CN115011630A - Application of tomato SlGID1L2 gene in regulation of tomato drought tolerance and cultivation of drought tolerant tomato - Google Patents

Abstract

The invention belongs to the field of cultivation of drought-tolerant tomato, in particular to the application of tomato SlGID1L2 gene in regulating the drought-tolerant tomato and cultivating the drought-tolerant tomato. The nucleotide sequence of the tomato SlGID1L2 gene is shown in SEQ ID NO: 1; the overexpression of the SlGID1L2 gene is regulated to improve the tomato drought tolerance. The present invention verifies the function of SlGID1L2 gene in drought response regulation for the first time, and plays an important role in the selection and breeding of drought-resistant tomato varieties.

Description

Application of tomato SlGID1L2 gene in regulating tomato drought tolerance and breeding drought tolerance tomato

technical field

The invention belongs to the field of cultivation of drought-tolerant tomato, in particular to the application of tomato SlGID1L2 gene in regulating the drought-tolerant tomato and cultivating the drought-tolerant tomato.

Background technique

Tomato (Solanum lycopersicum) is one of the most important vegetable crops worldwide. Drought is an important limiting factor for plant growth and development. Mild drought stress closes plant stomata, reduces _CO2 uptake, reduces photosynthesis, and changes cell wall elasticity. With the increase of drought intensity, it further leads to the production of toxic metabolites and aggravated osmotic stress, resulting in plant death. The water status of plants changes after drought stress, which affects the growth of plants, mainly including four aspects: nutrient absorption, photosynthesis, respiration and redox state.

When plants are subjected to drought stress, the water supply decreases, resulting in a decrease in the transpiration rate, which in turn reduces the utilization, uptake, transport and metabolism of nutrients by plants, and ultimately leads to a decrease in total nutrient absorption. Studies have shown that the reduction of photosynthesis in plants under drought stress is one of the main reasons for crop yield reduction. On the one hand, after plants are under drought stress, in order to prevent water loss caused by transpiration, the stomata are gradually closed, resulting in a decrease in _CO uptake and a subsequent decrease in net photosynthesis. On the other hand, non-stomatal factors limit plant photosynthesis after drought stress. When plants are subjected to drought stress, the content of energy substances ATP and ribulose diphosphate RuBP decreases. As the degree of drought stress increases, ribulose diphosphate carboxylase The carboxylation efficiency of RuBisCO/oxygenase is greatly reduced, which seriously affects plant photosynthesis. Respiration is an important metabolic process of plants, which consumes carbohydrates through respiration to generate CO ₂ and H ₂ O, which provide energy for plant growth and development. However, under drought stress conditions, alternate respiration is enhanced. Since the alternate respiration pathway does not require transmembrane transport of protons, only a small amount of ATP is produced, thereby affecting plant growth and metabolic processes. When plants are subjected to drought stress, the scavenging mechanism of reactive oxygen species is damaged, and reactive oxygen species accumulate in plants, resulting in toxic effects. Reactive oxygen species cause lipid peroxidation, leading to membrane damage, protein degradation and enzymatic inactivation, causing oxidative damage and affecting the normal function of cells.

Traditional breeding has extremely low success rate in drought tolerance research, high cost and long cycle. Since each generation in the breeding process needs to be identified for drought tolerance, special facilities or environmental conditions are required, and each generation cannot be identified by a single plant, but a population is required, so the cycle is longer than that of general traits. Conventional breeding cycles are at least twice as long.

Creating new drought-tolerant tomato materials and cultivating new drought-tolerant varieties are of great significance to ensuring stable and increasing tomato production.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of the tomato SlGID1L2 gene in regulating the drought tolerance of tomato, thereby improving the drought tolerance of tomato.

The second object of the present invention is to provide the application of tomato SlGID1L2 gene in breeding drought-tolerant tomato.

In order to achieve the above purpose, the technical scheme adopted in the present invention is:

Application of tomato SlGID1L2 gene in regulating tomato drought tolerance, the nucleotide sequence of tomato SlGID1L2 gene is shown in SEQ ID NO: 1; regulating the overexpression of SlGID1L2 gene to improve tomato drought tolerance.

The present invention verifies the function of SlGID1L2 gene in drought response regulation for the first time, and plays an important role in the selection and breeding of drought-resistant tomato varieties.

Preferably, the variety of tomato is Ailsa Craig.

The application of tomato SlGID1L2 gene in cultivating drought-tolerant tomato, the nucleotide sequence of tomato SlGID1L2 gene is shown in SEQ ID NO: 1; a transgenic tomato plant overexpressing tomato SlGID1L2 gene is constructed to improve tomato drought tolerance.

The experiments confirmed that the wilting degree of the control plants was aggravated in the SlGID1L2 gene overexpressing plants compared with the control plants. It indicated that SlGID1L2 gene was involved in the regulation of tomato drought resistance response and had the function of drought resistance regulation.

Preferably, the transgenic tomato plants are constructed by Agrobacterium-mediated methods.

Further preferably, the Agrobacterium-mediated method includes constructing a recombinant vector overexpressing the tomato SlGID1L2 gene, and using the recombinant vector to transform Agrobacterium, and the initial vector of the recombinant vector is pHellsgate8.

Preferably, the variety of tomato is Ailsa Craig.

Description of drawings

Fig. 1 is the tissue expression profile of SlGID1L2 gene;

Fig. 2 is the expression situation of SlGID1L2 gene after drought stress;

Fig. 3 is the expression level of SlGID1L2 gene in transgenic tomato plants;

Fig. 4 is the drought tolerance performance of SlGID1L2 gene overexpressing plants and control plants;

Figure 5 is a comparison of the number of tomato stomata between SlGID1L2 gene overexpressing plants and control plants;

Figure 6 is a comparison of the root development of SlGID1L2 gene overexpression plants and control plants.

Detailed ways

The implementation process of the present invention will be described in detail below with reference to specific embodiments.

Example 1 Application of tomato SlGID1L2 gene in regulating tomato drought tolerance

The nucleotide sequence of the tomato SlGID1L2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

1.1 Tissue expression profile of SlGID1L2 gene

Extraction of cultivar AC tomato root (RT), stem (ST), young leaf (YL), mature leaf (ML), flower (FL), young fruit (IM), green ripe fruit (GM), broken fruit (BR) , yellow ripe fruit (YR) and red ripe fruit (RR) RNA were reverse transcribed into cDNA, and the relative expression levels of tomato tissues were detected.

Green PCRMaster Mix 5μl,forward Primer 0.5μl Reverse Primer 0.5μl,cDNA 4μl。qRT-PCR反应程序为：预变性95℃5min；变性95℃20s；退火60℃30s，42次循环；融合曲线95℃10s，65℃1min；冷却40℃1s。Primer was designed with Primer 3.0 online software. Parameter setting: product length 80bp～200bp, GC content 40%～60%, Tm value 60℃～62℃, primer length 18bp～23bp. The qRT-PCR reaction was carried out according to the kit SYBR SelectMaster Mix (Applied Biosystems, Mardrid, CA, USA), and the instrument used was Bio-Rad Laboratories; the relative expression differences of the genes were analyzed by the SYBR Green I fluorescent dye method. The EF1a gene was used as the internal reference gene, and each sample was set up with 3 replicates, and the reaction system was

Green PCR Master Mix 5μl, forward Primer 0.5μl Reverse Primer 0.5μl, cDNA 4μl. The qRT-PCR reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20s; annealing at 60°C for 30s, 42 cycles; fusion curve at 95°C for 10s, 65°C for 1 min; cooling at 40°C for 1s.

qRT-PCR primers to amplify SlGID1L2:

Fw: 5'-GCCCTAGTCTTGGGATCAGC-3' (SEQ ID NO: 3);

Rev: 5'-ACCCATTGAAGAGCAGTCCA-3' (SEQ ID NO: 4).

qRT-PCR primers to amplify EF1a:

Fw: 5'-TGCTGTTCTCATTATTGACTCCAC-3' (SEQ ID NO: 5);

Rev: 5'-CTTCCTTCACGATTTCATCATACC-3' (SEQ ID NO: 6)

The results are shown in Figure 1. It can be seen from Figure 1 that the SlGID1L2 gene has the highest expression in roots, followed by flowers and fruits at the color-breaking stage, and the lowest expression in young fruits. It is speculated that SlGID1L2 affects root development.

1.2 Expression of SlGID1L2 gene after drought stress

After drought treatment of tomato cultivar M82 plants, the growing points were taken at 0h, 1d, 2d, 3d, 4d and 5d, respectively, and sent to Bio-Company for transcriptome sequencing (data from NCBI https://www.ncbi.nlm.nih. gov) and analysis.

The results are shown in Figure 2. It showed that the expression level of SlGID1L2 first increased and then decreased after drought stress.

1.3 Analysis of cis-elements of SlGID1L2 gene promoter

The cis-element analysis of the 2kb upstream ATG promoter sequence of the SlGID1L2 gene was performed on the PlantCARE website. The results are shown in Table 1. They are classified into four categories according to their functions: stress response, hormone response, light response, and tissue-specific expression. cis element. We speculate that the SlGID1L2 gene may be involved in the regulation of plant stress response.

Table 1 Analysis of cis-elements of SlGID1L2 gene promoter

Example 2 Application of tomato SlGID1L2 gene in breeding drought-tolerant tomato

The application of the tomato SlGID1L2 gene of the present embodiment in cultivating drought-tolerant tomato, the specific process is described as follows:

(1) Cloning of tomato SlGID1L2 gene

1.1 Materials

The tomato cultivar Ailsa Craig was used as the test material. The tomato seeds were sown in a light culture room for general cultivation management, and the mature leaves of the 4-week-old plants were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

1.2 Total RNA extraction and cDNA synthesis

1.2.1 Extraction of total RNA

RNA was extracted by TRIZOL method. Take 100 mg of the sample, grind it in liquid _N2 , add 1 ml of TRIZOL, invert and mix quickly, after a few minutes of extraction, add 200 μl of chloroform, fully extract for 15 s, place at room temperature for 3 min, centrifuge at 12,000 rpm at 2 °C ~ 8 °C for 10 min, suck up the Transfer 200 μl of the supernatant to a 1.5 ml clean RNAase-free centrifuge tube, add 400 μl of ice-cold isopropanol, invert and mix, place in a -20 °C refrigerator for 20 min, centrifuge at 12000 rpm at 2 °C ~ 8 °C for 10 min, carefully remove the supernatant, and get Colloidal RNA, add 1 ml of 75% ethanol, vortex and mix, after 10 minutes at room temperature, centrifuge at 5000rpm at 2℃～8℃ for 5 minutes, discard the supernatant, and let the colloid air dry (be careful not to be too dry, otherwise the RNA will be difficult to dissolve), Add 20 μl ~ 30 μl Diethylpyrocarbonate (DEPC) water to dissolve.

RNA quality was detected by 1% agarose electrophoresis. Soak the electrophoresis tank with 10% NaOH solution for 6-7 hours, the gel concentration is 1%, add an appropriate amount of bromine powder blue, load 1 μl, 130V voltage (the voltage should not be too low), and run the gel for about 10 minutes. Glue with a UV illuminator.

1.2.2 Synthesis of the first strand of cDNA

Using 1ug of tomato leaf RNA as a template, reverse transcription using HiFi-MMLV cDNA first-strand synthesis kit (Kang Wei Century). First, DNA was removed, and the system included 3 μl RNA, 1 μl 10×buffer, 1 μl DNAase I, and 1 μl DEPC water; 37°C for 30 min. Add 1 μl of EDTA and stop the enzyme reaction at 65°C for 10 min. Taking this as a template to carry out the next reaction, the system is shown in Table 2 below.

Table 2 Synthesis system of the first strand of cDNA

1.3 Cloning of tomato SlGID1L2 gene

Using the cDNA of tomato Ailsa Craig as a template, the coding sequence of the gene SlGID1L2 (gene ID: Solyc04g005230) was amplified, the cDNA sequence of the gene was downloaded from the SGN database (https://solgenomics.net/), and primers were designed using Primer 5.0. Forward primer: 5'-TCATTGTGAGCTAGGGTT-3' (SEQ ID NO: 7) Reverse primer: 5'-GACTCACTTGCTTTGGAT-3' (SEQ ID NO: 8) The designed primer is preceded by a homologous recombination linker, forward 5'-CATTTGGAGAGGACACGCTCGAG-3' (SEQ ID NO: 9) was added before the primer, and 5'-TCTCATTAAAGCAGGACTCTAGA-3' (SEQ ID NO: 10) was added before the reverse primer. KOD Plus high-fidelity DNA polymerase was amplified by PCR to obtain the full length of tomato SlGID1L2 gene.

PCR amplification reaction system: 1U/ul KOD Plus (TOYOBO) 0.5ul, 10X Buffer for KOD Plus 2.5ul, 2mM dNTPs 2.5ul, 25mM MgSO ₄ 1.5ul, 10uM forward and reverse primers each 0.75ul, template 50ng, sterilization Distilled water was made up to a total volume of 25ul. PCR amplification program: 94°C for 2min, 94°C for 15sec, 55°C for 30sec, 68°C for 1min, a total of 35 cycles, 68°C for 5min.

(2) Construction of overexpression vector and genetic transformation

2.1 Vector construction

The pHellsgate8 overload vector was subjected to double restriction enzyme digestion reaction with XbaI and XhoI restriction enzymes, and the restriction enzyme digestion vector was subjected to enzymatic ligation reaction with the target gene at 37°C for 30 minutes. The enzyme-linked product was added to the competent DH5α, and after being placed on ice for 30 min, it was heat-shocked on a metal bath at 42° C. for 45 s, and then transferred to ice for 3 min. Add 600 μL of LB culture without antibiotics to the centrifuge tube; rejuvenate on a shaker at 37°C for 45 min, and use a sterile spreader to evenly spread 100-200 μL of bacterial liquid on LB solid medium (LB+100mg/L Spec), After culturing for 12-18h in a 37°C incubator, white independent single colonies can be observed.

A single colony was picked and cultured in 500ul LB liquid medium (LB+100mg/L Spec) for 8 hours, and then PCR positive detection was performed. The positive monoclonal bacterial solution was sent to Shanghai Sangon Company for sequencing. The sequence alignment results were correct, and the vector was successfully constructed. The vector was named SlGID1L2-pHellsgate8, and the bacterial solution was stored for future use.

2.2 Agrobacterium transformation

The constructed overexpression vector SlGID1L2-pHellsgate8 was transformed into Agrobacterium. Clean the electroporation cup for later use, thaw 30ul of Agrobacterium competent cells on ice, add 1ul of the constructed recombinant plasmid SlGID1L2-pHellsgate8, flick and mix, and transfer to the bottom of the electroporation cup. Place the spinner cup on the stand of the spinner, click, quickly add 0.6ml of antibiotic-free LB liquid medium to the spinner cup, and gently pipette the suspended cells. Aspirate the cells into a 1.5ml sterilized centrifuge tube and culture at 28°C for 36-48h.

PCR identification and screening of positive bacteria, the primers used for PCR identification are:

Forward primer: 5'-ACGCACAATCCCACTATCCTTC-3' (SEQ ID NO: 11);

Reverse primer: 5'-GACTCACTTGCTTTGGAT-3' (SEQ ID NO: 8).

As a result, a positive Agrobacterium clone with a target fragment of about 1200 bp was obtained.

2.3 Genetic transformation of tomato

The constructed vector was transformed into tomato by Agrobacterium-mediated method. The seeds were sterilized and sown on 1/2 MS solid medium, and cultivated in a light culture room under the conditions of 16h light/8h dark, and a constant temperature of 26°C.

The positive Agrobacterium containing the target vector was inoculated into LB liquid medium (Kan: 100ug/mL; Rif: 50ug/mL) containing kanamycin and rifampicin, and shaken at 28°C overnight to OD600=1.5. 3000rpm Centrifuge at room temperature for 5 min and collect the cells. Bacteria were suspended in 0.2MS liquid medium. The 1-week-old tomato cotyledons were used as explants, infected for 2 min in the dark, the bacterial liquid was poured out, the residual bacterial liquid was blotted with filter paper, and the leaves were placed on the pre-medium for 2 days of dark culture and then transferred to the screening medium. After 10-20 days of culture, callus formation was induced. Transfer to redifferentiation medium to induce adventitious bud formation. When the adventitious bud grows more than 1 cm, it can be cut off and cultured in rooting medium. When the transgenic seedlings grow to an appropriate size, they are transplanted and colonized.

(3) Positive and expression detection of transgenic tomato plants

The target fragment of _SlGID1L2 gene was amplified by using DNA from young leaves of transgenic tomato plants as a template.

Green PCR Master Mix 5μl,forward Primer 0.5μl ReversePrimer 0.5μl,cDNA 4μl。qRT-PCR反应程序为：预变性95℃5min；变性95℃20s；退火60℃30s，42次循环；融合曲线95℃10s，65℃1min；冷却40℃1s。Twenty positive plants were identified, and the expression of SlGID1L2 gene in the transgenic lines was further detected. The qRT-PCR reaction was carried out according to the kit SYBR Select Master Mix (Applied Biosystems, Mardrid, CA, USA), and the instrument used was Bio-Rad Laboratories; the relative expression differences of genes were analyzed by SYBR Green I fluorescent dye method, and the selected The EF1a gene of tomato was used as an internal reference gene, and each sample was set up with 3 replicates, and the reaction system was

Green PCR Master Mix 5μl, forward Primer 0.5μl ReversePrimer 0.5μl, cDNA 4μl. The qRT-PCR reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20s; annealing at 60°C for 30s, 42 cycles; fusion curve at 95°C for 10s, 65°C for 1 min; cooling at 40°C for 1s.

qRT-PCR primers to amplify SlGID1L2:

Fw: 5'-GCCCTAGTCTTGGGATCAGC-3' (SEQ ID NO: 3);

Rev: 5'-ACCCATTGAAGAGCAGTCCA-3' (SEQ ID NO: 4).

qRT-PCR primers to amplify EF1a:

Fw: 5'-TGCTGTTCTCATTATTGACTCCAC-3' (SEQ ID NO: 5);

Rev: 5'-CTTCCTTCACGATTTCATCATACC-3' (SEQ ID NO: 6).

After the qRT-PCR reaction, data processing and analysis were performed.

The results are shown in Figure 3. The results showed that compared with the control, the expression of SlGID1L2 in transgenic lines OE1, OE2 and OE9 was significantly up-regulated, indicating that the SlGID1L2 overexpression system was successfully constructed.

(4) Drought tolerance of SlGID1L2 overexpressed plants improved

Drought stress treatment experiments were carried out on tomato SlGID1L2-OE gene overexpression lines and wild-type tomato Ailsa Craig. Planted in an artificial climate room at 25°C, 16h light/8h dark conditions, the seedlings were cultivated in light for 5 weeks, and 30 materials with the same growth were picked from each of the two materials and divided into two groups (control group and drought treatment group), one group (15 trees) The SlGID1L2-OE line and 15 Ailsa Craig) were watered normally as the control, and the other group was treated with drought. After one week, the phenotype was observed and photographed, and the water was rehydrated on the 11th day of the drought treatment. The results are shown in Figure 4. Show.

The experimental results showed that the wilting degree of the SlGID1L2 gene overexpressed plants was aggravated compared with the control plants. On the 6th day of the drought treatment, the control tomatoes started to wilt, while the transgenic plants remained firm; the transgenic plants showed leaf wilting on the 8th day of the drought treatment. After rehydration, the plants in the control group died and large areas of leaves were diseased and necrotic, while the transgenic plants recovered completely, with only a small area of leaves withered and dried up. This indicates that SlGID1L2 gene is involved in the regulation of tomato drought resistance response and has a drought resistance regulation function.

(5) Overexpression of SlGID1L2-OE and the number of stomata and root development in wild tomato

Water can be lost through the transpiration of the stomata, and the opening of the stomata also directly affects the loss of water. So we compared the stomatal density and stomatal diameter of the third leaf of transgenic and control tomatoes. SlGID1L2-OE overexpression and wild tomato (both corresponding to Figure 4 after rehydration) were observed by microscope, and the results are shown in Figure 5.

The results showed that the number of stomata per unit area of the transgenic line was about 49, and that of the control group was about 40.5, which indicated that under normal treatment, there was no significant difference in the number of stomata per unit area between the control and SlGID1L2-OE overexpressing tomato plants.

The root development of SlGID1L2-OE overexpression and wild tomato (both after rehydration in Figure 4) was counted, and the results are shown in Figure 6.

The results showed that under the root scanner we could observe that the root development of the three control plants was weaker than that of the transgenic plants. After analyzing the scanned data, we found that compared with the control, the total root length, total root volume, root tip number, and total surface area of the roots of SlGID1L2-OE overexpressed plants increased, that is, under drought stress, SlGID1L2 promoted tomato root growth. development.

The above results show that overexpression of SlGID1L2 gene can significantly improve the drought resistance of tomato, and can prepare drought-resistant tomato materials.

<110> Henan Agricultural University

<120> Application of tomato SlGID1L2 gene in regulation of tomato drought tolerance and cultivation of drought tolerant tomato

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 954

<212> DNA

<213> Tomato (Solanum lycopersicum)

<221> SlGID1L2 gene

<400> 1

atggctagtg aagacaatga aattgtttct gatttctatc cccattttcg aatctataaa 60

aatggacgcg ttgaacgttt ttatcattta caaaattgtt tttacgttcc accaacacat 120

gaaccagatt ctgacacagg tgtctcctct aaagatgttg caatcaactc tcatgtttca 180

gctaggctct ttttaccaaa tgttacaatt aacacaaata aaaaactccc aattattgta 240

ttttaccatg gtggagccct agtcttggga tcagcattct tcaacaaggt ttatcgtttc 300

cttaatcttc ttgtgtctga atcaaactca atagccgtag ctgttgatta caggttaacc 360

ccagaacatg atgtgtctac tgtgtatgaa gattgttgga ctgctcttca atgggttgct 420

agtagccaag attcatggct aacaagtcat ggtgattttg gtaaggtgtt tttgttggga 480

gaaagtgccg gagccaatat tgctttcaac atgataatca gagcagacag agaaaaatta 540

aatggtgatg tgaaaataaa tggtttaatt cttgcttgtc cttatttctt gatcccacat 600

gaaaatattg atgtggagaa tttgcttgct tacaaggcat ggagagagat tatttgtcca 660

aacttagaat ccccttttga ttgtccaatg attaatccac tttgtaaaac aagtcctaac 720

ttatcaaagc tagtatgttc aaaattattt gtgtgtttgg ctgagaaaga tgaattgatt 780

ccagtagaaa tgttgatgca atttgttgat agtgtgaaga aaagtggatg gaatggacag 840

tttgtgttac atgtggtgga aggagaaggt cattgttttt taattgacaa tcttgaaact 900

gagaaagcta gagatagcat taagagattt gcttctttca tccaaagcaa gtga 954

<210> 2

<211> 317

<212> PRT

<213> Tomato (Solanum lycopersicum)

<221> SlGID1L2 protein

<400> 2

Met Ala Ser Glu Asp Asn Glu Ile Val Ser Asp Phe Tyr Pro His Phe

1 5 10 15

Arg Ile Tyr Lys Asn Gly Arg Val Glu Arg Phe Tyr His Leu Gln Asn

20 25 30

Cys Phe Tyr Val Pro Pro Thr His Glu Pro Asp Ser Asp Thr Gly Val

35 40 45

Ser Ser Lys Asp Val Ala Ile Asn Ser His Val Ser Ala Arg Leu Phe

50 55 60

Leu Pro Asn Val Thr Ile Asn Thr Asn Lys Lys Leu Pro Ile Ile Val

65 70 75 80

Phe Tyr His Gly Gly Ala Leu Val Leu Gly Ser Ala Phe Phe Asn Lys

85 90 95

Val Tyr Arg Phe Leu Asn Leu Leu Val Ser Glu Ser Asn Ser Ile Ala

100 105 110

Val Ala Val Asp Tyr Arg Leu Thr Pro Glu His Asp Val Ser Thr Val

115 120 125

Tyr Glu Asp Cys Trp Thr Ala Leu Gln Trp Val Ala Ser Ser Gln Asp

130 135 140

Ser Trp Leu Thr Ser His Gly Asp Phe Gly Lys Val Phe Leu Leu Gly

145 150 155 160

Glu Ser Ala Gly Ala Asn Ile Ala Phe Asn Met Ile Ile Arg Ala Asp

165 170 175

Arg Glu Lys Leu Asn Gly Asp Val Lys Ile Asn Gly Leu Ile Leu Ala

180 185 190

Cys Pro Tyr Phe Leu Ile Pro His Glu Asn Ile Asp Val Glu Asn Leu

195 200 205

Leu Ala Tyr Lys Ala Trp Arg Glu Ile Ile Cys Pro Asn Leu Glu Ser

210 215 220

Pro Phe Asp Cys Pro Met Ile Asn Pro Leu Cys Lys Thr Ser Pro Asn

225 230 235 240

Leu Ser Lys Leu Val Cys Ser Lys Leu Phe Val Cys Leu Ala Glu Lys

245 250 255

Asp Glu Leu Ile Pro Val Glu Met Leu Met Gln Phe Val Asp Ser Val

260 265 270

Lys Lys Ser Gly Trp Asn Gly Gln Phe Val Leu His Val Val Glu Gly

275 280 285

Glu Gly His Cys Phe Leu Ile Asp Asn Leu Glu Thr Glu Lys Ala Arg

290 295 300

Asp Ser Ile Lys Arg Phe Ala Ser Phe Ile Gln Ser Lys

305 310 315

<210> 3

<211> 20

<212> DNA

<213> Artificial sequences

<221> qRT-PCR primer Fw for amplifying SlGID1L2

<400> 3

gccctagtct tgggatcagc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequences

<221> qRT-PCR primer Rev for amplifying SlGID1L2

<400> 4

acccattgaa gagcagtcca 20

<210> 5

<211> 24

<212> DNA

<213> Artificial sequences

<221> qRT-PCR primer Fw for amplifying EF1a

<400> 5

tgctgttctc attattgact ccac 24

<210> 6

<211> 24

<212> DNA

<213> Artificial sequences

<221> qRT-PCR primer Rev for amplifying EF1a

<400> 6

cttccttcac gatttcatca tacc 24

<210> 7

<211> 18

<212> DNA

<213> Artificial sequences

<221> SlGID1L2 gene cloning forward primer

<400> 7

tcattgtgag ctagggtt 18

<210> 8

<211> 18

<212> DNA

<213> Artificial sequences

<221> SlGID1L2 gene cloning reverse primer

<400> 8

gactcacttg ctttggat 18

<210> 9

<211> 23

<212> DNA

<213> Artificial sequences

<221> Forward primer homologous recombination linker

<400> 9

catttggaga ggacacgctc gag 23

<210> 10

<211> 23

<212> DNA

<213> Artificial sequences

<221> Reverse primer homologous recombination linker

<400> 10

tctcattaaa gcaggactct aga 23

<210> 11

<211> 22

<212> DNA

<213> Artificial sequences

<221> Positive Agrobacterium identification forward primer

<400> 11

acgcacaatc ccactatcct tc 22

Claims (6)

1. the application of tomato SlGID1L2 gene in regulating tomato drought tolerance, it is characterized in that, the nucleotide sequence of tomato SlGID1L2 gene is as shown in SEQ ID NO:1; Regulate the overexpression of SlGID1L2 gene, improve tomato drought tolerance. 2. The use according to claim 1, wherein the variety of tomato is Ailsa Craig. 3. the application of tomato SlGID1L2 gene in cultivating drought-tolerant tomato, it is characterized in that, the nucleotide sequence of tomato SlGID1L2 gene is as shown in SEQ ID NO:1; Construct the transgenic tomato plant of tomato SlGID1L2 gene overexpression, improve tomato drought tolerance sex. 4. The application of claim 3, wherein the transgenic tomato plant is constructed by an Agrobacterium-mediated method. 5. application as claimed in claim 4 is characterized in that, described Agrobacterium-mediated method comprises constructing the recombinant vector of tomato SlGID1L2 gene overexpression, utilizes described recombinant vector to transform Agrobacterium, and the initial vector of described recombinant vector is pHellsgate8. 6. The use according to any one of claims 3 to 5, wherein the variety of tomato is Ailsa Craig.

Images