CN104561025A - Tomato slml1 gene and application - Google Patents

Abstract

The invention discloses a tomato SlML1 gene and application, and belongs to the field of biological techniques. The cDNA sequence and the encoding amino acid sequence of the gene are as shown in SEQIDNO.1 and SEQIDNO.2. The transgenosis experiment shows that the gene not only has the functions of improving the quality and increasing the yield of tomatoes, but also has the properties of improving the drought resistance, the fungal disease resistance, bacterial disease resistance and TMV and insect resistance of tomatoes, and moreover the shelf life of tomatoes can be prolonged. Therefore, SlML1 can be used as a target gene introduction plant for improving the quality and the resistance and increasing the yield of plants and has good application prospect in breeding, quality improvement and resistance study on tomatoes.

Description

Tomato SlML1 Gene and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a tomato S1ML1 gene, and also relates to the application of the gene in tomato strain quality, biotic stress and abiotic stress resistance and shelf life. the

Background technique

The MYB transcription factor family refers to a class of transcription factors containing the MYB domain. The MYB domain is a DNA-binding domain containing 52 amino acids. MYB transcription factors in plants have a common feature of a MYB domain consisting of about 52 amino acids at their N-terminus. Clorless1 (C1) of maize is the first MYB transcription factor isolated and identified in plants (Paz-Areset et al., The re gulatory c1 locus of Zea may encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.EMBO J,1987,6:3553-3558), and subsequently, Arabidopsis (Romero et al.,More than80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana.The Plant Journal,1998,14:273- 284), cotton (Loguerico et al, Differential regulation of si x novel MYB-domain genes defines two distinct expression patterns in allotetra ploid cotton (Gossypium hirsutum L.). Mol Gen Genet, 1999, 261:660-671), apple ( Takos et al., Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol. 2006, 142: 1216-1232) and other plants have also isolated and identified MYB proteins with different functions. The results of functional research on MYB transcription factors show that the main functions of MYB proteins are: participating in plant secondary metabolic processes, regulating cell morphology, participating in plant responses to biotic and abiotic stress, playing a role in plant responses to hormones, and regulating plant The circadian clock of MYB plays a role in cell cycle and proliferation, etc. Vailleau et al. (Vailleau F et al., A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. PNAS, 2002, 99:10179-10184) found that in excess Arabidopsis thaliana expressing AtMYB30 exhibited disease-resistant responses during the interaction with pathogenic bacteria, while Arabidopsis thaliana inhibiting AtMYB30 expression decreased resistance to several pathogenic bacteria; Van et al. (Van SC, et al., Plant immune responses triggered by beneficial microbes[J].Cur rent Opinion in Plant Biology,2008,11:443-448) constructed MYB72-deficient Arabidopsis thaliana by gene knockout method, which is effective against Pseudomonas, Peronospora parasitica, Alternaria Bacteria and Botrytis cinerea were reduced, indicating that Arabidopsis MYB72 plays a role in plant disease resistance. At present, more and more research results indicate the function of MYB transcription factors in plant disease resistance. the

MIXTA is a MYB-related transcription factor, which was discovered by Glover BJ et al. (Glover BJ et al., Development of several epidermal cell types can be specified by the same MYB-related plant transcription factor. Development, 1998125:3497–3508) It can control the formation of cone cells of Antirrhinum and promote the formation of Antirrhinum trichomes, and Glover BJ et al. (Glover BJ et al., Convergent evolution within the genus Solanum: the spe cialised anther cone develops through alternative pathways. Gene, 2004 , 331:1-7.) also found that a MIXTA-LIKE gene can induce the differentiation of epidermis in Ou Baiying, while Avila J et al. (Avi la Jet al., Petunia hybrid genes related to the maize regulatory C1gene and to animal myb proto-oncogenes. Plant J, 1993, 3:553-562) also found a MIXTA-LIKE gene that can induce the differentiation of petunia epidermis. Studies on MIXTA have shown that MIXTA is necessary for the formation of epidermal hairs and can promote the differentiation of epidermal hairs. Cuticle hairs are a rich source of molecular breeding for stress-resistant plants, which play important roles in defense against biotic and abiotic stresses. The cloning of SlML1 (Solanum lycopersic um MIXTA-like1) gene, the quality, resistance and shelf life of transgenic materials provide an important theoretical and material basis for the use of genetic engineering to study tomato. Breeding and other aspects, has a good application prospect. Before the publication of the present invention, there has not been any publication or report on the functional research of the S1ML1 gene and its amino acid sequence in tomato mentioned in this patent application. the

Contents of the invention

The object of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provides a tomato S1ML1 gene, whose sequence is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2. the

Another object of the present invention is to provide a research application of tomato S1ML1 gene in improving tomato quality, biotic and abiotic stress resistance and extending shelf life. the

In order to achieve the above object, the present invention takes the following technical measures:

A kind of tomato SlML1 gene, its screening process is

Tomato total RNA was extracted, mRNA was separated using the mRNA Purification Kit (QuichprepTM Micro mRNA P urifiCation Kit, Pharmacia Company), and the Creator Smart cDNA Library Construction Kit (Cat. No. 634903) of Clontech Company was used to construct the library. (switch mechanism at 5′ end of mRNA template). Randomly pick 5000 single clones for colony PCR identification. According to the gel map, the size of the inserted fragment and the rate of small fragments were roughly identified. A single amplified product of the selected band was sent to Huada Gene Company for sequencing, and a tomato SlML1 gene was obtained, the sequence of which was shown in SEQ ID NO.1, and the encoded protein was shown in SEQ ID NO.2. the

A research application of tomato SlML1 gene in improving tomato quality, biotic and abiotic stress resistance and extending shelf life: the process is as follows:

The function of SlML1 gene was studied by synchronous experiment of tomato wild-type line, SlML1 gene overexpression line and SlML1 gene RNAi line. the

The present invention realizes the quality research through the following technical scheme: the quality research is carried out by changing the contents of lycopene, soluble sugar, etc. in the fruits of tomato wild-type strains, S1ML1 gene overexpression strains and S1ML1 gene RNAi strains. the

The present invention realizes the research of resistance through the following technical scheme: Carry out the following experiments on the tomato S1ML1 gene in biotic and abiotic stress resistance by carrying out the following experiments on the tomato wild-type strain, the S1ML1 gene overexpression strain and the S1ML1 gene RNAi strain Research applications in:

(1). Drought resistance research

(2).Anti-fungal diseases

(3). Anti-bacterial disease

(4). Insect resistance test

(5). Anti-TMV experiment

The present invention also conducts a comparative study on the color, thickness, hardness and shelf life of tomato wild-type strains, S1ML1 gene overexpression strains and S1ML1 gene RN Ai strains. the

Compared with prior art, the present invention has the following advantages:

The present invention conducts functional research on the tomato SlML1 gene for the first time, and successfully constructs a transgenic system. Through gene function research, it was found that the SlML1 gene plays a positive regulatory role in tomato resistance to biotic and abiotic stress, and its overexpression can improve tomato quality and resistance, promote the formation of epidermal hairs, increase fruit thickness and firmness, and increase shelf life. , RNAi showed the opposite result. the

Description of drawings

Figure 1 is the prediction analysis of SlML1 functional domain (from NCBI database). the

Figure 2 is a phylogenetic tree of SlML1 gene. the

Figure 3 is a schematic diagram of the construction of an expression vector pBI121-SlML1. the

Fig. 4 is a schematic diagram of the result of transgenic tomato epidermis. the

a is the schematic diagram of the result of leaf epidermis, b is the schematic diagram of the result of stem epidermis. the

Fig. 5 is a schematic diagram of fruit color results of a transgenic tomato. the

Fig. 6 is a schematic diagram of the research results of the total carotenoid content, total soluble matter and titratable acid of a transgenic tomato. the

a is a schematic diagram of the results of the total carotenoid content, b is a schematic diagram of the results of the total soluble content, and c is a schematic diagram of the results of the titratable acid content. the

Fig. 7 is a schematic diagram of the research results of the total sugar content of a transgenic tomato. the

Fig. 8 is a schematic diagram of the research results of the concentrations of lycopene, β-carotene, phytoene and lutein in a transgenic tomato. the

Fig. 9 is a schematic diagram of the research results on the drought resistance of a transgenic tomato. the

a is the control group without drought stress treatment, b is the result of the experimental group with drought stress treatment. the

Fig. 10 is a schematic diagram of the results of a transgenic tomato against fungal diseases. the

a is a schematic diagram of the results of tomato infestation infestation, b is a schematic diagram of the results of tomato Botrytis cinerea infection. the

Fig. 11 is a schematic diagram of the results of transgenic tomato resistance to bacterial diseases. the

Figure 12 is a schematic diagram of the results of a transgenic tomato insect resistance test. the

a and b are schematic diagrams of tomato leaves inoculated with noctuid moth larvae, and c is a schematic diagram of electron microscope scanning whitefly results. the

Fig. 13 is a schematic diagram showing the results of a transgenic tomato anti-TMV experiment. the

Fig. 14 is a schematic diagram of the shelf life comparison results of a transgenic tomato. the

Detailed ways

Example 1:

Construction of tomato cDNA library

1. Isolation and detection of tomato total RNA

Take 2 g of fresh tomato (tomato S. pennellii) leaves, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer to 10 mL of extraction buffer (CTAB (W/V) 2%, Tris-HCl) preheated at 65 °C (pH 8.0) 100mmol·L ^-1 , EDTA25mmol·L ^-1 , NaCl2.0mol·L ^-1 , PVP402%, spermidine 0.5g/L, mercaptoethanol 2%), shake and mix well; Chloroform extracted twice, centrifuged at 7500g for 15 minutes. Add 1/4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, and use 500 μL SSTE (SDS0.5%, NaCl1mol·L ^-1 , Tris-HCl (pH8.0) 10mmol·L ^-1 , EDTA1mmol·L ^-1 , dissolved at 65°C for 5 minutes, extracted with an equal volume of chloroform, centrifuged at 13,000g for 5 minutes; added 2 times the volume of absolute ethanol to the supernatant, and placed at -70°C for 2 hours; 4 Centrifuge at 13000g for 20 minutes, dry the precipitate at room temperature for 10 minutes, dissolve in 100 μL of DEPC-treated water, use 1.0% agarose electrophoresis to detect the integrity of RNA, and use GenQuant nucleic acid quantifier to measure the ratio and concentration of A260 and A280. Store at -80℃ Refrigerator for spare.

2. Construction of cDNA library

After the mRNA was isolated using the mRNA Purification Kit (QuichprepTM Micro mRNA PurifiCation Kit, Pharmacia Company), the Creator Smart cDNA Library Construction Kit (Cat. No. 634903) of Clontech Company was used to construct the library. 'end of mRNA template). the

Example 2:

Cloning of related genes in tomato

Primers P1: 5'-ATGGGAAGATCACCAT-3', P2: 5'-T TAAAATACTGATGAACCATCAAC-3' were designed according to the tomato genome sequence, and the PCR reaction was carried out using tomato cDNA as a template. The reaction system was as follows: Taq buffer 2.5 μL, MgCl ₂ (25 mM) 1.8 μL, dNTP (2.5mM) 1 μL, P1 primer (10 pmol) 1 μL, P2 primer (10 pmol) 1 μL, Taq enzyme 0.4 μL. The PCR reaction conditions were 5 minutes of pre-denaturation at 94°C, 40 seconds at 94°C, 40 seconds at 50°C, 4 minutes at 72°C, 35 cycles of extension at 72°C for 10 minutes, and storage at 4°C. Take 7ul of PCR product and add 3ul of bromofinland for agarose gel electrophoresis, take a picture half an hour later, observe the gel map, select the amplified product with a single band and the correct position and send it to Huada Gene Company for sequencing, and the sequence alignment is correct. Tomato tomato SlML1 gene.

Example 3:

Bioinformatics analysis of SlML1 gene

The length of the obtained tomato SlML1 full-length cDNA is 1005bp, the sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 1-1005bp, and the sequence of the encoded protein is shown in SEQ ID NO.2. The full-length tomato cDNA sequence was searched for nucleotide homology in the Non-redundant GenBank+EMBL+ DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR databases using the BLAST program. Compared with R1R3-MYB of other species, the gene is highly conserved at the amino acid level and has typical MYB_DNA-bind and SANT domains. Figure 1. the

Example 4:

Construction of transgenic SlML1 strains

1. Construction of overexpression vector

Using tomato cDNA as a template, the PCR reaction was carried out using primers P1: 5'-BamHI-ATGGGAAGATCACCAT-3', P2: 5'-SmaI-TTAAAATACTGATGAACCATCAAC-3', and the reaction system was the same as in Experimental Example 2. Take 5ul of the amplified product and add 3ul of bromofinland for agarose gel electrophoresis. After half an hour, take a picture and observe the gel map. The amplified fragment is 1017bp. The PCR product was connected with pMD18-T Vector overnight at 16°C, transformed into Escherichia coli DH5α competent cells by electric shock, and recombinants were screened on LB plates containing ampicillin. After PCR and DNA sequence analysis, the recombinant plasmid pMD18-SlML1 with the correct target sequence was preserved. Then, the recombinant plasmid pMD18-SlML was double-digested with BamHI and SmaI at 37°C for 12 hours, and the digested product was purified using a recovery kit (Takara, China). At the same time, BamHI and Sma I were used to digest the plant expression vector pBI121Vector at 37°C for 2 hours, and 5ul of brominated Finnish was added for agarose gel electrophoresis, the gel pattern was observed, and the vector fragment was recovered using a recovery kit. the

The recovered SlML1 and pBI121Vector fragments were ligated at 16°C to obtain a recombinant plasmid. After identification by PCR and restriction endonuclease electrophoresis and DNA sequence analysis, the expression vector containing the correct sequence of SlML1 was named pBI121-SlML1 (Figure 3)

2. RNAi vector construction

Using tomato cDNA as a template, PCR was performed using primers P3: 5'-AAAAAGCAGGCTTAATGGGAAGA TCACCAT-3', P4: 5'-AGAAAGCTGGGTATTAAAATACTGATGAACCATCAAC-3', and the reaction system was the same as in Example 2. Use the recovery kit (Takara Company, China) to recover the target fragment, and pDONR20750ng, recover the product 50ng, BP Clonase0.67ul, add TE to 3.33ul. After 3 hours at 25°C, add 0.35ul proteinase K, and stop the reaction at 37°C for 10 minutes. Transform DH5α competent by electric shock, screen recombinants on LB plates containing gentamicin, and save recombinant plasmids with correct target sequences by PCR and DNA sequence analysis. Press Entry Clone 50ng, pHellsgate2, 50ng, LR enzyme 0.67ul, add TE to 3.33ul, incubate at 25°C for more than 3h, add 0.35ul proteinase K, stop the reaction at 37°C for 10min. Through BP reaction for transformation and identification, the expression vector containing the correct sequence of SlML1 was named pHellsgate-SlML1

3. Research on genetically modified materials

The expression vectors pBI121-SlML1 and pHellsgate-SlML1 were transformed into Agrobacterium C58 by electroporation, and tomato (tomato Solanum lycopersicum) calli were infected, and cultured for resistance, differentiation induction and rooting. At the same time, a blank experiment was carried out as a control, and positive transgenic plants were verified by PCR and RT-PCR. T0 and T1 generation transgenic tomato lines were self-crossed, and T2 seeds were germinated on the selection medium containing kanamycin (100 mg·L-1), and 13 different overexpression and 5 RNAi lines were obtained, Three lines with high expression and two completely silenced genes were used as research objects by transcription detection analysis. The results showed that the overexpression line (OE) had significantly more epidermal hairs than the wild type (WT) and RNAi lines (Figure 4). the

The transgenic tomato variety in the embodiment of the present invention is S.lycopersicum Ailsa Craig (AC) (Eliza beth A.Bray, Drought-and ABA-Induced Changes in Polypeptide and mRNA Accumulation in Tomato Leaves.Plant physiol, 1988,88:1210-1214 ), the wild type is S. pennellii (LA0716) (Tieman et al., Identification of loci affecting flavor volatile emissions in tomato fruits. Journal of Experimental Botany, 2006, 57:887-896). T0 and T1 transgenic tomato lines were self-crossed, T2 seeds were germinated on the selection medium containing kanamycin (100 mg·L-1), and the correct transgenic SlML1 gene overexpression lines and SlML1 gene RNAi transgenic lines were screened system as the research object. the

Embodiment 5:

Study on the Quality of Transgenic Tomatoes

1. Research on the content of total carotenoids

Take 5 g of mature fruit samples, add hexane-acetone (1:1, v/v) and quartz sand to grind, then centrifuge to get the supernatant, and repeatedly extract the centrifuged residue until the supernatant is clear. The supernatants were combined, added petroleum ether, washed with distilled water, and then concentrated using a rotary evaporator at a temperature lower than 35°C. the

According to the method mentioned by Delia B.Rodriguez-Amaya (Delia B.Rodriguez-Amaya.A guide to car otenoid analysis in foods.Washington:ILSI Press,2001), the absorbance at 470nm was measured by UV spectrophotometry and the total carotenoids were calculated element content, where the absorption coefficient A ^1% _1cm =3450.

2. Total sugar content and total soluble solids, acidity titration research

Eight mature fruits were taken from wild-type (WT), overexpression (OE) and RNAi lines respectively for the following research: use RFM81 refractometer to detect the total soluble solids in the homogenate juice; use 0.1N NaOH to titrate the homogenate juice To pH8.1 to detect the titrated acidity; according to the method mentioned by Han YS (Han YS. Food chemistry experiment guidance. Beijing: China Agricultural University Press, 1996.) to detect the total sugar content in the homogenized fruit juice. the

3. Research results

Observing the color of tomato at the mature stage, it was found that the fruit of the overexpression line was cherry-colored, while the color of RNAi was yellowish, and the wild fruit was red (Figure 5); the detection of carotenoid content showed that the carotenoid content in OE was 4 times that of WT , while RNAi decreased by 2 times (Figure 6a); total soluble solids detection showed that the content of OE was significantly higher than that of WT and RNAi (p<0.05) (Figure 6b); acidity titration showed that OE was significantly higher than that of WT and RNAi ( Fig. 6c); the sugar content of OE in mature tomato was higher than that of WT and RNAi, and the sugar concentration did not change between RNAi and WT (Fig. 7). the

In order to further study the change of carotenoid concentration, the concentration accumulation of lycopene, β-carotene, phytoene and lutein in tomato was detected, and it was found that the content of lycopene was 2 times that of WT, which was the effect of RNAi. 4 times; in addition, compared with WT, the contents of β-carotene, phytoene and lutein in OE were all increased, but decreased in RNAi (Fig. 8). the

The results showed that the overexpression of SlML1 could improve tomato quality, and the change of carotenoid content in transgenic tomato, especially the change of lycopene concentration was consistent with the change of fruit color. the

Embodiment 6:

Study on Stress Resistance of Transgenic Lines

1. Drought resistance research

Drought resistance experiments were carried out on wild-type (WT), overexpression (OE) and RNAi lines cultured for more than 1 month. After 10 days without water, it was found that WT and RNAi showed wilting, while OE showed normal growth; after another week without water, it was found that WT and RNAi showed wilting and dehydration, while OE did not show wilting and dehydration. After returning to normal watering for 15 days, it was found that OE returned to normal faster and better than WT, while RNAi could not. The water loss rate experiments were carried out on the leaves of the three strains, and it was found that OE lost water more slowly than WT, while RNAi lost water the fastest. The seedlings of the three strains were transferred to MS containing mannitol (Mannietol, 200 μM), and after 15 days of culture, it was found that the roots and aerial parts of the RNAi strains were more severely inhibited than WT, while OE had no effect (Figure 9) . The above experiments showed that SlML1 can reduce water loss under drought conditions, and SlML1 can regulate mannitol signaling, and high expression of SlML1 can increase tomato mannitol resistance. the

2. Anti-fungal diseases

Inoculate the detached leaves of WT, OE and RNAi with P. infestans and Botrytis cinerea respectively (bacterial liquid turbidimetric method, the concentration of diluted bacterial liquid is 9×10 ⁹ /ml), at 20°C and 100% humidity Illuminated for 16 hours a day, observed symptoms once every 2 days, lasted for a week, and repeated 3 times under the same conditions. The results showed that both RNAi and WT leaves had symptoms of late blight and gray mold, and the RNAi was more severe, while the OE group had slower infection of late blight and no symptoms of gray mold. Experiments show that SlML1 can significantly improve tomato resistance to Botrytis cinerea infection (Figure 10a), and also has better resistance to tomato late blight (Figure 10b).

3. Anti-bacterial disease

According to the method of Somodi GC et al. (Somodi GC, Jones JB, Scott JW, (1993) Comparison of inoculation techniques for screening tomato genotypes for bacterial wilt resistance. Kaoshiung, Taiwan: ACIAR Proceedings), culture WT, O E and Adding 5 mL of R. solanacearum H4A-1 (10 ⁷ cfu/mL) to the roots of RNAi strains, Zhang Saiqun et al., Research on systemic acquired resistance induced by non-pathogenic strains of R. solanacearum. Journal of Xiamen University, 2008 ,47:sup2), observe the wilting condition of the plants after inoculation. The leaves of all lines were completely withered after inoculation, WT and RNAi began to wither on the 7th and 4th day after inoculation, and completely withered after the 10th and 8th day, and the leaves of OE withered after 21 days, which was significantly slower than that of WT and RNAi. Among the three strains, RNAi was infected the fastest and most severely, followed by WT, and OE was the latest and least infected (Fig. 11). Experiments show that SlML1 can enhance tomato bacterial wilt disease resistance.

4. Insect resistance test

Put the same number (15) of armyworm Spodoptera exigua into the WT, OE and RNAi strains that have been cultured for one month, and observe the growth of the plants after five days of cultivation under the same conditions. At the same time, the fresh leaves of three strains with similar morphology in the same period were placed on the culture medium, and whiteflies were placed in the petri dish, and the position of the larvae and the condition of the leaves were observed after 24 hours. The leaves of three strains cultivated in the greenhouse for February were observed by scanning electron microscope, and the situation of whitefly on the leaves was detected. the

The experimental results showed that WT had better insect resistance than RNAi, and OE strains were not affected by noctuid larvae at all (Fig. 12a, 12b). Similarly, whitefly pupae were only present in the leaves of WT and RNAi (Fig. ), indicating that SlML1 can enhance tomato resistance to noctuid moth and whitefly. Moreover, the change of tomato's ability to resist herbivorous organisms may have a great relationship with the change of the epidermis of transgenic HlML1 plants. the

5. Anti-TMV experiment

TMV-U1 (Zhou Xueping, Tobacco mosaic virus gene introduction and regeneration of transformed tobacco plants. Yunnan Botanical Research, 1989,3: 247-263) of tobacco cultivar SR1 (Li Fu et al., Tobacco mosaic virus coat protein gene introduction Cloning and sequence analysis of the coat protein gene and the 3' non-coding region of the virus broad bean strain. Acta Virus Sinica, 1997, Vol, 13: No3) infected leaves, referring to the method of S.M.Paul Khurana et al., using 20mM sodium phosphate buffer ( pH7.0) and corundum to prepare TMV suspension, and inoculate the leaves of WT, OE and RNAi strains cultivated for one month, light 16h a day, 30°C during the day, 24°C at night, and observe the disease of the plants after 17 days of continuous greenhouse cultivation . The results showed that the symptoms of RNAi were significantly more significant than those of WT, while OE showed stronger resistance to TMV (Figure 13), indicating that SlML1 can enhance the resistance of tomato to TMV. the

Embodiment 7:

Study on shelf life of transgenic tomato

1. Yield research of transgenic tomato

The seedlings of each genotype cultivated for six weeks were transplanted into the greenhouse for cultivation, and 10 plants were randomly selected at the fruit maturity stage to record the average fruit weight per plant (g) and fruit yield per plant (kg). the

2. Research on fruit firmness of transgenic tomato

Fruit firmness was detected according to the method of Fan et al. (Fan X, Blankenship SM, Mattheis JP. (1999) 1-Methylcycloprop ene inhibits apple ripening. J Am Soc Hortic Sci). 30 fruit samples were collected for each genotype, and each fruit was measured four times to get the average value. the

3. Study on shelf life of transgenic tomato

Transgenic and WT plants were harvested at maturity, rinsed with a large amount of water containing bleach (2.5mL/L), then rinsed with tap water, then decontaminated and surface dried and trimmed to remove deformed and damaged fruits. Fruits were stored at room temperature (temperature 23-25°C, relative humidity 55-60%), and the degree of tomato deterioration was evaluated twice a week using a 5-point scale. The score for all tomatoes on the first day was 0, and the score for fruit on the shelf was 1.0. the

4. Research results

Tomato yield research showed that the average fruit weight per plant and fruit yield per plant of OE lines were higher than those of WT, while RNAi was lower (Table 1); fruit firmness tests showed that the firmness of WT, OE and RNAi were 6N, respectively. /mm, 9N/mm and 4.5N/mm were found at the same time, and then the pericarp thickness of the wild type, overexpression strain and RNAi strain was found to be 100.71, 221.42 and 53.57 microns by scanning electron microscopy. Shelf life measurements showed that RNAi deteriorated after 10 days, while WT did not appear until 20 days later, while OE did not show signs of deterioration, and kept the original texture and hardness until 60 days (Figure 14), which shows that HlML1 Expression can significantly increase fruit yield, firmness and shelf life. the

Table 1

Different letters indicate the significance of the difference, P<0.05 (n=10 strains). the

SEQUENCE LISTING

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Gly Cys Gly Ser Trp Arg Ala Leu Pro Thr Lys Ala Gly Leu Lys Arg

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Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp

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Ile Lys Arg Gly Lys Phe Ser Leu Gln Glu Glu Gln Thr Ile Ile Gln

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Leu His Ala Leu Leu Gly Asn Arg Trp Ser Ala Ile Ala Thr His Leu

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Ala Asn Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu

100 105 110

Lys Lys Arg Leu Thr Lys Met Gly Ile Asp Pro Asn Thr His Lys Pro

115 120 125

Lys Ser Asn Ile Phe Gly Ser Ala Asn Leu Ser His Met Ala Gln Trp

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Glu Lys Ala Arg Leu Glu Ala Glu Ala Arg Leu Val Arg Glu Ser Lys

145 150 155 160

Lys Gln His Gln Gln Ile Ile Ser Asn Asn Asn Asn Asn Ile Asn Asn

165 170 175

Tyr Asn Asn Ile His Phe Ser Pro Asn Asn Leu Thr Thr Thr Thr Thr Thr

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Thr Asn Val Leu Pro Pro Leu Gln Thr Lys Leu Pro Ser Pro Pro Cys

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Leu Asp Val Leu Lys Ala Trp Gln Gly Gly Ala Asn Trp Ser Ile Met

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Pro Lys Ile Thr Lys Asp Asn Phe Phe Asp Asn Pro Pro Ile Ser Thr

225 230 235 240

Ser Asn Leu Ser Leu Ile Met Val Pro Asn Asn Asn Ser Ile Thr Gly

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Ala Gly Leu Ile Asp Asn Ser Cys Leu Ile Gly Thr Glu Asn Phe Met

260 265 270

Glu Asn Asn Ile Asn Gly Ile Ser Tyr Ser Asn Tyr Pro Asn Leu Asn

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Thr Ile Gln Gly Phe Thr His Leu Asp His Val Leu Gly Ser Arg Glu

290 295 300

Glu Glu Asp Asp Asn Asp Asn Asn Asp Asn Thr Tyr Trp Asn Thr Ile

305 310 315 320

Leu Lys Ser Cys Thr Ser Phe Val Asp Gly Ser Ser Val Phe Tyr

325 330 335

Claims (12)

1. An isolated tomato S1ML1 gene, whose sequence is shown in SEQ ID NO.1. 2. The protein encoded by the gene of claim 1, whose sequence is shown in SEQ ID NO.2. 3. the application of the gene described in claim 1 or the protein described in claim 2 in improving tomato carotenoids; The carotenoid is β-carotene; phytoene; or lutein. 4. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato drought resistance. 5. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato resistance to mannitol. 6. the application of the gene described in claim 1 or the protein described in claim 2 in improving tomato antifungal; The fungus is Phytophthora infestans or Botrytis cinerea. 7. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato resistance to R. solanacearum. 8. the application of the gene described in claim 1 or the protein described in claim 2 in improving tomato insect resistance; The worm is noctuid or whitefly. 9. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato resistance to tobacco mosaic virus. 10. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato yield. 11. The application of the gene according to claim 1 or the protein according to claim 2 in improving tomato fruit firmness. 12. The application of the gene according to claim 1 or the protein according to claim 2 in prolonging the shelf life of tomato.