CN110423753A - A kind of root knot specificity promoter T106-P and application by root-knot nematode induction - Google Patents

Abstract

The invention discloses a root-knot specific promoter T106-P induced by root-knot nematodes and its application. The root-knot-specific promoter T106-P induced by the root-knot nematode is composed of the nucleotide sequence shown in SEQ ID NO: 1. The primer pair includes a first primer and a second primer, the sequence of the first primer is shown in SEQ ID NO: 2, and the sequence of the second primer is shown in SEQ ID NO: 3. In the present invention, the underground parts and aerial parts of tomato inoculated with Elephant bean root-knot nematode for 21 days and the underground part of tomato without inoculation are used for transcriptome sequencing analysis, and it is screened that the root-knot nematode-inoculated specific expression and the difference in expression level are significant. The candidate gene T106 . The present invention successfully obtains a promoter T106-P that is specifically expressed in the root knot when the root knot nematode is infected, provides a suitable promoter for transgenic nematode breeding, and can be used for safe and efficient breeding of root knot resistance. Transgenic plant varieties of knot nematodes.

Description

A root-knot-specific promoter T106-P induced by root-knot nematodes and its application

technical field

The invention belongs to the technical field of plant genetics, in particular to a root-knot-specific promoter T106-P induced by root-knot nematodes and application thereof.

Background technique

Meloidogyne spp. is a kind of obligate sessile endoparasitic nematode that damages plant roots, with serious disease on lateral roots and fibrous roots, and induces root distortion of plant roots to form root knots. Root-knot nematodes are known as "the most destructive pathogen in the world" due to their strong adaptability, diverse transmission routes, wide host range and great harm. Southern root knot nematode ( M.incognita ) , northern root knot nematode ( M.hapla ) , Java root knot nematode ( M.Javanica ) and peanut root knot nematode ( M. arenaria ) are the representative species of the genus Root knot nematode , and they are also in China The common root-knot nematode species, in recent years, the occurrence of M.enterolobii has shown a gradual upward trend. It has a wide range of hosts and can overcome all the known resistance genes. It has been recognized internationally. Make it an important quarantine object.

Root-knot nematode disease mainly damages the roots of plants. In the early stage of root-knot nematode infection, the shoots are usually asymptomatic. When the damage is severe, the symptoms are dwarfing, yellowing, wilting, and weak growth. Root-knot nematode populations have strong reproductive capacity, are easy to spread, have multiple hosts, are stubborn and concealed, and are difficult to control and pose a serious threat to agricultural production. On the one hand, the damage caused by root-knot nematodes to crops is to absorb plant nutrients, secrete enzymes and toxins and other substances by invading the host plant tissues, and induce pathological changes in the host plants; Virus and other pathogens complex infection.

Root-knot nematodes can parasitize more than 3,000 kinds of plants, including more than 100 kinds of commercial crops, including herbs, woody plants, dicotyledonous plants and monocotyledonous plants, covering flowers, vegetables, economical, medicinal materials, fruit trees, grains, etc. Crops, distributed in tropical, subtropical and temperate regions, cause economic losses as high as $157 billion. In recent years, the incidence of root-knot nematode disease has been on the rise, and the yield has been reduced by 30%-50% after the occurrence, and even more than 70% in severe cases, even causing crop failure.

Due to the parasitic nature of root-knot nematodes, the control of root-knot nematode disease is extremely difficult. The traditional control concept is to apply chemical, physical and biological factors that are unfavorable to the nematodes in the soil, and act on the root-knot nematode eggs and nematodes exposed in the soil. The second instar larvae reduce the chance of the nematodes successfully invading the host, thereby achieving the purpose of disease control. At present, the main control methods are chemical control, biological control and agricultural control.

Chemical control has always been the main body of disease control due to its advantages of quick effect and strong pertinence. However, due to the negative effects of high toxicity, high residue, pathogen resistance, etc. Gradually ban or restrict use.

Biological control mainly uses the natural enemies and microbial resources of nematodes to control nematodes. It is a relatively environmentally friendly control method gradually developed in recent years. However, many studies are only carried out indoors, and there is no practical application in production practice.

Agricultural control includes planting disease-resistant varieties, rational crop rotation, planting predatory plants, and grafting. Among them, crop rotation is widely used for disease prevention. It is limited by the use of root-knot nematode hosts. However, crop rotation itself has certain limitations. It is not suitable for the control of root-knot nematodes with a wide host range.

Due to the obvious drawbacks of chemical, biological and agricultural control, the exploration of efficient and safe root-knot nematode control technology has become a joint effort of plant pathologists from all over the world. With the development of recombinant DNA technology, the establishment of exogenous DNA introduction system, the improvement of plant tissue culture technology, and the continuous deepening of research on nematode infection mode and plant self-defense defense system, the use of disease-resistant varieties is economical and safe. , high efficiency, environmental friendliness and other advantages, has become the first choice for research and development. Genetic engineering breeding has become the most concerned breeding method in recent years due to its advantages of short cycle, high efficiency, durable resistance, easy planting and cultivation, and broad-spectrum resistance. In the process of genetic engineering breeding, the promoter gene and the main nematode resistance gene are two necessary elements. Select the appropriate promoter gene and the main nematode resistance gene, so that the promoter can drive the nematode resistance gene to be expressed in plants according to the needs. , is the key to genetic engineering breeding.

Constitutive promoters can drive the high-efficiency expression of the target gene in all tissues of the organism, so that the expression level of the target gene is constant at a certain level, and it can be expressed in plants without being limited by time and space or induced by a certain substance. However, due to the characteristics of non-specific and continuous expression of exogenous genes driven by constitutive promoters in recipient plants, the burden on plants is increased, and in specific tissue parts or specific times where the gene needs to be expressed in large quantities, the expression level is too low. The expected effect is not achieved, and some deficiencies are gradually exposed in practical application.

Tissue-specific promoters are also called organ-specific promoters, which can regulate the efficient expression of exogenous genes in specific plant tissues and organs, and usually exhibit the characteristics of developmental regulation. Chemical physical signals are the basis of existence.

In the process of genetic engineering breeding, it is often expected that the inserted exogenous gene can be expressed in a specific plant tissue, improve the regional expression, avoid unnecessary waste of nutrients in the plant, thereby reducing the burden on the plant and reducing the impact on crop agronomic traits. In order to obtain useful traits, it is necessary to regulate the expression of target genes through effective tissue-specific promoters. Therefore, tissue-specific promoters have become the most promising promoter elements of exogenous genes in transgenic research, and have become a hot spot and frontier field of plant molecular biology research in recent years.

In terms of transgenic disease resistance breeding of root-knot nematodes, great progress has been made in the research on the mechanism of plant resistance to root-knot nematodes, such as using the structural resistance of plants to root-knot nematode disease, the plant's own disease resistance or defense-related genes, Root-knot nematode-inhibiting toxic protein genes, and dsRNA or siRNA structures capable of producing RNA interference with nematode pathogenic genes, although some transgenic plants that can significantly reduce root-knot nematode infection have been obtained, they have not yet formed a single transgenic plant. One of the important reasons for the commercial production of disease-resistant varieties is the lack of suitable gene promoters.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a root-knot-specific promoter T106-P induced by K. elegans; the second object is to provide a set of root-knot-specific promoters for amplifying the K. elegans-induced A primer pair for promoter T106-P; the third purpose is to provide a recombinant expression vector containing the root knot specific promoter T106-P induced by root knot nematodes; the fourth purpose is to provide an expression cassette; The fifth object is to provide the application of the root-knot specific promoter T106-P induced by root knot nematodes.

The first object of the present invention is achieved in that the root-knot-specific promoter T106-P induced by the root-knot nematode is constituted by the nucleotide sequence shown in SEQ ID NO: 1.

The second object of the present invention is achieved as follows: the primer pair includes a first primer and a second primer, the sequence of the first primer is shown in SEQ ID NO: 2, and the sequence of the second primer is shown in SEQ ID NO: 2 ID NO: 3.

The third object of the present invention is achieved in the following way. The recombinant expression vector is that the constructed recombinant plasmid T106-P-TA is digested with Hind III and Nco I double enzymes, and the T106-P promoter of the inserted fragment is recovered by glue. The pCambia1304 vector was double digested with Hind III and Nco I, the pCambia1304 vector fragment with the 35S promoter removed was recovered, and the inserted fragment T106-P promoter and the pCambia1304 vector fragment with the 35S promoter removed were connected to obtain the recombinant plasmid P1304-T106-P; In the recombinant expression vector, the root-knot-specific promoter T106-P induced by the root-knot nematode is connected to the upstream of the GUS gene.

The fourth object of the present invention is achieved by the expression cassette comprising the root knot specific promoter T106-P induced by the root knot nematode described in claim 1.

The fifth object of the present invention is achieved by the application of the root-knot-specific promoter T106-P induced by the root-knot nematode in obtaining a transgenic plant variety that is safe and highly resistant to the root-knot nematode.

Constitutive expression promoters are used more in transgenic plants in the past. They are the earliest and most widely used promoters in plant genetic engineering. They are characterized by continuous expression and basically constant expression. The expression efficiency of the source gene in the recipient plant is low, the expression product is unstable, and even gene inactivation or silencing occurs, which affects the normal growth and development of the plant, and even death, making the transgenic plant unable to be put into practical application. Selecting tissue-specific promoters to construct plant expression vectors to regulate the timing, localization and quantitative expression of exogenous genes in plants is an important strategy to solve the above problems.

In the interaction between root-knot nematodes and plants, the induction and maintenance of giant cells by root-knot nematodes is the key to its parasitic damage. Finding out the major plant genes that control the formation and maintenance of giant cells will help to deeply understand the pathogenicity of root-knot nematodes. Mechanism, at the same time cloning of the root-knot nematode-induced gene promoter specifically expressed at the root-knot, help to obtain high-efficiency root-knot nematode-resistant transgenic material.

In the present invention, the underground parts and aerial parts of tomato inoculated with Elephant bean root-knot nematode for 21 days and the underground part of tomato without inoculation are used for transcriptome sequencing analysis, and it is screened that the root-knot nematode-inoculated specific expression and the difference in expression level are significant. The candidate gene T106 .

The 1.5kb upstream fragment of T106 was intercepted, and bioinformatics software was used to predict the possible location of the transcription initiation site, and specific primers were designed to amplify the target region of the promoter to obtain a T106 gene promoter fragment of 2506bp.

The amplified predicted gene promoter fragment was used to replace the 35S promoter of pCambia1304 to construct a new plant expression vector P1304-106-P. Transform tomato to obtain transgenic-positive tomato seedlings with normal development.

The successfully screened transgenic tomato seedlings were transplanted into soil and inoculated with Elephant bean root-knot nematode and M. incognita 21 days after GUS gene staining to verify the activity of the T106-P promoter. It was found that the predicted promoter T106-P could promote GUS . Genes are specifically expressed at root knots. Fluorescence quantitative PCR test proved that compared with the 35S promoter, which can promote the stable expression of GUS gene in various parts of the plant, the relative expression level of GUS gene in the root knot of the T106-P promoter was significantly higher than that in the root system and the aerial part without root knot.

The experimental results show that the present invention successfully obtains a promoter T106-P that is specifically expressed in the root knot when the root knot nematode is infected, provides a suitable promoter for transgenic nematode breeding, and can be used for safe breeding. , Transgenic plant varieties with high resistance to root-knot nematodes.

The present invention provides a root knot specific promoter induced by root knot nematodes, the nucleotide sequence of which is the T106-P promoter sequence shown in SEQ ID No. 1.

Use specific primers to amplify the promoter fragment of gene T106 , the primer sequence is shown in SEQ ID No.2/SEQ IDNo.3, and the amplified T106-P promoter nucleotide sequence is shown in SEQ ID No.1 . The PCR product of the amplified T106-P promoter sequence was recovered by gel and used for the next step of TA cloning.

The TA cloning vector is pEASY-T1. After connecting the T106-P promoter gene fragment to the vector pEASY-T1, the recombinant plasmid T106-P-TA is obtained. T106-P-TA is double digested with Hind III and Nco I to verify The accuracy of the ligation of the promoter fragment is required for the construction of the plant expression vector in the next step.

The constructed recombinant plasmid T106-P-TA was digested with Hind III and Nco I, and the T106-P promoter of the inserted fragment was recovered by gelation. At the same time, the pCambia1304 vector was digested with Hind III and Nco I, and the pCambia1304 with the 35S promoter removed was recovered. The vector fragment was connected with the T106-P promoter of the inserted fragment and the pCambia1304 vector fragment from which the 35S promoter was removed to obtain the recombinant plasmid P1304-T106-P.

The constructed plant expression vector P1304-T106-P was verified by PCR to ensure that the T106-P promoter fragment was correctly connected to the front of the GUS gene, and the constructed plant expression vector P1304 -T106-P1304- T106- P was verified by double-enzyme digestion.

The successfully constructed P1304-T106-P plant expression vector was transformed into Agrobacterium strain EHA105 by electric shock method. Plate, and screen for resistance by YM medium plate (rifampicin 25 mg/L, kanamycin 50 mg/L).

The YM medium refers to weighing a quantitative dry powder of YM according to the instructions of the YM medium, dissolving in 1 L of deionized water, and sterilizing at 121 °C for 20 min. After sterilization, take it out and let it cool, and add 50 mg/L kanamycin and 50 mg/L rifampicin.

Pick a single colony and shake the bacteria at 28°C for 48 h until the OD value of the bacterial solution reaches 0.6-0.8.

Centrifuge and resuspend. When the OD value of the Agrobacterium liquid reaches between 0.6 and 0.8, take out the culture bottle, pour the bacterial liquid into a sterile 50 mL centrifuge tube in the ultra-clean bench, and centrifuge at 5000 r/min at 4 °C. 12 min. After centrifugation, take out the centrifuge tube, pour off the supernatant on the ultra-clean workbench, add the resuspended liquid medium, resuspend to OD value 0.3, add 100 μmol/L AS (acetosyringone) and mix well for use.

The resuspended liquid medium refers to adding 20 g/L D-glucose and 10 g/L sucrose to MS basal medium, and sterilizing at 117° C. for 20 min.

Use specific primers to screen the recombinant bacterial liquid by PCR. The primer sequences are shown in SEQ ID No.4/SEQ ID No.5. Pick 7 single colonies on the plate transformed with the P1304-T106-P vector, and the PCR verification is positive. There were 2 strains, and the positive strains were sent to a sequencing company for sequencing, and the recombinant strains with correct sequencing were stored at -80°C. A part of the streak plate was saved for co-cultivation with tomato callus in the next step to obtain transgenic tomato seedlings to verify the activity of the T106-P promoter.

Tomato seeds are sterilized using the sodium hypochlorite method.

Sterile tomato seeds were inoculated into seedling medium for cultivation.

The seedling growth medium refers to 1/2 MS+30 g/L sucrose+8 g/L agar, medium pH=5.8, and high temperature sterilization at 121°C for 20 min.

After the seeds have grown in the seedling medium for 10-15 days, cut the leaves of young sterile tomato seedlings into tissue blocks of about 2-5 mm × 2-5 mm, and place them on the induction medium at moderate intervals, about 5 mm in size. mm.

The induction medium refers to MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8 g/L agar, sterilized by high temperature at 121°C for 20 min.

Pick bright yellow dense callus every 10 days for subculture. After two generations, callus with good growth condition and bright yellow color is obtained, which can be used as a receptor for Agrobacterium tumefaciens infection and transformation.

The callus was taken and resuspended in Agrobacterium tumefaciens solution with AS (acetosyringone) added, 100 rpm, 20 ℃, soaked for 30 min. The callus material was air-dried in a petri dish covered with sterile filter paper in an ultra-clean workbench to complete the transfection of recombinant Agrobacterium.

The infected callus material was transferred to the co-culture medium and cultured at 18°C in the dark for 2-3 d.

The co-culture medium refers to MS+20 g/L sucrose+10 g/L glucose+8 g/L agar+100 μmol/L AS, sterilized by high temperature at 118°C for 20 min.

Transfer the callus to the screening medium, and the callus transformed with the plant expression vector with the resistance gene can grow on the medium containing the screening resistance, while the callus without the resistance gene can grow. Slowly brown and die.

The screening medium refers to MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8 g/L agar+600 mg/L cef+20 mg/L Hyg. Among them, IAA, Cef and Hyg should be added after high temperature sterilization at 121°C for 20 min.

New shoots will be differentiated on selection medium and transferred to rooting medium to induce rooting.

The rooting medium refers to 1/2MS+0.1 mg/L IBA+150 mg/L Cef, sterilized by high temperature at 121°C for 20 min.

PCR detection was performed on the obtained transgenic plants with the cross-sequence primers used to verify the plant expression vector. The primer sequences are shown in SEQ ID No.4/SEQ ID No.5 to verify that the T106-P promoter of the target gene was successfully transferred into the tomato genome.

The positive transgenic tomato plants that grew well in the rooting medium were transplanted into the cultivation soil to strengthen the seedlings.

The cultivation soil refers to the mixing of nutrient soil and sand in a ratio of 1:1, and high temperature sterilization at 121° C. for 20 min.

When the transgenic tomato plants were growing well, each pot of transgenic tomato seedlings was inoculated with 2,000 second-instar larvae of Elephant bean root-knot nematode and M. incognita. One month after inoculation, the transgenic tomato grew root and knot, and the tissue staining of GUS gene activity was carried out.

The results of GUS staining showed that the T106-P promoter was only stained blue in the root knots infected by Elephant bean root knot nematode and M. incognita, but not on tomato roots and shoot leaves that were not infected by root knot nematodes. None were dyed blue. 35S promoter transgenic tomato seedlings were stained blue at the root node and non-root node, that is, the entire root system after root-knot nematode infection.

The RNA of transgenic tomato plants was extracted, reverse transcribed into cDNA, and the relative expression of GUS gene was detected by real-time PCR.

The detection results showed that in T106-P promoter transgenic tomato plants, the relative expression level of GUS gene at root knots was significantly higher than that in roots and shoots without root knots. Basically no expression was detected in the root system at the root node. In the transgenic tomato plants with 35S promoter, there was no significant difference in the relative expression levels of GUS genes at root knots, roots without root knots and shoots, and the relative expression levels of GUS genes were basically the same.

The above results show that the T106-P promoter provided by the present invention and the 35S promoter have different expression conditions in various parts of plant tissues. Compared with the 35S constitutive promoter, the root knot specific promoter T106 induced by root knot nematodes -P is specifically and stably expressed at root nodes. This shows that the T106-P promoter provided by the present invention can not only be used for the disease-resistant breeding of Elephant bean root-knot nematode, but also can be applied to the disease-resistant breeding of the current dominant population of root-knot nematode M. incognita.

Description of drawings

Fig. 1 is the electrophoresis picture of T106-P promoter fragment;

Fig. 2 is the gel recovery electrophoresis diagram of T106-P promoter fragment;

Figure 3 is an electrophoresis image of T106-P-TA vector double-enzyme digestion;

Figure 4 is a structural diagram of the vector pCambia1304;

Figure 5 is a schematic diagram of constructing an expression vector P1304-T106-P;

Fig. 6 is the electrophoresis picture of plant expression vector P1304-T106-P PCR identification;

Fig. 7 is the electrophoresis picture of double enzyme digestion identification of plant expression vector P1304-T106-P and vector pCambia1304;

Fig. 8 is the electrophoresis picture of bacterial liquid PCR identification after plant expression vector P1304-T106-P is transformed into Agrobacterium;

Figure 9 is a transgenic tomato seedling;

Figure 10 is a PCR detection electropherogram of transgenic tomato plants;

Figure 11 is the inoculation of strong seedlings of transgenic tomato seedlings and Elephant bean root-knot nematode;

Figure 12 is GUS staining;

Figure 13 shows the relative expression of GUS gene detected by real-time PCR.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and the accompanying drawings, but the present invention is not limited in any way, and any transformation or replacement based on the teachings of the present invention belongs to the protection scope of the present invention.

The root-knot-specific promoter T106-P induced by the root-knot nematode is composed of the nucleotide sequence shown in SEQ ID NO: 1.

A set of primer pairs for amplifying root knot specific promoter T106-P induced by root knot nematodes according to the present invention, the primer pair includes a first primer and a second primer, and the first primer The sequence is shown in SEQ ID NO: 2, and the sequence of the second primer is shown in SEQ ID NO: 3.

The recombinant expression vector containing the root-knot-specific promoter T106-P induced by root knot nematodes described in the present invention is the constructed recombinant plasmid T106-P-TA is digested with Hind III and Nco I double enzymes, and the inserted fragment is recovered by glue T106-P promoter, digested pCambia1304 vector with Hind III and Nco I at the same time, recovered the pCambia1304 vector fragment with the 35S promoter removed, and connected the inserted fragment T106-P promoter and the pCambia1304 vector fragment with the 35S promoter removed to obtain a recombinant plasmid P1304-T106-P; in the recombinant expression vector, the root-knot-specific promoter T106-P induced by the root knot nematode is connected to the upstream of the GUS gene.

The expression cassette of the present invention comprises a root-knot-specific promoter T106-P induced by R. elegans.

The application of the root-knot-specific promoter T106-P induced by the root-knot nematode of the present invention is that the root-knot-specific promoter T106-P induced by the root-knot nematode is used in obtaining safe and efficient anti-root knot nematodes. application of transgenic plant varieties.

The present invention is further described below with specific embodiment:

biomaterials

Elephant ear and bean root-knot nematode and southern root-knot nematode are preserved in our laboratory.

The plant material tomato variety is Rutgers, which is preserved in this laboratory.

Main instruments and reagents

Instruments: PCR instrument, centrifuge, electrophoresis instrument, electric shock conversion instrument, constant temperature incubator, water bath, tissue culture bottle, petri dish, alcohol lamp, tweezers, scalpel, fluorescence quantitative PCR instrument (CFX96, BIO-RAD)

Reagents: Tris, concentrated HCl, EDTA-Na ₂ , NaCl, CTAB, mercaptoethanol, LA-Taq DNA polymerase, agarose, TAE buffer, complete gold gel recovery kit, pEASY-T1 Simple Cloning Kit (complete Gold), DH5α competent, LB medium (liquid/plate), Omega plasmid extraction kit, DNA A-Tailing KIT (TakaRa), pCAMBIA1304, restriction endonucleases EcoR I, Hind III and Nco I, T4 ligase , Agrobacterium EHA105, indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-Dichlorophenoxyacetic acid) , 2,4-D), zeatin (ZT), acetosyringone (Acetosyringone, AS), rifampin (Rifampin, Rif), kanamycin sulfate (Kanamycin Monosulfate, KM), ampicillin (Ampicillin) ), hygromycin (Hygromycin, Hyg) 10% sodium hypochlorite solution, alcohol (domestic analytical reagent), sucrose, glucose, MS medium, YM medium, GUS staining kit (coolaber), reverse transcription kit purchased from Dalian Bao Biological Company, SYBR dye and TRIZOL extraction box were purchased from Quanshijin Company.

Example 1

isolated promoter sequence

1. Plant DNA extraction

Extracted by CTAB method. The specific operation process is as follows:

Solution preparation: CTAB extract was prepared according to the following ratio: 100 mmol/L Tris-HCl (pH 8.0), 20 mmol/LEDTA-Na2, 1.4 mol/L NaCl, 2 % CTAB, and 0.1% (V/V) was added before use ) of β-mercaptoethanol. Among them, Tris-HCl and EDTA-Na2 need to be prepared into 1 M and 0.5 M solutions in advance, adjust the pH to the required value, and then sterilize together with other components to prepare CTAB solutions with corresponding concentrations.

1) Take out about 1.0 g of fresh tomato root tissue, put it in a clean mortar prepared in advance, quickly pour it into liquid nitrogen, quickly grind it into powder, add liquid nitrogen continuously in the middle, and transfer it to a 1.2 mL 65°C solution. Add 30 μL of β-mercaptoethanol to the centrifuge tube of the CTAB extract in a warm bath, mix well, and incubate at 65°C for 50 min.

2) Cool to room temperature and centrifuge at 12,000 rpm for 15 min at 4°C.

3) Take the supernatant into a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), vortex for 30s, 12000 rpm at room temperature, and centrifuge for 15 minutes.

4) Take the supernatant, add twice the volume of absolute ethanol, slowly turn it upside down, see the milky white flocculent precipitate, remove the supernatant after centrifugation, wash it with 70% and 100% ethanol, and air dry it naturally.

5) Add 600 μL ddH2O to dissolve the crude DNA precipitate, add 2 μL 10 mg/mL RNase, and incubate at 37°C for 1 h.

6) Take 500 μL of supernatant into a new centrifuge tube, add 350 μL of isopropanol, invert several times, place at room temperature for 10 min, and centrifuge at 13,000 rpm for 15 min at 4°C.

7) Take the supernatant, add 1/3 volume of 3M NaAc (pH 5.2) and 2 times volume of absolute ethanol, and place at -20°C for 2 h or overnight.

8) Centrifuge at 13,000 rpm for 10 min at 4°C. A milky white flocculent precipitate can be seen at the bottom of the tube. Carefully pour off the ethanol, gently suck off the excess liquid with a pipette tip, and dry.

9) Add 30-60 μL sterile ddH2O to dissolve the DNA pellet, and store at -20°C for later use.

2. Promoter sequence amplification and recovery

2.1 Primer design and determination of restriction enzyme sites

The plant expression vector pCambia1304 was selected to verify the predicted promoter activity, and the cloned promoter fragment was required to replace the 35S promoter of the vector pCambia1304. According to the map of the plasmid vector pCambia1304 (Figure 1) and the target sequence of the promoter, the T106-P restriction sites of the promoter were selected as HindIII and NcoI sites. Primers were designed using primer design software Primer Premier and Oligo7, and the promoter sequence T106-P was amplified after adding the restriction enzyme cleavage site sequence and protective base. The final designed amplification primers are as follows:

T106-P-F: 5'-CCCAAGCTTGAGCATGGAGCATGACGTATAACG-3' (SEQ ID No. 2)

T106-P-R: 5'-CATGCCATGGAACTTTGGAATCCTATGCCTTTG-3' (SEQ ID No. 3)

PCR reaction system:

PCR reaction program: 95°C, 3 min; 35 cycles of 94°C, 1 min, 60°C, 1 min, 72°C, 3 min; 72°C, 10 min.

The PCR product obtained after amplification was detected by 1.2% agarose gel electrophoresis. As shown in Figure 1, the electrophoresis of the T106-P promoter fragment was shown in Figure 1. The length of the T106-P promoter sequence was 2506 bp, which was used as the promoter of the downstream experiment. sequence.

2.2 Recovery of target fragments

Use the gel recovery kit to recover the target fragments. The specific operation process is as follows:

1) Use 1xTAE as a buffer to configure an agarose gel with a concentration of 0.8%, and perform agarose gel electrophoresis on the PCR stock solution to separate the target gene product.

2) In the UV gel cutter, cut the gel block where the target fragment is located, about 0.1 g, and put it into a clean and sterile 1.5 mL centrifuge tube.

3) Add 3 times the volume of GSB solution, that is, 300 mL, and melt the gel in a water bath at 55°C for 5-10 min until the gel block is completely melted. Add one volume of isopropanol, that is, 100 mL, into the melted gel solution. , which can increase the amount of DNA recovered.

4) When the gel solution to be melted drops to room temperature, add the solution to the spin column and let it stand for 1 min, centrifuge at 13,000 rpm for 1 min, and discard the effluent.

5) Add 650 μL of solution WB, centrifuge at 13,000 rpm for 1 min, and discard the effluent.

6) Repeat step 5) and centrifuge the empty tube for 2 min to completely remove the residual WB.

7) Put the spin column in a clean 1.5 mL centrifuge tube, open the lid and let it stand for 3 minutes, thoroughly dry the ethanol, add 30 μL of preheated deionized water to the center of the column, and let it stand for 3 minutes at room temperature.

8) Centrifuge at 13,000 rpm for 2 min to elute DNA and store at -20°C.

The results of gel recovery of T106-P promoter fragments are shown in Figure 2. The electropherogram of gel recovery of T106-P promoter fragments is shown in Figure 2. The size of the target fragment recovered by gel was 2506 bp, which was used as the next step of TA cloning.

3. Promoter sequence TA cloning

3.1 Connection of the target fragment to the vector

The specific operation process is as follows:

PCR recovery product 4.5 μL

pEASY-T1 vector (10 ng/μL) 0.5 μL

Solution I 5.0 μL

Mix gently and ligate at 25°C for 30 min.

3.2 Transformation and screening of ligation products

The specific operation process is as follows:

1) Take out the DH5α competent cells from the ultra-low temperature freezer and thaw them on ice. Gently add 10 μL of the ligation product to 100 μL of E. coli DH5α competent cells, mix gently, and place on ice for 30 min.

2) Heat shock at 42°C for 90 s, and immediately stand in an ice bath for 3 min.

3) Add 500 μL of pre-warmed LB liquid medium and incubate at 37°C with shaking at 220 r/min for 1 h.

4) Centrifuge at 3000 rpm for 1 min, aspirate 200 μL of supernatant, take 100 μL of the revived bacterial solution and spread it on the LB medium plate containing Amp (100 μg/mL), invert the plate, and incubate it in a constant temperature incubator at 37 °C for 12 -16h.

5) Pick a single white plaque on the ultra-clean workbench, put the plaque into the liquid LB medium containing antibiotic Amp, and shake the bacteria at 37°C and 190 rpm for 8-12 h.

3.3 Plasmid extraction

Use the full gold plasmid DNA mini-extraction kit to extract the plasmid in the E. coli bacterial liquid. The specific operation process is as follows:

1) Take the bacterial liquid cultured overnight, centrifuge at 13,000 rpm for 1 min, take out the supernatant, and collect it by centrifugation several times.

2) Add 250 μL of colorless solution RB (containing RNase A), and shake to suspend the bacterial pellet until the bacterial block is completely suspended.

3) Add 250 μL of blue solution LB, gently invert and mix for 4-6 times to fully lyse the cells, and the color changes from translucent to translucent blue, indicating that the bacterial solution is completely lysed, generally within 5 minutes.

4) Add 350 μL of yellow solution NB, and mix gently for 5-6 times until the color turns from blue to yellow completely, forming a yellow agglomerate, and let it stand at room temperature for 2 min.

5) Centrifuge at 13,000 rpm for 10 min, carefully pipette the supernatant into the spin column, centrifuge at 13,000 rpm for 1 min, and discard the effluent.

6) Add 650 μL of solution WB, centrifuge at 13,000 rpm for 1 min, and discard the effluent.

7) Repeat step 6, and centrifuge the empty spin column at 13,000 rpm for 2 min to remove residual WB.

8) Put the spin column in a clean centrifuge tube, dry it, add 30 μL of sterile deionized water to the center of the column, let it stand for 3 minutes, and centrifuge at 13,000 rpm for 2 minutes to elute the plasmid DNA.

3.4 Double enzyme digestion detection

The extracted plasmid T106-P-TA was detected by double restriction endonucleases HindIII and NcoI. The specific operation process is as follows:

Enzyme cleavage system:

TA plasmid 4 μL

10xK 2 μL

0.1% BSA 2 μL

HindⅢ/NcoⅠ 1 μL

Make up to 20 μL

After gentle mixing, overnight at 37°C, the double-digested fragments were recovered by 0.8% agarose gel. Figure 3 shows the results of double-enzyme digestion of T106-P-TA vector. The electrophoresis diagram of double-enzyme digestion of T106-P-TA vector verifies the accuracy of the ligated fragments and is used for the construction of plant expression vectors in the next step.

Example 2

Plant expression vector construction

Construction of plant expression vector The vector pCambia1304 is used as the backbone. The structure of the vector pCambia1304 is shown in Figure 4. The plant expression vector P1304-T106-P is constructed.

1. Enzyme digestion of pCambia1304 vector

The vector pCambia1304 was double digested with restriction enzymes HindIII and NcoI to obtain P1304 H/N. The enzyme digestion operation was carried out according to the enzyme digestion system and reaction conditions described in 3.4 in Example/1.

2. T4 enzyme ligation and transformation

2.1 Connection

Use T4 ligase to connect the target fragment and the double-enzyme cut linear vector. The connection system is as follows:

Promoter fragment 16.5 μL

P1304 H/N 4 μL

10×T4 ligase buffer 2.5 μL

T4 ligase 2 μL

2.2 Conversion

The total reaction volume was 25 μL, mixed gently, and ligated at 16°C overnight. The constructed plasmid was introduced into E. coli DH5α competent. The transformation operation was carried out as described in 3.2 in Example 1.

3. Identification of recombinant plasmids

3.1 PCR identification

Design primers for PCR identification of the constructed plant expression vector P1304-106-P, to check whether the promoter sequence correctly replaces the 35S promoter in the vector pCambia1304, and is correctly inserted in front of the GUS region of the reporter gene. Therefore, the designed front primer should be located on the target sequence of the promoter, and the rear primer should be located in the GUS region of the pCambia1304 vector. Primers were designed using the software Primer Premier and Oligo7. The final designed primer sequences are as follows:

P1304-106-P-F: 5'-TTTCTCAAGTAAAATCTACCCAA-3' (SEQ ID No. 4)

P1304-106-P-R: 5'-TTCTACAGGACGTAAACTAGCT-3' (SEQ ID No. 5)

PCR reaction system:

PCR reaction program: 95°C, 3 min; 35 cycles of 94°C, 1 min, 54°C, 1 min, 72°C, 1 min; 72°C, 10 min.

The PCR products obtained after amplification were detected by 1.2% agarose gel electrophoresis. The results of PCR identification of plant expression vector P1304-T106-P are shown in Figure 6. The electropherogram of plant expression vector P1304-T106-P PCR identification is shown in Figure 6. P1304-T106-P is verified to ensure that the promoter fragment is correctly connected to the upstream of the GUS gene .

3.2 Double enzyme digestion identification

The constructed binary vector P1304-106-P was identified by double restriction endonucleases HindIII and NcoI. The enzyme digestion operation was carried out according to the enzyme digestion system and reaction conditions described in 3.4 in Example 1. Figure 7 shows the results of double-enzyme digestion identification of plant expression vector P1304-T106-P and vector pCambia1304.

Example 3

Transformation of recombinant plasmids into EHA105 Agrobacterium

1. Preparation of electroporated EHA105 Agrobacterium

The specific operation process is as follows:

1) Take out the strain EHA105 from the -80°C refrigerator, and streak Agrobacterium EHA105 on a YM plate (containing Rif 25 mg/L).

2) Pick a single colony into 2 mL of YM liquid medium (containing 25 mg/L Rif), and activate it overnight at 28°C.

3) Take 1 mL of the activated bacterial solution and inoculate it into 50 mL of YM liquid medium, and cultivate it at 28°C and 200 rpm until the OD value is 0.4-0.5.

4) Transfer the bacterial solution to a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min at 4°C, discard the liquid, and suspend the Agrobacterium cells in 20 mL of sterilized and pre-cooled 0.1 M NaCl.

5) After 30 minutes on ice, centrifuge at 5000 rpm and 4°C for 10 minutes, discard the liquid, and collect the cells.

6) Add 2 mL of sterilized and pre-cooled 10 % glycerol, dispense 100 μL into clean sterile 1.5 mL centrifuge tubes, quickly freeze in liquid nitrogen, and store at -80°C.

2. Transformation of Agrobacterium EHA105

The correctly constructed recombinant plasmid was transformed into Agrobacterium EHA105 by electric shock method, and the specific operation process was as follows:

1) Soak the electric shock cup in 200 mL of 70% alcohol, then rinse it with sterile water for 3-5 times, dry it and place it on ice for later use.

2) Take out an EP tube containing 100 μL of competent Agrobacterium EHA105 cells from the liquid nitrogen tank and thaw in an ice bath.

3) Add 2 μL of recombinant plasmid, mix gently, and take an ice bath for 30 min.

4) Take out the mixed solution, transfer it into the pre-cooled electric shock cup, put the electric shock cup into the electric shock tank, shock with 2100V high voltage, press and hold the electric shock button on the device with your hand, when you hear a pop, it is an electric shock Finish.

5) Take out the electric shock cup, add 700 μL of pre-cooled YM liquid medium, gently pipette and mix, aspirate the bacterial liquid and transfer it to a clean and sterile 1.5 mL centrifuge tube.

6) 28 ℃, 200 rpm shaker for recovery for 2-4 h.

7) Centrifuge at 10,000 rpm for 1 min, collect the bacteria, aspirate 500 μL of the supernatant, resuspend the bacteria by pipetting with a pipette tip, and then take 100 μL of the bacteria solution and spread it on a solution containing Rif 25 mg/L and kana 50 mg/L. The YM plate was incubated upside down at 28°C.

3. PCR identification of plant expression vector into Agrobacterium

Colony PCR was used to detect whether the recombinant plasmid was successfully transferred into Agrobacterium, and the plasmid was extracted. Primers P1304-106-P-F (SEQ ID No. 4)/P1304-106-P-R (SEQ ID No. 5). The PCR reaction was carried out according to the PCR reaction system and reaction program described in Example 2 3.1. Figure 8 shows the results of PCR identification of bacterial liquid after plant expression vector P1304-T106-P was transformed into Agrobacterium.

Example 4

Plant Tissue Culture

1. Seed disinfection

First, take a sterilized 50 mL centrifuge tube, take tomato seeds into the centrifuge tube, add 75% ethanol, shake gently for 30 s, pour off the ethanol, add sterile water to rinse 1-2 times, and pour out Then add an appropriate amount of 5% sodium hypochlorite (NaClO) solution to sterile water and shake gently for 20 min. Then, rinse with sterile water 5-6 times until you can no longer smell the sodium hypochlorite. Finally, put the sterilized seeds on a petri dish covered with sterile filter paper, open the lid and blow dry in an ultra-clean workbench.

2. Tomato seedlings

The medium for culturing sterile tomato seedlings was named FM. The medium formula is: 1/2 MS+30 g/L sucrose+8 g/L agar, medium pH=5.8. Packed into tissue culture bottles, 30 mL per bottle, and placed in a sterilizing pot for 20 min at 121 °C. After the medium was solidified, each bottle was inoculated with 20 sterile tomato seeds, and cultivated in an artificial climate box at 25°C. Seedlings of tomato seeds are shown in Figure 9A.

3. Cultivation of callus

After the seeds have grown in the seedling medium for about 10-15 days, the leaves of the tomato seedlings are taken for cutting. Pour a small amount of sterile water into a glass petri dish covered with sterile filter paper, moisten the filter paper in the petri dish, take out the tomato seedling leaves in the tissue culture bottle with tweezers, put them on the filter paper, and cut the leaves into about 2-5 mm×2-5 mm in size, with incisions on 4 sides of tomato leaves. The cut leaves were connected to induction medium (YD), and the interval between tissue blocks was about 5 mm. The formula of callus-inducing medium was MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8 g/L agar. After the parafilm is sealed, mark the name of the variety, the name of the medium and the date, etc., cultivate in the tissue culture room, and replace the medium every 1 week. After culturing for 3-4 weeks, the callus can be used for subsequent genetic transformation operations. . Induction of tomato callus as shown in Figure 9B.

Example 5

Transfection and co-culture of recombinant Agrobacterium

1. Preparation of recombinant Agrobacterium

1.1 Preparation of the medium

According to the instructions of the YM medium, a quantitative amount of YM dry powder was weighed, dissolved in 1 L of deionized water, and poured into a 1 L bottle for sterilization at 121 °C for 20 min. After sterilization, take it out and let it cool, and add 50 mg/L kanamycin and 50 mg/L rifampicin. The resuspension was a liquid medium, supplemented with 20 g/L D-glucose and 10 g/L sucrose in MS basal medium, and sterilized at 117 °C for 20 min.

1.2 Shake bacteria

When the callus is well cultured, take a sterile tissue culture bottle, add about 50 mL of YM liquid medium, add 500-1000 μL of Agrobacterium stock solution to the liquid medium, and seal with a parafilm at 200 r/min, Incubate overnight at 28°C.

1.3 Centrifugal resuspension

When the OD value of the Agrobacterium solution reaches 0.6-0.8, take out the culture bottle, pour the bacterial solution into a sterile 50 mL centrifuge tube in the ultra-clean bench, and centrifuge at 5000 r/min at 4 °C for 12 min. After centrifugation, take out the centrifuge tube, pour off the supernatant on the ultra-clean workbench, add the resuspension solution, resuspend to an OD value of about 0.3, add 100 μmol/L AS (acetosyringone) and mix well for use.

2. Transfection

Take a sterile 100 mL wide-mouthed conical flask, put the callus into the conical flask on the ultra-clean workbench, and pour an appropriate amount of resuspended and AS-added bacterial solution. The Agrobacterium bacterial solution exceeds 3 mm of the callus. After sealing with parafilm, shake at 100 rpm and 20 °C for 30 min. Air dry the tissue material in a Petri dish lined with sterile filter paper on a clean bench.

3. Co-cultivation

The medium used for co-cultivation was named GP. The formula of GP medium is MS+20 g/L sucrose+10 g/L glucose+8g/L agar+100 μmol/L AS. Sterilize in an autoclave at 118°C for 20 min, in which AS needs to be added after sterilization. Before screening, spread a sterile filter paper on each co-cultivation medium, then attach the infected material to the medium, and cultivate in the dark at 18°C for 2-3 days.

4. Regeneration and Rooting

4.1 Screening

The screening medium was named SX. The screening medium formula was MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8 g/L agar+600 mg/L cef+ 20 mg/L Hyg. Among them, IAA, Cef and Hyg should be added after the medium is sterilized. The callus after co-cultivation was inoculated into the screening medium and cultured in a light incubator, and the screening medium was replaced once every 15 days, for a total of 2 times. Resistance screening is shown in Figure 9C.

4.2 Differentiation

Differentiation medium SR1 is: MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8 g/L agar+400 mg/Lcef. Subculture every 10 days until new shoots appear.

4.3 Rooting

When the shoots grow to 3-5 cm, transfer the shoots to rooting medium (ST). The rooting medium formula is: 1/2MS+0.1 mg/LIBA+150 mg/L Cef. After sterilization, IBA and cef were added, and they were packaged into tissue culture flasks, and cultured at 26°C under daily light for 16 h to induce rooting. Rooting was induced as shown in Figure 9D.

5. Molecular detection of transformed tomatoes

5.1 Transformed tomato DNA extraction

The DNA of transgenic tomato seedlings was extracted by CTAB method. The specific operation process was performed as described in 1 in Example 1 for DNA extraction.

5.2 PCR verification of transgenic tomato seedlings

To verify the transgenic tomato seedlings, primers P1304-106-P-F (SEQ ID No. 4)/P1304-106-P-R (SEQ ID No. 5) were used. The PCR reaction was carried out according to the PCR reaction system and reaction program described in Example 2 3.1. The PCR detection results of the transgenic tomato plants are shown in Figure 10. The PCR detection electrophoresis of the transgenic tomato plants shows that the promoter T1061-P target gene has been successfully transferred into the tomato genome. A total of 18 P1304-106-P transgenic tomato seedlings were obtained. Among them, 12 were positive seedlings, the positive rate was 66.7%; there were 30 transgenic seedlings obtained from P1304-35S-P, 22 were positive seedlings, and the positive rate was 73.3%. The positive rate of the transgenic tomato plants obtained from the T106-P plant binary expression vector and the 35S control group both exceeded 60%.

6. Transplantation of transgenic tomato seedlings

The well-developed and successfully transformed positive tomato seedlings in the rooting medium were transferred to the soil for cultivation. Rinse the medium adhered to the roots with clean water, prepare a 1:1 mixture of nutrient soil and sand, and transplant the transgenic tomato seedlings into the cultivating soil. Strong seedlings of transgenic tomato seedlings are shown in Figure 11A.

7. Transgenic tomato seedlings inoculated with root-knot nematodes

When the transgenic tomato plants to be transplanted into the cultivation soil are growing well, under the same conditions, 2000 second instar larvae of Elephant bean root knot nematode and M. incognita are respectively inoculated. As shown in Figure 11B, the transgenic tomato seedlings were inoculated with root knot nematodes for 1 month.

Experimental example 6

Tissue staining assay for GUS activity

1. Tissue staining

The transgenic tomato seedlings were inoculated with Elephant bean root-knot nematode for one month. After the tomato roots grew root knots, the tissue staining of GUS activity was performed. T106-P promoter and 35S promoter transgenic tomato root tissue and shoot tissue with root knots were taken and placed in a 1.5 mL centrifuge tube, stained with GUS staining solution, and kept at 37°C overnight.

The results of GUS staining showed that the T106-P promoter was stained blue in the root knots infected by Elephant bean root-knot nematode, as shown in Figure 12A, while the tomato roots and grounds that were not infected with Elephant bean root-knot nematode None of the leaves were stained blue as shown in Figure 12A B. The root knots infected by M. incognita were blue-stained as shown in Figure 12C, while the roots and shoots of tomato that were not infested by M. incognita were not stained blue, as shown in Figure 12C D . The 35S promoter transgenic tomato seedlings were stained blue at the root node and non-root node, that is, the entire root system after the infection of Elephant bean root-knot nematode, as shown in Figure 12C.

2. Fluorescence quantitative PCR detection of GUS gene

2.1 RNA extraction from transgenic plants

Plant tissue RNA was extracted using TransZol Up Plus RNA Kit. The specific operation process is as follows:

1) After weighing the ultra-low temperature frozen plant material, quickly transfer it to a mortar pre-cooled with liquid nitrogen, fully grind the plant material into powder with a pestle, and continuously replenish liquid nitrogen in the middle.

2) Transfer the ground powder to a centrifuge tube, add 1 mL of Transzol Up per 100 mg of sample, and mix well.

3) Let stand for 5 minutes at room temperature.

4) Add 0.2 mL of chloroform to each tube, shake vigorously for 30 s, and incubate at room temperature for 3 min.

5) Centrifuge at 12000 r at 4°C for 15 min, and collect the uppermost colorless supernatant (about 500 μL) into a new 1.5 mL centrifuge tube.

6) Add an equal volume of absolute ethanol and mix by gently inverting.

7) Add all the mixed liquid to the spin column, centrifuge at 14000 r for 30 s at room temperature, and discard the effluent (2-3 times).

8) Add 500 μL of CB9, centrifuge at 14,000 r for 30 s at room temperature, and discard the flow-through.

9) Repeat step 8.

10) Add 500 μL of WB9, centrifuge at 14,000 r for 30 s at room temperature, and discard the flow-through.

11) Repeat step 10.

12) Centrifuge at 14000 r for 2 min at room temperature, and let stand at room temperature for 10 min.

13) Take out the spin column and put it in an RNase-free Tube, add 30 μL of RNase-free Water to the center of the spin column, let it stand at room temperature for 1 min, and centrifuge at 14,000 r for 1 min at room temperature.

14) Detect quality on BioMateTM3S spectrophotometer (Thermo Fisher Scientific, USA), Agilent2100 (Agilent, USA) and Qubit2.0 (Thermo Fisher Scientific, USA) to ensure the quality and integrity of RNA, the OD standard reaches 1.8≤OD260/ 280≤2.2.

15) Store the RNA solution at -80°C for later use.

The part that formed root knots after inoculation of promoter T106-P transgenic positive plants was sample No. 1, the root system that did not form root knots was sample No. 2, and the aerial part was sample No. 3. Among the 35S transgenic positive plants, the root system with root knot formation was sample No. 4, the root system without root knot formation was sample No. 5, and the aerial part was sample No. 6.

2.2 cDNA synthesis

The PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) kit of Bao Bio (Dalian) Engineering Co., Ltd. was used for reverse transcription reaction, and the cDNA used for the detection was synthesized, and the amount of reversed total RNA was 1 μg. The specific operation process is as follows:

1) Prepare the following reaction system:

2) 42°C, 2 min (or 5 min at room temperature)

3) Store at 4°C

4) Prepare the following reaction system:

After mixing the reagents, place them in a PCR machine for the reaction. The reaction conditions are 37 °C, 15 min; 85 °C, 5 s; 4 °C, 1 min. After the reaction, the reverse transcribed cDNA was stored at -20°C for later use.

2.3 Fluorescence quantitative PCR detection

The primer sequences for fluorescence quantitative PCR detection of GUS gene are as follows:

GUS -F: ACACCGACATGTGGAGTGAA (SEQ ID No. 6)

GUS -R: TCATTGTTTGCCTCCCTGCT (SEQ ID No. 7)

Actin -F: TGTCCCTATTTACGAGGGTTATGC (SEQ ID No. 8)

Actin -R: AGTTAAATCACGACCAGCAAGAT (SEQ ID No. 9)

Fluorescence quantitative PCR reaction system

Fluorescence quantitative PCR reaction parameter settings

Amplification cycle parameters: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 20 s; annealing and extension at 59°C for 20 s; fluorescence signal collection at 65°C; the number of cycles was 44 times.

Dissolution curve parameters: the temperature was increased from 59 °C, and the fluorescence signal was collected for one cycle for each temperature increase of 0.5 °C, and a total of 80 cycles of fluorescence signals were collected. Three replicates were set for each sample, Ct values were recorded, and relative expression levels were calculated.

Fluorescence quantitative PCR data processing, using 2- ^△Ct method to calculate the expression of GUS gene. Relative expression level of GUS gene = GUS gene CT value - Actin CT value.

The results of real-time quantitative PCR are shown in Figure 13. The relative expression of GUS gene was detected by real-time quantitative PCR.

After T106-P promoter transgenic positive tomato plants were inoculated with Elephant bean root-knot nematode, the relative expression of GUS gene in the root knot of sample No. 1 was 0.0057, and the relative expression of GUS gene in the root system of non-root knot, that is, sample No. 2 was 0.0015, the relative expression of GUS gene in the shoots of T106-P transgenic positive tomato plants after inoculation with Elephant bean root-knot nematode, that is, sample No. 3 was 0.0007;

After 35S promoter transgenic positive tomato plants were inoculated with Elephant bean root-knot nematode, the relative expression of GUS gene in the root knot of sample No. 4 was 0.0052, and the relative expression of GUS gene in the root system of non-root knot, that is, sample No. 5 was 0.0048. The relative expression level of 35S promoter transgenic positive tomato plants after inoculation of Elephant bean root-knot nematode, that is, sample No. 6, was 0.0051.

From the results of fluorescence quantitative PCR detection of the relative expression of GUS gene, it can be seen that in T106-P promoter transgenic tomato plants, the relative expression of GUS gene in root knots is significantly higher than that in roots and shoots without root knots. Substantially no expression was detected in the shoots and root systems of T106-P transgenic tomato plants. In the transgenic tomato plants with 35S promoter, there was no significant difference in the relative expression levels of GUS genes at root knots, roots without root knots and shoots, and the relative expression levels of GUS genes were basically the same.

The above results show that the expression of T106-P promoter and 35S promoter in different parts of plant tissues is different. specific and stable expression.

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced. Without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Yunnan Agricultural University

<120> A root-knot-specific promoter T106-P induced by root-knot nematodes and its application

<130> 2019

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 2626

<212> DNA

<213> T106-P promoter sequence

<400> 1

tgttcttttt catcatatat ttgttcgtat tatatggtgt ttttctattt ttataattag 60

ttaaaaataa ttttttttaaa tcaagaatta tggtcctagt tttagtattt tattttatat 120

aaatttttttt ttatatctag gcatgtacac atgtgatctt ttatattttt cccaagatgc 180

aacccatctt tagttttaag tatgcattta taaatttaat ttttgtttca aattttattt 240

gacttttggg tagattttac ttgagaaaaa aatgaaaacc aaaaaagtag tcgctttaat 300

atcaaagtag tagttttatt gttgtttttc ttttttctaa aaggaaccaa attttttttta 360

aaatcatacc cgataacatg ttttttttttc tttttcgata acatgttttt aggtgattgt 420

agacattgaa aataactcat atttgattaa tgagatatct ttctttccta ttacttatat 480

aattctatta aatatataaa atttcactca tacgtttatg ttattcatat aaaattaaaa 540

agaaaaaaat tatcattttg aatttatttc ttttaaaaga cttatgctga ggatgaacaa 600

attatatatt tgaaacatgt gaaaagataa tattagcaac ttgttattgt tgttgactaa 660

caaatcttta taatattcgg ttaaaatctt ataatcacaa atattcattt ataaaagaaa 720

tataaaatta tgtgattcat aaatatgaca ttttaaacta ttttttatgtc ttgtttatga 780

actataattg attatttagt agtattatat aaattttacc agttattaca atttatggag 840

tttttttttta taaaaaaata tacaacttta aattttaaat tacttaaata aaatttcaac 900

tttatattat aatttaattt aacatcataa tttcatactg tctgtccaat gacatatcgt 960

gtttgacttg gttggggaca ttttgttgtt catcttgtat ttcaaacata cttagaacaa 1020

aactttgaac atctttgtaa gaaaacaaag tttaagaaca ttttcacaac tagaaaatta 1080

aaactagtag cgaaactaga atcagaaaca gatttaaaaa aaatatacga aatatttttc 1140

ttaaagaaga attgttatta atatgtgtct aaagaatctt actgattcca acaaactttg 1200

cagaaacacg aagttaccaa gtttaatgaa ccagtgaaga acaaaagaat agaagaactg 1260

aaattaattc acaaatctaa agtttgtaaa acacgtacca gaatttggaa aattttaaaa 1320

ggaaaaggat caagtccact gaattcacag tgtcccctta aggaaattat tcccctctag 1380

tatccgaggt ttgatttgga atatgacctc ccagggtaaa atgatctcaa tcaccagagt 1440

atagatacca aaaactccgg tgtcagcgag ccactcaacg gcaataaagt acacttagca 1500

tactagattt agtagttgaa taagaaatcc atgaattcta tttaaaatga gaggaaatcc 1560

ctcaatttat agaaaacaaa gaaaagtgcg aaaaggttct tattgtgcct taccggaaag 1620

gtcacaaacc tttggaaaag tcacaatgtt tcagaaaggt cgtcaccttt cataaaagtc 1680

acaacttacc ataaaagtca caacttttca taaaagtcac aacatttcat aaaagtcacg 1740

actcttcata aaagtcacaa catttcataa aagtcacaac tcttcataaa agtcttcata 1800

aaagtcgcaa ctatttattt tccattcaca cctttttaaa atccaacaat cccttgcatg 1860

aatgtggaat gactcgaaga caaagaaacg gacaagtatg tgtactttac aagcaagaac 1920

taattgcatc tggataagta ggtttctcct tggactttcc gtagtgaaca tatgttggat 1980

atactcgaaa aatcggtaga tgcgatattt ttgaaccgtc gaactttggt gtatacctag 2040

acaaccatat gtcacacaat taactcttta ccatttgtgg ttcttacggt tgtgttcgtt 2100

ttatcaatga acacctcctg gtttcatgag tgtatagaga gatggacttt tgacaatcat 2160

cttctttgaa gcggcttaca cttcacactc acataggtga tttctaaccg tgttatcgcg 2220

tagatatact atttggtcaa ctttgccaaa cttagcaaat cattaaaacc attaatcttt 2280

attaactcat taacaaacct taatgttgta tccttgtccc tgagcattgt cttcatcatg 2340

agaatggatt gagtttattg acaatgctga accgtcattc acaactttat ttttctcctt 2400

gaatctagct cttgggatct ccagtctgct agatagagtt atcgccatga tgacttgtcc 2460

taagccgtaa attcattctc ttggatgatc ttttaacttt ctctctagtt aggtcttttt 2520

gtaagtggat ccgacacatt atcctttaac tttacatagt caattctgat aattccacta 2580

gagagtagtt ttctaacagt atcatgtcta tgtcgtatat gacgag 2626

<210> 2

<211> 33

<212> DNA

<213> Primer T106-P-F

<400> 2

cccaagcttg agcatggagc atgacgtata acg 33

<210> 3

<211> 33

<212> DNA

<213> Primer T106-P-R

<400> 3

catgccatgg aactttggaa tcctatgcct ttg 33

<210> 4

<211> 23

<212> DNA

<213> Primer P1304-106-P-F

<400> 4

tttctcaagt aaaatctacc caa 23

<210> 5

<211> 22

<212> DNA

<213> Primer P1304-106-P-R

<400> 5

ttctacagga cgtaaactag ct 22

<210> 6

<211> 20

<212> DNA

<213> GUS-F

<400> 6

acaccgacat gtggagtgaa 20

<210> 7

<211> 20

<212> DNA

<213> GUS-R

<400> 7

tcattgtttg cctccctgct 20

<210> 8

<211> 24

<212> DNA

<213> Actin-F

<400> 8

tgtccctatt tacgagggtt atgc 24

<210> 9

<211> 23

<212> DNA

<213> Actin-R

<400> 9

agttaaatca cgaccagcaa gat 23

Claims (5)

1. A root-knot-specific promoter T106-P induced by root-knot nematode is characterized in that the root-knot-specific promoter T106-P induced by root-knot nematode is represented by SEQ ID NO: 1 Nucleotide sequence composition. 2. a group of primer pairs for amplifying the root knot specific promoter T106-P induced by root knot nematode according to claim 1, it is characterized in that described primer pair comprises the first primer and the second primer, The sequence of the first primer is shown in SEQ ID NO: 2, and the sequence of the second primer is shown in SEQ ID NO: 3. 3. a kind of recombinant expression vector containing the root knot specific promoter T106-P induced by root knot nematode according to claim 1, it is characterized in that described recombinant expression vector is the recombinant plasmid T106-P- TA was double digested with Hind III and Nco I, and the T106-P promoter of the inserted fragment was recovered by gelation. At the same time, the pCambia1304 vector was double digested with Hind III and Nco I, and the pCambia1304 vector fragment with the 35S promoter removed was recovered, and the inserted fragment T106-P was connected. Promoter and pCambia1304 vector fragment with 35S promoter removed to obtain recombinant plasmid P1304-T106-P; in the recombinant expression vector, the root-knot-specific promoter T106-P induced by root knot nematodes is connected to GUS upstream of the gene. 4. An expression cassette, characterized in that the expression cassette comprises the root knot specific promoter T106-P induced by the root knot nematode described in claim 1. 5. the application of the root knot specific promoter T106-P induced by the root knot nematode of claim 1, it is characterized in that the root knot specific promoter T106-P induced by the root knot nematode is in Use in obtaining safe and highly effective root-knot nematode resistant transgenic plant varieties.