A method for transferring LbbHLH36 gene to Ningxia wolfberry mediated by Agrobacterium rhizogenes

Abstract

The invention discloses a method for LbbHLH36 gene transfer of Ningxia wolfberry mediated by Agrobacterium rhizogenes, and relates to the technical field of plant genetic engineering. The method for LbbHLH36 gene transfer of Ningxia wolfberry mediated by Agrobacterium rhizogenes disclosed by the invention is to scratch the hypocotyl of Ningxia wolfberry seedlings with a sterile blade, inoculate the incision with the activated Agrobacterium rhizogenes K599 bacterial solution carrying the target gene recombinant plasmid (35S::eGFP or pRI101-LbbHLH36-eGFP), and cultivate to obtain Ningxia wolfberry transgenic plants. The rolB, NeoR, eGFP, and LbbHLH36 genes in the induced transgenic fluorescent hairy roots of Ningxia wolfberry can all amplify bands, indicating that the above are all transgenic hairy roots induced by Agrobacterium rhizogenes, and it is fast and feasible to transfer the target gene into the root system of Ningxia wolfberry using this system.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a method for transferring the LbbHLH36 gene of Ningxia wolfberry mediated by Agrobacterium rhizogenes. Background Art

[0002] Lycium L. belongs to the Lycium Wettst. subtribe of the Solanaceae family, the Solaneae Reichb. family. There are about 80 species of Lycium L. plants, which are widely distributed all over the world. Lycium barbarum is also widely distributed in my country. The 1978 "Flora of China" divided the genus Lycium into 7 species and 3 varieties, namely Lycium chinense Mill, Ningxia Lycium barbarum L, Xinjiang Lycium dasystemum Pojark, Black Lycium ruthenicum Murr, Lycium truncatum YCWang, Lycium cylindricum Kuang et AMLu, Yunnan Lycium yunnanense Kuanget AMLu; varieties of northern wolfberry (Lycium chinense Mill.var.potaninii(Pojark)AMLu), yellow wolfberry (Lycium barbarum L.var.auranticarpum KFChing), red wolfberry (Lycium dasystemum Pojark.var.rubricaulium AMLu).

[0003] From the perspective of vegetation, Ningxia wolfberry is a species distributed in grassland desert areas. It often grows on ditches, hillsides, ridges and houses with deep soil layers. It is resistant to salinity, sand and drought, which provides an ideal ecological environment for the healthy growth of wolfberry. Its soil quality is rich in nutrients and maintains a suitable water supply, providing the necessary growth resources for wolfberry plants.

[0004] The establishment of the genetic transformation system of Ningxia wolfberry is not only conducive to in-depth research on its function under different abiotic stress adversities, but also conducive to further expanding the production of Ningxia wolfberry, improving quality and variety improvement, and thus providing a theoretical basis for the further development of my country's wolfberry industry. The wolfberry transgenics reported so far are all genetic transformation mediated by Agrobacterium tumefaciens. Although the genetic transformation technology of wolfberry mediated by Agrobacterium tumefaciens has promoted the development of research on the function of wolfberry genes to a certain extent, the genetic transformation method mediated by Agrobacterium tumefaciens has long transformation cycle, low efficiency and cumbersome operation, making it difficult to establish an efficient genetic transformation system. Therefore, it is particularly necessary to explore a genetic transformation system with high efficiency and simple operation to improve the efficiency and quality of genetic transformation of Ningxia wolfberry. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for transferring the LbbHLH36 gene of Ningxia wolfberry mediated by Agrobacterium rhizogenes, so as to establish an efficient genetic transformation system of Ningxia wolfberry.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for transferring the LbbHLH36 gene to Ningxia wolfberry mediated by Agrobacterium rhizogenes, comprising:

[0008] 1) Sterilize and culture Ningxia wolfberry seeds until they germinate to obtain seedlings, and scratch the hypocotyl of the seedlings with a sterile blade;

[0009] 2) The recombinant plasmid carrying the target gene is transferred into Agrobacterium rhizogenes by freeze-thaw method to prepare bacterial solution;

[0010] 3) The Agrobacterium rhizogenes glycerol bacteria were streaked and cultured, and a single colony was picked for PCR verification. The single colony that was positive for PCR was spotted with a sterile pipette tip onto a new LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate and grown at 28°C for 24-36 hours for later use;

[0011] 4) scratching the hypocotyl of the Ningxia wolfberry seedlings with a sterile blade, and inoculating the cut with activated Agrobacterium rhizogenes for infection;

[0012] 5) Cultivate the infected Lycium barbarum seedlings under a 16h / 8h photoperiod for 3 weeks at a temperature of 23-25°C;

[0013] 6) Observe with a handheld fluorescence observer, extract the induced fluorescent hairy root genomic DNA and amplify the target gene.

[0014] The Agrobacterium rhizogenes is K599 Agrobacterium rhizogenes, MSU440 Agrobacterium rhizogenes or C58C1 Agrobacterium rhizogenes.

[0015] The Agrobacterium rhizogenes is K599 Agrobacterium rhizogenes.

[0016] The activation method of the K599 rhizogenes Agrobacterium tumefaciens liquid is as follows: a recombinant plasmid carrying a target gene is transferred into the rhizogenes Agrobacterium tumefaciens K599 by a freeze-thaw method, a positively verified single clone is picked and placed in an LB liquid culture medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28° C. and 200 rpm for 16-24 hours to obtain an activated rhizogenes Agrobacterium tumefaciens K599 liquid.

[0017] The target genes are eGFP gene and LbbHLH36 gene.

[0018] The recombinant plasmid of the eGFP gene is 35S::eGFP.

[0019] The recombinant plasmid of the LbbHLH36 gene is pRI101-LbbHLH36-eGFP.

[0020] The method for transferring the LbbHLH36 gene to Ningxia wolfberry mediated by Agrobacterium rhizogenes comprises the following specific steps:

[0021] 1) Soak the Ningxia wolfberry fruit in water overnight, take out the seeds and place them in a 1.5mL centrifuge tube, soak them in 70% ethanol for 2 minutes, pour out the alcohol, soak them in 0.5% NaClO for 15 minutes, pour out the NaClO, and wash them with sterile water for 3-5 times; sow the disinfected seeds in a tissue culture bottle containing MS solid culture medium, vernalize at 4°C for 48 hours, and culture them in the dark at 22°C for 48 hours; then transfer the tissue culture bottle to a culture room and continue to culture until the seeds germinate; transfer the Ningxia wolfberry seedlings that have germinated for 10 days in the tissue culture bottle to nutrient soil, then cover them with a transparent plastic cover and place them in a light incubator for growth;

[0022] 2) The recombinant plasmid 35S::eGFP or pRI101-LbbHLH36-eGFP with the target gene was transferred into K599 Agrobacterium rhizogenes, MSU440 Agrobacterium rhizogenes or C58C1 Agrobacterium rhizogenes by freeze-thaw method, and the positive single clone was selected and transferred to LB liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28°C and 200 rpm for 16-24 hours, and the shaken bacterial solution was mixed with an equal volume of 50% glycerol, After quick freezing with liquid nitrogen, place in -80℃ ultra-low temperature refrigerator for standby use; streak the stored Agrobacterium rhizogenes K599 glycerol bacteria on LB solid medium containing 50mg / L kanamycin sulfate and 25mg / L streptomycin sulfate, invert and culture at 28℃ for 48h, pick a single colony for PCR verification, and use a sterile pipette tip to spot the single colony that is positive for PCR on a new LB solid medium containing 50mg / L kanamycin sulfate and 25mg / L streptomycin sulfate and grow at 28℃ for 24-36h for standby use;

[0023] 3) The Ningxia wolfberry that had been transferred to soil culture for 2 weeks was scratched at the hypocotyl with a sterile blade, and the cut was inoculated with K599, MSU440 or C58C1 positive colonies grown in LB solid medium. After the infection was completed, the plastic cover was covered to keep moisture and the infected plants were transferred to an incubator at a culture temperature of 23-25°C and a 16h / 8h photoperiod;

[0024] 4) Observe the hairy roots of the transformed plants using a LUYOR-3415G handheld fluorescence observer;

[0025] 5) The induced fluorescent hairy roots were quickly frozen in liquid nitrogen and ground into powder. The genomic DNA was extracted using the CTAB method for PCR amplification detection. Bands of the rolB gene, NeoR gene, eGFP gene, and LbbHLH36 gene in the induced transgenic fluorescent hairy roots of Ningxia wolfberry were amplified.

[0026] The transgenic root line of Ningxia wolfberry was obtained by the LbbHLH36 gene transfer method mediated by Agrobacterium rhizogenes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The invention discloses a method for transferring the LbbHLH36 gene of Ningxia wolfberry mediated by Agrobacterium rhizogenes. The method comprises the following steps: scratching the hypocotyl of a Ningxia wolfberry seedling with a sterile blade, inoculating the incision with an activated Agrobacterium rhizogenes K599 bacterial solution, and culturing the transgenic Ningxia wolfberry plant. The recombinant plasmid carrying the target gene, 35S::eGFP or pRI101-LbbHLH36-eGFP, is contained in the Agrobacterium rhizogenes K599 bacterial solution.

[0029] The rolB gene, NeoR gene, eGFP gene and LbbHLH36 gene in the induced transgenic fluorescent hairy roots of Ningxia wolfberry were able to amplify bands, indicating that the transgenic hairy roots induced by Agrobacterium rhizogenes were successfully obtained, indicating that it is rapid and feasible to transfer the target gene into the root system of Ningxia wolfberry using this system. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the genetic modification process of Ningxia wolfberry mediated by Agrobacterium rhizogenes;

[0031] Figure 2 The hairy roots are induced by transformation of Ningxia wolfberry mediated by different Agrobacterium rhizogenes strains;

[0032] Figure 3 It is the transgenic hairy roots induced by Ningxia wolfberry after infection with Agrobacterium rhizogenes;

[0033] Figure 4 The results of PCR detection of transgenic hairy roots in Example 1 (WT represents negative control; P represents positive control; T plus numbers represent transgenic hairy roots of different strains);

[0034] Figure 5 This is a graph showing the results of PCR detection of hairy roots transfected with the LbbHLH36 gene in Example 2 (WT represents the negative control; P represents the positive control; T plus numbers represent transgenic hairy roots of different strains); DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0036] The K599, MSU440 and C58C1 Agrobacterium rhizogenes competent cells used in this application were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0037] Example 1

[0038] 1. Seed germination

[0039] The Ningxia wolfberry fruit is soaked in water overnight, the seeds are taken out and placed in a 1.5mL centrifuge tube, soaked and disinfected with 70% ethanol for 2 minutes (keeping the seeds suspended), the alcohol is poured out, and the seeds are soaked and disinfected with 0.5% NaClO for 15 minutes (the centrifuge tube is turned upside down to keep the seeds suspended), the NaClO is poured out, and the seeds are washed with sterile water for 3-5 times; the disinfected seeds are sown in a tissue culture bottle containing MS solid culture medium, vernalized at 4°C for 48 hours, and cultured in the dark at 22°C for 48 hours, and then the tissue culture bottle is transferred to a culture room for continued culture until the seeds germinate; the Ningxia wolfberry seedlings that have germinated for 10 days in the tissue culture bottle are transferred to nutrient soil, and then covered with a transparent plastic cover and placed in a light incubator for growth.

[0040] 2. Activation and cultivation of Agrobacterium rhizogenes

[0041] The pRI101 plasmid carrying the 35S::eGFP reporter gene was transferred into Agrobacterium rhizogenes K599, MSU440 and C58C1 by freeze-thaw method, and the positive clones were selected and transferred to LB liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28°C and 200 rpm for 16-24 h. The activated bacterial solution was mixed with an equal volume of 50% glycerol, and then quickly frozen with liquid nitrogen and placed in a -80°C ultra-low temperature refrigerator for use. The stored Agrobacterium rhizogenes (including K599, MSU440 and C58C1) glycerol bacteria were streaked on LB solid culture medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, inverted and cultured at 28°C for 48 h, single colonies were picked for PCR verification, and single colonies that were PCR-positive were spotted with a sterile pipette tip onto new LB solid culture medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate and grown at 28°C for 24 to 36 h for use.

[0042] 3. Seedling infection and induction of hairy roots

[0043] Ningxia wolfberry that had been transferred to nutrient soil and cultured for 2 weeks was scratched at the hypocotyl with a sterile blade, and the cut was inoculated with positive colonies of K599, MSU440 and C58C1 grown in LB solid medium, respectively, and the hypocotyl without inoculation was used as a control. After the infection was completed, the plastic cover was covered to keep moisture and the infected plants were transferred to an incubator with a culture temperature of 23-25℃ and a 16h / 8h photoperiod to observe the induction of hairy roots after infection with different rhizobacterium.

[0044] The results are as follows Figure 2As shown in the figure, different types of Agrobacterium rhizogenes have significant differences in their ability to induce transgenic hairy roots of Ningxia wolfberry, among which K599-mediated transformation efficiency is the highest, at 65%; MSU440 is second, at 15.79%; C58C1 only produces callus after infection, and no hairy roots are produced after 3 weeks of growth, with an induction efficiency of 0%. In summary, K599-mediated transformation efficiency of Ningxia wolfberry is the highest, so K599-mediated transformation of target genes of Ningxia wolfberry was used in subsequent experiments.

[0045] 4. eGFP fluorescence detection

[0046] The infected Ningxia wolfberry culture pots were covered with pot covers and placed back in the incubator for 3 weeks under a 16h / 8h photoperiod at a temperature of 23-25°C. The hairy roots of the transformed plants were observed using a LUYOR-3415G handheld fluorescence observer.

[0047] The results are as follows Figure 3 As shown, the root system of the plant transformed with the 35S::eGFP gene showed strong green fluorescence throughout the root.

[0048] 5. PCR testing

[0049] The hairy roots with strong green fluorescence were quickly frozen in liquid nitrogen and ground into powder. The genomic DNA was extracted using the CTAB method for PCR amplification detection.

[0050] The primers for rolB gene detection are:

[0051] rolB-FW: 5'-ACGAAGAAGGTGCAGGCTAC-3',

[0052] rolB-RV: 5′-TCAACGCTTTCAACCACGAG-3′, PCR product of 500 bp;

[0053] The primers for NeoR gene detection are:

[0054] NeoR-FW: 5'-ACCGGATCTGGATCGTTTCG-3',

[0055] NeoR-RV: 5′-TGGACTCCAACGTCAAAGGG-3′, PCR product of 1399 bp;

[0056] The gene-specific primers are:

[0057] eGFP-FW: 5'-ATGGTGAGCAAGGGCGAGG-3',

[0058] eGFP-RV: 5′-TTACTTGTACAGCTCGTCCATGCC-3′, product size is 720 bp.

[0059] The results are as follows Figure 4 As shown, the rolB gene, NeoR gene and eGFP gene in the induced transgenic fluorescent hairy roots of Ningxia wolfberry can all be amplified, further indicating that these are all transgenic hairy roots induced by Agrobacterium rhizogenes, indicating that it is rapid and feasible to transfer the target gene into the root system of Ningxia wolfberry using this system.

[0060] Example 2

[0061] 1. Grind the leaves, stems and roots of Lycium barbarum into powder in liquid nitrogen, weigh 50 mg of the sample and extract total RNA using an RNA extraction kit, reverse the extracted RNA to obtain cDNA, and the obtained cDNA is used to amplify the CDS sequence of the target gene LbbHLH36.

[0062] 2. Design of gene-specific primers:

[0063] LbbHLH36-FW: 5'-ATGTTTTCTTTACAAGGAAGTGATGACA-3',

[0064] LbbHLH36-RV: 5'-CAGGTCCAAGTTGATCATATCAA-3'.

[0065] The CDS of the LbbHLH36 gene was amplified from the mixed cDNA of leaves, stem segments and roots of Lycium barbarum, and its amino acid sequence is shown in SEQ ID NO. 1. The sequence successfully sequenced was connected to the N-terminus of the eGFP tag in the pRI101-eGFP vector to obtain the pRI101-LbbHLH36-eGFP overexpression vector.

[0066] 3. The recombinant plasmid with the target gene pRI101-LbbHLH36-eGFP was transferred into Agrobacterium rhizogenes K599 by freeze-thaw method, and the positive single clone was selected and placed in LB liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28°C and 200 rpm for 16-24 hours. The shaken bacterial solution was mixed with an equal volume of 50% glycerol, and then quickly frozen with liquid nitrogen and placed in a -80°C ultra-low temperature refrigerator for standby use. The preserved Agrobacterium rhizogenes K599 glycerol bacteria were streaked on LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and inverted at 28°C for 48 hours. Single colonies were selected for PCR verification, and single colonies with positive PCR verification were spotted with a sterile pipette tip on a new LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and grown at 28°C for 24-36 hours for standby use.

[0067] 4. Use a sterile blade to scratch the hypocotyl of the Ningxia wolfberry plant that has been transferred to soil culture for 2 weeks, and inoculate the cut with K599 positive colonies grown in LB solid medium. Use the hypocotyl without inoculation as a control. After infection, cover with a plastic cover to keep moisture and transfer the infected plants to an incubator at a temperature of 23-25°C and a photoperiod of 16h / 8h.

[0068] 5. The hairy roots of the transformed plants were observed using a LUYOR-3415G handheld fluorescence observer. The roots of the plants transformed with 35S::eGFP showed strong green fluorescence throughout the roots, while the untransformed roots had no green fluorescence.

[0069] 6. The induced fluorescent hairy roots were quickly frozen in liquid nitrogen and ground into powder. The genomic DNA was extracted using the CTAB method for PCR amplification detection.

[0070] The primers for LbbHLH36 gene detection are:

[0071] LbbHLH36-FW: 5'-GACGCACAATCCCACTATCC-3',

[0072] LbbHLH36-RV: 5'-CAGGTCCAAGTTGATCATATCAA-3';

[0073] The primers for rolB gene detection are:

[0074] rolB-FW: 5'-ACGAAGAAGGTGCAGGCTAC-3',

[0075] rolB-RV: 5'-TCAACGCTTTCAACCACGAG-3';

[0076] The primers for NeoR gene detection are:

[0077] NeoR-FW: 5'-ACCGGATCTGGATCGTTTCG-3',

[0078] NeoR-RV: 5'-TGGACTCCAACGTCAAAGGG-3'.

[0079] The results are as follows Figure 5 As shown, the LbbHLH36, rolB and NeoR genes in the induced fluorescent hairy roots of Ningxia wolfberry can all be amplified, further indicating that these fluorescent hairy roots are transgenic hairy roots induced by Agrobacterium rhizogenes, and it is rapid and feasible to use this system to transfer the target gene into the root system of Ningxia wolfberry.

[0080] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method of Agrobacterium rhizogenes-mediated transformation of Ningxia wolfberry LbbHLH36 A genetic method, characterized in that include: 1) Sterilize the Ningxia wolfberry seeds and culture them until they germinate to obtain seedlings, and scratch the hypocotyl of the seedlings with a sterile blade; 2) The recombinant plasmid carrying the target gene is transferred into Agrobacterium rhizogenes by freeze-thaw method to prepare bacterial solution; 3) Plate the Agrobacterium rhizogenes glycerol bacteria, pick a single colony for PCR verification, and use a sterile pipette tip to spot the single colony that is positive for PCR onto a new LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate and grow it at 28°C for 24-36 hours for later use; 4) Use a sterile blade to scratch the hypocotyl of the Ningxia wolfberry seedlings, and inoculate the cut with activated Agrobacterium rhizogenes for infection; 5) Cultivate the infected Ningxia wolfberry seedlings under a 16h / 8h photoperiod for 3 weeks at a temperature of 23-25°C; 6) Observe with a handheld fluorescence observer, extract the induced fluorescent hairy root genomic DNA and amplify the target gene; The Agrobacterium rhizogenes is K599 Agrobacterium rhizogenes; The activation method of the K599 Agrobacterium rhizogenes bacterial liquid is as follows: the recombinant plasmid carrying the target gene is transferred into the Agrobacterium rhizogenes K599 by freeze-thaw method, and the positively verified single clone is selected and placed in LB liquid culture medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28° C. and 200 rpm for 16-24 hours to obtain the activated Agrobacterium rhizogenes K599 bacterial liquid; The target gene is eGFP Gene 、LbbHLH36 Gene; Said eGFP The recombinant plasmid of the gene is 35S::eGFP; Said LbbHLH36 The recombinant plasmid of the gene is pRI101- LbbHLH36 -eGFP.

2. The Agrobacterium rhizogenes-mediated transformation of Lycium barbarum according to claim 1 LbbHLH36 A genetic method, characterized in that The specific steps include: 1) Soak the Ningxia wolfberry fruit in water overnight, take out the seeds and place them in a 1.5mL centrifuge tube, soak and disinfect them in 70% ethanol for 2 minutes, pour out the alcohol, soak and disinfect them in 0.5% NaClO for 15 minutes, pour out the NaClO, and wash them with sterile water 3-5 times; sow the disinfected seeds in a tissue culture bottle containing MS solid culture medium, vernalize at 4℃ for 48 hours, and culture in the dark at 22℃ for 48 hours; then transfer the tissue culture bottle to the culture room and continue to culture until the seeds germinate; transfer the Ningxia wolfberry seedlings that have germinated for 10 days in the tissue culture bottle to nutrient soil, then cover them with transparent plastic covers and place them in a light incubator for growth; 2) The recombinant plasmid 35S:: eGFP or pRI101- LbbHLH36 -eGFP was transferred into K599 rhizogenes Agrobacterium, and the positive single clones were picked and placed in LB liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and cultured at 28°C and 200 rpm for 16-24 hours. The shaken bacterial solution was mixed with an equal volume of 50% glycerol, and then quickly frozen with liquid nitrogen and placed in a -80°C ultra-low temperature refrigerator for standby use; the preserved rhizogenes Agrobacterium K599 glycerol bacteria were plated on LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and inverted cultured at 28°C for 48 hours, and single colonies were picked for PCR verification. The single colonies with positive PCR verification were spotted with a sterile pipette tip on a new LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate, and grown at 28°C for 24-36 hours for standby use; 3) Use a sterile blade to scratch the hypocotyl of the Ningxia wolfberry that has been transferred to soil culture for 2 weeks, and inoculate the cut site with K599 positive colonies grown in LB solid medium. After infection, cover with a plastic cover to keep moisture and transfer the infected plants to an incubator at a temperature of 23-25°C and a photoperiod of 16h / 8h. 4) Observe the hairy roots of the transformed plants using the LUYOR-3415G handheld fluorescence observer; 5) The induced fluorescent hairy roots were quickly frozen in liquid nitrogen and ground into powder. The genomic DNA was extracted using the CTAB method for PCR amplification detection. eGFP Gene, LbbHLH36 All genes could be amplified.

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