CN114940997B - Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth - Google Patents

Abstract

The invention discloses the application of GmBBE-like43 gene in regulating plant adaptation to low phosphorus and acid aluminum stress and promoting growth. The research of the present invention shows that the cell wall protein GmBBE-like43 is induced to up-regulate expression in soybean roots by aluminum stress and low phosphorus stress; under the conditions of different concentrations of phosphorus treatment and aluminum treatment, the excessive expression of GmBBE-like43 significantly promotes the growth of transgenic soybean hairy roots in vitro. and Arabidopsis growth; GmBBE‑like43 gene has the function of positively regulating soybean or Arabidopsis root system to adapt to low phosphorus stress and aluminum toxicity, thereby promoting root growth; at the same time, GmBBE‑like43 gene has the function of regulating Arabidopsis root growth. Therefore, GmBBE‑like43 plays an important role in plant adaptation to low phosphorus and acid aluminum stress, and can be used to regulate plant adaptability to acid soil low phosphorus and acid aluminum stress through transgenic technology.

Description

GmBBE-like43 gene regulates plant adaptation to low phosphorus and aluminum stress and promotes growth application in

technical field

The invention belongs to the technical field of genetic engineering. More specifically, it relates to the application of GmBBE-like43 in regulating plant adaptation to low phosphorus and acid aluminum stress and promoting growth.

Background technique

Acidic soils are widely distributed in the world. The total area of acidic soils in the world accounts for 30-40% of the world's cultivated land, and more than 50% of the potential cultivated land is acidic soils, mainly distributed in tropical, subtropical and temperate regions. The inhibitory effect of acidic soil on crop growth is not only manifested in the damage of crops itself due to excessive H ⁺ concentration, but also often manifested in the synergistic inhibition of plant growth and yield by the lack of available phosphorus and aluminum toxicity. Therefore, improving crop tolerance to low phosphorus stress and aluminum toxicity in acidic soil through genetic improvement is considered to be an important way to develop sustainable agriculture.

During the long-term evolution process, plants have formed a series of morphological, physiological and molecular cooperative adaptive mechanisms to overcome obstacles such as available phosphorus deficiency and aluminum toxicity in acidic soils. According to reports, the mechanism of plants adapting to aluminum toxicity mainly includes internal tolerance and efflux mechanisms (for example, the secretion of organic acids); the mechanism of adapting to low phosphorus stress mainly includes changing the morphological configuration of roots and increasing the secretion of organic acids in roots. As well as increasing the enzyme activity of purple acid phosphatase, inducing the expression of high-affinity phosphorus transporters and forming symbiosis with rhizosphere microorganisms such as mycorrhizal fungi (Raghothama, 1999; Vance et al., 2003; Liang et al., 2014). Since available phosphorus deficiency and aluminum toxicity coexist in acidic soil, it suggests that plants may have a common adaptive mechanism to overcome available phosphorus deficiency and aluminum toxicity. Early studies have found that there are 28 BBE-Likes proteins in Arabidopsis, among which argoline peroxidase AtBBE-Like15 is involved in the synthesis of plant cell wall lignin (Daniel et al., 2015). Later studies reported that there are four peroxidase proteins AtBBE-Like1/2/20/21 in Arabidopsis that can oxidize oligogalacturonic acid OGs to generate H ₂ O ₂ , and they are named OGOX4/ 3/1/2 (Benedetti et al., 2015).

Soybean is an important economic crop and plays a very important role in my country's agricultural production. It is a traditional leguminous crop that is used for both grain, oil and feed. At present, in the report of soybean berberine family protein-related (binding berberine family protein-related, BBE), the specific function of its protein has not been studied. Although in the existing research, in the soybean BBE-Likes protein family, two BBE-Likes proteins in response to aluminum stress and one BBE-Like protein in response to low phosphorus stress were found (Wu et al., 2018; Zhao et al. , 2020), but so far the specific function and role of the BBE-Likes protein family gene in soybean has not been studied and analyzed; the key genes related to the soybean genotype in southern acidic soil have not been reported yet.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above problems, and provide the application of the soybean GmBBE-like43 gene in regulating plant adaptation to low phosphorus and acid aluminum stress and promoting growth.

The first object of the present invention is to provide the application of the GmBBE-like43 gene and its protein.

The second object of the present invention is to provide a product that promotes plant growth and/or improves plant tolerance to low phosphorus and/or aluminum toxicity stress.

The third object of the present invention is to provide a method for promoting plant growth and/or improving plant tolerance to low phosphorus and/or aluminum toxicity stress.

The above object of the present invention is achieved through the following technical solutions:

The present invention found a cell wall protein GmBBE-like43 that was significantly up-regulated at the protein level by low phosphorus stress in the results of proteomic analysis of soybean roots treated with high and low phosphorus, and selected the GmBBE-like43 gene as a candidate gene for our research, and then passed qRT- PCR verification found that GmBBE-like43 was also significantly up-regulated by low phosphorus stress at the gene transcription level, and was also significantly up-regulated by aluminum stress. Subsequently, the specific function of GmBBE-like43 gene in the process of soybean synergistic response to low phosphorus stress and aluminum toxicity in acidic soil was analyzed and studied.

The present invention clones a gene GmBBE-like43 that is synergistically regulated by exogenous phosphorus and aluminum by means of real-time fluorescent quantitative PCR and homologous cloning. Then, soybean isolated hairy roots and Arabidopsis transgenic plant materials with excessive and suppressed GmBBE-like43 expression were obtained through soybean isolated hairy root transformation and Arabidopsis transgenic technology. The results showed that GmBBE-like43 gene has the ability to regulate soybean root system to adapt to low phosphorus stress and aluminum toxicity to promote root growth; GmBBE-like43 gene has the function of regulating Arabidopsis root growth and regulating Arabidopsis root adaptation to low phosphorus stress and aluminum toxicity to promote root growth.

Therefore, the present invention provides the GmBBE-like43 gene shown in SEQ ID NO: 1 or the GmBBE-like43 protein shown in SEQ ID NO: 2 or its expression promoter in positively regulating the ability of plant roots to tolerate low phosphorus and/or aluminum toxicity stress and/or Or root growth, promoting plant root growth, improving plant root tolerance to low phosphorus and/or aluminum toxicity stress, cultivating plants resistant to low phosphorus and/or aluminum toxicity, preparing plant growth promoters or improving plant resistance Adaptation to acidic soils and/or application of acidic soil growth promoters.

The invention provides a product for promoting plant growth and/or improving plant tolerance to low phosphorus and/or aluminum toxicity stress, which contains a GmBBE-like43 protein expression promoter.

The invention provides a method for promoting plant growth and/or improving plant tolerance to low phosphorus and/or aluminum toxicity stress, through gene editing technology, positive regulation of GmBBE-like43 gene expression level or protein activity in plants to promote plant root system Growth and/or increased plant tolerance to low phosphorus and/or aluminum toxicity stress.

Preferably, by overexpressing the GmBBE-like43 gene in the plant, the plant root growth is promoted and/or the plant's tolerance to low phosphorus and/or aluminum toxicity stress is improved.

Preferably, an expression vector for overexpressing the GmBBE-like43 gene is constructed, and the plants are transformed to obtain transgenic plants that promote plant root growth and/or improve plant tolerance to low phosphorus and/or aluminum toxicity stress.

The present invention has the following beneficial effects:

The invention discloses the application of soybean GmBBE-like43 gene in regulating plant adaptation to low phosphorus and acid aluminum stress and promoting growth. The research of the present invention shows that the expression of GmBBE-like43 gene is induced and up-regulated by aluminum stress, and its expression is up-regulated by low phosphorus stress, and its expression level increases obviously with the prolongation of phosphorus treatment time. Under different phosphorus concentration treatment conditions, overexpression of GmBBE-like43 significantly increased the biomass of transgenic plants; at the same time, under aluminum treatment conditions, overexpression of GmBBE-like43 significantly increased the growth rate of transgenic plants, indicating that GmBBE-like43 positively regulates The ability of plant roots to adapt to low phosphorus and acid aluminum stress. Therefore, GmBBE-like43 plays an important role in plant adaptation to low phosphorus and acid aluminum stress, and can be used to regulate the adaptability of plants to acid soil low phosphorus and acid aluminum stress through transgenic technology.

Description of drawings

Fig. 1 is the pattern analysis of soybean GmBBE-like43 (A: the effect of low phosphorus treatment time on the expression pattern of GmBBE-like43 in soybean root system; B: the effect of aluminum treatment time on the expression pattern of GmBBE-like43 in soybean root tip; the data is 3 Mean and standard error of repeated times, asterisks indicate significant difference between control (+P/-Al) and treatment (-P/+Al) (Student'st-test), *: P<0.05, **: P<0.01, ***: P<0.001);

Figure 2 is the tissue localization and subcellular localization analysis of GmBBE-like43 (A: Histochemical localization analysis of GmBBE-like43 in Arabidopsis thaliana, the scale of the first row of above-ground pictures is 2mm, and the scale of the last two rows of root pictures is 0.5 mm; B: The subcellular localization analysis results of GmBBE-like43 fusion GFP protein in tobacco leaves; C: The subcellular localization analysis results of GmBBE-like43 fusion GFP protein in kidney bean hairy roots; the first row in Figures B and C is transformation The subcellular localization map of tobacco or bean with empty vector (35S::GFP); the second row is the subcellular localization map of GmBBE-like43 fusion GFP protein in tobacco leaf or bean hairy root (35S::GmBBE-like43-GFP); The pictures in B are the observation of the green fluorescent channel (GFP), the light mirror channel (bright field) and the overlapped picture (fusion) under the laser confocal microscope, and the pictures in C are the green fluorescent channel under the laser confocal microscope (GFP), red fluorescent channel (PI staining) and overlapping pictures (fusion) observation, the scale bar is 20 μm);

Figure 3 shows the effects of excessive or inhibited GmBBE-like43 expression on the growth of transgenic soybean hairy roots in vitro under aluminum treatment conditions (A, B: GmBBE-like43 in the empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX-CK/RNAi-CK) /RNAi) expression pattern analysis; C: phenotypes of empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) under aluminum treatment conditions, scale bar = 2cm; D, F: empty Growth of control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); E, G: Relative growth rate of empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) ; Asterisks indicate significant differences between empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) (Student's t-test); *: P<0.05, **: P<0.01, ** *: P<0.001);

Figure 4 is the effect of excessive or inhibited GmBBE-like43 expression on the growth of transgenic soybean hairy roots in vitro under high and low phosphorus conditions (A, B: GmBBE-like43 in the empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX-CK) /RNAi) expression pattern analysis; C: phenotypes of empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) under high and low phosphorus treatment conditions, scale bar = 2cm; D, F: empty Dry weight of control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); E, G: total roots of empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) Long; asterisks indicate significant differences between empty control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) (Student's t-test), *: P<0.05, **: P<0.01, * **: P<0.001);

Figure 5 is the effect of overexpressing GmBBE-like43 on the growth of transgenic Arabidopsis (A: GmBBE-like43 expression pattern analysis in wild type (WT) and transgenic Arabidopsis plants (OX1, OX2); B: wild type Phenotypes of (WT) and transgenic Arabidopsis lines (OX1, OX2) under aluminum treatment conditions, the scale bar in the figure is 0.5cm; C: wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) Root growth under aluminum treatment conditions; D: relative root growth rate of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) under aluminum treatment conditions; E: wild type (WT) and transgenic Arabidopsis lines Phenotypes of Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments, the scale bar in the figure is 1cm; F: Roots of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments Fresh weight; G: Taproot length of wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments; asterisks indicate wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2 ) were significantly different (Student'st-test), *: P<0.05, **: P<0.01, ***: P<0.001).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Expression pattern analysis of embodiment 1GmBBE-like43 gene

The present invention found a cell wall protein GmBBE-like43 that was significantly up-regulated by low-phosphorus stress in the results of proteomic analysis of high- and low-phosphorus-treated soybean roots, so the GmBBE-like43 gene was selected as a candidate gene for our research, and then through qRT- PCR verification found that GmBBE-like43 was also significantly up-regulated by low phosphorus stress at the gene transcription level, and was also significantly up-regulated by aluminum stress. Subsequently, the specific function of GmBBE-like43 gene in the process of soybean synergistic response to low phosphorus stress and aluminum toxicity in acidic soil was analyzed and studied.

1. Effects of low phosphorus stress on the expression pattern of GmBBE-like43 in soybean roots

Using the rolling paper seedling method, select seeds with undamaged seed coats and uniform sizes, disinfect with chlorine gas generated by the reaction of 100ml sodium hypochlorite and 4.2ml hydrochloric acid for 12 hours, and then blow on the ultra-clean workbench for 1 hour for later use. Cut 20×20cm square filter paper, prepare 1/4 soybean complete nutrient solution with pH 5.8 and sterile water, and sterilize for use.

When carrying out roll paper cultivation, spread the plastic wrap on the test table, soak the filter paper with the prepared 1/4 soybean nutrient solution, and place 7 sterilized beans about 1cm away from one side of the filter paper, with the navel of the beans facing down, Roll from the first bean to the end. Put the end of the rolled filter paper with no beans facing down into a 500mL beaker filled with 1/4 of the complete soybean nutrient solution, and wrap the upper end of the filter paper rolled with beans with plastic wrap. Put the beaker into an incubator at 24-26°C, culture in the dark for 1 day, then light/dark (12h/12h) for 3-4 days until the radicle reaches 5-6cm.

Seedlings with consistent growth were selected and transferred to +P (250 μM KH ₂ PO ₄ ) and -P (5 μM KH ₂ PO ₄ ) nutrient solution, with 4 replicates for each treatment, and 8 seedlings for each replicate. The pH of the nutrient solution was adjusted to about 5.8 every two days, and the nutrient solution was changed once a week. The root samples were harvested on the 3d, 6d, 9d and 12d respectively, frozen in liquid nitrogen, and stored in a -80°C refrigerator for future use.

2. Effect of aluminum treatment on the expression pattern of GmBBE-like43 in soybean root tips

Using the rolling paper seedling method, culture for 3-4 days until the radicle is 5-6 cm, select the seedlings with consistent growth, and transfer them to -Al (pH 4.2, 0.5mM CaCl ₂ ) and +Al (pH 4.2, 50μM AlCl ₃ , 0.5mM CaCl ₂ ) solution, the soybean root tip (0-2 cm) samples were harvested after 12, 24, 48, 72 and 96 hours of treatment, were quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator until use.

3. Real-time fluorescent quantitative PCR (qRT-PCR) analysis

The total RNA of the above-mentioned treated plant samples was respectively extracted using TRIzol kit (Invitrogen, USA). The RNA treated with DNase I was synthesized into cDNA by reverse transcription using the MMLV-reverse transcription kit (Promega, USA). The SYBR (Promega, USA) kit was used for qRT-PCR analysis. After the reverse transcription was completed, the sample was diluted 15 times and then analyzed by real-time fluorescent quantitative PCR by Applied Biosystems StepOnePlus Real-Time PCR system.

Preparation of standard curve: First, pipette 1-2 μL sample from the cDNA stock solution of each sample into a new PCR centrifuge tube, then dilute the mixed solution 10 times to the first standard sample S1, and then the first standard sample Dilute 10 times as the second standard sample S2, and so on to dilute to 5 concentration gradient standard samples, namely S1, S2, S3, S4, S5.

Quantitative PCR primers use GmBBE-like43 gene quantitative primers and the internal reference gene is the housekeeping gene primer of soybean as follows:

GmBBE-like43-RT1-F (SEQ ID NO.3): 5'-CGTGAACCATTCTGAGCCTTC-3';

GmBBE-like43-RT1-R (SEQ ID NO.4): 5'-AATGGAACCTCTGCCACGTAAG-3';

EF1-α-F (SEQ ID NO.5): 5'-TGCAAAGGAGGCTGCTAACT-3';

EF1-α-R (SEQ ID NO. 6): 5'-CAGCATCACCGTTTCTTCAAA-3'.

Prepare the reaction mixture: add 10 μl SYBR Premix Ex Taq (2×), 0.5 μl forward/reverse primers, 7 μl ddH ₂ O and 2 μl template to every 20 μl reaction system. First calculate the required reaction volume, mix the reagents except cDNA, aliquot 18 microliters into each tube, and then add 2 microliters of cDNA template to make the final volume 20 microliters.

The quantitative PCR reaction program was: pre-denaturation at 95°C for 30 seconds, PCR reaction (denaturation at 95°C for 30 seconds, renaturation at 60°C for 15 seconds, extension at 72°C for 30 seconds) for 40 cycles. The detection results were calculated by using Rotor-Gene's Real-Time Analysis Software to calculate the expression level of each sample.

The results are shown in Figure 1. Within 96 hours of aluminum treatment, the expression of GmBBE-like43 was up-regulated by aluminum stress, and its expression level showed a trend of increasing first and then decreasing with the prolongation of aluminum stress time, among which GmBBE-like43 was in The expression level was the highest at 12h, followed by 24h, which were 3.8 times and 25.5 times that of the control treatment respectively (Fig. 1A). In addition, under high and low phosphorus treatment conditions, compared with high phosphorus treatment, low phosphorus treatment for 3 days had no significant effect on the expression level of GmBBE-like43. However, after 6 days of low phosphorus treatment, the expression level of this gene was significantly up-regulated by low phosphorus stress, and with the extension of phosphorus treatment time, its expression level increased significantly. That is, compared with the normal phosphorus condition, the expression levels of the low phosphorus treatment on the 6th, 9th and 12th day were 2, 3.1 and 5.6 times that of the control treatment, respectively (Fig. 1B).

Example 2 GmBBE-like43 histochemical localization and subcellular localization analysis

1. Histochemical localization analysis of GmBBE-like43

(1) According to the GmBBE-like43 gene sequence, design specific primers pGmBBE-like43::GUS-F (SEQ ID NO.7): 5'-CGGAATTCCACGATGGAGTGCAAAAGCAT-3', pGmB BE-like43::GUS-R (SEQ ID NO. 8): 5'-AAGGATCCTTTGGCTTATCCCAATGATGAG-3'. Using the root DNA of soybean genotype YC03-3 as a template, PCR amplification was performed to amplify the 2000 bp sequence at the start codon of GmBBE-like43. The reaction conditions were pre-denaturation at 98°C for 5 minutes, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, renaturation at 72°C for 1-3 minutes (depending on the size of the fragment), this process was cycled 30 times, and extension at 72°C for 10 minutes.

II将PCR产物与限制性内切酶EcoR I和BamH I双酶切法切割的pTF102载体进行重组连接反应。反应体系为20μL，包含PCR产物6μL，线性化载体质粒8μL，用重组连接酶Exnase II 2μL，反应缓冲液4μL。将试剂混合物于37℃反应30min，将重组质粒转化大肠杆菌并进行测序，获得大豆pGmBBE-like43::GUS融合基因的植物表达载体。最后将目的载体进行GV3101农杆菌转化，检测阳性克隆并于-80℃保存菌液。(2) Vector construction: The PCR amplified product is subjected to gel electrophoresis, recovered and purified using a kit, and after the purified PCR product is obtained, a homologous recombination kit is used to

II The PCR product was subjected to a recombination ligation reaction with the pTF102 vector cleaved by restriction endonucleases EcoR I and BamH I. The reaction system is 20 μL, including 6 μL of PCR product, 8 μL of linearized vector plasmid, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer. The reagent mixture was reacted at 37° C. for 30 min, and the recombinant plasmid was transformed into Escherichia coli and sequenced to obtain a plant expression vector of soybean pGmBBE-like43::GUS fusion gene. Finally, the target vector was transformed with GV3101 Agrobacterium, the positive clones were detected and the bacterial liquid was stored at -80°C.

(3) Obtaining transgenic Arabidopsis: Inflorescence infection method was adopted. The main steps are: transfer the above constructed pGmBBE-like43::GUS vector plasmid into Agrobacterium GV3101, pick positive clones in 5mL YEP culture medium (containing spectacle and rifampicin), and culture overnight at 28°C; then transfer into 100mL YEP culture solution was expanded to OD ₆₀₀ of 1.6-2.0; then, centrifuged at 6000rpm for 10min, discarded the supernatant to collect the bacteria, resuspended the bacteria with an equal volume of 5% sucrose water or 1/2MS culture solution, and added 0.005- 0.02% SilwetL-77 was made into the transformation solution; when transforming, all the flocs of Arabidopsis thaliana (watered the day before the transformation to keep the plant moist) were immersed in the transformation solution for 1 min, and after taking it out, wipe off the excess transformation solution with filter paper, and then cover it to keep fresh The film was used to keep the plants moist, and the black bag was used to cover the dark culture for 18 hours, then transferred to normal culture conditions and cultivated until the seeds were harvested (transformation every 1 week during the cultivation period, a total of 3 transformations).

After the transformed Arabidopsis thaliana seeds were harvested from the T ₀ generation, about 100 μL of the seeds were taken for seed propagation identification. The specific steps are as follows: the whole operation is completed in the ultra-clean workbench, first rinse with 70% ethanol for 1 min, centrifuge to remove the ethanol, and then wash once with sterilized secondary water; then pre-wash once with 10% sodium hypochlorite , centrifuge to remove sodium hypochlorite, then shake and rinse with 1mL of 10% sodium hypochlorite for 5 minutes, centrifuge to remove sodium hypochlorite, rinse 5-6 times with sterile water; finally resuspend the seeds with sterile water, and spread the seeds evenly on the After 1 day of low-temperature treatment at 4°C, break dormancy, and move into a plant light incubator with a photoperiod of 16h/8h (light/dark) and a temperature of 22°C/20°C (day/night); about 2 weeks later , the normal surviving T1 generation seedlings were moved into the matrix to continue to grow, and after the plants grew up, a few leaves were picked for DNA extraction, and positive plants were identified by PCR.

(4) GUS staining of transgenic Arabidopsis: After the homozygous transgenic Arabidopsis was treated with high and low phosphorus for 6 days, the treated Arabidopsis was first taken out, washed 3 times with secondary water, and then fixed in 90% acetone for 30 minutes. Then wash with the prepared washing solution for 3 times, and place them in GUS staining solution (0.1M Na ₂ HPO ₄ /NaH ₂ PO ₄ , pH 7.2, 1mM X-Gluc), draw a vacuum for 20min, and then transfer to 37°C In a constant temperature incubator, protect from light for 6-8 hours. After the root system was colored, the hairy root was moved into 75% (v/v) ethanol for storage, and the GUS staining of the root system was observed and photographed under a stereomicroscope (Leica, Germany).

The results are shown in Figure 2A, no matter under low phosphorus or aluminum treatment conditions, the expression level of GmBBE-like43 promoter fusion GUS reporter gene in Arabidopsis thaliana was significantly higher than that in the control treatment, and mainly in the old leaves and lateral roots of Arabidopsis. and up-regulated expression in the main root tip.

2. GmBBE-like43 subcellular localization analysis

(1) Design specific primers GmBBE-like43-GFP-F (SEQ ID NO.9): 5'-CTCTAGCGCTACCGGTATGGGAGTCCTTTTCTTCTCA-3', GmBBE-like43-GFP-R (SEQ ID NO.10): 5'-CATGGTGGCGACCGGTCGACCCTTCCTATATGACAGCG-3 '. Using the root cDNA of soybean genotype YC03-3 as a template, the full-length ORF of soybean GmBBE-like43 gene was amplified. The reaction conditions were pre-denaturation at 98°C for 5 min, denaturation at 98°C for 30 s, annealing at 58°C for 30 s, annealing at 72°C for 1-3 min, this process was cycled 30 times, and extension at 72°C for 10 min.

II将PCR产物与经Age I酶切后的线性化载体pEGAD进行重组连接反应。反应体系为20μL，包含PCR产物6μL，pEGAD线性化载体质粒8μL，用重组连接酶Exnase II 2μL，反应缓冲液4μL。将试剂混合物于37℃反应30min，将重组质粒转化大肠杆菌并进行测序，无误后提取质粒。将35S::GmBBE-like43-GFP质粒转化农杆菌GV3101和发根农杆菌K599，检测无误后保存备用。(2) The PCR amplified product is recovered and purified by gel electrophoresis using a kit, and after obtaining the purified PCR product, use a homologous recombination kit

II The PCR product was subjected to a recombination ligation reaction with the linearized vector pEGAD digested by Age I. The reaction system was 20 μL, including 6 μL of PCR product, 8 μL of pEGAD linearized vector plasmid, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer. The reagent mixture was reacted at 37°C for 30 minutes, the recombinant plasmid was transformed into Escherichia coli and sequenced, and the plasmid was extracted after being correct. The 35S::GmBBE-like43-GFP plasmid was transformed into Agrobacterium GV3101 and Agrobacterium rhizogenes K599, and stored for future use after the detection was correct.

(3) GmBBE-like43 subcellular localization analysis

To analyze the subcellular localization of transient expression in tobacco epidermal cells, 35S::GmBBE-like43-GFP or pEGAD empty (35S:GFP) was introduced into Agrobacterium strain GV3101 through the Agrobacterium transformation method. After successful transformation, GV3101 containing the vector was inoculated in YEP medium, shaken at 28°C for 16 hours, centrifuged (5000rpm, 10min), and resuspended to OD with infiltration solution (containing 10mM MgCl ₂ , 10mMMES and 10mM acetosyringone, pH=5.6) The spectrophotometric value of ₆₀₀ is 0.4-0.5. After the bacterial cell suspension was kept at 43° C. in the dark for 3 hours, the lower epidermis of tobacco leaves of 5-6 weeks old was transformed by the method of infiltrating the bacterial liquid through a syringe. After the transformed tobacco was cultured normally for 3 days, the distribution of fluorescent signals in tobacco epidermal cells was observed with a laser converging scanning microscope (Zeiss LSM780, Germany). For subcellular localization of transgenic bean hairy roots, 35S::GmBBE-like43-GFP or pEGAD empty (35S::GFP) was introduced into Agrobacterium rhizogenes strain K599 for transformation of bean hairy roots. The specific method refers to the transformation system of kidney bean hairy root established in our laboratory. The constructed transgenic material was observed under a laser scanning microscope (Zeiss LSM780, Germany) to observe the localization of the GFP signal in the cells.

The results are shown in Figure 2. After the 35S::GmBBE-like43-GFP vector was transferred into tobacco leaf epidermal cells for transient expression, the empty control (35S::GFP) had the green color of GFP in the nucleus, cytoplasm and plasma membrane. Fluorescent signal, while 35S::GmBBE-like43-GFP had strong GFP fluorescence at the cell edge, which coincided with the cell outline, indicating that GmBBE-like43 might be localized on the cell wall (Fig. 2B).

In order to further verify the subcellular localization of GmBBE-like43, the 35S::GmBBE-like43-GFP vector was introduced into Agrobacterium rhizogenes K599, and the 35S:: Transgenic hairy roots stably expressing GmBBE-like43-GFP fusion protein. The results were similar to those of tobacco epidermal cells. GFP fluorescence signal detection found that the GFP fluorescence of the unloaded transgenic control kidney bean hairy root spread throughout the cells. However, the GFP fluorescence of 35S::GmBBE-like43-GFP transgenic kidney bean hairy roots was only at the cell edge, and it was fused with the red fluorescence excited by PI dye (Fig. 2C). Therefore, GmBBE-like43 may be localized on the cell wall.

Example 3 Effects of GmBBE-like43 expression on the growth of isolated soybean hairy roots after phosphorus treatment under different conditions

1. Construction of GmBBE-like43 functional analysis vector

(1) Construction of overexpression vector (OX-GmBBE-like43-pTF101s)

II kits连接到经XbaⅠ酶切后的线性化载体pTF101s上，得到的重组载体转化大肠杆菌并测序验证，测序结果正确后，抽取重组质粒35S::GmBBE-like43转入发根农杆菌K599和农杆菌GV3101备用。Using the root cDNA of soybean genotype YC03-3 as a template, GmBBE-like43 specific primers were designed, and the upstream specific primer 5'-GTACCCGGGGATCCTCTAGAATGGGAGTCCTTTTCTTCTCA-3'(SEQ ID NO:11) and the downstream specific primer 5'-GCCTGCAGGTCGACTCTAGACTAACCCTTCCTATATGACAGCG-3'(SEQ ID NO:12) amplified the GmBBE-like43 coding region fragment. The PCR reaction system is: a total of 50 μL system, including 43 μL Master mix (2×), 0.5 μL gene forward/reverse primers, 2 μL cDNA template, 1 μL dNTPs (10 μM) and 1 μL high-fidelity enzyme, and the rest is made up with water. The PCR program was: pre-denaturation at 98°C for 5 minutes, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, renaturation at 72°C for 1-3 minutes, this process cycled 30 times, and extension at 72°C for 10 minutes. After the PCR fragment was recovered and sequenced correctly, the amplified CDS fragment of GmBBE-like43 was subjected to homologous recombination kit

The II kits were connected to the linearized vector pTF101s digested with XbaⅠ, and the resulting recombinant vector was transformed into Escherichia coli and verified by sequencing. After the sequencing results were correct, the recombinant plasmid 35S::GmBBE-like43 was extracted and transformed into Agrobacterium rhizogenes K599 and Agrobacterium rhizogenes K599. Bacillus GV3101 spare.

(2) Construction of interference expression vector (RNAi-GmBBE-like43-pFGC5941)

Using the root cDNA of YC03-3 as a template, two specific primers RNAi-GmBBE-like43-pFGC5941-F/R (SEQ ID NO: 13-16) were designed. The specific sequences are as follows:

Primer RNAi-GmBBE-like43-pFGC5941-Asc I-F (SEQ ID NO: 13):

5'-ACAATTACCATGGGGCGCGCCGGTGTGTCTTACGTGGCAGA-3';

Primer RNAi-GmBBE-like43-pFGC5941-Asc I-R (SEQ ID NO: 14):

5'-AAATCATCGATTGGGCGCGCCCAAAAGCTAGCTCCACCACCA-3';

Primer RNAi-GmBBE-like43-pFGC5941-BamH I-F (SEQ ID NO: 15):

5'-ATTTGCAGGTATTTGGATCCCAAAGCTAGTCCACCACCA-3';

Primer RNAi-GmBBE-like43-pFGC5941-BamH I-R (SEQ ID NO: 16):

5'-TCTAGACTCACCTAGGATCCGGTGTGTCTTACGTGGCAGA-3'.

Amplify the open reading frame sequence of about 300bp of GmBBE-like43, and then digest the target vector with restriction endonuclease Asc I to obtain the linearized binary vector pFGC5941; the 15bp-20bp sequence at the end of the linearized vector is used as the homologous sequence, And add them to the 5' end of the gene-specific forward/reverse amplification primer sequence respectively, and use this primer pair to amplify the insert fragment with homologous sequence, and measure the concentration of the recovered product. According to the ligation method of one-step cloning (Novazyme Company), the recombination reaction: mix the linearized vector and the insert in a ratio of 1:2, under the catalysis of Exnase II, react at 37°C for 1 h to complete the recombination reaction, and then pass the recombination product through After the restriction endonuclease BamH I digested the binary vector pFGC5941, the amplified second fragment sequence was recombined according to the one-step cloning ligation method (Novazyme Company). After the recombination reaction, pipette 3 μL of the left rear The recombinant product (depending on the concentration of the product) was added to 50 μL Escherichia coli Trans T1 competent for transformation, and a single clone was selected for later positive screening, and then sequenced for verification after detection. The positive single clone was expanded and cultivated, the bacterial liquid was preserved and the plasmid was extracted to obtain the soybean GmBBE-like43 interference expression vector. Transform the target vector into Agrobacterium K599, detect positive clones and store the bacterial solution at -80°C.

2. Obtaining genetically modified materials

Agrobacterium rhizogenes-mediated transformation of soybean cotyledon nodes in vitro hairy roots. The main steps include:

(1) Seed sterilization and germination: select soybean seeds with undamaged seed coats and the same size, and sterilize the surface in chlorine for 12-14 hours, and then place the seeds in an ultra-clean work table to blow for 30 minutes to remove excess chlorine; then sow them for germination culture medium for 4 days at 28°C under light conditions.

(2) Preparation of bacterial solution: Agrobacterium rhizogenes K599 containing OX-GmBBE-like43-pTF101s or RNAi-GmBBE-like43-pFGC5941 plasmids were streaked on a plate, single clones were picked, and placed at 28°C. Cultivate at 200rpm/min for 12h until the OD ₆₀₀ is about 1.0.

(3) Cotyledon node infection and co-cultivation: first cut off the germinated seeds in the hypocotyl region of 0.5 cm in ion leaf with a scalpel, then cut the seeds vertically along the back with a scalpel, and cut off the seed sprouts . In addition, use a scalpel to dip in the bacterial solution, and cut multiple wounds perpendicular to the cotyledon nodes and hypocotyls. The cut exosomes were moved horizontally upwards to a petri dish containing wet filter paper; after that, the petri dish was sealed with a plastic wrap, transferred to an incubator (24° C.), and incubated under light for 5 days. The co-cultured explants were transferred to the medium containing herbicide and carbenicillin and grown for 14 days.

3. Positive identification of transgenic soybean hairy roots

Extract control group hairy root and transgenic hairy root RNA to be detected, reverse cDNA, use GmBBE-like43 fluorescent quantitative PCR primers in Example 1 to detect the expression level of the overexpressed GmBBE-like43 transgenic line, and use the housekeeping gene EF1-α( Glyma17g23900) was used as the internal reference gene to design primers GmBBE-like43-RT2-F (SEQ ID NO: 17) and GmBBE-like43-RT2-R (SEQ ID NO: 18) to amplify, and to detect the interference of GmBBE-like43 expression transgenic lines. The expression level, the specific sequence is as follows:

Primer GmBBE-like43-RT2-F (SEQ ID NO: 17):

5'-GGACGTTGTGAACGGTACACG-3';

Primer GmBBE-like43-RT2-R (SEQ ID NO: 18):

5'-AGGATGCAATGTCTATGTTGTCC-3'.

4. Functional verification of transgenic soybean hairy roots

(1) Aluminum treatment of transgenic soybean hairy roots

Transgenic hairy roots with fresh growth, good condition and consistent growth were selected and photographed, and transferred to 1/4MS liquid medium (pH 4.5, without adding 100 μM AlCl ₃ (filtered and added to the sterilized and cooled culture solution)) to take pictures. Add KH ₂ PO ₄ ,). In a shaker at 28°C, 100rpm/min was processed for 48 hours before harvesting, photographs of the root system were taken, and the root length was measured using the software Image J.

The results are shown in Figure 3. Quantitative PCR analysis showed that, compared with the empty vector control (OX-CK/RNAi-CK), GmBBE-like43 was overexpressed in GmBBE-like43 ( OX) The expression levels in isolated soybean hairy roots increased by about 10-fold and 4-fold, respectively (Fig. 89% (Fig. 3B). The results of MS culture experiments showed that under normal treatment conditions, excessive or inhibited expression of GmBBE-like43 had no significant effect on soybean hairy root growth. However, under aluminum treatment conditions, overexpression of GmBBE-like43 (OX) significantly promoted the growth of transgenic soybean hairy roots in vitro, and the root growth volume and relative growth rate increased by about 67% and 77% (Fig. 3C-E). In contrast, interference with GmBBE-like43 (RNAi) expression significantly inhibited the growth of transgenic soybean hairy roots in vitro under aluminum treatment conditions, specifically, the amount and relative growth of roots compared with the empty vector control (RNAi-CK). The rates were reduced by about 31% and 22%, respectively (Fig. 3F and G).

(2) High and low phosphorus treatment of transgenic soybean hairy roots

Select transgenic hairy roots with fresh growth, good condition, similar root morphology, and a weight of about 0.1 g, and transfer them to high-phosphorus (+P: 1250 μM KH ₂ PO ₄ ) or low-phosphorus (-P: 10 μM KH ₂ PO ₄ ) respectively. In MS solid medium, grow for 14 days. Harvest and measure the dry weight and total root length of the hairy roots.

The results are shown in Figure 4. Quantitative PCR analysis showed that compared with the empty vector control (OX-CK/RNAi-CK), GmBBE-like43 was overexpressed in GmBBE-like43(OX) soybean in vitro under high and low phosphorus treatment conditions. The expression levels in hairy roots increased by about 1.7-fold and 1.8-fold, respectively (Figure 4A), while the expression levels in isolated soybean hairy roots that interfered with GmBBE-like43 expression (RNAi) decreased by about 80% and 77%, respectively (Figure 4B ). The results of MS culture experiments showed that overexpression of GmBBE-like43(OX) significantly promoted the growth of transgenic soybean hairy roots in vitro no matter under high phosphorus or low phosphorus treatment conditions. At the same time, the total root length of soybean hairy roots overexpressing GmBBE-like43 (OX) under high and low phosphorus treatment conditions was 1.6 times and 2.3 times that of the control treatment (OX-CK) respectively (Fig. 4C- E). On the contrary, compared with the control, the dry weight in the isolated hairy root of GmBBE-like43 was reduced by about 43% and 38% respectively under the high and low phosphorus treatment conditions, and the total root length and the total root length were respectively reduced by about 38% compared with the control. 59% and 35% (Figure 4F and G).

Example 4 Effect of Overexpression of GmBBE-like43 on the Growth of Arabidopsis

1. Obtaining transgenic Arabidopsis

The overexpression vector (OX-GmBBE-like43-pTF101s) plasmid constructed in Example 3 was transformed into Agrobacterium GV3101, and then the inflorescence dipping method and herbicide screening in Example 2 were used to obtain T3 transgenic Arabidopsis seeds Finally, quantitative PCR was used to confirm the different transgenic Arabidopsis lines with high expression of GmBBE-like43 for subsequent gene function research. Arabidopsis GmBBE-like43 quantitative primers are: GmBBE-like43-RT3-F (SEQ ID NO: 19) and GmBBE-like43-RT3-R (SEQ ID NO: 20). Taking Arabidopsis thaliana housekeeping gene EF1-α as an internal reference gene, the Arabidopsis housekeeping gene uses EF1-α quantitative primers (SEQ ID NO: 21-22), and the specific primer sequences are as follows:

GmBBE-like43-RT3-F (SEQ ID NO: 19):

5'-CTCCTTTCCCTCATCGAGCTG-3';

GmBBE-like43-RT3-R (SEQ ID NO:20):

5'-TCCATAAACTCTCCCTTCAGCG-3';

EF1-α-F (SEQ ID NO:21):

5'-GTCGATTCTGGAAAGTCGACC-3'

EF1-α-R (SEQ ID NO:22):

5'-AATGTCAATGGTGATACCACGC-3'.

2. Functional verification of transgenic lines

(1) Aluminum treatment of transgenic Arabidopsis

Two Arabidopsis transgenic lines (OX1 and OX2) overexpressing GmBBE-like43 were obtained through the Arabidopsis heterologous gene expression transformation system. The plump seeds of appropriate amount of wild type and two overexpressed GmBBE-like43 positive Arabidopsis lines OX1 and OX2 were selected. After disinfection, they were first sown on normal MS medium. After about 3 to 4 days, the root system of Arabidopsis thaliana grew to about 1 cm. The uniform seedlings were selected and photographed, and then moved to _1/2 5 Hoagland nutrient solution (pH 4.5, without KH ₂ PO ₄ , 0.5 mM CaCl ₂ ) for cultivation. Culture conditions: room temperature 23°C, light intensity 120 μmol·m ^-2 ·S ^-1 , light time 16 hours a day. Seedlings were harvested 48 hours after transplanting, photographs of seedlings were taken, and root lengths were measured using ImageJ.

It can be seen from Figure 5A that the expression level of GmBBE-like43 in the transgenic Arabidopsis was significantly higher than that in WT. At the same time, overexpression of GmBBE-like43 significantly promoted the growth of transgenic Arabidopsis roots no matter under the treatment conditions of adding aluminum or not adding aluminum (Fig. 5B). Under normal treatment conditions, compared with WT, the root growth of transgenic Arabidopsis lines OX1 and OX2 overexpressing GmBBE-like43 increased by about 35%. The root growth of OX2 increased by 72% and 133%, respectively, compared with WT (Fig. 5C). Meanwhile, relative root growth rates of transgenic lines OX1 and OX2 overexpressing GmBBE-like43 were increased by about 27% and 72%, respectively, compared with WT (Fig. 5D).

(2) High and low phosphorus treatment of transgenic Arabidopsis

The plump seeds of an appropriate amount of wild type and two positive lines OX1 and OX2 overexpressing GmBBE-like43 were selected. After disinfection treatment, first sow in normal MS medium. After about 3 to 4 days, the root system of Arabidopsis thaliana grows to about 1 cm. Pick out uniform seedlings and move them to the correspondingly treated 1/2 MS medium square dish. Set up two Phosphorus level treatment: normal phosphorus supply treatment (1250 μM _{KH 2} PO ₄ ) and low phosphorus treatment (6.25 μM KH ₂ PO ₄ ), each square dish is a repetition, and each treatment has 5 repetitions. The samples were collected on the 9th day after treatment, and the fresh weight and tap root length of the plants were measured.

The results were shown in Fig. 5, no matter under high phosphorus or low phosphorus treatment conditions, the overexpression of GmBBE-like43 significantly promoted the root growth of transgenic Arabidopsis (Fig. 5E). Under normal phosphorus treatment conditions, compared with WT, the root fresh weight of transgenic lines OX1 and OX2 overexpressing GmBBE-like43 increased by about 23%, while under low phosphorus treatment conditions, the transgenic lines OX1 and OX2 Compared with the WT, the root fresh weight increased by 20% and 47%, respectively (Fig. 5F). At the same time, under normal phosphorus treatment conditions, compared with WT, the tap root lengths of transgenic lines OX1 and OX2 overexpressing GmBBE-like43 increased by about 9%, while under low phosphorus treatment conditions, the transgenic lines OX1 and The taproot length of OX2 was increased by 17% and 23%, respectively, compared with WT (Fig. 5G).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

sequence listing

<120> Application of GmBBE-like43 gene in regulating plant adaptation to low phosphorus and acid aluminum stress and promoting growth

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cctttccctc atcgagctgg gaacctatgg aagatccaat accaagcgaa ttggaataag 1320

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Claims (6)

1. SEQ ID NO.1GmBBE-like43Application of gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in positively regulating and controlling growth of soybean root system under low phosphorus-resistant condition.

2. SEQ ID NO.1GmBBE-like43Genes or SApplication of GmBBE-like43 protein shown in EQ ID NO. 2 in positively regulating and controlling growth of soybean root system under the condition of resisting aluminum toxicity stress.

3. SEQ ID NO.1GmBBE-like43Application of the gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in improving the growth capacity of soybean root systems under the condition of low phosphorus or aluminum toxicity stress.

4. SEQ ID NO.1GmBBE-like43Application of gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in cultivation of low phosphorus resistant or aluminum toxicity resistant soybean.

5. A method for promoting soybean growth and/or improving soybean tolerance to low phosphorus or aluminum toxicity stress is characterized by positive regulation of soybean by gene editing technologyGmBBE-like43The gene expression level or the protein activity is used for promoting the growth of soybean root systems, so that the tolerance of the soybean to low phosphorus or aluminum toxicity stress is improved;GmBBE-like43the sequence of the gene is shown as SEQ ID NO.1, and the sequence of the GmBBE-like43 protein is shown as SEQ ID NO. 2.

6. The method of claim 5, wherein the over-expression is constructedGmBBE-like43The expression vector of the gene is transferred into a soybean plant body to obtain transgenic soybean which promotes the growth of soybean root systems and improves the tolerance of the soybean to low phosphorus or aluminum toxicity stress.

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