WO2021004098A1

WO2021004098A1 - Method for rapidly detecting plant disease-resistant genes

Info

Publication number: WO2021004098A1
Application number: PCT/CN2020/081955
Authority: WO
Inventors: 张颖; 刘锐涛; 刘崇怀; 樊秀彩; 姜建福
Original assignee: 中国农业科学院郑州果树研究所
Priority date: 2019-07-05
Filing date: 2020-03-29
Publication date: 2021-01-14
Also published as: CN110408648A

Abstract

Provided is a method for rapidly detecting plant disease-resistant genes, the method comprising the following steps: introducing disease-resistant genes into agrobacterium to obtain recombinant bacteria; infecting the back side of grape leaves with the recombinant bacteria, transforming the disease-resistant genes into the grape leaves, and inoculating the front side of the grape leaves with pathogenic bacteria to achieve the detection of the disease-resistant genes; introducing the disease-resistant genes into the agrobacterium through a recombinant vector, which is a vector obtained by cloning a target gene into the multiple cloning site of an expression vector. The method for rapidly detecting plant disease-resistant genes is suitable for the expression detection of rapidly and transiently transformed foreign genes of various transgenic plants.

Description

A method for rapid detection of plant disease resistance genes

Technical field

The invention belongs to the field of agricultural biotechnology, and specifically relates to a method for rapidly detecting plant disease resistance genes

Background technique

Grape (Vitis vinifera L.) is one of the most widely cultivated fruits in my country and has always been one of the most important fruits in the world. Its economic value is very high. It can not only be eaten directly, but also be processed into raisins, wine, etc. Currently the world's wine-growing area is very large, the cultivation area accounted for 10% of all types of fruit, at least, by the end of 2013, viticultural area of China has reached 714 600 hm ^2, grape yield 11.55 million tons.

Transferring foreign genes into plants is the main method for studying gene functions. Plant genetic transformation includes stable transformation and transient transformation. The stable transformation technology mediated by Agrobacterium has been applied to many crop species and is currently a common method for studying the function of genes in vivo. However, the transformation and regeneration efficiency of stable transformation technology is low, and it takes a long time. It will take a lot of time and energy to carry out functional research on multiple genes. Transient expression (transienent gene expression) The foreign DNA introduced into the cell by transient expression does not integrate with the chromosomal DNA of the host, but these DNA can generally be expressed within 12 hours after the vector enters the cell, and can be stably maintained for a certain period of time. Compared with the stable expression method, the transient expression method has many outstanding advantages such as simple operation, short cycle, reflecting the working environment in the plant, high expression efficiency, safety and efficiency.

The methods for realizing transient expression include injection infiltration method and infection method. Infection method can infect different parts of plant explants according to different requirements. For example: soak plant seedlings, seeds, inflorescences or callus in a bacterial liquid containing exogenous genes to realize the transformation of exogenous genes. At the same time, in order to shorten the expression time of exogenous genes, mechanical The external force caused certain mechanical damage to infect the damaged parts of the plant with the bacterial liquid. Therefore, whether it is the injection method or the transformation method through mechanical damage, it causes certain damage to the explants and is easy to cause infection. When performing disease resistance tests, it interferes with the results and cannot be applied to disease resistance genes. Functional testing; while the seedlings, seeds and other parts of soaked plants cannot guarantee the transfer and expression of foreign genes. At the same time, the seedlings and seeds need to be cultivated under light for about a week. At the same time, the light intensity is too high, the seedlings are easy to die, and the light intensity If it is too low, the growth conditions of seeds and seedlings will not be reached, and light affects gene expression and translation. There is a contradiction between the light demand of seedlings and seeds and the protection of gene expression from light. Therefore, it is difficult to infect seeds and seedlings through transformation methods. It is applied to the detection of plant exogenous gene expression.

Therefore, finding a method to quickly detect the expression of plant exogenous genes has become an urgent problem in this technical field.

Summary of the invention

The purpose of the present invention is to overcome the above shortcomings of the prior art and provide a method for rapid detection of plant disease resistance genes.

In order to achieve the above objectives, the technical solutions adopted by the present invention are:

A method for rapidly detecting plant disease resistance genes, comprising the following steps: introducing disease resistance genes into Agrobacterium to obtain recombinant bacteria; infecting the back of grape leaves with the recombinant bacteria, and transforming disease resistance genes into grape leaves, The front side of grape leaves is inoculated with pathogenic bacteria to realize the detection of disease resistance genes;

The disease resistance gene is introduced into Agrobacterium through a recombinant vector;

The recombinant vector is a vector obtained by cloning the target gene into the multiple cloning site of the expression vector.

In the above method, the disease resistance gene is VdWRKY70 gene.

In the above method, the method for infecting the back of grape leaves by the recombinant bacteria includes the following steps: floating the back of the grape leaves on the recombinant bacteria liquid and treating them in a vacuum, so that the cells on the back of the leaves are completely infiltrated by Agrobacterium Then, take it out and place it in an incubator for dark culture.

In the above method, the vacuum treatment parameters are: vacuum degree 20hPa (20mbar), 30°C treatment for 5 min.

In the above method, the culture condition is: a temperature of 24° C. dark culture for 48 hours.

In the above method, the method of inoculating the front side of the grape leaves with pathogenic bacteria includes the following steps: inoculating the front side of the leaves with pathogenic bacteria, and incubating in a dark at 28°C for 24 hours.

In the above method, the OD600 of the recombinant bacteria infecting the back of the grape leaves by the recombinant bacteria is 0.4.

The beneficial effects of the present invention: the method for rapidly detecting plant disease resistance genes of the present invention is suitable for the expression detection of transiently transformed exogenous genes in various plants. In this method, the expression of exogenous genes can be observed in 48 hours, and phenotypic changes can be observed in 72 hours, and the expression efficiency can reach 100%. At the same time, moderate vacuum can help Agrobacterium to carry exogenous genes to infect leaves from the stomata. Ensuring the integrity of leaf cells is conducive to gene expression and phenotype identification compared to other methods. Based on the characteristics of the method, the method has the advantages of simplicity, speed, high expression, and economy. The transiently transformed plant body obtained by this method can satisfy the application of plant in gene function analysis and recombinant protein, etc., laying a good foundation for further research on the molecular mechanism of grape disease resistance genes.

Description of the drawings

Figure 1 is a schematic diagram of the GUS gene expression comparison result of the present invention (A is the GUS gene expression result; B is the pBI101 negative control).

Figure 2 is a schematic diagram of the comparison results of the expression of disease resistance genes of the present invention (a is that the genetically modified grape leaves show disease-resistant symptoms, while the grape leaves without the genetically modified grape leaves are seriously affected, and the untreated leaves remain fresh; b is the western blot After detection, the expression of the GFP-labeled protein on the VdWRKY70 vector was higher, and no similar expressed protein was detected in the non-transgenic grape leaves).

Detailed ways

In order to show the technical solutions, objectives and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings.

Example 1

Step 1. Construction of plant expression vector carrying GUS gene

1.1 E. coli DH5α competent E. coli, Omega gel extract kit, LB medium, etc. were purchased from Henan Baosai Technology Company.

1.2 Use of plant expression vector of GUS gene

1.3 Preparation of Agrobacterium LBA4404 Competent: Scratch a plate on the LB solid medium containing 50mg/L rifampicin, pick the cells and shake them for 48h in the LB liquid medium containing 50mg/L rifampicin until the bacteria liquid becomes turbid , According to the ratio of 1:100 to inoculate 50ml fresh LB liquid medium containing 50mg/L rifampicin for 5h. Place the bacterial solution on ice in an ice bath for 30 minutes, 4°C, and centrifuge at 5000g for 5 minutes; discard the supernatant and add 1ml 0.1% calcium chloride suspension, ice bath for 5 minutes, 4°C, and centrifuge at 5000g for 5 minutes; discard the supernatant and add 800μL of 0.02% Calcium chloride was resuspended and divided into 8 tubes each with 100μL ice bath for later use.

1.6 Add 2μg of the plasmid to be transformed into the prepared Agrobacterium competence, mix well, quick-freeze in liquid nitrogen for 1min, 37℃ water bath for 5min, add 1ml LB liquid medium, 28℃, 200rpm, shake for 5h. After centrifugation and concentration, spread it on an LB plate containing 50 mg/L rifampicin and 50 mg/L kanamycin, culture at 28°C for 48 hours, and pick a single colony for verification.

1.7 Transform into Agrobacterium GV1301, Agrobacterium GV3101 strain contains PBI121 plasmid (containing 35S-GUS) and PBI101 (without 53S promoter, can not express GUS) vector, at 28 ℃, culture in 3ml LB containing card sodium antibiotics In the base, shake at a speed of 200 rpm overnight. The overnight culture was inoculated into 200ml LB medium and cultured overnight at 28°C. The Agrobacterium cells were centrifuged, the cells were collected, and resuspended in buffer (the buffer ratio was 10mM MgCl2, 10mM MES (pH 5.6), 100μM acetosyringone), and the concentration of the Agrobacterium solution was OD600nm (OD600) 0.4.

Step 2. Instant transformation of grape leaves

2.1 Store the bacteria at room temperature for 2-3 hours. Collect 24 days of tissue culture seedling'Beauty Finger' grape leaves, float the leaves on the Agrobacterium liquid with their backs facing down, and place them in a Concentrator plus at a vacuum degree of 20hPa (20mbar), processing at 30℃ After the next 5 minutes, the cells on the back are completely infiltrated with Agrobacterium, and then take it out.

2.2 After wiping clean, place it in a petri dish, moisten filter paper to moisturize, culture in the dark at 24°C for 48 hours, and perform GUS color detection after gene protein expression. The results are shown in Figure 1, compared to the control group (carried PBI101 Agrobacterium Infected leaves), grape leaves with GUS gene expression (leaves infected with Agrobacterium carrying the PBI121 vector) showed a dark blue color after GUS expression.

Example 2

Step 1. Construct a vector carrying VdWRKY70 disease resistance gene

1.1 The young leaves of grape grape 0943 were selected, E.coli DH5α competent E. coli, Omega gel extract kit, LB medium, etc. were purchased from Henan Baosai Technology Company; Bioteke plant RNA extraction kit was purchased from Beijing Biotech Company, PhusionTM ultra-fidelity enzyme was purchased from Zhengzhou Bomei Company, and pGEMT-easy vector kit, ExTaq enzyme, DL2000Marker, etc. were purchased from TaKaRa (Dalian) Company.

1.2 Extraction of total RNA using Bioteke plant RNA extraction kit. Use Thermo Scientific Nanodrop 1000 Micro UV Visible Spectrophotometer to measure the concentration and confirm the integrity of RNA by agarose gel electrophoresis. The first strand of cDNA was obtained using Fermentas' reverse transcription kit.

1.3 VdWRKY70 gene sequence Use primer find in Vector NTI 11 to design primers to clone mRNA sequence. The primers are shown in Table 1. PCR amplification uses NEB’s PhusionTM ultra-fidelity enzyme amplification, the reaction system is: HF buffer 10μL, 2.5mmol/L dNTPs 2.5μL, template 2μL, upstream and downstream primers (10mmol/L) each 2.5μL, Phusion ultra-fidelity Enzyme 0.5μL, double distilled water to make up to 50μL. The PCR reaction program is: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 58°C for 10s, extension at 72°C for 30s, a total of 31 cycles; finally extension at 72°C for 10min, and storage at 4°C. The PCR product was subjected to 2% agarose gel electrophoresis, and the amplified product was recovered using the Omega Gel extract recovery kit. The purified product was purified using TaKaRa EXTaqTM polymerase plus A. Reaction system: 10x buffer 2μL, 2.5mmol Mg ²⁺ 2μL , 2.5mmol/L dNTPs 2μL, template 14μL. Reaction procedure: react at 72°C for 10 min. Refer to the pGEMT-easy TM vector construction instructions to construct the VdWRKY49-T vector, transform E.coli DH5α Escherichia coli for blue and white spot screening, pick a single clone and send it to Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequencing primers are T7 and T7Terminal general primer .

Table 1: Gene cloning primers

1.4 The plant expression vector is pCAMBIA1303, the restriction sites are NCOⅠ and BstEⅡ, the enzyme is NEB's HF super-fidelity enzyme, and the restriction system refers to the instructions. PGW series vectors are digested with NCOⅠ, and PW series vectors are digested with NCOⅠ and BstEⅡ. After digestion, it was electrophoresed through 2% agarose gel, and recovered using Promega gel extract kit. Use the T vector of VdWRKY70 verified by sequencing as a template, add adapters respectively, and the adapter primers are shown in Table 2. PW70 carrier uses P70F and PW70R; PGW70 carrier uses P70F and PGW70R. Use NEB's PhusionTM ultra-fidelity enzyme PCR amplification, system reference materials and methods described in 1.3. Promega gel extract was used to recover the target fragment. According to NEB's Gibson

The instructions were used to construct the plant expression vector PGW70 and send it to workers for sequencing verification. Use Axygen Small Amount Plasmid Extraction Kit to extract plasmids for use.

Table 2: Vector construction of primer adapters

1.5 Competent preparation of Agrobacterium LBA4404: Scratch a plate on the LB solid medium containing 50mg/L rifampicin, pick out the cells and shake them for 48h in the LB liquid medium containing 50mg/L rifampicin until the bacterial liquid becomes turbid , According to the ratio of 1:100 to inoculate 50ml fresh LB liquid medium containing 50mg/L rifampicin for 5h. Place the bacterial solution on ice in an ice bath for 30 minutes, 4°C, and centrifuge at 5000g for 5 minutes; discard the supernatant and add 1ml 0.1% calcium chloride suspension, ice bath for 5 minutes, 4°C, and centrifuge at 5000g for 5 minutes; discard the supernatant and add 800μL of 0.02% Calcium chloride was resuspended and divided into 8 tubes each with 100μL ice bath for later use.

1.7 Transform into Agrobacterium GV1301, Agrobacterium GV3101 strain containing pCAMBIA1303 (control) and pCAMBIA1303-VdWRKY70 vector, at 28°C, in 3ml LB medium containing card sodium antibiotic, shake overnight at 200rpm. The overnight culture was inoculated into 200ml LB medium and cultured overnight at 28°C. The Agrobacterium cells were centrifuged, the cells were collected, and resuspended in buffer (the buffer ratio was 10mM MgCl2, 10mM MES (pH 5.6), 100μM acetosyringone), and the concentration of the Agrobacterium solution was OD600nm (OD600) 0.4.

Step 2. Instant transformation of grape leaves

2.2 After wiping clean, place it in a petri dish, moisten filter paper and incubate in the dark at 24°C for 48h. After the gene protein is expressed, the front of the leaf is inoculated (the pathogen of grape white rot). After 24 hours in the dark at 28°C, the result is as shown As shown in 2, compared to the control group (leaves infected with Agrobacterium carrying pCAMBIA1303), grape leaves with transgene expression (leaves infected with Agrobacterium carrying pCAMBIA1303-VdWRKY70 vector) showed a disease-resistant phenotype.

The results show that the detection method of the present invention can quickly detect the expression results of exogenous genes in plants in 68 hours.

Comparative example 1

Using the Agrobacterium containing foreign genes in Example 1 to transform seedlings, the specific steps are as follows:

(1) Take grape seedlings that have grown for about 7 days (before the true leaves appear), wash away the culture solution and other impurities on the seedlings with water, and lightly spray alcohol (75%, v/v) before putting it in Clean the workbench with 0.1% (w/v) HgCl ₂ for full washing, and then soak the seedlings in ddH2O for 5 minutes, shake slightly to wash away the residual HgCl ₂ , replace ddH ₂ O repeatedly, and wash 4 to 5 times Make sure to clean;

(2) Place the cleaned and disinfected seedlings in a hypertonic solution for hypertonic pretreatment for 3 minutes. The hypertonic solution is specifically: 1/2MS solution containing 165μM acetosyringone and 3% sucrose, pH=5.8, hypertonic solution The osmotic pressure is about 0.7～0.75kg/cm2 (it should be noted that too high osmotic pressure is easy to damage the seedlings, but too low can not effectively promote gene transformation).

(3) Put the seedlings after the medium-hypertonic treatment into the transient transformation solution at 25°C, 120rpm, provide suitable light, the light intensity is about 80μmol m-2s-1, cultivate for 5h, cultivate under too long culture time and without light. The seedlings are easy to die.

(4) Wash the transformed seedlings twice with ddH ₂ O, remove the Agrobacterium on the seedlings, and place them containing antibiotics (50 mg/L kanamycin and 50 mg/L rifampicin) and 0.8% ( w/v) Growth of agar powder on 1/2MS medium; culture conditions are: photoperiod 16h light/8h dark, light intensity 80-90 μmol m-2s-1, culture temperature: 26°C daily/20°C at night.

(5) After 7 days of cultivation, inoculate the pathogenic bacteria and cultivate them. The culture conditions are: photoperiod 16h light/8h dark, light intensity 80～90μmol m-2s-1, culture temperature: 26℃/night 20°C. The results showed that compared to the control group (seedlings infected with Agrobacterium carrying pCAMBIA1303), grape leaves with transgene expression (seedlings infected with Agrobacterium carrying pCAMBIA1303-VdWRKY70 vector) showed a disease-resistant phenotype.

The results show that the detection method of Comparative Example 1 takes 5 to 7 days to detect the foreign gene expression results of plants. At the same time, the seedlings need to be pretreated with hypertonicity, otherwise the effect of gene transformation will be low.

Comparative example 2

Using the Agrobacterium containing foreign genes in Example 1 to transform Arabidopsis thaliana inflorescence, the specific steps are as follows:

(1) Soak the inflorescence in the Agrobacterium bacterium solution for 1 min, and remove the excess bacterium solution on the stem with filter paper. Place it in the dark at 22°C for two days, pay attention to moisturizing, and transform again every 7 days. About 3 weeks after transformation, the seeds are collected when the fruit clips are mature.

(2) Place the dried seeds in a 2 ml EP tube, add 900 μL of 70% ethanol containing 0.2% Tween 20 and shake for 9 min. Discard the supernatant and add 90% ethanol to wash 3 times, and finally suspend it in 100% ethanol and pour it on sterilized filter paper to dry. Configure 1/2MS medium, add hygromycin B to a final concentration of 25mg/L before inverting the plate. The sterilized Arabidopsis seeds were evenly sprinkled on 1/2MS medium, and after 7 days, they would normally grow into transgenic Arabidopsis positive plants. The transgenic Arabidopsis thaliana with good growth condition was selected, and the white rot fungi were inoculated by acupuncture method, and their disease resistance phenotypes were observed after 7 days.

The results show that the detection method of Comparative Example 2 takes about 28 days to detect the foreign gene expression results of the plant.

Comparative example 3

The Agrobacterium containing exogenous genes in Example 1 was used to transform grape leaves by injection. The specific steps are as follows:

Take pCAMBIA1303-VdWRKY70 bacterial solution with OD600 of 0.4 to prepare injection bacterial solution;

Use a 1ml needleless syringe to inject the bacterial solution into the grape leaves. The whole process is extremely difficult, and water stains can only fill part of the leaves. After injection, place it in a petri dish, moisten filter paper to moisturize, and cultivate in the dark at 24°C for 48 hours. After the gene protein is expressed, the front of the leaf is inoculated (the pathogen of grape white rot) and placed in an incubator in the dark at 28°C for 24 hours. The results showed that the leaves in Comparative Example 1 failed to show gene expression and disease-resistant phenotypes. The reason may be that the leaves were mechanically damaged by the syringe, and some repairs were required to their own damage before expressing foreign genes, which prolonged The expression time of exogenous disease resistance genes. Therefore, Comparative Example 3 adopts the same inoculation and culture method as in Example 1, and it takes longer to express the foreign gene.

The above-mentioned embodiments only express several embodiments of the present invention, and the descriptions are more specific and detailed, but they should not be understood as limiting the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A method for rapidly detecting plant disease resistance genes, which is characterized in that it comprises the following steps: introducing the disease resistance gene into Agrobacterium to obtain recombinant bacteria; infecting the back of grape leaves with the recombinant bacteria to transform the disease resistance gene into In the grape leaves, the front of the grape leaves are inoculated with pathogens to realize the detection of disease resistance genes;

The disease resistance gene is introduced into Agrobacterium through a recombinant vector;

The recombinant vector is a vector obtained by cloning the target gene into the multiple cloning site of the expression vector.
The method for rapid detection of plant disease resistance genes according to claim 1, wherein the disease resistance gene is the VdWRKY70 gene.
The method for rapid detection of plant disease resistance genes according to claim 1, wherein the method for the recombinant bacteria to infect the back of grape leaves comprises the following steps: floating the back of the grape leaves on the recombinant bacteria liquid After the cells on the back of the leaves are completely infiltrated with Agrobacterium, they are placed in a vacuum and placed in an incubator for dark cultivation.
The method for rapid detection of plant disease resistance genes according to claim 3, wherein the vacuum treatment parameters are: vacuum degree 20hPa (20mbar), 30°C for 5 min.
The method for rapid detection of plant disease resistance genes according to claim 3, wherein the culture conditions are: a temperature of 24° C. dark culture for 48 hours.
The method for rapid detection of plant disease resistance genes according to claim 1, wherein the method for inoculating the front side of the grape leaves with pathogenic bacteria comprises the following steps: inoculating the front side of the leaves with pathogenic bacteria, and incubating in a dark at 28°C for 24 hours.
The method for rapid detection of plant disease resistance genes according to claim 1, wherein the OD600 of the recombinant bacteria infecting the back of grape leaves by the recombinant bacteria is 0.4.