Application of HbACS2 gene in improving plant abiotic stress tolerance

Abstract

The application clones a rubber tree HbACS2 gene from a rubber tree for the first time, and the gene is shown as SEQ ID NO: 1. Research shows that the protein coded by the gene is a functional protein, which can catalyze acetic acid and coenzyme A to generate acetyl coenzyme A under the energy supply of ATP. HbACS2 The gene can improve the tolerance of the strain to various stresses, such as HbACS2 The gene transformed into E. coli can improve the resistance of the E. coli to sodium chloride, heavy metal ions copper and zinc, acetic acid and the like. HbACS2 The gene can also promote plant growth and improve the tolerance of the plant to various stresses, such as HbACS2 The gene transformed into Arabidopsis thaliana can increase the stem length, fresh weight, fruit pod number, fruit pod length and the like of the Arabidopsis thaliana, and also improve the resistance of the Arabidopsis thaliana to mannitol and alkaline environment and the like, and under the mannitol or alkaline environment, increase the leaf number, main root length, lateral root number and whole plant fresh weight and the like of the plant.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the HbACS2 gene in improving plant tolerance to abiotic stress. Background Technology

[0002] Plants, or different tissues or cells of specific plants, may encounter hypoxia / anaerobic or other stresses in different habitats, triggering anaerobic respiration metabolism. Examples include root cells and cells inside tree stems. This patent focuses on analyzing the role of anaerobic respiration metabolism initiated by plants to respond to stress and improve survival. Existing literature shows that anaerobic respiration metabolism can replenish cellular energy in a timely manner, increasing the plant's ability to resist stress. However, the accumulation of acetaldehyde / acetic acid, intermediate products of anaerobic respiration metabolism, can damage cells and lead to carbon source loss. Through millions of years of evolution, plant cells have developed ingenious solutions: either excreting ethanol or oxidizing ethanol to acetic acid, which, under the action of acetyl-CoA synthase, activates acetic acid into the crucial pivotal substance acetyl-CoA. Acetyl-CoA synthase (ACS) is the key enzyme catalyzing the activation and utilization of acetic acid. Under the action of this enzyme, anaerobic respiration is promoted in a direction favorable to energy production and the synthesis of other metabolites, even secondary metabolites. Therefore, anaerobic respiration is an indispensable respiratory metabolic mode in plants. While there has been some research on ACS in plants such as Arabidopsis thaliana, rice, poplar, and cotton, the function of ACS in rubber trees still needs further investigation. The HbACS2 of this invention belongs to the short-chain acetyl-CoA ligase family. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the HbACS2 gene in improving the tolerance of plants to abiotic stress.

[0004] The first aspect of the present invention is to provide a rubber tree HbACS2 gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0005] A second aspect of the present invention is to provide a protein encoded by the HbACS2 gene of the rubber tree as described in the first aspect of the present invention.

[0006] A third aspect of the present invention is to provide a recombinant vector containing the coding region of the rubber tree HbACS2 gene as described in the first aspect of the present invention.

[0007] The original vector for the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus or plasmid. This invention does not limit this. In one specific embodiment of this invention, the original vector uses pET32a, pMAL-c5E, pCAMBIA1301, pET43.1a+MysB, etc., but it should be understood that other plasmids or viruses can also be used.

[0008] A fourth aspect of the invention is to provide a host bacterium or expression cassette containing the coding region of the rubber tree HbACS2 gene as described in the first aspect of the invention.

[0009] The fifth aspect of the present invention is to provide the use of the rubber tree HbACS2 gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacteria or expression cassette as described in the fourth aspect of the present invention in improving the resistance of Escherichia coli to acetic acid stress, and / or resistance to heavy metal copper stress, and / or resistance to heavy metal zinc stress, and / or resistance to sodium chloride.

[0010] The sixth aspect of the present invention is to provide the use of the rubber tree HbACS2 gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacteria or expression cassette as described in the fourth aspect of the present invention in improving the plant's resistance to mannitol stress and / or alkali stress.

[0011] The seventh aspect of the present invention is to provide the application of the rubber tree HbACS2 gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacteria or expression cassette as described in the fourth aspect of the present invention, in increasing the number of plant leaves and / or increasing the length of the taproot and / or increasing the number of lateral roots and / or increasing the fresh weight of the whole plant under mannitol stress and / or alkali stress.

[0012] The eighth aspect of the present invention is to provide the use of the rubber tree HbACS2 gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacteria or expression cassette as described in the fourth aspect of the present invention in increasing plant stem length and / or increasing plant fresh weight, and / or increasing plant pod number, and / or increasing pod length.

[0013] The plant is preferably Arabidopsis thaliana.

[0014] A ninth aspect of the present invention is to provide an HbACS2-specific antibody, the amino acid sequence of which is AAREALAVQISPVVFD, an antigenic epitope oligopeptide.

[0015] This invention is the first to clone the HbACS2 gene from the rubber tree. Studies show that the protein encoded by this gene is a functional protein capable of catalyzing the reaction of acetic acid and coenzyme A to produce acetyl-CoA under ATP-powered conditions. The HbACS2 gene can enhance the tolerance of plant strains to various stresses. For example, transforming Escherichia coli with the HbACS2 gene can improve its resistance to sodium chloride, heavy metal ions such as copper and zinc, and acetic acid. The HbACS2 gene can also promote plant growth and improve plant tolerance to various stresses. For instance, transforming Arabidopsis thaliana with the HbACS2 gene can increase stem length, fresh weight, number of pods, and pod length. It can also improve Arabidopsis thaliana's resistance to mannitol and alkaline environments. Under mannitol or alkaline conditions, it increases the number of leaves, taproot length, number of lateral roots, and total plant fresh weight. This invention provides a new candidate gene for research on improving the stress resistance of plant strains, promoting plant growth, and enhancing plant adaptability, and has promising application prospects. Attached Figure Description

[0016] Figure 1 for HbACS2 The organizational expression characteristics.

[0017] Figure 2 for HbACS2 Expression characteristics in different rubber-producing strains.

[0018] Figure 3 This is the modified prokaryotic expression protein vector.

[0019] Figure 4 Enzymatic curves for HbACS2 substrates acetic acid (top) and CoA (bottom).

[0020] Figure 5 The results of sodium chloride stress experiments (top) and acetic acid stress experiments (bottom) on Escherichia coli transfected with the HbACS2 gene are shown.

[0021] Figure 6 The results of copper chloride (top) and zinc chloride (bottom) stress experiments on Escherichia coli transgenic with the HbACS2 gene.

[0022] Figure 7 To validate transgenic Arabidopsis plants that overexpress HbACS2.

[0023] Figure 8 The results show the activity of ACS enzymes in Arabidopsis thaliana plants overexpressing HbACS2.

[0024] Figure 9Results of mannitol stress experiments on Arabidopsis seedlings overexpressing HbACS2. (A) Growth of control and transgenic plants on 1 / 2 MS medium; (B) Growth of control and transgenic plants on 1 / 2 MS medium + 100 mM mannitol medium.

[0025] Figure 10 The results of the experiment on the growth of Arabidopsis thaliana seedlings overexpressing HbACS2 on pH 8.0 medium. (A) shows the growth of the control and transgenic seedlings on the control medium; (B) shows the growth of the control and transgenic seedlings on the alkaline stress medium.

[0026] Figure 11 Characterization of Arabidopsis thaliana overexpressing HbACS2.

[0027] Figure 12 Arabidopsis plants overexpressing HbACS2. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific examples to better understand the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0029] Example 1: Cloning of the HbACS2 gene in rubber trees Using rubber tree latex cDNA as a template, and HbACS2F “ACCGACGACGACGACAAGATGAAGGTGGCGAGTTCACTATC” and HbACS2R “GCAGCCGGATCTCAGTGGTCAGCGATCAGCAAGTTCTATAAG” as primers, PCR amplification was performed. The PCR amplification reaction system consisted of a 50 µL reaction volume. The PCR amplification program was as follows: 25 µL of 2×Primestar HS DNA polymerase high-fidelity enzyme premix, 1 µL each of forward and reverse primers to a final concentration of 0.4 mM, 2 µL of cDNA, and ddH2O added to a final volume of 50 µL. The amplification program was: 95℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 2 min, 28 cycles, 72℃ final adjustment for 5 min, and storage at 12℃. The amplified product was recovered and sequenced to obtain an ACS gene, named HbACS2, whose full-length CDS sequence is shown in SEQ ID No:1 (excluding primer sequence).

[0030] Example 2: Expression characteristics of the HbACS gene

[0031] 1. Tissue expression characteristics of HbACS2 Based on the cDNA obtained from the random reverse transcription of total RNA from flowers, young stems, latex, bark, and leaves of the rubber tree cultivar 7-33-97, the expression level of the HbACS2 gene in each tissue was analyzed. Expression levels were expressed as Reads Per Kilobase Per Million mapped reads (RPKM), representing the number of reads per kilobase length from a particular gene per million reads, and plotted. Results are shown below. Figure 1 As shown, tissue expression analysis revealed significant specific expression of HbACS2. HbACS2 was expressed in all tissues of the rubber tree, and also in the bark and leaves. HbACS2 High expression.

[0032] 2. Expression characteristics of HbACS2 in different gum-producing lines To explore the capabilities of HbACS2, we examined HbACS2 The expression of HbACS2 in two rubber varieties representing different yields, PR107 and Reyan 8-79, was investigated. Using cDNA obtained from random reverse transcription of total latex RNA from both varieties as templates, real-time quantitative PCR was performed using HbACS2 gene-specific primers (qHbACS2f: 5'-CTCCGCCTCTCGTCTTTG-3'; qHbACS2r: 5'-CTCGGACACAACCGATTC-3'). The reaction system consisted of a total volume of 20 µL, including 10 µL of TBGreen Premix Ex Taq II FAST qPCR mixture (Takara), 2 µL of 0.3 mM primers, 2 µL of 10 ng cDNA, and 6 µL of ddH2O, totaling 20 µL. After an initial denaturation step of 3 min at 95°C, 40 cycles of amplification were performed at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s, followed by fluorescence readings. Finally, an incubation at 72°C for 5 min yielded melting curves from 90°C to 60°C. The threshold cycle (CT) was manually adjusted by subtracting the CT value of the housekeeping control gene elF1Ab (specific primers: F: 5'- TGGTGTTGGTGTGGTGATAG -3'; R: 5'- TATTCCTGCCCATCTTCCTTG -3') amplified in parallel on each plate to generate a normalized CT value (ΔCT). The relative expression level was calculated using the ΔΔCT method. Results showed... HbACS2 The expression level was higher in the relatively high-yielding thermal research 8-79 strain than in the relatively low-yielding PR107 strain. Figure 2 ).

[0033] Example 3 HbACS2 protease activity assay

[0034] 1. Prokaryotic expression and purification of HbACS2 To better express and purify the HbACS2 protein, we first modified the prokaryotic expression vector. Based on the pET43.1a vector, we constructed the pET43.1a+MysB prokaryotic expression vector, in which MysB replaced NusA in pET43.1a.

[0035] A. pET43.1a+MysB vector construction method 1) Use the empty vector of pET43.1a plasmid with Nde I and Spe I double enzyme digestion removes the NusA lysis-aiding gene.

[0036] 2) Using E. coli DH5α genomic DNA as a template, primers MysB-NdeF“tCta were designed. catATG ACCATGTACGCAACGct" and MysB-Spe-R "GATG actagt The reverse primer, "CGGACCCTGGAACAGCACCTCCAGacgttcatcccactcatcag", contains an HRV 3C protease cleavage site and terminal protective bases. The MysB gene was amplified by PCR, and an HRV 3C protease cleavage site was successfully added to its C-terminus. The PCR product was double-digested with Nde I and Spe I, recovered, and then ligated into the Nde I and Spe I linearized pET43.1a vector using T4 DNA ligase (10 µL ligation system: 1 µL 10×T4 DNA ligase buffer, 2 µL 20 ng linearized pET43.1a vector, 3 µL 10 ng MysB gene fragment, 1 µL T4 DNA ligase, 3 µL ddH2O). 3) Ligation was performed overnight at 16°C. The top 10 cloned bacteria were transformed, and colony PCR was performed. Single colonies of the correct size were selected for sequencing to confirm successful construction of the pET43.1a+MysB vector. Its nucleotide sequence is shown in SEQ ID No:3.

[0037] B. pET43.1a+MysB+HbACS2 vector construction method 1) The method and procedure are the same as above. Based on the vector pET43.1a+MysB, the full-length CDS of HbACS2, along with the following protease cleavage sites and six histidine His tags, is constructed into the vector.

[0038] 1) First use Sac I and Sal I. The linearized vector pET43.1a+MysB was double-digested with enzymes. The enzyme digestion system was as follows: 1 µl of SacI enzyme, 1 µl of SalI enzyme, 10 µl of 10x FastDigest Buffer, 1 µl of Fast AP, 10 µl of the total 2 µg of vector, and the remainder was added to 100 µl with ddH2O. After mixing the system, it was placed in a 16℃ water bath for 12 h for enzyme digestion. The digested vector was recovered and homologously ligated with the target fragment.

[0039] 2) The full-length CDS region of HbASC2 was amplified using homologous primers ACS2-EnthoF: “CGATTGATGACGACGACAAGATGAAGGTGGCGAGTTCAC” and ACS2-43hoR: “GAACGCGTATCGATGGTACCTTAGCGATCAGCAAGTTCTATAAG”. The PCR product recovered from the gel was then used for in vitro recombination (homological ligation reaction system: 2µl of 5×CE II Buffer, 1µl of Exnase II, and 50ng of [unclear text] in a sterile PCR centrifuge tube). 1 µl of the digested vector and 3 µl of the target fragment (the amount added depends on the length ratio of the vector to the target fragment) were added, and the remainder was supplemented with ddH2O to a final volume of 10 µl. The mixture was then placed in a 37°C water bath for homologous arm ligation. After incubation for 40 min, the ligation product was transformed into TOP10 competent cells (after thorough mixing). The cells were then placed on ice for at least 30 min and cultured on ampicillin-containing agar medium for 12 h. Single colony PCR was performed on selected colony clonings. Positive clones were sent for testing. Subsequently, the plasmid “pET43.1a+pMysB+HbACS2” vector sequence was extracted from the correctly sequenced bacteria and is shown in SEQ ID No:4.

[0040] A schematic diagram of the renovated carrier segment is shown below. Figure 3 As shown: MysB replaced NusA in pET43.1a, and the full-length CDS of HbACS2, along with the following protease cleavage sites, was constructed into the vector.

[0041] C. Expression and purification of 6×His-HbACS2 fusion protein The plasmid pET43.1a+MysB+HbACS2 was transformed into the expression strain BL21(DE3)pLysS, while the empty vector plasmid pET43.1a+MysB was also transformed into the expression strain BL21(DE3)pLysS as a control. Then, using the strains with the empty vector and the expression strain, 5-10 mL of LB liquid medium supplemented with carbenicillin and chloramphenicol was cultured in a small batch at 37°C for approximately 16 hours at 250 rpm. The next day, the control and expression strains were transferred to 200 mL of medium at a 1:50 ratio and cultured for another 2 hours. When the OD... 600 When the measured value reached 0.4, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium until the final IPTG concentration was 0.4 mM. The culture was then induced at 160 rpm and 16°C for 20 hours. Then, collect the bacteria (10000 rpm for 8 minutes), resuspend them thoroughly in (50 mM Tris-HCl, pH 8.0 + 5 mM KCl buffer), sonicate thoroughly (until the bacterial solution becomes clear; note that the sonication amplitude should be at maximum and the time should be 3 seconds of sonication followed by 5 seconds of pause to prevent air bubbles; use ice water to cool), centrifuge (13000 rpm for 25 minutes), collect the supernatant, and digest it overnight with HRV3C (HRV3C enzyme 10 U / mg bacteria) at 4°C. Then, purify the His-tagged protein and elute the target protein with 250 mM imidazole solution to obtain a single, pure fusion protein, His-HbACS2. After dialysis to remove imidazole, it is used for HbACS2 protease activity assay.

[0042] 2. HbACS2 protease activity assay Principle: Based on the following reaction equation and the reaction of DTNB with coenzyme A to produce a yellow substance, this substance in OD... 412 Based on the principle that nm has a specific absorbance value, OD is measured using an enzyme-linked immunosorbent assay (ELISA) reader. 412 The change in absorbance at nm was used to calculate the amount of substrate CoA consumed, and then the ACS enzyme activity was calculated.

[0043] Reaction equation: (1) Acetyl-CoA → (ACS catalyzed) Acetyl-CoA (2) CoA-SH + DTNB → TNB + CoA-SS-TNB In enzyme activity assays, the change in ACS-catalyzed reactions was observed while keeping the coenzyme A content constant. When acetic acid reacts with CoA to generate acetyl-CoA, the amount of remaining CoA was measured. This mixture was then mixed with DTNB chromogenic solution, and the reaction product CoA-SS-TNB showed a strong absorbance at 412 nm. Therefore, the absorbance at 412 nm was measured using a microplate reader. The amount of CoA participating in the reaction was obtained by subtracting the remaining CoA from the total amount, and the ACS enzyme activity was calculated.

[0044] a) First determine the optimal pH and temperature for HbACS2. (1) At 37℃, the enzyme activity of HACS2 was compared at 5 different pH gradients to obtain the optimal reaction pH value of 8.2. Then, the enzyme activity of HACS2 was compared at 6 different temperature gradients (26 ℃, 28 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃) to calculate the optimal reaction temperature of HACS2 as 35℃. The enzyme activity reaction system was as follows: 110 µL of Tris-HCl buffer, pH=8.0, 10 µL of 10 mM KCl, 10 µL of 10 mM potassium acetate, 10 µL of 10 mM coenzyme A, 10 µL of 20 mM ATP, 30 µL of ddH2O, and 20 µL of purified HbACS2 protein. After reacting for 1 hour, 45 µL of the reaction solution was taken from each reaction and thoroughly mixed with 90 µL of 5% trichloroacetic acid (TCA) to precipitate the protein and terminate the reaction. Centrifuge the solution after the reaction is terminated at 13,000 rpm for 3 minutes to remove the precipitated protein. Then, take 120 µL of the supernatant and add it to 680 µL of 0.1 mM DTNB (diluted with 0.5 M potassium phosphate buffer at pH 7.5), and incubate at 30°C for about 10 minutes.

[0045] Three 200µL samples were transferred to three wells of a 96-well microplate for three replicate measurements. The absorbance at OD412nm was measured using a Thermo Multiskan Go full-wavelength microplate reader. The absorbance of the sample reaction system was subtracted from the absorbance of the control (containing no substrate potassium acetate, but all other components were the same). Based on the amount of substrate CoA consumed and the molar absorption constant ε = 13600 M / cm, and defining 1 U as the amount of enzyme required to consume 1 µM CoA per minute, the measurement differences were incorporated into the calculation to determine the ACS enzyme activity.

[0046] b) The reaction curves of the substrate acetic acid and CoA were determined at the optimal pH and temperature. In the acetic acid substrate experiment, acetic acid existed in the reaction system as acetate; therefore, potassium acetate was used. The CoA concentration was kept constant during the reaction, and similarly, the acetate concentration was kept constant when measuring CoA. The results are shown in Table 1 and... Figure 4 .

[0047] Table 1 shows the final determination results of acetic acid substrate and CoA substrate. Figure 4 The top and bottom graphs are Michaelis-Menten equation curves of HbACS2 enzyme on two substrates. The horizontal axis represents the substrate concentration (from low to high mM), and the vertical axis represents the enzyme activity measured at different concentrations.

[0048] Table 1

[0049]

[0050] The Michaelis equations for the two substrates of HbACS2 are as follows: Figure 3 As shown: At the optimal pH of 8.2 and the optimal temperature of 35℃, the Km of the acetic acid substrate at different concentrations was determined to be 0.2781 mM, and the Vmax was 117.3 nmol / min / mg protein. Figure 4 (See above); Similarly, the Km of the CoA substrate is 0.6456 mM, and Vmax is 150.7 nmol / min / mg protein ( Figure 4 (See figure below); This experiment demonstrates that HbACS2 catalyzes the reaction of acetic acid and coenzyme A to produce acetyl-CoA, and is a functional protein that is an important enzyme for acetic acid activation.

[0051] Example 4: Escherichia coli experiment Using seamless cloning technology, a pair of homologous primers, HbACS2F “ACCGACGACGACGACAAGATGAAGGTGGCGAGTTCACTATC” and HbACS2R “GCAGCCGGATCTCAGTGGTCAGCGATCAGCAAGTTCTATAAG”, were designed to construct the full-length CDS sequence of HbACS2 into the pET32a vector. The vector was then transformed into the Top10 cloned bacteria, plated, and colony PCR was performed. Three bacteria with the correct size were selected for sequencing, and plasmids were extracted from the correctly sequenced bacteria to obtain the pET32a-HbACS2 vector. This vector was then transformed into the expression bacterium BL21(DE3)pLysS. At the same time, the empty pET32a vector plasmid was also transformed into the expression bacterium BL21(DE3)pLysS. Then, using the strain carrying the pMAL-c5E empty vector as the control strain and the strain carrying the correct pET32a-HbACS2 vector as the expression strain, 5-10 mL of LB liquid medium supplemented with carbenicillin was used for small-scale incubation at 37°C at 250 rpm for about 16 hours. The next day, the control strain was stored at 4°C, and the expression strain was transferred to 100 mL of medium at a 1:50 ratio and incubated for another 2 hours. When its OD... 600When the measured value reached 0.4, isopropyl-β-D-thiogalactoside (IPTG) was added to the expression strain culture medium until the final IPTG concentration was 0.4 mM. Induction culture was then performed at 160 rpm and 20°C for 10 hours. Then, the OD values ​​of the preserved control strain and the expression strain were compared. 600 The measured values ​​were adjusted to be close to 0.4. Then, growth stress experiments were carried out using this culture in sequence with 300 mM NaCl solution, 10 mM CuCl2 solution, 1 mM acetic acid solution, and 10 mM ZnCl2 solution.

[0052] Specifically, 300 mM NaCl solution, 10 mM CuCl2 solution, 1 mM acetic acid solution, 10 mM ZnCl2 solution, and 10 mM copper chloride solution were added to the culture medium, and the OD was measured hourly. 600 Cell growth was monitored throughout the experiment, and growth curves were plotted based on the results. The control and expression bacteria were cultured under the same conditions. The results of the NaCl stress experiment are as follows: Figure 5 As shown in the figure above, under sodium chloride stress, E. coli carrying the HbACS2 gene grew better than the control strain with empty vector.

[0053] The concentrations of *E. coli* were diluted 10,000, 1,000, 100, and 10 times, respectively, and spotted onto control and LB solid medium supplemented with acetic acid (final concentration 1 mM). Their growth was observed. The results of the acetic acid stress experiment are as follows: Figure 5 As shown in the figure below, compared with the control strain transgenic empty vector, E. coli carrying the transgenic HbACS2 gene grew better under acetic acid stress.

[0054] The results of the copper chloride stress experiment are as follows Figure 6 As shown in the figure above, compared with the control strain transgenic empty vector, E. coli carrying the transgenic HbACS2 gene grew better under copper chloride stress.

[0055] The results of the zinc chloride stress experiment are as follows Figure 6 As shown in the figure below, compared with the control strain transgenic empty vector, E. coli carrying the transgenic HbACS2 gene grew better under zinc chloride stress.

[0056] Example 5: Transgenic Experiment

[0057] 1. Construction of transgenic plants Using seamless cloning technology, a pair of homologous primers, ACS2-ATG-1301 homo:CACGGGGGACTCTTGACCATGAAGGTGGCGAGTTCACTA and ACS2-TGA-1301 homo:CTGGTCACCTGTAATTCACACTCAGCGATCAGCAAGTTCTATA, were designed to construct the full-length CDS sequence of HbACS2 into the pCAMBIA1301 vector. The vector was then transformed into the Top 10 clones, plated, and subjected to colony PCR detection. Three correctly sized bacteria were selected for sequencing. Plasmids and empty vectors were extracted from the correctly sequenced bacteria and transformed into Agrobacterium LB4401, respectively. The Agrobacterium strains with the empty vector and the correctly expressed strains were then cultured in 5-10 mL of LB liquid medium supplemented with the appropriate antibiotics at 28°C for approximately 250 rpm for 20 hours. They were then transferred to 200 mL of culture medium for large-scale culture. When their OD... 600 When the measured value reached 1.0-1.2, bacteria were collected at 4000 rpm. Infection solutions were prepared according to the Arabidopsis infection method. The collected bacteria were resuspended in the infection solution and used to infect Arabidopsis flower buds in the flowering stage. Each bacterium was used to infect at least 8 small pots of Arabidopsis seedlings (4 seedlings per pot). After treatment, the plants were kept in the dark and moist for one day, and then cultured normally the next day. The obtained seeds were the T0 generation. These seeds were then screened on 1 / 2 MS medium with hygromycin. Seedlings that developed roots and true leaves were used for inoculation and single-plant seed harvesting until no further segregation occurred, resulting in a pure line.

[0058] Three independent transgenic pure lines were obtained after three generations of screening and named Ox-ACS-1, Ox-ACS-2, and Ox-ACS-3, respectively. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to analyze Arabidopsis gene expression. After preparing cDNA, specific primers qHbACS2f (5'-CTCCGCCTCTCGTCTTTG-3'), qHbACS2r (5'-CTCGGACACAACCGATTC-3'), and the Arabidopsis internal reference gene AtUBC21 were used as controls (forward and reverse primers were 5'-CTTAACTGCGACTCAGGGAATCT-3' and 5'-GGCGAGGCGTGTATACATTTGT-3', respectively). The reaction system consisted of 10 µL of TB Green Premix Ex Taq II FASTqPCR mixture (Takara), 2 µL of 0.3 mM primers, 2 µL of 10 ng cDNA, and 6 µL of ddH2O, for a total of 20 µL. After an initial denaturation step of 3 min at 95°C, 40 cycles of amplification were performed at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s, followed by fluorescence readings. Finally, after incubation at 72°C for 5 min, melting curves from 90°C to 60°C were generated. The threshold cycle (CT) was manually adjusted by subtracting the CT value of the housekeeping control amplified in parallel on each plate from the CT value of each target gene, thus generating a normalized CT value (ΔCT). The ΔΔCT method was used to calculate the overexpression fold of the transgenic plants. qRT-PCR analysis revealed that the three obtained pure lines showed HbACS2 overexpression, with relative expression levels ranging from 354 to 618 fold (…). Figure 7 This proves that it is the correct overexpression line.

[0059] 2. Enzyme activity and stress resistance of transgenic plants The experiment was conducted using transgenic pure lines obtained through three generations of screening.

[0060] ① Enzyme activity First, the harvested transgenic pureline seeds were sterilized at room temperature for 10 hours with 2% plant tissue culture antibacterial agent (Plant Preservative Mixture, PPM). After rinsing five times with sterile water, the seeds were sown on 1 / 2 MS medium and vernalized at 4°C for 2 days. Then, they were placed in a plant incubator for pre-culture. The incubator temperature was 22°C, with a light-to-dark cycle of 12 hours. When the taproot length was about 1-1.5 cM, the seeds were transplanted into culture soil and placed in a greenhouse for further cultivation. When the Arabidopsis had 8 true leaves, 1g of the whole plant from the control and transformed lines was taken and rapidly ground with 50 mM Tris-HCl, pH 8.0 buffer, and a small amount of quartz sand. The mixture was then centrifuged at 12,000 rpm for 15 minutes at low temperature to remove impurities and prepare the crude ACS protein enzyme solution.

[0061] The enzyme activity assay system and procedure are as follows: (2) First, prepare 200 µL of HbACS reaction premix, including 25 µL of 500 mM Tris-HCl (pH 8.0) (final concentration 50 mM), 25 µL of 100 mM MgCl2 (final concentration 10 mM), 25 µL of 100 mM ATP (final concentration 10 mM), 15 µL of 10 mM potassium acetate (final concentration 0.6 mM), and 15 µL of 10 mM coenzyme A (final concentration 0.6 mM). Make up the volume to 200 µL with sterile water. At the same time, set up a control CK without potassium acetate (make up the excess with water). Mix the reaction premix prepared according to the above components thoroughly, centrifuge slightly, and incubate in a water bath at 37°C for 3 min. After taking it out, add 25 µL of purified acetyl-CoA synthase to initiate the reaction, mix thoroughly, centrifuge slightly, and incubate in a water bath at 37°C for 1 h.

[0062] (3) For each reaction, take 45 µL of the reaction solution and mix it thoroughly with 90 µL of 5% trichloroacetic acid (TCA) to precipitate the protein and terminate the reaction. Centrifuge the solution after terminating the reaction at 13000 rpm for 3 min to remove the precipitated protein.

[0063] (4) Next, take 120µL of supernatant and add it to 680µL of 0.1mM DTNB (diluted with 0.5M potassium phosphate buffer at pH 7.5), and incubate at 30°C for about 10 minutes.

[0064] (5) Take 200µL three times and add it to three wells of a 96-well microplate for three replicate measurements. Measure the absorbance at OD412nm using a ThermoMultiskanGo full-wavelength microplate reader. Subtract the absorbance of the sample reaction system from the absorbance of the control (containing no substrate potassium acetate, but other components are the same). Based on the amount of substrate CoA consumed and the molar absorption constant ε = 13600M / cm, define 1 U as the amount of enzyme used per minute to consume 1µM CoA. Substitute the measurement difference into the constant to calculate the ACS enzyme activity. Figure 8 The absorbance of the complete reaction system was subtracted from the absorbance of the control (CK) without potassium acetate. Compared with the control (WT), the ACS enzyme activity was higher in the three Arabidopsis plants overexpressing HbACS2: ox-ACS-1, ox-ACS-2, and ox-ACS-3.

[0065] ② Mannitol stress First, the harvested transgenic pureline seeds were disinfected with 75% ethanol for 2 minutes, rinsed 3 times with sterile water, then disinfected with a 20-fold diluted bleach solution (containing 15% effective hypochlorous acid) for 10 minutes, rinsed 5 times with sterile water, and then disinfected with 2% plant tissue culture antibacterial agent (Plant Preservative Mixture, PPM) at room temperature for 10 hours, rinsed 5 times with sterile water. The seeds were then sown on 1 / 2 MS medium and vernalized at 4°C for 2 days. After vernalization, they were placed in a plant incubator for pre-culture. The incubator temperature was 22°C, with a 12-hour light-12-hour dark culture. When the taproot length was approximately 1-1.5 cM, seedlings of similar growth were selected and placed on control medium (normal medium) and 1 / 2 MS + 100 mM mannitol stress medium, and cultured vertically in the same incubator. Observations were made every 2 days, and photographs and analysis were performed after 6 days. Figure 9 Compared with the control, Arabidopsis seedlings overexpressing HbACS2 (ox-HbACS2) grew better on a medium containing 100 mM mannitol.

[0066] ③Alkali stress On March 8, 2024, the harvested seeds were first disinfected with 75% ethanol for 2 minutes, rinsed 3 times with sterile water, then disinfected with a 20-fold diluted bleach solution (containing 15% effective hypochlorous acid) for 10 minutes, rinsed 5 times with sterile water, and then disinfected with 2% plant tissue culture antibacterial agent (Plant Preservative Mixture, PPM) at room temperature for 10 hours, rinsed 5 times with sterile water, and then sown on 1 / 2 MS medium. After vernalization at 4℃ for 2 days, the seeds were placed in a plant incubator. The incubator temperature was 22℃, with a light-to-dark cycle of 16 hours of light and 8 hours of darkness. After 3 days of cultivation, seedlings with similar growth were selected and placed on control medium (normal medium) and pH 8.0 alkaline stress medium in the same incubator for vertical cultivation. The temperature in the cultivation room was controlled at 22-23℃, with a light-to-dark cycle of 12 hours of light and 12 hours of darkness. Observations were made every 2 days, and photographs, measurements, statistics, and analysis were performed on March 24.

[0067] The results are as follows Figure 10 As shown. Under alkaline stress at pH 8.0, seedlings overexpressing HbACS2 (ox-HbACS2) adapted to growth better than the control, as evidenced by the average number of leaves per transgenic plant being 8, the taproot length being 1.4 cM, the number of lateral roots per plant being 12, and the total fresh weight being 0.0056 g per plant, while the control plant had 6 leaves per plant, the taproot length being 1.0 cM, the number of lateral roots per plant being 4, and the total fresh weight being 0.0032 g per plant.

[0068] 3. Phenotype of plants overexpressing HbACS2 We selected fourth-generation pure Arabidopsis seeds that had been tested by qRT-PCR, and each generation had been screened and verified with hygromycin. On January 14, 2024, the harvested seeds were first disinfected with 75% ethanol for 1 minute, rinsed three times with sterile water, and then disinfected for 2 days and night with 2% plant tissue culture antibacterial agent (Plant Preservative Mixture, PPM) at room temperature. After rinsing five times with sterile water, the seeds were sown on 1 / 2 MS medium. After vernalization in a 4℃ refrigerator for 2 days, they were placed in a plant incubator. The incubator temperature was 22℃, with a light-to-dark cycle of 16 hours of light and 8 hours of darkness. After 5 days of cultivation, on January 21, 2024, the seeds were transplanted into small pots, four plants per pot, and placed in a plant cultivation room with uniform fertilization and watering. The temperature in the cultivation room was controlled at 22-23℃, with a light-to-dark cycle of 16 hours of light and 8 hours of darkness. On March 12, 2024, we photographed, counted, weighed, and analyzed the transgenic plants and the control group. The results are as follows: Figure 11-12 And as shown in Table 2 ( Figure 11(The average of the data in Table 2) The stem length of the transgenic plants increased significantly, the fresh weight increased, the number of pods increased significantly, and the pod length also increased significantly, indicating that overexpression of HbACS2 can promote plant growth.

[0069] Table 2

[0070] Example 6 HbACS2-specific antibody We selected the HbACS2-specific amino acid oligopeptide C-AAREALAVQISPVVFD ("C" indicates that this oligopeptide is located at the C-terminus of the protein), synthesized it, conjugated it with a tag protein for expression and purification, and then injected it into rabbits (three times) to prepare a specific polyclonal antibody. After purification, the antibody's specificity was tested. Western blot results showed that the antibody could specifically recognize the HbACS2 protein in rubber latex at a dilution of 1:2000.

[0071] The specific examples of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific examples described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of the rubber tree HbACS2 gene, or the protein encoded by the rubber tree HbACS2 gene, or a recombinant vector containing the coding region of the rubber tree HbACS2 gene, or an expression cassette containing the coding region of the rubber tree HbACS2 gene in improving plant resistance to mannitol stress and / or alkali stress, wherein the nucleotide sequence of the rubber tree HbACS2 gene is shown in SEQ ID NO:

1.

2. The application of the rubber tree HbACS2 gene, or the protein encoded by the rubber tree HbACS2 gene, or a recombinant vector containing the coding region of the rubber tree HbACS2 gene, or an expression cassette containing the coding region of the rubber tree HbACS2 gene, in improving plant growth under mannitol stress and / or alkali stress; or The application of the rubber tree HbACS2 gene, or the protein encoded by the rubber tree HbACS2 gene, or a recombinant vector containing the coding region of the rubber tree HbACS2 gene, or an expression cassette containing the coding region of the rubber tree HbACS2 gene, in increasing the number of plant leaves, and / or increasing the length of the taproot, and / or increasing the number of lateral roots, and / or increasing the fresh weight of the whole plant under mannitol stress and / or alkali stress; the nucleotide sequence of the rubber tree HbACS2 gene is shown in SEQ ID NO:

1.

3. The application of the rubber tree HbACS2 gene, or the protein encoded by the rubber tree HbACS2 gene, or a recombinant vector containing the coding region of the rubber tree HbACS2 gene, or an expression cassette containing the coding region of the rubber tree HbACS2 gene in increasing plant stem length, and / or increasing plant fresh weight, and / or increasing the number of plant pods, and / or increasing pod length, wherein the nucleotide sequence of the rubber tree HbACS2 gene is shown in SEQ ID NO:

1.

4. The application according to claim 2 or 3, characterized in that, The plant in question is Arabidopsis thaliana.

5. An HbACS2-specific antibody, characterized in that, The amino acid sequence of its antigenic epitope oligopeptide is AAREALAVQISPVVFD.

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