CN108715852B - Tomato fruit mature gene Sl0658 and application thereof - Google Patents

Abstract

The invention discloses a tomato fruit mature geneSl0658And use thereof, tomatoSl0658The gene and the nucleotide sequence are shown in SEQ ID NO 1; it has a typical NAC family conserved domain, encoding NAC protein; the invention adopts related technology of functional genomics to prove that the tomatoSl0658The gene plays an important role in the ripening of tomato fruits; the fruit mature gene of the inventionSl0658The transgenic tomato can show the capability of delaying fruit ripening and can be directly used for controlling the ripening process of fruits in production.

Description

Tomato fruit mature gene Sl0658 and application thereof

Technical Field

The invention belongs to the field of molecular biology and genetic engineering, and particularly relates to a tomato extractSolanum lycopersicum ) To obtain a new fruit mature geneSl0658And applications thereof.

Background

Tomato (A)Lycopersicon esculentum Mill.) has a long history of cultivation, and is a model plant for researching the development and maturity of fleshy fruits because of small genome, short growth cycle and far genetic relationship with model plants such as Arabidopsis, rice, corn and the like. A large number of tomato germplasm resources, mutant libraries, high-density genetic maps and EST resources are obtained, and the transient expression and stable transformation technology is mature. In addition, the fine sequence analysis of the whole genome of cultivated tomatoes, which is completed in 5 months of 2012, greatly promotes the research of tomato functional genomics and molecular genetics. The fruit ripening process involves regulation of a large number of metabolic pathways and significant change of physiological and biochemical properties, and requires precise transcriptional regulation of time-sequence expression of a series of genes, so that the functional research of tomato ripening related transcription factors becomes one of the research hotspots in the field of botany.

The transcription factor is a group of specific combination with the upstream specific sequence (promoter region) of the 5' end of the gene, so as to ensure that the target gene expresses protein molecules at specific time and space with specific strength, and the biological behavior of the cell is influenced by regulating the transcription level of the target gene. Bioinformatics predicts that at least 62 transcription factor families exist in tomato, and play an important role in regulation and control in multiple aspects such as plant hormone synthesis, tissue cell expansion, cell wall substance metabolism, regulation of fruit ripening, pigment accumulation and the like (Zhang et al, 2010). NAC transcription factor, its name is based on the first discovered acronym for several genes of this family: N AM (NO APICAL MERISTEM) 、 A TAF1-2 、 C UC2 (CUP-SHAPED COTYLEDON). NAC transcription factor generally consists of two introns and three exons; the first two exons encode primarily the DNA binding domain of NAC, while the last exon encodes the transcriptional activation domain.NACTranscription factors play an extremely important role in various aspects of plant development, such as low temperature, drought, pathogen and mechanical damage (Souer)et al.，1996；Takada et al., 2001; Olsen et al., 2005; Hu et al., 2006; Catherine et al., 2010). And its report on fruit ripening has been limited to dateLeNAC-NOR(Cantu et al.,2009). Tomato mutant nor matures normally, with no peak in respiration release and ethylene synthesis late in fruit development, but can synthesize ethylene when damaged (Osorio et al, 2011).

Based on in tomatoLeNAC-NOR Similar exploration of upstream transcription factor of ethylene, the invention clones a new NAC gene from the chip of tomato nor mutant-Sl0658. InterferenceSl0658The transgenic tomato shows that the fruit ripening process becomes slow, the marketing time of the tomato can be adjusted by reasonably utilizing the transgenic material, and no report of the gene is found at present for production service.

Disclosure of Invention

The invention aims to provide a tomato fruit ripening geneSl0658The nucleotide sequence is shown as SEQ ID NO. 1; it has a typical NAC family conserved domain, encoding NAC protein.

Another object of the present invention is to provide tomato fruit maturation genesSl0658In controlling the ripening process of tomatoes and other climacteric fruits (climacteric fruits refer to the period between the cessation of growth and the onset of senescence in certain fleshy fruits, with a sudden increase in their respiration rate, such as apples, bananas, tomatoes, avocados, mangoes, etc.); in particular, the geneSl0658Constructing on plant expression vector and introducing into tomato to expressSl0658Interfering the transgenic tomato to carry out the physiological and biochemical and molecular biological detection related to fruit ripening, and confirmingSl0658Can change the ripening process of tomato fruits and lay a foundation for improving the capability of the ripening process of tomato and other climacteric fruits by utilizing the gene in the later period.

The complete C-terminal specific terminal sequence fragment of a tomato fruit ripening gene carried in the tomato Ailsa Craig is cloned, the C-terminal specific terminal sequence fragment is transferred into a receptor tomato by an agrobacterium-mediated method for interference expression, and the effect of the gene in the tomato fruit ripening process is verified through experiments, so that a foundation is laid for the capability of improving the ripening process of tomatoes and other climacteric fruits by utilizing the gene in the later period.

To pairSl0658The gene is subjected to sequence analysis, and the result shows thatSl0658The full-length cDNA was 1152 bp, having an Open Reading Frame (ORF) of 783 bp, a 5 'untranslated region of 50 bp and a 3' untranslated region of 309bp, encoding a protein containing 260 amino acids.Sl0658The coding protein has a conserved structural domain of NAC family, and is derived from apple (Malus pumila L.) (Malus domestica) Peach (a), (b)Prunus persica) NAC protein of other species is highly similar and interferes with expressionSl0658 The 3' end specific end can significantly delay the progress of tomato fruit ripening.

The gene can be used for controlling the ripening process of tomato and other climacteric fruits, and the specific operation is as follows:

(1) obtaining of genes: extracting total RNA of tomato fruits in red ripe stage by using tomato wild type Ailsa Craig as material, and obtaining the total RNA by RT-PCR amplificationSl0658 3' end specific end, then connected to pMD18-T vector, through sequencing to obtain the clone with target fragment.

(2) Construction and genetic transformation of plant expression vectors:

after the sequence is subjected to enzyme cutting site analysis, the sequence is subjected to enzyme cutting site analysisSl0658 Inserting the 3' end specific terminal forward sequence into the multiple cloning site of the expression vector 1300-35S-XBamHI-SpeBetween I, the reverse sequence is inserted into the multiple cloning site of the expression vector 1300-35S-XSac I-KpnAnd I, performing sequencing verification to successfully construct the tomato interference expression vector.

Extracting plasmid of recombinant expression vector bacterial liquid, introducing agrobacterium tumefaciens C58 by an electric transformation method, transforming wild tomato Ailsa Craig by an agrobacterium-mediated genetic transformation method, screening antibiotic (kanamycin) and RT-PCR (reverse transcription-polymerase chain reaction) for positive transgenic plants of transgenic tomatoes, and performing subsequent analysis by taking tomato plants of wild type and transformed empty vectors as controls.

(3) Fruit ripening characteristic analysis of transgenic tomatoes: the strongly growing transgenic plants and the control wild plants are planted in flowerpots respectively. Recording the time when the fruit reaches maturity, detectingThe fruit ripening related indexes (including fruit ethylene release amount, fruit respiration rate, fruit hardness, soluble solid content and the like) are tested, and the expression level of the fruit ripening related gene is determined, so that the fruit ripening related indexes are verifiedSl0658The fruit ripening of the interference transgenic tomato is obviously delayed.

The invention provides a new method for controlling the ripening process of tomato fruits, and the defect of traditional breeding can be overcome by cultivating transgenic plants by means of genetic engineering, and the breeding period can be shortened. Will be provided withSl0658The gene is expressed in tomato in an interference manner, the fruit ripening process can be remarkably delayed, and the fruit hardness is moderately increased, so that the fruit marketing time can be artificially controlled, the taste characteristics of the fruit are not changed, compared with the fruit treated by artificial hormone, the fruit treatment method is more environment-friendly, the effect is more stable, and the tomato fruit treatment method has obvious advantages and irreplaceable importance. The method can provide a convenient way for the large-scale production of the climacteric fruit, can greatly reduce the economic loss caused by actual softening and rotting, can save the cost for agricultural production and improve the management level, and therefore, the method has wide market application prospect.

Drawings

FIG. 1 is a drawing of the present inventionSl0658Full-length and 3' end-specific fragments (sequences in black box), wherein the start codon (ATG) and stop codon (TAA) are indicated in bold;

FIG. 2 is a multi-cloning site diagram (A) of an interference expression vector 1300-35S-X employed in the present invention and a schematic diagram (B) of the insertion of a target fragment into the expression vector;

FIG. 3 shows the regeneration stage (A), tissue culture seedling stage (B), control tomato red-maturing stage (C) andSl0658interfering with the red-ripe period (D) picture of the transgenic tomato;

FIG. 4 is a graph showing comparison results of days after flowering of control tomato (Wt) and interference expression (Sl0658-RNAi) transgenic tomato fruits in different maturity stages (green stage, brokening stage, yellow stage, red stage) and comparison results of ethylene release amount of fruits (B);

FIG. 5 is a graph showing the comparison result of the respiration rate of the fruit and the fruit hardness of the control tomato (Wt) and the interference expression (Sl0658-RNAi) transgenic tomato of the invention;

FIG. 6 is a graph (A) showing the comparison result of the soluble solid content of the fruit of the control tomato (Wt) and the interference expression (Sl0658-RNAi) transgenic tomato of the invention and a graph (B) showing the expression level of the mature related gene.

Detailed Description

The invention will be further explained in the following with reference to the accompanying drawings, but the scope of the invention is not limited to the above description; the method used in the present invention is a conventional method unless otherwise specified.

Example 1:Sl0658cloning of 3' end specific fragments

Grinding the tomato Ailsa Craig red-ripe fruit into powder by using liquid nitrogen, transferring the powder into a centrifugal tube, and extracting the total RNA by adopting a Trizol method. The first strand cDNA is synthesized by reverse transcriptase M-MLV (Tiangen) and total RNA as a template, and the reaction system and the operation process are as follows: mu.g of total RNA was taken, 50 ng oligo (dT) 15 and 2. mu.L dNTP (2.5mM each) were added in this order, and ddH was added₂O (No RNase) to a reaction volume of 13.5. mu.L; after mixing uniformly, heating and denaturing at 70 ℃ for 5 min, then quickly cooling on ice for 5 min, then sequentially adding 4 muL of 5 XFirst-stand buffer, 0.5 muL of RNase inhibitor (200U), 1 muL of reverse transcriptase (200U) and 1 muL of dimercaptothreitol (0.1M), mixing uniformly and centrifuging for a short time, bathing at 42 ℃ for 1.5 h, taking out, heating at 95 ℃ for 5 min, and stopping reaction; the first strand cDNA is synthesized and stored at-20 deg.C for further use.

Amplifying the first strand cDNA as a templateSl0658 3 ' end specific fragment (primer sequences 5'-TCGATGTTTATTCTGGGATCTG-3' and 5'-TTTTCCCTAAATTTTTCCCTTTC-3'). Adopting high-fidelity DNA polymerase ex taq of Dalibao biology to amplify target genes, wherein the PCR reaction conditions are as follows: 3 min at 94 ℃; 94 ℃ for 45 s, 58 ℃ for 30 s, 72 ℃ for 45 s, 30 cycles; 5 min at 72 ℃. The reaction system (20. mu.L) was 1. mu.L of cDNA, 2. mu.L of 10 XBuffer, 0.4. mu.L of dNTP (10 mM each), 0.1. mu.L of forward primer (20. mu.M), 0.1. mu.L of reverse primer (20. mu.M), 0.2. mu.L of ex Taq DNA polymerase (5U/. mu.L), 16.2. mu.LL ddH₂O; after the PCR was completed, 5. mu.L of the DNA fragment was subjected to agarose gel electrophoresis to detect the amplified fragment of the desired size.

Obtained by PCRSl0658 TA cloning is carried out on the specific fragment (shown in figure 1) at the 3' end, the kit used is pMD-18T vector kit (Dalianbao organism), and the reaction system and the operation process are as follows: mu.L of the PCR product was taken, and 1. mu.L of pMD18-T vector (50 ng/. mu.L) and 2.5. mu.L of 2 × Ligation solution I were added in this order, mixed well and allowed to react overnight at 16 ℃. The ligation product was transformed into E.coli DH 5. alpha. using a heat shock transformation method. Positive clones were screened in LB solid medium plated with beta-galactosidase (X-Gal), Isopropylthiogalactoside (IPTG), ampicillin (Ampicillin, Amp). Selecting several white colonies, shaking, and amplifyingSl0658 Primers for the 3' specific fragment identified the cloning vector with the correct insertion direction.

Example 2: construction of plant interference expression vector

Sl0658After the PCR product of the 3' end specific fragment was recovered, it was dissolved in TE buffer to carry out BP reaction. The reaction system included PCR product 50-100 ng, 1300-35S-X vector 2. mu.L, 5 XBP clonase reaction buffer 4. mu.L, BP clonase mix 4. mu.L, and TE buffer (pH 8.0) supplemented to 20. mu.L. The reaction system was incubated at 25 ℃ for 16 h, then 2. mu.L of proteinase K solution was added, and incubated at 37 ℃ for 10 min to terminate the BP reaction. Thereby obtaining a fragment carrying the inverted repeat target gene (as shown in FIG. 2); positive clones were screened.

Transferring the constructed plant expression vector into the prepared agrobacterium tumefaciens C58 competent cell by adopting a liquid nitrogen freeze-thawing method; the operation steps are as follows: adding 2 mu g of expression vector plasmid into a centrifuge tube containing 200 mu L of competent cells, gently mixing uniformly, performing ice bath for 5 min, then transferring into liquid nitrogen for freezing for 1 min, then rapidly placing in a water bath at 37 ℃ for 5 min, immediately performing ice bath for 2 min, adding 800 mu L of LB liquid medium, and performing shake culture at 28 ℃ for 4 h. Coating the activated agrobacterium on an LB solid culture medium containing 50 mg/L kanamycin, and performing static culture at 28 ℃; selecting single colony shake bacteria, and amplifyingSl0658 PCR is carried out by the primer of the specific segment at the 3' end, and whether the expression vector is transferred into agrobacterium is detectedPerforming the following steps; for positive clones, glycerol was added and stored at-80 ℃ for future use.

Example 3: genetic transformation of tomato

The genetic transformation system adopted is an agrobacterium-mediated genetic transformation method, the agrobacterium strain is C58, and the transformation acceptor material is a tomato wild species Ailsa Craig. Soaking plump tomato seeds in sodium hypochlorite solution (with effective chlorine content of 2%) for 15 min, cleaning the seeds with distilled water, sowing the seeds on 1/2 MS culture medium, culturing at 25 deg.C under 16 h light/8 h dark condition, cutting cotyledon when aseptic seedling grows to 7-9 d, and culturing in the dark for one day on 1/2 MS culture medium. The method comprises the steps of taking agrobacterium tumefaciens with the resuspension concentration of OD600 & lt approximately 0.5 to dip and stain leaves for 5 min, sucking the redundant bacterial liquid with sterilizing filter paper, then placing the liquid on a pre-culture medium paved with the filter paper again, transferring the liquid to a screening culture medium (a basic culture medium MS + zeatin 2.0 mg/L + kanamycin 100 mg/L + cephalosporin 100 mg/L) to perform differentiation culture on the leaves after dark culture for 2 d, performing subculture once for two weeks, transferring the obtained callus to the screening culture medium (the basic culture medium MS + zeatin 0.2 mg/L + kanamycin 100 mg/L + cefuroxime 100 mg/L) after forming callus, cutting the resistant buds after growing to a certain size, placing the obtained resistant buds into a rooting culture medium to root the resistant buds, transferring the rooted resistant seedlings to the sterilized medium to acclimatize for one week, and finally transferring the tomato seedlings to a greenhouse or a greenhouse to cultivate (as shown in figure 3).

Example 4: PCR positive detection of transgenic tomato plants

After the regenerated seedlings are transplanted to survive, extracting total RNA of transgenic single plants and young leaves of the Ailsa Craig as a control, carrying out reverse transcription to generate first-chain cDNA, taking the cDNA of the Ailsa Craig as a negative control, and carrying out positive detection on the obtained transgenic material by using NPT II primers (the primer sequences are 5'-AGACAATCGGCTGCTCTGAT-3' and 5'-TCATTTCGAACCCCAGAGTC-3') contained in an expression vector; the total PCR reaction system is 25 μ l, and comprises 1 XPCR buffer (containing Mg)²⁺) 0.38 mmol/L dNTPs, 0.62 mu mol/L forward and reverse primers respectively,TaqEnzyme 1.2U, template about 60 ng; the reaction program is pre-denaturation at 95 ℃ for 5 min, pre-denaturation at 94 ℃ for 45 s, pre-denaturation at 58 ℃ for 45 s, pre-denaturation at 72 ℃ for 1 min, 32 cycles, and extension at 72 ℃ for 10 min; for final PCR productAnd (3) analyzing the agarose gel electrophoresis with the concentration of 0.8%, observing and recording an electrophoresis pattern by a gel imaging system, and selecting a positive transgenic plant.

Example 5: statistics of ripening time after fruit blossom

Artificial supplementary pollination is carried out after the tomato material blooms, and the date is marked by listing, and the time for different transgenic materials to reach different maturity stages (green maturity stage, color breaking stage, yellow maturity stage and red maturity stage) of the fruit is counted (as shown in figure 4A). The fruit ripening of the interference expression transgenic tomato is obviously delayed compared with that of a control tomato, and the final ripening time is delayed by 10 days compared with that of the control tomato; this explains the interferenceSl0658The ripening process of tomato fruits is significantly delayed after the gene.

Example 6: determination of fruit ethylene Release

Transgenic material was randomly picked and ethylene measurements were performed against different maturity fruits. The fruits were sealed with a polypropylene film and placed at 25 ℃ for 48 h, 1 ml of gas was aspirated therefrom with a syringe, ethylene content was detected by injecting into a gas chromatograph (Agilent 6890N, USA), peak time and peak area were recorded, and ethylene release of the samples was calculated in ul/L in comparison with standard ethylene. Three fruits per sample were assayed per period and three biological replicates were made (as shown in fig. 4B); the detection result shows that the control tomato has obvious peak of ethylene release during the fruit ripening process, and the interference transgenic tomato has no significant peak of ethylene release during the fruit ripening process, which indicates thatSl0658After the gene is intervened, the fruit ripening process is significantly inhibited.

Example 7: fruit respiration rate detection

Observing mature period of transgenic fruit, randomly picking transgenic material and fruit of control fruit in different mature periods (green mature period, color breaking period, yellow mature period and red mature period), and adopting CEA-800 type infrared CO₂The analyzer measures the respiration intensity of the fruit and the result is expressed as CO₂ml/kg/h, three fruits per sample per period were assayed and three biological replicates were made (as shown in FIG. 5A). The results show that the control tomato has a significant breath during fruit ripeningThe peak of suction and release shows the typical characteristics of fruit with respiratory jump; however, the respiration rate is not changed significantly in the whole process of interfering the fruit development and maturity of the transgenic tomato, and the peak of the respiration jump does not appear. Show thatSl0658After the gene is intervened, the fruit ripening process is retarded.

Example 8: measurement of fruit hardness

Transgenic and control fruits at different maturity stages (green, broken, yellow and red) were harvested and tested for four points of symmetry between the top and equator of each fruit using a durometer (Model GY-1, Cany Precision Instruments co., Ltd, Shanghai, China) as indicated by the force (N) expended on piercing a 5mm peel with a cylindrical probe (fig. 5B). The result shows that the fruit hardness of the interference expression transgenic tomato is not obviously different from that of a control tomato at the early stage of development, but is half of that of the control tomato at the final stage of fruit development, which shows that the softening speed of the interference expression transgenic tomato is reduced at the later stage of development. This indicates that the interference with the ripe softening of transgenic tomato fruits is inhibited.

Example 9: determination of soluble solid content in fruit

The soluble solids content of the fruits at different ripening stages (green, broke, yellow and red ripening stages) of the transgenic and control fruits was determined in percent using a hand-held refractometer (Atago PAL-1, Co. Ltd., Tokyo, Japan) (as shown in FIG. 6A). The results show that the soluble solid content of the control tomato is increased along with the ripening of the fruit, while the soluble solid content of the interference transgenic tomato is also increased, but the increase is obviously smaller than that of the control tomato; during the red-ripe stage of the fruit, the soluble solids content of the intervention transgenic tomato is 81% of the control.

The Primer for real-time fluorescent quantitative PCR detection is designed by using online software Primer 5, the length of the amplified target gene fragment is controlled to be 80-200 bp, and the mature genes are selected from ACO1, ACS2, ACS4, PE, PG2A, RIN and NOR. Primer sequences are shown in the following table; extracting RNA of tomato material, and reverse transcription to obtain cDNA. This cDNA was diluted 10-fold and used as a template for the reaction. Real time RT-PCR amplification was performed using a Real-time fluorescent quantitative PCR instrument (Roche, USA) model Light Cycler 480. The reaction was performed in a total volume of 20. mu.l, containing 5. mu.l of template, 10. mu.l of Fast SYBR Green I Master Mix (Roche, USA), 1.0. mu.l (10. mu.M) of forward and reverse primers, respectively, and 3.0. mu.l of PCR water; the PCR reaction condition is 95 ℃ for 10 min; 95 ℃ for 10s, 58 ℃ for 15 s and 72 ℃ for 20 s for 45 cycles. Tomato housekeeping gene β -actin served as an internal reference (as shown in FIG. 6B). The results show that the expression of fruit ripening genes ACO1, ACS2 and PE in the intervening transgenic tomatoes is significantly reduced compared to the control, to 14%, 54.5% and 27% of the control, respectively. PG2A decreased to 80% of the control. RIN and NOR varied significantly from the control. This shows that Sl0658 has an extremely important relationship with the ripening of tomato fruits, and Sl0658 plays an important role in tomato fruits;

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sequence listing

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Claims (1)

1. Tomato fruit mature geneSl0658Application in controlling tomato fruit ripening process, tomato fruit ripening geneSl0658The nucleotide sequence of (A) is shown as SEQ ID NO. 1.

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