CN103320464B - Method for generating hairy roots by efficiently inducing trifoliate orange by agrobacterium rhizogenes and application of method - Google Patents

Abstract

The invention discloses a method for efficiently inducing hairy roots in trifoliate oranges by using Agrobacterium rhizogenes and its application. The method is easy to implement and easy to operate. The frequency of co-transformation hairy roots is high, and the isolated hairy roots can be grown on MT basic medium. Self-growth, easy control of growth conditions, and convenient experimental observation. Because the hairy root system contains DR5:YFP:GUS reporter gene, and the DR5 promoter is induced by auxin, it can be visually observed according to the results of YFP fluorescence and GUS staining. The distribution and expression pattern of endogenous auxin during the growth and development of trifoliate roots; it overcomes the problems existing in the conventional methods of studying the root development of trifoliate oranges, such as the roots growing in the soil, inconvenient to observe and obtain materials, and difficult to control the growth environment.

Description

A method and application of using Agrobacterium rhizogenes to efficiently induce trifoliate oranges to produce hairy roots

technical field

The invention belongs to the field of biotechnology, and specifically relates to a method for efficiently inducing hairy roots in trifoliate oranges by using Agrobacterium rhizogenes, and also relates to the application of the hairy roots prepared by the method in the research on the development of the root system of trifoliate oranges.

Background technique

Citrus is one of the most important fruit tree crops in the world. At present, the research on citrus is mostly focused on the aboveground organs, such as flower bud differentiation, fruit quality formation mechanism, etc., but there are very few reports on the underground root system. The root system is an important organ for fruit trees to absorb water and nutrients, and plays a vital role in the growth and development of the aboveground parts of fruit trees. Because the root system grows in the soil, it is extremely inconvenient to observe and obtain materials, and the isolated root organs cannot be cultured and subcultured on the medium, which is the main reason why the research on citrus root system is relatively backward.

Agrobacterium tumefaciens mediated genetic transformation has been widely used in the genetic improvement research of citrus (G.A.Moore et al., 1992.Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants. 1Plant Cell Rep, 11:238 -242); however, the genetic transformation system has problems such as transformation mosaicism and low transformation rate, which increases the workload of transformation operations; in addition, the cycle of fruit tree transformation and regeneration is long, which also limits the research related to gene function identification. bring inconvenience.

Agrobacterium rhizogenes and Agrobacterium tumefaciens are Gram-negative bacteria. Agrobacterium rhizogenes can infect plant explants with wounds, and the T-DNA on the Ri plasmid contained in it can be integrated into the recipient nucleus genome; because the Ri plasmid T-DNA contains rolA, rolB, Genes such as rolC and rolD also contain genes related to auxin synthesis, which can promote the recipient tissue cells to spontaneously produce hairy roots, and the isolated hairy roots can be subcultured in the medium without adding hormones. The hairy roots induced by Agrobacterium rhizogenes are usually derived from single cells, and there is no chimera; at the same time, studies have shown that the Ri plasmid of Agrobacterium rhizogenes can drive the transfer of T-DNA on the exogenous binary expression vector, thereby interacting with The T-DNA of the Ri plasmid itself was transformed into recipient cells together to obtain co-transformed hairy roots (Wu Jiao et al., 2012. High-efficiency regeneration of Agrobacterium rhizogenes-induced hairy root in apple rootstock Malus baccata (L.) Borkh. Plant Cell Tiss Organ Cult, 111:183-189.).

The DR5 promoter is a synthetic auxin-induced expression promoter. After recombining the DR5 promoter with a reporter gene such as GUS or GFP, it can be used as a visual marker for the concentration and distribution of auxin in plants. It has been successfully applied to poplar In the study of endogenous auxin response and distribution (Chen Yiru et al., 2013. DR5 as a reporter system to stdudy auxin response in Poulus. Plant Cell Rep, 32:453-463).

Hovenia trifoliate is a commonly used rootstock in citrus cultivation, but there are few reports on the root development of Hovenia trifoliate. The object of the present invention is to utilize the Agrobacterium rhizogenes containing DR5::YFP:GUS on the binary carrier to establish a high-efficiency induction and in vitro culture system for trichotrium trifoliate roots, and based on this to carry out root growth in the trifoliate root system and the study of auxin response patterns during development.

Contents of the invention

Aiming at the chimera phenomenon and low transformation rate in the transformation of Agrobacterium tumefaciens, and the problems existing in the research of citrus roots, the purpose of the present invention is to provide a method for efficiently inducing hairy roots in trifoliate oranges by Agrobacterium rhizogenes. The method, the method is easy to operate, the co-transformation hairy root induction frequency is high, the isolated hairy root can grow independently on the MT basic medium, the growth conditions are easy to control, and the experimental observation is convenient.

Another object carrier of the present invention provides a kind of application in root development research of the hairy root induced by A. The promoter is induced by auxin, so the distribution and expression pattern of endogenous auxin during the growth and development of hairy roots can be visually observed according to the results of YFP fluorescence and GUS staining; it overcomes the problems existing in conventional methods for the study of trifoliate root development, such as The root system grows in the soil, it is inconvenient to observe and obtain materials, and it is difficult to control the growth environment.

A method utilizing Agrobacterium rhizogenes to efficiently induce trifoliate oranges to produce hairy roots, the steps of which are as follows:

(1) Peel the seeds of Hovenia citrus fruit, soak in 1M NaOH for 10 minutes to remove pectin, rinse with distilled water for 3-5 times, then disinfect with 3% sodium hypochlorite for 20 minutes, rinse with sterilized distilled water for 3 times, remove the inner and outer seed coats and sow on MT seeding medium, 24 Cultivate in the dark at -26°C for 30-45 days to obtain aseptic seedlings, take them out under light intensity of 1500-2000lx for about 5-7 days, select robust aseptic seedlings after turning green, cut epicotyls into sections of about 1.5cm, and wait for use;

(2) DR5 consists of seven 11bp (5'-CCTCGTGTCTC-3') tandem repeats (Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements . Plant Cell, 1997, 9(11):1963–1971). Endonucleases XhoⅠ and NotⅠ digested SlIAA14::GUS+YFP vector (Li Xiaojing, Chen Yinhua, Zhang Junhong, Ye Zhibiao, Cloning and analysis of tomato SlIAA14 gene promoter, Acta Horticultural Science, 2010, 37 (10): 1605-1612) and recovered, The DR5 sequence was ligated into the recovery vector with T4 DNA ligase, and the DR5::YFP:GUS binary expression vector was constructed. The binary expression vector plasmid containing DR5::YFP:GUS was introduced into Agrobacterium rhizogenes MSU440 by electroporation (Limpens E et al., RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.J Exp Bot.2004May;55( 399):983-92.), to obtain Agrobacterium rhizogenes containing binary vectors;

(3) Pick a single colony of activated Agrobacterium rhizogenes, streak on LB solid medium containing 50mg/L spectinomycin, culture in dark at 28°C for 36-48h, scrape the cultured bacteria with a scalpel In the MT suspension medium added with 20mg/L acetosyringone, shake culture at 180rpm, 28°C for 1.5-2h, adjust the OD ₆₀₀ value of the bacterial solution to 0.6-0.8, and set aside;

(4) Transfer the sectioned epicotyls of sterile seedlings to MT suspension medium containing Agrobacterium rhizogenes, soak for 20-30 minutes, and shake gently 2-3 times during the period;

(5) Blot up excess bacterial liquid with sterile filter paper and transfer to MT co-cultivation medium containing 20 mg/L acetosyringone, and co-cultivate in the dark at 20-22°C for 3-4 days;

(6) Take the explants after co-cultivation, rinse them with sterilized distilled water for 3 times, dry them with sterile filter paper, and transfer them to the hairy root induction medium containing 50 mg/L kanamycin and 400 mg/L cephalosporin , cultured in the dark at 25±1°C for about 14 days, white hairy roots can be seen successively emerging from the cut epicotyl wound;

(7) When the hairy root grows to about 2 cm, cut about 1.5 cm of the hairy root containing the root tip and subculture it in MT basic medium containing 400 mg/L cephalosporin. 3 times to get sterile hairy roots;

(8) Observing the YFP fluorescence of hairy roots with a stereofluorescence microscope and identifying co-transformed hairy roots with GUS histochemical staining;

A kind of application of the hairy root that utilizes Agrobacterium rhizogenes to efficiently induce trifoliate orange to produce in root system development research, and its steps are:

(1) Observation of the expression pattern of auxin in hairy roots: the hairy roots were observed by stereofluorescence and GUS histochemical staining, and the endogenous auxin content of hairy roots could be judged according to the YFP fluorescence and GUS staining sites. expression pattern;

(2) Observation of auxin expression pattern in the process of lateral root generation: the co-transformed hairy roots with lateral roots were stained with GUS, treated with chloral hydrate transparent agent for 1-2 days, and then observed on a universal microscope. According to the location of GUS staining and Deep and shallow, the auxin expression pattern information in the process of lateral root primordium occurrence, growth and lateral root formation can be obtained;

(3) Effects of treatment with auxin analog NAA and auxin transport inhibitor on auxin expression pattern in hairy roots: cut out co-transformed hairy roots, and transfer them to MT solid medium containing 5 μM and 50 μM NAA respectively for 12 h ;The concentration and method of TIBA treatment are the same as those of NAA. After GUS staining for 2 hours, the chloral hydrate transparent agent is transparent and treated for 1-2 days, and then observed on a universal microscope. According to the depth and location of GUS staining, the effects of NAA treatment and TIBA treatment on hairy root growth can be obtained. information on the effects of protein expression patterns.

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

Advantages in hairy root induction: Compared with the method of Agrobacterium tumefaciens, the co-transformation hairy root induction frequency mediated by A. rhizogenes is high, which can reach about 60% (the total number of co-transformed hairy roots accounts for The percentage of the total number of plants), reducing the transformation workload; the reason for the increase in the transformation rate is that the infectivity of Agrobacterium rhizogenes is stronger than that of Agrobacterium tumefaciens; in addition, 20mg/L of acetyl Syringone is also beneficial to increase the infection activity of Agrobacterium rhizogenes, and the obtained hairy roots are of single-cell origin, without transformation chimeras, and can be further induced to regenerate into complete regenerated plants;

The advantages of using the hairy root system for root development biology research are: isolated hairy roots can grow independently on MT medium, and the culture conditions are easy to control; hairy roots have no secondary growth, are thin in diameter, and are easy to observe under a microscope; In addition, the cycle of inducing hairy roots is short, and usually the hairy roots can be seen about 14 days after the end of co-cultivation, which saves research time.

Description of drawings

Fig. 1 is a schematic diagram of the induction, fluorescence and GUS staining identification of a citrifolia root.

Wherein a: sterile water infection control; b: hairy root induction; c: GUS staining of hairy root without DR5::YFP:GUS transfection; d: GUS staining of hairy root transfected with DR5::YFP:GUS; e: YFP fluorescence of untransformed DR5::YFP:GUS hairy roots; f: Fluorescence observation of 35S:DsRed1 co-transformed hairy roots; g: Fluorescence observation of DR5::YFP:GUS co-transformed hairy roots; "-" is the scale, where a and b are 1 cm, and c to g are 1 mm.

Fig. 2 is a schematic diagram showing the endogenous auxin response mode of hairy roots transduced with DR5::YFP:GUS by GUS staining.

Among them: a: single transformation hairy root control (without DR5::YFP:GUS); b: hairy root transformed with DR5::YFP:GUS; c: hairy root with lateral root primordium transformed with DR5::YFP:GUS; d: Transformed DR5::YFP:GUS hairy roots with lateral roots; scale "-" = 1mm

Fig. 3 is a schematic diagram of GUS staining of a transgenic DR5::YFP:GUS hovenia trifoliate root-showing the auxin response mode of the root tip.

Among them: a is the root tip of the hairy root (adventitious root) from the epicotyl section - the root tip is transparent as a whole; b is the root tip of the hairy root side - paraffin section; scale "-" = 0.1mm

Fig. 4 is a schematic diagram of GUS staining during the lateral root development of a hairy root transfected with DR5::YFP:GUS—showing the auxin response mode during lateral root development.

Among them: a: the arrow indicates that there is auxin accumulation at the initial part of the lateral root primordium; b: the arrow indicates that the growing lateral root primordium has auxin at the top of the primordium; c: the arrow indicates that the lateral root primordium that breaks through the cortex has auxin at the top of the primordium Accumulation; d: Arrows indicate auxin accumulation at the apex of newly formed lateral roots; scale "-" = 0.1mm

Fig. 5 is a schematic diagram of the effect of NAA and TIBA treatment on the auxin response mode of trifoliate root for 12 h

Among them: a and d distilled water treatment control ck; b: 5 μM NAA treatment; c: 50 μM NAA treatment; e: 5 μM TIBA treatment; f: 50 μM TIBA treatment

Detailed ways

Unless otherwise specified, the experimental procedures in the examples of the present invention are conventional experimental procedures.

Each step of the present invention and application thereof are described in further detail as follows now:

Example 1:

A method utilizing Agrobacterium rhizogenes to efficiently induce trifoliate oranges to produce hairy roots, the steps of which are as follows:

1.1 Obtaining Agrobacterium rhizogenes containing binary expression vector MSU440: conventional alkaline lysis method (see the third edition of "Molecular Cloning Experiment Guide", J. Sambrook, Science Press) to extract DR5 from C58 Agrobacterium tumefaciens :: YFP: The binary expression vector plasmid of GUS was introduced into the competent Agrobacterium rhizogenes MSU440 by electroporation, and the resistant transformants were screened on LB solid medium supplemented with 50mg/L spectinomycin. % glycerol was stored in the ultra-low temperature refrigerator at -80°C in our laboratory for later use; in addition, the cheap carrier (with 35S::DsRed1) Agrobacterium rhizogenes MSU440 (Limpens E et al., RNA interference in Agrobacterium rhizogenes-transformed rootsof Arabidopsis and Medicago truncatula.J Exp Bot.2004May;55(399):983-92.) as a control for the expression pattern of DR5 promoter;

The binary expression vector plasmid of described DR5::YFP:GUS, its construction process is:

DR5 consists of seven 11bp (5'-CCTCGTGTCTC-3') tandem repeats (Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.Plant Cell, 1997, 9(11):1963–1971). Endonucleases XhoⅠ and NotⅠ digested SlIAA14::GUS+YFP vector (Li Xiaojing, Chen Yinhua, Zhang Junhong, Ye Zhibiao, Cloning and analysis of tomato SlIAA14 gene promoter, Acta Horticultural Science, 2010, 37 (10): 1605-1612) and recovered, The DR5 sequence was ligated into the recovery vector with T4 DNA ligase, and the DR5::YFP:GUS binary expression vector was constructed.

1.2 Preparation of aseptic seedling explants of Hovenia trifoliate: The fruit of Hovenia trifoliate was collected from the Citrus Breeding Center of Huazhong Agricultural University, the seeds of Hovenia trifoliate fruit were stripped, soaked in 1M NaOH for 10 minutes to remove pectin, rinsed with distilled water for 3-5 times, and placed on a clean bench Disinfect with 3% (v/v) sodium hypochlorite solution for 20 minutes, rinse with sterilized distilled water for 3 times, 5 minutes each time; remove the inner and outer testa, sow in MT seeding medium, and cultivate in the dark at 24-26°C for 30-45 days to obtain sterile seedlings ;Take out 1500-2000lx light intensity and light it for about 5-7d, after turning green, select robust aseptic seedlings, cut the epicotyls into sections of about 1.5cm, and set aside;

1.3 Preparation of Agrobacterium rhizogenes bacteria liquid: pick MSU440 Agrobacterium rhizogenes (including two strains containing DR5::YFP:GUS and containing 35S::DsRed1) preserved in 30% glycerol with the inoculation loop, and streak on the line containing 50mg/L Spectinomycin LB solid medium plate, 24-26 ℃ dark place inverted culture for 36-48h; pick a single colony of Agrobacterium rhizogenes on the plate, and streak again on LB solid medium plate containing 50mg/L spectinomycin Inverted culture at 24-26°C in the dark for 36-48h; Scrape the amplified bacteria on the LB plate with a sterile scalpel, transfer to 50ml MT suspension medium containing 20mg/L acetosyringone, and store in the dark at 28°C Shake culture at 180rpm for 1.5-2h, adjust OD ₆₀₀ =0.6-0.8, and set aside;

1.4 Transformation of explants

①Infection: Transfer the epicotyl section of sterile seedlings to the MT suspension medium containing Agrobacterium rhizogenes, infect for 20-30min, and shake gently 2-3 times during the period; The MT suspension medium of bacillus replaces the bacterial liquid, and the other treatments are the same;

② Co-cultivation: Blot up the excess bacterial liquid with sterile filter paper, transfer to MT co-cultivation medium containing 20 mg/L, and co-cultivate in the dark at 20-22°C for 3-4 days;

③Hairy root induction: take the explants after co-cultivation, rinse them with sterilized distilled water for 3 times, each time for 5 minutes; blot them dry with sterile filter paper and transfer them to a medium containing 50 mg/L kanamycin and 400 mg/L cephalosporin. On MT basic medium, cultured in the dark at 24-26°C for about 14 days, white hairy roots could be seen emerging from the cut epicotyl wounds, while no adventitious roots emerged from the uninfected control (Fig. 1a, b).

④Hairy root subculture: when the hairy root grows to about 2cm, cut about 1.5cm of the hairy root containing the root tip and subculture in the basic medium containing 400mg/L MT, and subculture once every 20 days or so;

1.5 Hairy root fluorescence and GUS staining observation

①Fluorescence observation: Fluorescence observation of hairy roots was carried out using a stereofluorescence microscope. The results showed that the single-transformed hairy roots (without DR5::YFP:GUS) had no fluorescence (Fig. 1e), and the hairy roots transformed with 35S::DsRed1 could be seen The whole root had strong red fluorescence (Fig. 1f), and the hairy roots transfected with DR5::YFP:GUS contained a specific expression promoter responsive to auxin, and yellow fluorescence was only observed at the root tip (Fig. 1g); The results showed that the expression pattern of DR5::YFP:GUS in Hovenia trifoliate roots was consistent with the expression characteristics of DR5 promoter, which could be used as a visual marker for the distribution and concentration of endogenous auxin in hairy roots.

② GUS histochemical staining observation

The hairy roots were cut out and stained in GUS staining solution at 37°C until a clear blue color appeared, and it took about 2 hours; the results showed that the GUS staining of single-transformed hairy roots was negative (Figure 1c), and the apex of co-transformed hairy roots Positive GUS staining was seen (Fig. 1d);

2. The application of transgenic DR5::YFP:GUS Hovenia trifoliate root in the study of root development biology

Using the auxin-induced expression characteristics of DR5::YFP:GUS, the hairy root was used as a system to study the endogenous auxin response mode in the growth and development of hairy root. Specifically, it includes the observation of endogenous auxin expression patterns in hairy roots, the observation of auxin expression patterns in hairy root lateral root development, and the effects of exogenous auxin analog NAA and auxin transport inhibitors on hairy root auxin expression patterns. Influence.

2.1 Observation on the expression pattern of endogenous auxin in hairy roots

GUS-stained hairy roots were treated with chloral hydrate transparent agent for 1-2 days and then observed on a universal microscope. According to the results of GUS staining, the distribution of endogenous auxin in hairy roots can be indirectly indicated; at the same time, the GUS-stained After the hairy roots were fixed with FAA fixative solution, the conventional paraffin sections could observe the depth of GUS staining in different cells, so as to have a more detailed understanding of the distribution of endogenous auxin in the hairy roots.

The results of GUS staining showed that the hairy root tips, lateral root primordia and lateral root tips of transgenic DR5::YFP:GUS had auxin response peaks (Fig. YFP:GUS) GUS staining results were not observed (Fig. 2a); the auxin concentration in the root cap columella and quiescent center was the highest in the root tip of the hairy root, and the auxin concentration in the lateral root cap and the base of the stele was relatively low (Fig. 3, a) ; The auxin response mode of hairy root lateral root tip was similar to the result of hairy root tip (Fig. 3, b);

2.2 The relationship between hairy root lateral root primordia and auxin distribution

After the hairy roots with lateral roots were stained with GUS, the chloral hydrate became transparent for 1-2 days. DIC observation on a universal microscope showed that the lateral root primordia started from the pericycle cells in the root tip, and auxin accumulated at the initial site (Fig. 4a ); GUS staining was the deepest at the top of the primordium during the growth of the primordium, indicating that the auxin accumulation was the highest at the top of the primordium (Fig. The highest auxin levels were maintained throughout (GUS staining was the deepest) (Fig. 4c,d).

2.3 Effects of NAA and TIBA treatments on auxin distribution in hairy roots

Add filter-sterilized naphthaleneacetic acid (NAA) and auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) to the sterilized MT basic medium, and cultivate hairy roots at 24-26°C for 12 hours in the dark , after GUS staining, chloral hydrate was treated for 1-2 days, observed under a universal microscope, and no NAA or TIBA was added to the control medium. The results showed that, compared with the control, after 12 hours of treatment with 5 μM NAA, the GUS staining at the root tip of the hairy root was obviously deepened, the staining range was enlarged, the lateral root cap was strongly stained, and the staining of the epidermis, cortex and stele was also enhanced, indicating that Application of NAA changed the distribution pattern of auxin in the root tip; this effect became more obvious with further increase of NAA concentration (Fig. 5, a,b,c).

The result of treatment with auxin transport inhibitor TIBA was opposite to that of NAA. 5 μM TIBA treatment of hairy roots for 12 hours reduced the concentration of auxin in the root tip, and with the increase of TIBA concentration, the accumulation of auxin in the root tip was less, but the root cap column cells The auxin accumulated in the quiescent center and quiescent center is still the most compared to other parts, indicating that the auxin synthesis ability of these two parts is the strongest (Fig. 5d, e, f).

The main medium and reagents in the present invention are as follows, wherein the pH value of the MT medium is 5.85; the pH value of the LB medium (which can also be replaced with YEB medium) can not be adjusted after dissolution:

The formulation of MT basal medium was formulated according to the literature Murashige T, Tucker DPH (Growth factor requirements of citrus tissue culture. Proc1st Int Citrus Symp, 1969, 3: 1155-1161).

MT seeding medium: MT+25g/L sucrose+8g/L agar

MT suspension medium: MT+0.5g/L malt extract+1.5g/L glutamine+40g/L sucrose+20mg/L acetosyringone

MT co-cultivation medium: MT+40g/L sucrose+8g/L agar+20mg/L acetosyringone

Hairy root induction medium: MT+40g/L sucrose+8g/L agar+50mg/L kanamycin+400mg/L+cephalosporin

LB solid medium: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, agar 15g/L

Chloral hydrate transparent agent: chloral hydrate 50g + distilled water 15mL + glycerin 10mL.

Claims (1)

1. a method utilizing Agrobacterium rhizogenes to efficiently induce trifoliate orange to produce hairy roots, the steps are as follows: (1) Peel the seeds of Hovenia citrus fruit, soak in 1M NaOH for 10 minutes to remove pectin, rinse with distilled water for 3-5 times, then disinfect with 3% sodium hypochlorite for 20 minutes, rinse with sterilized distilled water for 3 times, remove the inner and outer seed coats and sow on MT seeding medium, 24 Cultivate in the dark at -26°C for 30-45 days to obtain sterile seedlings, take them out under light intensity of 1500-2000 lx for about 5-7 days, select robust sterile seedlings after turning green, cut epicotyls into 1.5 cm, and set aside; (2) The binary expression vector plasmid containing DR5::YFP:GUS was introduced into Agrobacterium rhizogenes MSU440 by electroporation to obtain Agrobacterium rhizogenes containing the binary vector; (3) Pick a single colony of activated Agrobacterium rhizogenes, streak on LB solid medium containing 50 mg/L spectinomycin, culture in dark at 28°C for 36-48 hours, scrape the cultured bacteria with a scalpel In the MT suspension medium added with 20mg/L acetosyringone, shake culture at 180rpm, 28°C for 1.5-2h, adjust the OD ₆₀₀ value of the bacterial solution to 0.6-0.8, and set aside; (4) Transfer the sectioned epicotyls of sterile seedlings to the MT suspension medium containing Agrobacterium rhizogenes, soak for 20-30min, and shake gently 2-3 times during the period; (5) Blot up excess bacterial liquid with sterile filter paper and transfer to MT co-cultivation medium containing 20 mg/L acetosyringone, and co-cultivate in the dark at 21°C for 3-4 days; (6) Take the explants after co-cultivation, rinse them with sterilized distilled water for 3 times, dry them with sterile filter paper, and transfer them to the hairy root induction medium containing 50 mg/L kanamycin and 400 mg/L cephalosporin , 24-26°C cultured in the dark for about 14 days, white hairy roots can be seen successively emerging from the cut epicotyl wound; (7) When the hairy root grows to 2cm, cut off the hairy root of about 1.5cm containing the root tip and subculture it in MT basic medium containing 400mg/L cephalosporin, subculture once every 20 days, and subculture 2-3 times Sterile hairy roots can be obtained; Described culture medium formula is as follows: MT seeding medium: MT+25g/L sucrose+8g/L agar MT suspension medium: MT+0.5g/L malt extract+1.5g/L glutamine+40g/L sucrose+20mg/L acetosyringone MT co-cultivation medium: MT+40g/L sucrose+8g/L agar+20mg/L acetosyringone Hairy root induction medium: MT+40g/L sucrose+8g/L agar+50mg/L kanamycin+400mg/L+cephalosporin LB solid medium: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, agar 15g/L; The binary expression vector plasmid of described DR5::YFP:GUS, its construction process is: DR5 consists of seven 11bp 5'-CCTCGTGTCTC-3' tandem repeats; the endonucleases Xho Ⅰ and Not Ⅰ digest the SlIAA14 ::GUS + YFP vector and recover it. The DR5 sequence is ligated into the recovery vector with T4 DNA ligase, That is, the DR5::YFP:GUS binary expression vector was constructed.