primer to amplify DWARF gene ORF to obtain a target gene fragment;

and step A2, connecting the target gene fragment to a linearized pHellsgate8 framework vector, and transforming to obtain the overexpression vector of the DWARF gene.

Furthermore, the sequence of the upstream primer of the amplification primer is shown as SEQ.NO.2, and the sequence of the downstream primer is shown as SEQ.NO. 3.

The application of the overexpression vector of the DWARF gene in lengthening tomato fruits or overexpressing the DWARF gene.

A biomaterial containing the overexpression vector of the DWARF gene, which is different from the previous biomaterial in that the biomaterial comprises:

a recombinant microorganism comprising an overexpression vector for the DWARF gene;

or

A transgenic plant cell line comprising an overexpression vector for the DWARF gene;

or

Transgenic plant tissue comprising an overexpression vector for the DWARF gene;

or

A transgenic plant organ comprising an overexpression vector for the DWARF gene.

Drawings

FIG. 1 shows DWARF gene test results of transgenic plants and AC wild plants;

FIG. 2 is the result of fruit longitudinal diameter/transverse diameter ratio statistics of transgenic plants and AC wild plants;

FIG. 3 is a comparison of fruits of transgenic plants and AC wild plants.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1

The embodiment provides a construction method of a DWARF gene overexpression vector, which comprises the following steps of adopting a primer FW:

CATTTGGAGAGGACACGCTCGAGATGGCCTTCTTCTTAATTTTTCT (shown in SEQ. NO. 2) and

RV：

TCTCATTAAAGCAGGACTCTAGATTAGTGAGCTGAAACTCTAATCC (shown in SEQ. NO.3), cloning DWARF gene by using cDNA reverse transcription of total RNA of each tissue of cultivated tomato Ailsa Craig (abbreviated as AC and purchased from tomato genetic center TGRC (https:// TGRC. ucdavis. edu /)) as a template to obtain target fragment.

The PCR amplification system adopted in the step is

The amplification procedure was:

recovering target fragments by using cutting gel after amplification;

the pHellsgate8 vector (from CSIRO Plant Industry) was digested with XhoI and XbaI, and the linearized vector was recovered by cutting the gel. And carrying out homologous recombination on the recovered target fragment and the linearized vector. The concentrations of the insert and the linear vector were adjusted so that the number of base pairs of the linearized vector used was 0.02 times the amount of the linearized vector used, and the number of base pairs of the insert used was 0.04 times the amount of the insert used, in ng. The volume to be used can be calculated from the amount used and the measured concentration. In a 10. mu.L system, 2. mu.L of 5 × CE II Buffer, 1. mu.L of Exnase II, calculated insert and linearized vector were added, the remainder using ddH₂And (4) supplementing and finishing. Adding various reaction solutions in sequence, sucking and pumping the mixed reaction solution gently, and placing the mixed reaction solution in a PCR instrument for reaction at 37 ℃ and 3 DEG C0mim was subjected to homologous recombination.

Transferring the ligation product into escherichia coli, carrying out amplification on the escherichia coli monoclonal with correct sequencing result, and extracting plasmids by using a DNA miniprep kit to obtain the overexpression vector DWARF-pHellsgate8 of DWARF genes.

Example 2

Genetic transformation for overexpression of DWARF genes

The specific operation steps are as follows:

(1) the constructed recombinant vector with correct sequencing is transferred into an agrobacterium competent state (C58) for standby.

(2) The cotyledon explants of tomato variety AC were infected with Agrobacterium containing the DWARF-pHellsgate8 vector.

(3) Dark culture of infected explant.

(4) The explants were screened until callus was developed.

(5) And subculturing the callus to a growing point.

(6) Transferring the grown growing points into a rooting culture medium for rooting.

(7) Transplanting the rooted plants into nutrient soil for growth, and carrying out PCR detection by using a sequencing primer, wherein the PCR product strip accords with the DWARF gene length, so as to obtain a positive transgenic line.

In example 1 and example 2, taq-PCR was used for positive plant detection, positive escherichia coli detection, and positive agrobacterium detection, and the positive plants and clones were detected using primer pairs (seq. No.4 and seq. No.3), and the positive clones were sequenced using sequencing primer pairs (seq. No.4 and seq. No.5), with the upstream primer sequence of the sequencing primers being:

ACGCACAATCCCACTATCCTTC (SEQ. NO. 4);

the sequence of the downstream primer is as follows:

CATAAAAATACGATAGTAACGGGTG (shown in SEQ. NO. 5).

the taq-PCR detection system is as follows:

the reaction procedure is as follows:

the sequence of the sequencing fragment is as follows:

ttcatttggagaggacacgctcgagatggccttcttcttaatttttctttcatccttttttggcctatgtatcttttgtactgctttattaagatggaatcaagtcaagtataaccaaaaaaacttgccccctggtactatgggttggccactttttggtgaaactactgagtttcttaaacttggtccaagtttcatgaaaaaccaaagagccagatatgggagtttttttaaatcacacatacttggttgtccaacaattgtttcaatggattcagaactgaacagatatatactagtgaatgaagcgaaaggactggtcccaggatacccacagtctatgatagatattttaggaaaatgtaatattgcagctgtcaatggttcagctcacaagtacatgaggggtgcattgttatccctaattagccctacaatgatcagagaccaacttttgcctaaaattgatgagtttatgagatcccacttaaccaattgggataataaagttattgacattcaagagaaaaccaataagatggcatttctatcatcgttgaagcaaattgctggtattgaatctacctctttagctcaagaattcatgtctgaatttttcaatctagtgctaggcactctttcactacctatcaatcttccaaacaccaactatcatcgcggatttcaggcaaggaaaattattgtgaacttattacgaacactcatagaagagagaagagcttcaaaggaaattcaacatgatatgcttggttacctgatgaatgaggaagcaacacgattcaaattaacagatgatgagatgattgatttaattataactattttgtactctggatatgaaactgtttccaccacttctatgatggctgtgaaatatcttcatgatcatccaaaagttcttgaagaacttagaaaagaacacatggctattagagaaaagaaaaaacctgaggatcctatcgattacaacgattacaggtcaatgcggttcacacgagctgtgattttagagacctccaggttagcaacaatagtaaatggggttttgagaaaaacaactcaagatatggaaataaatgggtatatcattcctaaaggatggagaatatacgtatatacaagggagttgaattacgatccaagactttatcctgatccatattcgttcaatccatggagatggatggataagagcctggaacaccaaaactcatttttggtatttggaggtggtactagacaatgtcctggaaaggaacttggtgtagcagaaatttccacatttcttcattacttcgtaacaaaatacagatgggaagaaataggtggagataaactgatgaaattcccaagagttgaagcaccaaatggtctacggattagagtttcagctcactaatctagagtcctgctttaatgagatatgcgaga

the extraction method of the plant tissue DNA comprises the following steps:

1. placing 0.1g of young leaves of plant seedlings in a 2mL centrifuge tube, grinding in about 750 μ L CTAB extract (added with beta-mercaptoethanol), placing in 65 deg.C water bath for more than half an hour after grinding;

2. after water bath, adding about 750 mu L of chloroform-isoamylol mixed solution (the volume ratio is 24:1) into a centrifuge tube, reversing the centrifuge tube from top to bottom for 100 times, and centrifuging the centrifuge tube at 10000r/min for 10 min;

3. absorbing 500 mu L of supernatant fluid 450-one into a new centrifugal tube of 1.5mL, adding cold isopropanol with the same volume, uniformly mixing, placing into a refrigerator of-20 ℃, centrifuging for 10min at 12000 r/min;

4. discarding the supernatant, adding 800 μ L75% ethanol, washing the precipitate, centrifuging instantly, discarding ethanol, and drying to colorless and transparent;

5. add 200. mu.L ddH₂O (adding different amount for different purpose), dissolving DNA, and storing at-20 deg.C for use.

Example 3

DWARF gene expression test

3.1 expression level test of transgenic Positive plants by the following method

Taking young leaves of the transgenic plant, and extracting RNA.

The expression level of DWARF gene in the positive transgenic lines (DWARF-OE-11, DWARF-OE-16 and DWARF-OE-19) was examined.

The expression quantity detection step comprises RNA extraction, RNA reverse transcription and expression quantity measurement.

The RNA extraction method comprises the following steps:

1. grinding plant tissues by using liquid nitrogen, putting about 100mg of a sample into a 2.0mL centrifuge tube frozen by using the liquid nitrogen, adding 1mL of trizol, reversely extracting for a few minutes, and standing for 5 minutes;

2. adding 200 μ L chloroform, extracting under severe condition for 15s, and standing at room temperature for 2-3 min;

centrifuging at 3.4 deg.C and 13000r/min for 10 min;

4. sucking 300-;

centrifuging at 13000r/min for 10min at 5.4 ℃;

6. discarding the supernatant, adding 1mL of 75% ethanol (DEPC water dilution), and suspending the precipitate;

7.12000r/min for 1 min;

8. discarding the supernatant (6 and 7 can be repeated once to improve the RNA purity), placing the centrifuge tube in a ventilation kitchen, and air-drying for 5-10 min;

9. adding about 20 mu L (increased or decreased according to the extracted RNA amount) DEPC water for dissolving;

10. detecting RNA quality by agarose gel electrophoresis, determining RNA concentration by absorbance method, and storing at-80 deg.C for use.

RNA reverse transcription step:

the RNA samples were uniformly diluted to specific concentrations using DEPC water according to the concentrations determined for the RNA samples. Reverse transcription was performed using a reverse transcription kit, and the reverse transcription process was as follows:

1) genomic DNA removal

Preparing the following mixed solution in an RNase-free centrifuge tube:

gently pipet and mix by using a pipette, and then place in a PCR instrument for 2min at 42 ℃.

2) First Strand cDNA Synthesis

Mu.l of 10 XT Mix and 2. mu.l of HiScript II Enzyme Mix were added to the mixture in the previous step.

Gently pipet and mix the mixture by using a pipette, and then the mixture is placed in a PCR instrument. Setting a reaction program by the PCR instrument: 50 ℃ for 15 min; 85 ℃ for 2 min.

After the reaction program is finished, the product can be immediately used for qPCR reaction or stored at-20 ℃.

Determination of relative expression amount of DWARF

The relative expression of DWARF was determined according to the following procedure and SYBR mix system, the selected reference gene was Solyc11g005330, and the relative expression of the gene was determined according to the routine detection method in the art.

The reaction system is as follows:

SYBR mix system of

In the table, Q _ FW and Q _ RV are an upstream primer and a downstream primer of the expression level detecting primer, respectively.

The primer for detecting the expression quantity of the reference gene Solyc11g005330 is as follows:

Q_FW:GTCCTCTTCCAGCCATCCA(SEQ.NO.6)

Q-RV:ACCACTGAGCACAATGTTACCG(SEQ.NO.7)

the detection primers of the expression level of DWARF gene are as follows:

Q_FW:AGATGGAATCAAGTCAAGTATAACC(SEQ.NO.8)

Q_RV:CAGTCCTTTCGCTTCATTCACT(SEQ.NO.9)

the test results are shown in FIG. 1, the relative expression level of DWARF gene of the tomato plant transferred with DWARF-pHellsgate8 vector is greatly improved compared with that of the control wild plant (AC), and the relative expression level test of specific DWARF gene is shown in Table 1.

TABLE 1 relative expression amount test results of DWARF genes

3.2 statistics of fruit conditions of DWARF gene overexpression plants

The transverse and longitudinal diameters of the fruits of the positive transgenic lines are detected, and the fruit shape index (fruit shape index) is calculated, and the statistical results are shown in tables 2 to 4.

TABLE 2 tomato transverse diameter statistics

TABLE 3 tomato longitudinal diameter statistics

TABLE 4 fruit shape statistics

In Table 4, the fruit shape index is L/W, wherein L is the longitudinal diameter of the tomato fruit, and W is the transverse diameter of the tomato fruit.

The results in tables 1-4 and fig. 2-3 show that the fruit shape index of DWARF over-expressed plants was significantly improved compared to the fruit shape index of control AC plants, the transverse diameter was not significantly changed, but the longitudinal diameter was significantly increased, and finally the fruit shape index was greater than 1, and the visual appearance was lengthened.

Furthermore, the DWARF expression level of the DWARF-OE-11 strain is increased to 70-90 times, and as can be seen from FIG. 3, the fruit of the DWARF over-expression plant has a more visually obvious elongation effect than the fruit of AC wild tomato.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Claims

1. A method for prolonging the shape of tomato fruits is characterized in that overexpression of DWARF genes in tomatoes is regulated and controlled to prolong the shape of the tomato fruits, and the ORF sequence of the DWARF genes is shown as SEQ ID No. 1.

2. Method for shape-lengthening tomato fruits according to claim 1, characterized in that it comprises the following steps:

step S1, constructing an overexpression vector of the DWARF gene;

and S3, selecting the tomato explants stained in the step S2, growing callus, and further culturing to obtain the tomato germplasm with long fruits.

3. The method of claim 2, wherein the overexpression vector of DWARF gene comprises the desired fragment containing ORF sequence of DWARF gene in step S1.

4. The method according to claim 2 or 3, wherein in step S3, after the callus grows into seedlings, DWARF gene expression level detection is performed on plant tissues, and plants with DWARF gene expression level increased by 50-200 times are selected by using the gene with the sequence number of Solyc11g005330 as an internal reference gene.

5. Method for shape-elongating tomato fruits according to claim 2 or 3, characterized in that the overexpression vector backbone vector for DWARF gene is pHellsgate8 vector.

6. The method for shape elongation of tomato fruit as claimed in claim 5, wherein the method for constructing the overexpression vector of DWARF gene comprises:

a1, using cDNA reverse transcribed from total RNA of tomato tissue as a template, and adopting an amplification primer to amplify the ORF region of DWARF gene to obtain a target gene fragment;

7. The method for shape-enlarging tomato fruits according to claim 6, wherein the sequence of the upstream primer of the amplification primer is shown as SEQ ID No.2, and the sequence of the downstream primer is shown as SEQ ID No. 3.