A method for cultivating drought-resistant gene-transformed poplars

Abstract

The invention discloses a method for cultivating a transgenic drought-resistant poplar, and belongs to the technical field of plant genetic engineering. The invention uses excellent poplar varieties as plant materials, and obtains transgenic drought-resistant poplars through target gene cloning, vector construction, preparation of Agrobacterium infection solution, and genetic transformation of poplar leaves. Among them, the Agrobacterium infection solution contains an AAM resuspension medium improved according to the nutritional requirements of the tree species, and the culture medium in the genetic transformation stage of the poplar leaves contains TDZ and Carb. The improved AAM resuspension medium can fully improve the activity of the bacterial liquid and the transformation efficiency, accelerate the growth of buds and increase the budding rate and positive rate. The special combination with Carb can effectively ensure the positive rate and inhibit the excessive growth of Agrobacterium. TDZ accelerates leaf cell differentiation, can directly grow bud points without callus tissue, shortens the transformation cycle, and achieves efficient transformation. The positive rate can be as high as 62.5%, which can accelerate the breeding process of drought-resistant poplars.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a method for cultivating drought-resistant gene-transformed poplars. Background Art

[0002] Poplar (Populus) is one of the most widely distributed and largest timber species in northern my country. It has the characteristics of fast growth, high yield and strong adaptability. However, in arid and semi-arid areas, due to the lack of irrigation conditions, water deficit and drought severely limit the productivity of poplar plantations, restricting the economic value and ecological benefits of poplars. Therefore, cultivating excellent forest tree strains that are resistant to drought stress is an important way to improve the survival rate of afforestation and the productivity of plantations in my country. The core goals of conventional breeding are mainly the rapid growth and material properties of wood, which cannot meet the resistance of trees to a certain extent and cannot efficiently meet the needs of forestry development. The use of genetic engineering technology to improve the purposefulness and operability of breeding at the genetic level provides an important way to improve the resistance of trees and cultivate new poplar varieties with strong resistance.

[0003] In recent years, the Agrobacterium tumefaciens-mediated method has been often used to genetically transform poplars, which has accelerated the progress of cultivating drought-resistant transgenic poplars. However, this method still has problems such as slow callus induction, long cycle, low transformation efficiency and low positive rate, and drought resistance needs to be further improved. Therefore, providing a method for cultivating drought-resistant gene-transgenic poplars with a short transformation cycle, high efficiency and rapid induction of adventitious buds, and creating excellent transgenic drought-resistant poplar varieties is of great significance to promoting the development of forest molecular breeding and the construction of forestry ecological civilization. Summary of the invention

[0004] 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 cultivating transgenic drought-resistant poplars without directly growing buds from callus tissue.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for cultivating drought-resistant gene-transformed poplars comprises the following steps:

[0007] 1) Construction of plant expression vector of poplar drought stress tolerance gene;

[0008] 2) transferring the constructed plant expression vector of the poplar drought stress tolerance gene into Agrobacterium;

[0009] 3) Resuspend the recombinant Agrobacterium containing the poplar drought stress tolerance gene in the improved AAM resuspension medium until the OD 600When the concentration of AS is 0.6-0.9, 200 μmol / L AS is added to prepare the Agrobacterium infection solution; the formula of the improved AAM resuspension medium is: sucrose 68500 mg / L+glucose 36000 mg / L+KCl 3000 mg / L+glutamine 900 mg / L+acid hydrolyzed casein 500 mg / L+MgSO4 250 mg / L+KH2PO4 185 mg / L+arginine 176 mg / L+CaCl2·2H2O 150 mg / L+inositol 100 mg / L+FeNaEDTA 36.7 mg / L+VB1 10 mg / L+MnSO4·H2O 7.58 mg / L+glycine 7.5 mg / L+H3BO33 mg / L+ZnSO4·7H2O 2 mg / L+nicotinic acid 1 mg / L+VB6 1 mg / L+KI 0.8mg / L+Na2MoO4·2H2O 0.25mg / L+CoCl2·6H2O 0.025mg / L+CuSO4·5H2O 0.025mg / L;

[0010] 4) Select poplar tissue culture seedlings with good growth status, take the young leaves at the top as plant materials, remove the main veins and leaf tips, perform wound treatment on the veins, and place them in MS differentiation medium for pre-culture for 24 hours; take out the pre-cultured leaves, place them in the infection solution for infection, take out the leaves after the infection, dry them with filter paper, put them back in MS differentiation medium, and invert them in the dark for 3 days;

[0011] 5) Take out the leaves from the MS differentiation medium, rinse them with sterile water, then rinse them with water containing Carb, dry them with filter paper, and transfer them to the MS selection medium for 7 days;

[0012] 6) The leaves in the MS selection medium were transferred to the MS screening medium for culture. The screening medium was changed every 10 days. During this period, buds were grown directly without passing through callus tissue. When the medium was changed for the third time, they were transferred to a sterile tissue culture bottle containing transformation plant rooting medium to obtain transgenic drought-resistant gene poplar seedlings.

[0013] Further, in step 1), the poplar drought stress resistance gene used in the present application is the PIP1;1 gene, but is not limited thereto, and other poplar drought stress resistance genes can be selected according to actual needs.

[0014] Furthermore, in step 2), the Agrobacterium used is GV3101 Agrobacterium.

[0015] Furthermore, in step 4), the formula of the MS differentiation medium is: MS+NAA 0.05 mg / L+6-BA 0.5 mg / L+TDZ 0.01 mg / L+sucrose 30 g / L+phytagel 4.2 g / L+AS 200 μmol / L, pH=5.8-6.0.

[0016] Furthermore, in step 4), select poplar tissue culture seedlings that are 45-55 days old and in good condition.

[0017] Furthermore, in step 4), the infection time is 15 minutes, and shaking is performed for 1 minute every 3 minutes during the infection.

[0018] Furthermore, the leaves in the MS differentiation medium were taken out, rinsed with sterile water for 5-6 times, and then rinsed with water containing 400 mg / L Carb.

[0019] Further, in step 5), the MS selection medium formula is: MS+NAA 0.05mg / L+6-BA 0.5mg / L+TDZ 0.01mg / L+sucrose 30g / L+phytagel 4.2g / L+AS 200μmol / L+Carb 400mg / L, pH=5.8-6.0; the culture conditions are: culture for 7d under 16h light and 8h dark conditions.

[0020] Further, in step 6), the formula of the MS screening medium is MS+NAA 0.05 mg / L+6-BA 0.5 mg / L+TDZ 0.01 mg / L+sucrose 30 g / L+phytagel 4.2 g / L+AS 200 μmol / L+Carb 400 mg / L+Hyg 1.5 mg / L, pH=5.8-6.0.

[0021] Furthermore, in step 6), the formula of the transformation plant rooting medium is 1 / 2MS+NAA 0.05 mg / L+sucrose 30 g / L+agar 7 g / L+Hyg 1.5 mg / L, pH=5.8-6.0.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The improved AAM resuspension medium can fully improve the activity of bacterial liquid and transformation efficiency, accelerate the growth of buds and increase the budding rate and positive rate. The special combination with Carb can effectively ensure the positive rate and inhibit the excessive growth of Agrobacterium. TDZ accelerates the differentiation of leaf cells, can directly grow buds without callus tissue, shorten the transformation cycle, and achieve efficient transformation;

[0024] 2) The method for preparing drought-resistant poplars of the present invention has the characteristics of easy operation, high efficiency, short cycle, etc., and the positive rate can be as high as 62.5%. The obtained drought-resistant gene overexpressed poplars show stronger drought resistance and growth ability under soil cultivation conditions compared with non-transgenic poplars. The overexpressed poplars prepared by the method of the present invention have excellent genetic stability, which provides a mature and stable genetic transformation system for cultivating new poplar varieties and can accelerate the breeding process of drought-resistant poplars. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a diagram of the genetic transformation process of poplar in the method of the present invention, wherein: Figure 1 A in the figure is the leaf co-cultivation picture; Figure 1 B in the figure is the bud point diagram after changing the screening medium once; Figure 1 C in the figure is the growth state of the buds after the screening medium was replaced twice; Figure 1 D in the figure is the initial state diagram after the bud is transferred into the rooting medium; Figure 1 E in the figure is the bud rooting diagram; Figure 1 F in the figure is the seedling picture; Figure 1 G in the figure is the transformed strain diagram after propagation;

[0026] Figure 2 The gel electrophoresis diagram of the transformed strain detection in the embodiment of the present invention, wherein the marker is DLMarker2000; the plasmid is the positive control, H2O is the blank control; WT (non-transformed strain) is the negative control; 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 are transformed strains;

[0027] Figure 3 The figure is a graph of the real-time fluorescence quantitative PCR detection results of each strain in the embodiment of the present invention; wherein WT is a non-transformed strain, and 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 are transformed strains;

[0028] Figure 4 This is a phenotypic comparison diagram of the non-transformed strain (WT) and the transformed strains (OE1, OE2) after 20 days of drought stress in the example of the present invention;

[0029] Figure 5 To remove the AAM resuspension medium during genetic transformation ( Figure 5 A) and AAM medium before improvement ( Figure 5 B) budding state after infection;

[0030] Figure 6 This is a diagram of the budding state of leaves in which TDZ was removed from the genetic transformation medium;

[0031] Figure 7 The status diagram of the transformed strains for the genetic transformation process to remove Carb;

[0032] Figure 8 This is a diagram of the leaf status after removing AAM resuspension medium, TDZ and Carb during genetic transformation. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments.

[0034] The strains and plasmids used in the following examples are: Escherichia coli DH5α, DB3.1 Ultracompetent cells, Agrobacterium tumefaciens GV3101 transformed with the plant expression vector PMDC32 containing the target gene; plant materials: 84K poplar (Populus alba × Populus glandularis) leaves; hormones and antibiotics: 1-naphthylacetic acid (NAA), 6-benzyladenine (6-BA), Thidiazuron (TDZ), Acetosyringone (AS), Kanamycin (Kan), Rifampicin (Rif), Carbenicillin (Carb), and Hygroscopious (Hyg).

[0035] The culture media and their formulations used in the following examples are: LB liquid medium, LB solid medium, MS differentiation medium, MS selection medium, MS screening medium, wild-type plant rooting medium and transformed plant rooting medium formulations are shown in Table 1; the improved AAM resuspension medium is shown in Table 2; the above culture media are all sterilized at 121°C for 20 min, and the antibiotics Hyg, Kan, Rif, Carb, and AS contained therein are added to the culture medium after sterilization.

[0036] Table 1 Different culture medium types and their compositions

[0037]

[0038]

[0039] Table 2 Improved AAM resuspension medium formula

[0040] drug Content (mg / L) drug Content (mg / L) sucrose 68500 glucose 36000 KCl 3000 Glutamine 900 Acid hydrolyzed casein 500 <![CDATA[MgSO4]]> 250 <![CDATA[KH2PO4]]> 185 Arginine (Arg) 176 <![CDATA[CaCl2·2H2O]]> 150 Inositol 100 FeNaEDTA 36.7 VB1 10 <![CDATA[MnSO4·H2O]]> 7.58 Glycine 7.5 <![CDATA[H3BO3]]> 3 <![CDATA[ZnSO4·7H2O]]> 2 niacin 1 VB6 1 KI 0.8 <![CDATA[Na2MoO4·2H2O]]> 0.25 <![CDATA[CoCl2·6H2O]]> 0.025 <![CDATA[CuSO4·5H2O]]> 0.025

[0041] Example 1

[0042] 1. PIP1;1 gene cloning, vector digestion recovery and plasmid recombination

[0043] 1. Cloning of the drought-tolerant water channel protein gene PIP1;1 in poplar

[0044] The specific primers of the coding region of the target gene PIP1;1 (Potri.008G065600) were designed by using the software Primer Permier 5.0, DNAMAN and Oligo 7 and were synthesized by a biological company. The primer sequences were: upstream primer sequence 5'-ATGGAGGGCAAAGAAGAAGATGTTAGGC-3', downstream primer sequence 5'-TCACTTCT TGAAAGGAATGGCTCTGATC-3'; the amplification primers were primers with sticky ends added to the 5' ends of the upstream primer and the downstream primer, respectively, and the final primers were: upstream primer sequence 5'-CGGG CCCCC CCTCGAGGCGCGCC ATGGAGGGCAAAGAAGAAGATGTTAGGC-3', downstream primer sequence 5'-TATCCAGTCACTATGGTCGACTCACTTCT TGAAAGG AATGGCTCTGATC-3'; 100 mg 84K poplar leaves were ground into powder to extract total RNA. The detailed steps of RNA extraction refer to the instructions of the OminiPlant RNAKit (DNase I) RNA extraction kit of Kangwei Century, which are as follows: 1) Take 100 mg poplar leaves and quickly grind them into powder in liquid nitrogen, add 500 μL Buffer RLS (add β-mercaptoethanol before use), and immediately vortex and shake vigorously for 30 seconds; 2) Centrifuge at 4°C, 12000rpm for 2 minutes; 3) Transfer the supernatant to a filter column with a collection tube, centrifuge at 4°C, 12000rpm for 1 minute, and aspirate the supernatant into a new RNase-Free centrifuge tube, using a pipette tip to prevent cell debris from precipitating in the tube; 4) Slowly add 0.5) Add 5 times the volume of anhydrous ethanol of the supernatant, shake well, transfer to RM, centrifuge at 4℃, 12000rpm for 1min, discard the waste liquid, and put the adsorption column back into the collection tube; 5) Add 350μL Buffer RW1 to the adsorption column, centrifuge at 4℃, 12000rpm for 1min, discard the waste liquid and put it back into the adsorption column; 6) Prepare DNase I mixed solution (take 52μLRNase-Free Water, 8μL 10×Reaction Buffer and 20μL DNase I and mix well); 7) Add 80μL DNase I mixed solution to the adsorption column and incubate at 25℃ for 15min; 8) Add 350μL Buffer RW1 to RM, centrifuge at 4℃, 12000rpm for 1min, discard the waste liquid and put it back into the adsorption column; 9) Add 500μL Buffer to RM RW2 (with anhydrous ethanol added), centrifuge at 4℃, 12000rpm for 1min, discard the waste liquid and put it back into the adsorption column; 10) Repeat step 9); 11) Centrifuge at 4℃, 12000rpm for 2min; 12) Load RM into new RNase-Free Centrifuge Tubes, add 40μL RNase-Free Water to the middle part of the adsorption membrane, place at room temperature for 2min, centrifuge at 4℃, 12000rpm for 1min, and perform electrophoresis and concentration detection on the extracted RNA. .

[0045] Two clear and bright bands detected by electrophoresis were considered qualified RNA; qualified RNA was used for reverse transcription to synthesize cDNA. The detailed steps refer to Kangwei Century HiFiScript gDNA Removal cDNA Synthesis Kit, which are as follows: 1) Dissolve the RNA template, Peimer Mix, dNTP Mix, DTT, KT Buffer, HiFi Script and RNase-Free Water and place them on ice for later use; 2) Add 4μL dNTP Mix, 2μL Peimer Mix, 3μL RNA, 4μL Buffer, 2μL DTT, 1μL HiFiScript and 4μL RNase-Free to 20μL; 3) Vortex to mix, and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube; 4) Incubate at 42℃ for 45min and 85℃ for 5min; 5) Centrifuge briefly after the reaction is completed and cool on ice. PCR amplification was performed using cDNA as a template. Samples were added according to the following amplification system. After adding the samples, they were slowly blown and mixed, centrifuged to the bottom of the PCR tube, and the target gene was amplified by a PCR instrument. The amplification system was: Primer Star Mix 25μL; Forward Primer 10μM 1μL; Reverse Primer 10μM 1μL; Template DNA 2μL; ddH2O 21μL. The reaction procedure was: 98℃ 1min; 98℃ 10s, 64℃ 15s, 72℃ 1min, a total of 30 cycles; 72℃ 2min.

[0046] After PCR amplification, the PCR product was tested for concentration and electrophoresis, and the target DNA fragment with a single band was recovered by gel recovery. The recovered target fragment was stored at -20°C and used for step 3, recombinant plasmid; wherein, the recovery step refers to MonPure TMGel&PCR Clean Kit Gel&PCR product recovery kit, specifically: 1) Cut the target DNA band from the agarose gel (try to cut off the excess gel), put it into a sterile centrifuge tube and weigh it; 2) Add 3 times the volume of sol solution Buffer PW (with anhydrous ethanol added) to the gel, place it in a 65℃ water bath, and gently turn the centrifuge tube upside down every 2-3 minutes. 100mg gel block can be regarded as 100μL; 3) After the sol solution reaches room temperature, add it to the adsorption column, leave it at room temperature for 2 minutes, centrifuge it at 13000rpm for 1 minute, discard the waste liquid and put the adsorption column back; 4) Add 600μL Buffer to the adsorption column PW, let it stand for 2-5 minutes, then centrifuge at 13000rpm for 1 minute, discard the waste liquid and put the adsorption column back; 5) Repeat step 4); 6) After putting the adsorption column back, centrifuge at 13000rpm for 1 minute, put the adsorption column into a new sterilized centrifuge tube, and let it stand in a ventilated place for 10 minutes; 7) Add 30-50μL Buffer EB to the adsorption column membrane in the air, let it stand at room temperature for 2 minutes, and centrifuge at 13000rpm for 1 minute; 8) Repeat step 4).

[0047] 2. Vector digestion and recovery

[0048] The pMDC32 plant overexpression vector Gateway vector was transformed in DB3.1, and the plasmid was extracted after the ccdB suicide gene was removed. Specifically: 1) After DB3.1 was melted on ice, the target DNA (i.e., the PCR product after gel recovery) was added, and the content did not exceed 1 / 10 of the total volume of DB3.1 in the tube, and the bottom of the EP tube was gently tapped by hand to mix it thoroughly, and it was placed in ice for 25 minutes; 2) The EP tube was placed in a 42°C water bath for 45 seconds, and quickly returned to ice for 2 minutes; 3) 700 μL of no Sterile LB liquid culture medium containing antibiotics, mix well and resuscitate at 37℃, 200rpm for 60min; 4) Centrifuge at 5000rpm for 1min to collect bacteria, keep 100μL supernatant, gently blow to resuspend the bacteria and spread it on LB solid culture medium containing corresponding antibiotics; 5) Invert the plate in a 37℃ incubator and culture overnight for 36h; 6) After the plaque grows, pick out a single clone and mix it in LB culture medium added with Kan, and culture it at 220rpm at 37℃ overnight for plasmid extraction. For detailed steps of plasmid extraction, please refer to the instructions of Tiangen Plasmid Extraction Kit.

[0049] The extracted PMDC32 plasmid was double-digested with Asc I and SaI I restriction sites respectively; 17 μL μg DNA, 5 μL 10×Customer Buffer, 1 μL Asc I restriction endonuclease, 1 μL SaII restriction endonuclease and 33 μL Nuclear-free water were added to enzyme-free PCR tubes respectively; the above system was linearized at 37°C for 30 min, heat-inactivated at 80°C for 25 min, placed on ice until cooled and then connected to the target fragment.

[0050] 3. Plasmid recombination

[0051] The recovered target fragment and the enzyme-cut plasmid vector were recombined by recombinase, and 1μL linear vector, 4μL target fragment, and 5μL 2×Mix were added to the enzyme-free PCR tube respectively, and the sample addition process was carried out on ice; the above system was connected at 50℃ for 30min, and E. coli transformation was carried out after the reaction was completed; after DH5α melted in ice, it could be directly transformed, which was consistent with the DB3.1 transformation process (the competent state was changed from DB3.1 to DH5α); after the plaque grew, the bacteria were tested by electrophoresis, and the bacteria test system was: 2×Taq Mix 5μL; Forward Primer 10μM 0.4μL; Reverse Primer 10μM 0.4μL; Template DNA 1μL; ddH2O 4.2μL. The bacteria test procedure was: 95℃ 3min; 95℃ 15s, 57℃ 15s, 72℃ 60s, a total of 30 cycles; 72℃ 5min. The products were tested by electrophoresis, and qualified plaques were marked and sent to Qingke Biotechnology Co., Ltd. for sequencing. Sequence comparison was performed through DNAMAN, and bacterial liquid and plasmids consistent with the target gene sequence were returned.

[0052] 2. Agrobacterium transformation, preparation of infection solution and genetic transformation of poplar leaves

[0053] 1. Agrobacterium transformation

[0054] After the GV3101 Agrobacterium competent cells are thawed, the returned plasmid can be directly transformed with Agrobacterium, specifically: 1) take the GV3101 competent cells and wait for them to partially melt at room temperature, and insert them into ice when they are in an ice-water mixed state; 2) for every 100 competent cells + (0.01-1) μg plasmid DNA (3 μL is added in this embodiment); 3) add 700 μL LB liquid culture medium without Kan and Rif, and culture at 28°C, 220rpm for 3h; 4) collect the bacterial solution by centrifugation at 6000rpm for 1min, retain 100 μL of supernatant, gently blow and resuspend the bacterial block, and spread it on an LB plate containing the corresponding antibiotics, invert it and culture it at 28°C for about 60h.

[0055] 2. Preparation of infection solution

[0056] After the plaques grow, the monoclonal strains are tested again. The test system and procedure are the same as those in Example 1, PIP1;1 Gene cloning, vector enzyme digestion recovery and plasmid recombination, 3, plasmid recombination; the monoclonal strains with qualified test results are picked and pipetted into LB liquid culture medium containing 50 mg / L Kan and 20 mg / L Rif. After expansion culture at 28°C and 250 rpm for 12 hours, 500 μL of the bacterial solution is taken and added to 100 ml of LB liquid culture medium to continue expansion culture in a tissue culture bottle. When the bacterial solution OD 600 When the value reaches 1.2-1.8, stop shaking and resuspend with improved AAM resuspension medium: take 50ml of bacterial solution into a sterile centrifuge tube, centrifuge at 12000rpm for 10min, discard the supernatant, add 50ml of improved AAM resuspension medium, dilute the bacteria with a sterile pipette tip until the precipitate dissolves, pour it back into the tissue culture bottle, and add 50ml of improved AAM until the OD reaches 1.2-1.8. 600 When the concentration of AS was 0.6-0.9, 200 μmol / L AS was added to prepare the infection solution, which was used to infect the sterile leaves of poplar. The formula of the improved AAM resuspension medium is shown in Table 2.

[0057] 3. Genetic transformation of poplar leaves

[0058] During the expansion culture period, the poplar tissue culture seedlings that were 45-55 days old and in good condition were taken, the young leaves at the top were taken as plant materials, the main veins and leaf tips were removed, 4-5 wounds were made at the veins, and the leaves were placed in MS differentiation medium for pre-culture for about 24 hours. The formula of MS differentiation medium is shown in Table 1.

[0059] The pre-cultured leaves were taken out and placed in the infection solution for 15 minutes, during which they were shaken for 1 minute every 3 minutes. After 15 minutes of infection, the leaves were taken out, the excess bacterial solution was absorbed with filter paper, and they were put back into the MS differentiation medium with the back of the leaves facing up and inverted in the dark for 3 days. The formula of the MS differentiation medium is shown in Table 1. The state of the leaves during this process is as follows: Figure 1 As shown in A.

[0060] Take out the leaves in the MS differentiation medium, put them in a tissue culture bottle, rinse them with sterile water for 6 times, and then rinse them once with water containing 400 mg / L Carb to thoroughly rinse out the excess bacteria and inhibit their excessive growth while Agrobacterium is transferred. After rinsing, carefully take out the leaves with tweezers to avoid tearing the leaves from the scratches.

[0061] The leaves were dried with filter paper and transferred to MS selective medium, and cultured for 7 days under 16 h light and 8 h dark conditions; wherein the formula of MS selective medium is shown in Table 1.

[0062] The leaves in the selection medium were transferred to the MS screening medium containing Hyg. The MS screening medium was changed every 10 days. After about one change of the MS screening medium, buds appeared. Figure 1 As shown in B; after replacing the MS screening medium twice, the buds grew to 0.5-1.5 cm. Figure 1 As shown in C, when the MS screening medium is replaced for the third time, it is transferred to the rooting medium, such as Figure 1 As shown in D; roots formed after about 10 days in the rooting medium, as shown in Figure 1 As shown in E; wherein, the formula of MS screening medium and rooting medium is shown in Table 1. When the rooted seedlings grow to 5 cm, tissue culture propagation and positive identification can be carried out, such as Figure 1 As shown in F.

[0063] 3. PCR identification of transformed strains, expression level detection of transformed strains and phenotypic observation of positive strains

[0064] 1. PCR identification of transformed strains

[0065] About 100 mg of young leaves can be ground into powder for positive identification, genomic DNA can be extracted and the PCR products of each transformed strain can be detected by electrophoresis. The detailed steps of DNA extraction refer to the instructions of NuClean Plant Genomic DNA kit, which are as follows: 1) Take 100 mg of leaves and add liquid nitrogen to fully grind into powder; 2) Add 400 μL Buffer LP1 and 6 μL RNase A, vortex for 1 minute, and leave at room temperature for 10 minutes; 3) Add 130 μL Buffer LP2, mix well, and vortex for 1 minute; 4) Centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube; 5) Add 1.5 times the volume of Buffer LP3 (with anhydrous ethanol added) and mix well; 6) Add all the solution and precipitate to the adsorption column loaded in the collection tube, centrifuge at 12000 rpm for 1 minute, discard the waste liquid and put the adsorption column back; 7) Add 500 μL Buffer to the adsorption column GW2, centrifuge at 12000rpm for 1min, discard the waste liquid and put the adsorption column back; 8) repeat step 7); 9) centrifuge at 12000rpm for 2min, discard the waste liquid and place the adsorption column at room temperature to dry; 10) place the adsorption column in a new centrifuge tube, add 80μL Buffer GE in mid-air, place at room temperature for 5min, centrifuge at 12000rpm for 1min, and collect the DNA solution.

[0066] The plasmid was used as a positive control, the non-transformed poplar strain (WT) was used as a negative control, and ddH2O was used as a blank control for positive identification; the stem segment was used as an explant for propagation, and the method used was plant tissue culture, and the formula was shown in Table 1; after about 30 days, a new complete plant was formed, such as Figure 1 G in the figure. The PCR products of each transformed strain were detected by electrophoresis, with the plasmid as the positive control, the non-transformed poplar strain (WT) as the negative control, and ddH2O as the blank control. The positive identification primer sequence was: upstream primer sequence 5'-GGAGGGCAAAGAAGAAGATGTTAGA-3', downstream primer sequence 5'-GATCCCTTGAATTCCAACCGTTG TG-3', and the PCR reaction system was: 2×Taq Mix 12.5μL; Forward Primer 10μM 1μL; Reverse Primer 10μM 1μL; Template DNA 1μL; ddH2O 9.5μL. The reaction program was: 95℃3min; 95℃ 15s, 58℃ 15s, 72℃ 60s, a total of 30 cycles; 72℃ 5min.

[0067] The identification results are as follows Figure 2 As shown; among them, among the 8 strains identified in this example, 5 were preliminarily determined to be positive strains, namely strains No. 1, No. 2, No. 4, No. 5 and No. 6, and the positive rate was as high as 62.5%.

[0068] 2. Expression level detection of transformed strains

[0069] About 100 mg of young leaves were ground into powder to extract total RNA. The detailed steps of RNA extraction refer to the instructions of OminiPlant RNA Kit (DNase I) of Kangwei Century RNA Extraction Kit; the quality of RNA was detected by electrophoresis, and the qualified RNA was used for reverse transcription and synthesis of cDNA. The detailed steps 1)-3) were consistent with the steps 1)-3) of Example 1, PIP1;1 gene cloning, vector enzyme digestion recovery and plasmid recombination, and the reaction conditions of step 4) were incubation at 42°C for 15 min and incubation at 85°C for 5 min; 5) after the reaction, centrifuge briefly and cool on ice.

[0070] Actin was used as the internal reference gene and 84K poplar (Populus alba x Populus glandularis) cDNA was used as the sample control. Real-time RT-qPCR experiment was completed by CFX96 quantitative PCR instrument to determine the expression characteristics of each strain, and each sample was repeated at least 3 times. Among them, the primer sequence of Actin was: upstream primer sequence 5'-GAAGTCCTCTTCCAGCCTTCTC-3', downstream primer sequence 5'-CTTGATCTTCATGCTGCTTGGG-3'; the primer sequence of the transformed strain was: upstream primer sequence 5'-CAAGAAGTGATGATTGTGATGC-3', downstream primer sequence 5'-ACGTCACTGTTATAGCACGC-3'; the reaction system was: 2×UltraSTBR Mixture 10μL; Forward Primer 10μM0.5μL; Reverse Primer 10μM 0.5μL; Template DNA 1μL; ddH2O 8μL. The reaction program was: 95°C 45 s; 95°C 15 s, 60°C 15 s, 72°C 45 s, for a total of 40 cycles.

[0071] According to R=2 -ΔΔCT Calculate the expression multiples of the transformed strains and the non-transformed strains; screen and identify two transgenic strains with high expression levels, such as Figure 3 As shown ( Figure 3 The figure is the result of real-time fluorescence quantitative PCR detection of each strain in the embodiment of the present invention (the expression multiple ratio of each strain to WT). It is used as an alternative material for potted test together with the non-transformed strain and propagated. The method adopted for propagation is plant tissue culture. The medium formula used for the first and second propagation is consistent with the rooting medium formula in Example 1, II, Agrobacterium transformation, preparation of infection solution and genetic transformation of poplar leaves, 3, and genetic transformation of poplar leaves. The rooting medium formula for the third and subsequent times is the rooting medium without Hyg. The results show that the expression level of strain No. 5 is the highest, which is 6.04 times that of WT; the expression level of strain No. 2 is second, which is 4.31 times that of WT, so strain No. 5 and strain No. 2 are selected for tissue culture propagation.

[0072] 3. Phenotypic observation of positive strains

[0073] After the explants grew for about 35 days, 10 tissue culture rooted seedlings with consistent growth and good condition were selected from each strain as transgenic test materials for pot experiments, of which No. 5 was OE1 and No. 2 was OE2. When the candidate materials for the pot experiment grew to 5 cm, the rooted seedlings with consistent growth morphology and good condition were selected for seedling hardening in a light incubator. On the first day, the tissue culture bottle cap was unscrewed once, on the second day, it was unscrewed twice, on the third day, the bottle cap was completely unscrewed and placed upside down on the bottle mouth, and transplanted on the fourth day; the non-transformed poplars and transgenic poplars were hardened and transplanted into plastic pots filled with mixed soil (fine sand: nutrient soil = 2:1), and after growing in the greenhouse for 50 days, drought stress tests were carried out to analyze the drought resistance of transgenic strains; after 20 days of drought stress treatment, the phenotypes were observed, and the potted phenotypes after treatment were as follows: Figure 4 After drought stress treatment, all leaves of the non-transformed lines showed obvious wilting, while only the lower leaves of the transformed lines OE1 and OE2 became chlorotic and drooping, while the upper leaves showed good growth status.

[0074] Comparative Example 1

[0075] The same as Example 1, except that the improved AAM resuspension medium in the preparation of Agrobacterium infection solution in Example 1 was replaced with LB liquid medium and the AAM medium before improvement (PM1961 AAM medium base salt (containing microorganisms, not containing acetosyringone, product number PM1961-50L) purchased by the company, respectively. The budding state of the leaves after replacing the MS screening medium twice in the genetic transformation of poplar leaves is as follows Figure 5 As shown, Figure 5 A is the budding state after resuspension in LB liquid medium, Figure 5 B is the budding state after resuspension using the AAM medium purchased by the company. As can be seen from the figure, compared with the leaves cultured in the improved AAM resuspension medium ( Figure 1 C), the budding rate and positive rate of leaf blade at the same stage after LB liquid medium resuspending are lower than the budding rate and positive rate of improved AAM resuspending medium, and the bud growth rate is slower than the bud growth rate after improved AAM resuspending medium infection. The positive rate of transformed strain after using LB liquid medium resuspending and the improved AAM medium purchased by the company is only 30-40%. In summary, the improved AAM resuspending medium can accelerate the growth of buds and improve budding rate and positive rate.

[0076] Comparative Example 2

[0077] The same as Example 1, except that the culture medium at each stage of the genetic transformation step of poplar leaves in Example 1, II. Agrobacterium transformation, preparation of infection solution and genetic transformation of poplar leaves was replaced with a culture medium without TDZ. The budding state of the leaves after replacing the screening culture medium twice was as follows: Figure 6 As shown, compared with leaves cultured in the medium supplemented with TDZ ( Figure 1 C), the budding rate and positive rate of leaves cultured in the medium without TDZ at the same stage were lower than those in the medium containing TDZ. The positive rate of the transformed strain was 42.9%. In summary, TDZ can increase the budding rate and positive rate, but has no significant effect on the growth rate of the buds.

[0078] Comparative Example 3

[0079] The method is basically the same as Example 1, except that the genetic transformation of poplar leaves in Example 1, II. Agrobacterium transformation, preparation of infection solution and genetic transformation of poplar leaves, is co-cultured for 3 days and then washed with sterile water, without the need to rinse with water containing 400 mg / L Carb, and the culture medium at each stage is replaced with a culture medium without Carb. The growth status of the transformed strain is shown in FIG. Figure 7 As shown in the figure, it can be seen that the transformed strains that removed Carb during the genetic transformation process had residual Agrobacterium, and the positive rate did not decrease significantly, while the transformed strains that used Carb water-washed bacteria could grow normally, and there was no Agrobacterium residue in the tissue culture bottle ( Figure 1 In summary, Carb can effectively inhibit the excessive growth of Agrobacterium.

[0080] Comparative Example 4

[0081] The method is basically the same as Example 1, except that the resuspended culture medium in the preparation of the Agrobacterium infection solution in Example 1 is replaced with LB liquid culture medium, and the culture medium at each stage in the genetic transformation steps of poplar leaves in Example 1, II. Agrobacterium transformation, preparation of infection solution and genetic transformation of poplar leaves is replaced with a culture medium without TDZ, and after 3 days of co-cultivation, the bacteria are washed with sterile water only, without the need to use water containing 400 mg / L of Carb for washing, and the culture medium at each stage is replaced with a culture medium without Carb. The state of the leaves after the MS screening culture medium is replaced twice is as follows: Figure 8 As shown, there were no obvious buds on the leaves without AAM resuspension medium, TDZ and Carb, and the budding rate and positive rate were 0.

[0082] In summary, the genetic transformation results of the present invention show that buds can grow about 20 days after leaf infection, without long-term callus differentiation, and the PCR identification positive rate of the transformed strain is as high as 62.5%. The present invention shortens the transformation cycle and improves the transformation efficiency and positive rate.

Claims

1. A method for cultivating drought-resistant gene-transgenic poplars, characterized in that: The following steps are involved: 1) Construction of plant expression vector of poplar drought stress tolerance gene; 2) transferring the constructed plant expression vector of the poplar drought stress tolerance gene into Agrobacterium; 3) Resuspend the recombinant Agrobacterium containing the poplar drought stress tolerance gene in the improved AAM resuspension medium until the OD 600 When the pH value is 0.6-0.9, 200 μmol / LAS is added to prepare the Agrobacterium infection solution; the formula of the improved AAM resuspension culture medium is: sucrose 68500 mg / L+glucose 36000 mg / L+KCl 3000 mg / L+glutamine 900 mg / L+acid hydrolyzed casein 500 mg / L+MgSO4 250 mg / L+KH2PO4 185 mg / L+arginine 176 mg / L+CaCl2·2H2O 150 mg / L+inositol 100 mg / L+FeNaEDTA 36.7 mg / L+VB1 10 mg / L+MnSO4·H2O 7.58 mg / L+glycine 7.5 mg / L+H3BO33 mg / L+ZnSO4·7H2O 2 meg / L+nicotinic acid 1 mg / L+VB6 1 mgg / L+KI 0.8mgg / L+Na2 MoO4·2H2O0.25mg / L+CoCl2·6H2O 0.025mg / L+CuSO4·5H2O 0.025mg / L; 4) Select poplar tissue culture seedlings with good growth status, take the young leaves at the top as plant materials, remove the main veins and leaf tips, perform wound treatment on the veins, and place them in MS differentiation medium for pre-culture for 24 hours; take out the pre-cultured leaves, place them in the infection solution for infection, take out the leaves after the infection, dry them with filter paper, put them back in MS differentiation medium, and invert them in the dark for 3 days; 5) Take out the leaves from the MS differentiation medium, rinse them with sterile water, then rinse them with water containing Carb, dry them with filter paper, and transfer them to the MS selection medium for 7 days; 6) The leaves in the MS selection medium were transferred to the MS screening medium for culture. The screening medium was changed every 10 days. During this period, buds were grown directly without callus tissue. When the medium was changed for the third time, the leaves were transferred to a sterile tissue culture bottle containing a transformation plant rooting medium to obtain drought-resistant gene-transfected poplar seedlings; In step 1), the poplar drought stress tolerance gene used is Potri .008G065600; In step 2), the Agrobacterium used is GV3101 Agrobacterium; In step 4), the formula of the MS differentiation medium is: MS+NAA0.05mg / L+6-BA0.5mg / L+TDZ0.01mg / L+sucrose 30g / L+phytagel 4.2g / L+AS 200μmol / L, pH=5.8-6.0; In step 5), the leaves in the MS differentiation medium were taken out, rinsed with sterile water for 5-6 times, and then rinsed with water containing 400 mg / L Carb.

2. The method for cultivating drought-resistant gene-transformed poplars according to claim 1, characterized in that: In step 4), select poplar tissue culture seedlings that are 45-55 days old and in good condition.

3. The method for cultivating drought-resistant gene-transformed poplars according to claim 1, characterized in that: In step 4), the infection time is 15 minutes, and shaking is performed for 1 minute every 3 minutes during the infection.

4. The method for cultivating drought-resistant gene-transformed poplars according to claim 1, characterized in that: In step 5), the MS selection medium formula is: MS+NAA 0.05mg / L+6-BA 0.5mg / L+TDZ 0.01mg / L+sucrose 30g / L+phytagel 4.2g / L+AS 200μmol / L+Carb 400mg / L, pH=5.8-6.0; the culture conditions are: culture for 7 days under 16h light and 8h dark conditions.

5. The method for cultivating drought-resistant gene-transformed poplars according to claim 1, characterized in that: In step 6), the formula of the MS screening medium is MS+NAA 0.05mg / L+6-BA 0.5mg / L+TDZ 0.01mg / L+sucrose 30g / L+phytagel 4.2g / L+AS 200μmol / L+Carb 400mg / L+Hyg 1.5mg / L, pH=5.8-6.

0.

6. The method for cultivating drought-resistant gene-transgenic poplars according to claim 1, characterized in that: In step 6), the formula of the transformation plant rooting medium is 1 / 2MS+NAA 0.05 mg / L+sucrose 30 g / L+agar 7 g / L+Hyg 1.5 mg / L, pH=5.8-6.0.

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