CN115109767B - Method for improving polyethylene glycol resistance of bacteria by amino-acylase-1 and application - Google Patents

Abstract

The invention provides an application of aminoacylase-1 in improving the anti-polyethylene glycol of escherichia coli. The protein of the amino-acylase-1 is selected from the proteins shown in SEQ ID NO. 1.

Description

Method for improving polyethylene glycol resistance of bacteria by amino-acylase-1 and application

The present application is a divisional application of patent application having application number 202010088971.1 and application name "use of aminoacylase-1", which is application number 2020, 02 and 12.

Technical Field

The invention belongs to the technical field of agricultural biology, and relates to application of a corn aminoacylase-1 gene in improving polyethylene glycol resistance of bacteria and improving plant growth performance.

Background

Aminoacylase-1 having Zn-binding activity ²⁺ An ionic zinc binding domain and a primary domain that promotes dimerization of the zinc binding domains, the active site of which is located between two zinc binding domains ^[1-2] . Binding zinc causes conformational transfer by promoting binding of N-acyl-L-amino acid substrates, with subunits of the protein aggregating around the substrateThereby allowing catalysis to occur ^[3] 。

Amino-acylase-1 (acylase I; N-acyl-L-amino acid amidase) is involved in the hydrolysis of N-acylated or N-acetylated amino acids (except L-aspartic acid), which when the N-terminus of the L-amino acid is covalently bound to an acyl group, forms an N-acyl-L-amino acid. Acyl groups provide stability to amino acids, making them more resistant to degradation. However, N-acyl-L-amino acids cannot be used directly as building blocks for building proteins, and must first be converted to L-amino acids by the enzyme aminoacylase-1, and the L-amino acid products can be used for biosynthesis or catabolic energy.

The aminoacylase-1 is involved in regulating the oxidative stress of cells in an animal body and participates in amino acid metabolism and urea circulation, and the significance of urea circulation in plants is that arginine is synthesized, urea can be produced by individual plants, and ammonia is decomposed under the action of urease to synthesize nitrogen-containing compounds including nucleic acid, hormone, chloroplast, heme, amine, alkaloid and the like. However, little research has been done on aminoacylase-1 in plants. Shi Huafang, etc ^[4] The rice aminoacylase purified from rice has the greatest activity on N-acetyl-L-methionine, and then N-acetyl-DL-serine and N-acetyl-L-alanine.

Reference to the literature

[1]Lindner-HA,Lunin-VV,Alary-A,et al.Essential Roles of Zinc Ligation and Enzyme Dimerization for Catalysis in the Aminoacylase-1/M20 Family[J].Journal of Biological Chemistry,2003,278(45):44496-44504.

[2]Fones-WS,Lee-M.Hydrolysis of N-acyl derivative of alanine and phenylalanine by acylase I and carboxypeptidase[J].Journal of Biological Chemistry,1953,201(2):847-856.

[3]Lindner-H,Alary-A,Wilke-M,et al.Probing the acyl-binding pocket of aminoacylase-1[J].Biochemistry,2008,47(14):66-75.

[4] Shi Huafang, huang Weida, li Hongjie classification, purification and characterization of Rice aminoacylases [ J ]. J.Biochem., 1997,13 (1): 54-58.

[5]Fougere-F,Le-RD,Streeter-JG.Effects of salt stress on amino acid,organic acid,and carbohydrate composition of roots,bacteroids,and cytosol of alfalfa(Medicago sativa L.)[J].Plant Physiology,1991,96(4):1228-1236.

[6]Zorb-C,Schmitt-S,Neeb-A.The biochemical reaction of maize(Zea mays L.)to salt stress is characterized by a mitigation of symptoms and not by a specific adaptation[J].Plant Science,2004,167(1):91-100.

[7]Zhi-Z,Cui-YM,Zheng-S,et al.The amino acid metabolic and carbohydrate metabolic pathway play important roles during salt-stress response in tomato[J].Frontiers in Plant Science,2017,8:1231.

[8]Khedr-AH,Abbas-MA,Wahid-AA,et al.Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of pancratium maritimum L.to salt-stress[J].Journal of Experimental Botany,2003,54(392):2553-2562.

[9]Abbasi-AR,Hajirezaei-M,Hofius-D,et al.Specific roles of-and gamma-ocopherol in adminbiotic stress responses of transgenic tobacco[J].Plant Physiology,2007,143(4):1720-1738.

Disclosure of Invention

In a first aspect the present invention provides the use of an aminoacylase-1 to improve a plant trait selected from the group consisting of: the germination rate of plant seeds is improved; the plant height is improved; increasing leaf area; increasing the stem thickness; root length is increased; increasing the number of blades; increasing root area; increasing pod weight; and increasing any of the fresh weight of the plant. In some embodiments, the aminoacylase-1 is a plant-derived aminoacylase-1. In some embodiments, the aminoacylase-1 is maize aminoacylase-1. In some embodiments, the protein of aminoacylase-1 is selected from the group consisting of the protein set forth in SEQ ID NO.1, or a protein having more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) sequence homology to the protein set forth in SEQ ID NO.1 and having aminoacylase-1 enzymatic activity. In the present invention, aminoacylase-1 proteins having homology to the protein shown in SEQ ID NO.1 include, but are not limited to: proteins with aminoacylase-1 protein activity are naturally occurring, genetically engineered (e.g., fusion proteins) or mutated in artificial environments (e.g., radiomutated) as found in corn and other plants (e.g., sorghum). When large fragments of fusion proteins, such as green fluorescent protein and aminoacylase-1 proteins, are fused, the homology may be nearly 50%, and the present invention is not limited to more than 90% sequence homology. In some embodiments, the nucleic acid sequence encoding the aminoacylase-1 is set forth in SEQ ID NO. 2. In some embodiments, the improving a plant trait is improving a tobacco trait. In some embodiments, the improved plant trait is a trait that is improved by increasing the expression level of the NBEXPA1 gene and/or the NBEIN2 gene.

In a second aspect, the present invention provides a method for improving a plant trait by transferring an expressible aminoacylase-1 gene into a plant, the improvement of the plant trait selected from the group consisting of: the germination rate of plant seeds is improved; the plant height is improved; increasing leaf area; increasing the stem thickness; root length is increased; increasing the number of blades; increasing root area; increasing pod weight; and increasing any of the fresh weight of the plant. In some embodiments, the aminoacylase-1 is a plant-derived aminoacylase-1. In some embodiments, the aminoacylase-1 is maize aminoacylase-1. In some embodiments, the protein of aminoacylase-1 is selected from the group consisting of the protein set forth in SEQ ID NO.1, or a protein having more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) sequence homology to the protein set forth in SEQ ID NO.1 and having aminoacylase-1 enzymatic activity. In some embodiments, the nucleic acid sequence encoding the aminoacylase-1 is set forth in SEQ ID NO. 2. In some embodiments, the improving a plant trait is improving a tobacco trait. In some embodiments, the improved plant trait is a trait that is improved by increasing the expression level of the NBEXPA1 gene and/or the NBEIN2 gene.

In a third aspect, the invention provides the use of an aminoacylase-1 to increase bacterial anti-polyethylene glycol. In some embodiments, the bacterial anti-polyethylene glycol is the bacteria are grown in a liquid medium containing 1wt% to 20wt% (e.g., 2wt%, 3wt%, 5wt%, 8wt%, 10wt%, 12wt%, 15wt%, 18 wt%) polyethylene glycol, preferably 5wt% to 15 wt%. In some embodiments, the aminoacylase-1 is a plant-derived aminoacylase-1. In some embodiments, the aminoacylase-1 is maize aminoacylase-1. In some embodiments, the protein of aminoacylase-1 is selected from the group consisting of the protein set forth in SEQ ID NO.1, or a protein having more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) sequence homology to the protein set forth in SEQ ID NO.1 and having aminoacylase-1 enzymatic activity. In some embodiments, the nucleic acid sequence encoding the aminoacylase-1 is set forth in SEQ ID NO. 2. In some embodiments, the bacterium is escherichia coli, preferably escherichia coli BL21.

In a fourth aspect, the present invention provides a method for increasing the polyethylene glycol resistance of a bacterium by transferring an expressible aminoacylase-1 gene into the bacterium. In some embodiments, the bacterial anti-polyethylene glycol is the bacteria are grown in a liquid medium containing 1wt% to 20wt% (e.g., 2wt%, 3wt%, 5wt%, 8wt%, 10wt%, 12wt%, 15wt%, 18 wt%) polyethylene glycol, preferably 5wt% to 15 wt%. In some embodiments, the aminoacylase-1 is a plant-derived aminoacylase-1. In some embodiments, the aminoacylase-1 is maize aminoacylase-1. In some embodiments, the protein of aminoacylase-1 is selected from the group consisting of the protein set forth in SEQ ID NO.1, or a protein having more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) sequence homology to the protein set forth in SEQ ID NO.1 and having aminoacylase-1 enzymatic activity. In some embodiments, the nucleic acid sequence encoding the aminoacylase-1 is set forth in SEQ ID NO. 2. In some embodiments, the bacterium is escherichia coli, preferably escherichia coli BL21.

In a fifth aspect, the present invention provides a recombinant genetic engineering vector comprising an expressible aminoacylase-1 gene. In some embodiments, the aminoacylase-1 is a plant-derived aminoacylase-1. In some embodiments, the aminoacylase-1 is maize aminoacylase-1. In some embodiments, the protein of aminoacylase-1 is selected from the group consisting of the protein set forth in SEQ ID NO.1, or a protein having more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) sequence homology to the protein set forth in SEQ ID NO.1 and having aminoacylase-1 enzymatic activity. In some embodiments, the nucleic acid sequence encoding the aminoacylase-1 is set forth in SEQ ID NO. 2. In some embodiments, the recombinant genetic engineering vector is a recombinant prokaryotic gene expression vector; preferably, the recombinant prokaryotic gene expression vector is pET28a. In some embodiments, the recombinant genetic engineering vector is a recombinant eukaryotic gene expression vector; preferably, the vector of the recombinant eukaryotic gene expression vector is pCAMBIA1300. In some embodiments, the recombinant genetic engineering vector is a recombinant gene expression shuttle vector.

In a sixth aspect, the present invention provides a host comprising the recombinant genetic engineering vector described above. In some embodiments, the host is a bacterium or a plant; preferably, the bacterium is escherichia coli, more preferably escherichia coli BL21. The plant is preferably tobacco.

The aminoacylase-1 of the invention is a zinc-binding enzyme involved in urea cycle and ammonia (NH) ₄ ⁺ ) Clearance and regulation of cellular responses to oxidative stress are well studied in plants. The invention takes the corn inbred line Zheng 58 as a material, clones the corn aminoacylase-1 gene ZmACY-1, and carries out biological function analysis and research on the ZmACY-1 at the prokaryotic and eukaryotic levels, and the result is as follows:

1. bioinformatics analysis shows that ZmACY-1 (LOC 100283955) belongs to the zinc peptidase superfamily, M20 aminoacylase-1 subfamily genes, the length of an open reading frame sequence encoded by the ZmACY-1 gene is 1317bp, 439 amino acids are encoded, the relative molecular weight of the encoded protein is 48.33KD, and the theoretical isoelectric point is 6.02.

2. The prokaryotic expression vector pET28a-ZmACY-1 is constructed and transferred into escherichia coli BL21 for salt-tolerant anti-polyethylene glycol analysis. The results show that: compared with the pET28a control group strain, the recombinant pET28a-ZmACY-1 escherichia coli strain shows a certain resistance capacity under 5 percent, 10 percent and 15 percent of PEG6000 stress; under the stress of 0.4mol/L,0.6mol/L and 0.8mol/L NaCl, along with the increase of salt concentration, the over-expression of pET28a-ZmACY-1 in the escherichia coli strain seriously inhibits the salt tolerance of the strain. From this, it is speculated that recombinant E.coli BL21 (pET 28 a-ZmACY-1) has different tolerance patterns against NaCl stress and polyethylene glycol stress.

3. A plant binary expression vector pCAMBIA1300-ZmACY-1 is constructed, agrobacterium is utilized to infect the tobacco of the present tobacco, and the phenotype identification is carried out on the transgenic tobacco. The results show that: overexpression of ZmACY-1 in this raw tobacco compared to wild type promotes the growth and development of transgenic plants, including increasing seed germination rate, increasing leaf area, plant height, stem thickness, aerial part weight, subsurface part weight, root length, root area, and pod size of mature plants. In the transgenic primary smoke of one month, the expression quantity of the genes NbEXPA1 and NbEIN2 related to plant growth is obviously higher than that of the wild type.

4. For tobacco T ₃ The transgenic strain and the wild type are subjected to NaCl treatment and natural drought treatment, and the fresh weight and chlorophyll content of the overground parts of the transgenic strain are obviously lower than those of the wild type after salt stress treatment; after drought stress treatment, the fresh weight of the overground part of the transgenic strain is obviously lower than that of the wild type, and the wilting degree is more serious. In addition, the measurement of stress-related physiological indexes shows that under salt stress and drought stress, the contents of protective enzymes POD, SOD and CAT in a transgenic strain are lower than that of a wild type strain, and MDA and relative conductivity are higher than that of the wild type strain. These results indicate that the overexpression of ZmACY-1 in Nicotiana benthamiana negatively regulates drought and salt tolerance of transgenic plants. Meanwhile, the over-expression of ZmACY-1 in escherichia coli and tobacco is shown to have different anti-polyethylene glycol modes.

All the results show that ZmACY-1 participates in various vital activities of plants and responds to stress, and the drought resistance and salt tolerance of the plants are inhibited although the growth and development of the plants are promoted.

Drawings

FIG. 1 is a gel photograph of PrimerSTAR Max DNA Polymerase amplified ZmACY-1 gene.

Note that: m: takara DL 2000DNA marker; lanes 1-4: amplification results of ZmACY-1 gene.

FIG. 2 shows a photograph of a pMD19T-ZmACY-1 plasmid colony PCR identification gel (A) and a photograph of a double enzyme cut verification gel (B).

Note that: lanes M1, M2: takara DL 2000DNA marker; lanes 1-7: PCR identification of pMD19T-ZmACY-1 plasmid colonies; lanes 8-11: double enzyme digestion of pMD19T-ZmACY-1 plasmid.

FIG. 3 is a schematic diagram showing the prediction of the conserved regions of ZmACY-1 protein.

FIG. 4 is a schematic diagram showing the analysis of the ZmACY-1 gene evolution.

FIG. 5 pET28a-ZmACY-1 PCR-verified gel (A) and double cleavage results gel (B).

Note that: m1: takara DL 2000DNA marker,M2: takara DL 5000DNA marker; lanes 1-4: PCR identification of pET28a-ZmACY-1 plasmid colony; lanes 5-6: and (3) carrying out double enzyme digestion verification on the pET28a-ZmACY-1 plasmid.

FIG. 6 is a photograph of a gel of analysis of the prokaryotic expression product of pET28 a-ZmACY-1.

Note that: m: protein markers; 1 and 2: induced supernatant and precipitate of pET28a-ZmACY-1 strain respectively; lanes 3 and 4: uninduced supernatant and pellet of pET28a-ZmACY-1 strain, respectively; lanes 5, 6 are the induction supernatant and pellet, respectively, of the empty vector strain; lanes 7, 8 are the induction supernatant and pellet, respectively, of the empty vector strain; the protein of interest is indicated by the arrow.

FIG. 7 shows the growth curves of bacteria under stress of different concentrations of NaCl or different concentrations of PEG 6000.

Note that: A-C, growth curves of recombinant bacteria and control bacteria under the stress of NaCl concentration of 0.4mol/L, 0.6mol/L and 0.8mol/L respectively.

D-F, growth curves of recombinant bacteria and control bacteria were obtained in PEG6000 aqueous solutions (5%, 10% and 15%), respectively.

FIG. 8 is a photograph of a gel of the results of PCR identification (A) and plasmid double digestion (B) of a plant expression vector pCambia1300-ACY-1 plasmid.

Note that: lane M1: takara DL 2000DNA marker, lane M2: takara DL15000 DNA marker; lanes 1-6: the PCR result of the positive clone; lanes 7-9: double cleavage results of recombinant plasmid.

Fig. 9 is a schematic illustration of an infection and screening process for a leaf of a raw tobacco.

Note that: a, in-vitro blade B: callus; C-D: adventitious buds; e, seedling; f: and (5) transplanting the tobacco plants.

FIG. 10 shows the result of PCR amplification of the selected seedling genome (A) and the relative expression level of ZmACY-1 in T3 generation transgenic benthames (B).

A: lane M: takara DL 2000DNA marker; lane 1, wild type raw tobacco; lanes 2-7: screening Miao Ben raw smoke PCR results by converting the T0 generation into ZmACY-1; b: WT is wild type benthamiana, OE1, OE3, OE5 are three transgenic lines T3 generation ZmACY-1.

FIG. 11 shows the phenotype identification results of the wild type and ZmACY-1 transgenic tobacco seedlings.

Note that: the germination rate of tobacco in a plate added with MS culture medium; B-D: tobacco vigor grown for 10 days in plates supplemented with MS medium; "*": p <0.05, "x": p <0.01.

FIG. 12 shows the phenotypic identification of one month old wild type and ZmACY-1 transgenic tobacco plants.

Note that: a: tobacco growth vigor for one month; B-H: identifying the phenotype of the tobacco of the age of one month; "*": p <0.05, "x": p <0.01.

FIG. 13 is a schematic diagram showing the expression level of a growth-related gene in transgenic and wild-type benthamic cigarettes.

Note that: a: the relative expression level of NbEXPA1 gene; b: relative expression level of NbEIN2 gene; "*": p <0.05, "x": p <0.01.

FIG. 14 shows the results of the phenotype identification of the wild type and ZmACY-1 transgenic tobacco pods.

Note that: a: photographs of fully developed pods of wild type and transgenic lines; b: pod weights of wild-type and transgenic lines; "*": p <0.05, "x": p <0.01.

FIG. 15 shows the results of salt tolerance analysis of the ZmACY-1 transgenic tobacco.

Note that: 350mmol/L NaCl stress and phenotype of each tobacco strain under the water treatment; b: fresh weight of the overground part of each tobacco strain under the stress treatment of 350mmol/L NaCl and the clear water treatment; c, the chlorophyll content of each tobacco strain under the stress treatment of 350mmol/L NaCl and the clear water treatment; "*": p <0.05, "x": p <0.01.

FIG. 16 is a photograph showing drought resistance analysis of ZmACY-1 gene-transferred smoke.

Note that: a, phenotype of each tobacco strain under natural drought and clear water treatment; b: fresh weight of overground parts of each tobacco strain under natural drought and clear water treatment.

FIG. 17 is a schematic diagram of the measurement results of physiological indexes related to wild type and transgenic Nicotiana benthamiana stress under salt stress and drought stress.

Note that: POD activity; b, SOD activity; CAT activity; d: MDA activity; e: relative conductivity; "*": p <0.05, "x": p <0.01.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

Materials and methods

Test material: corn inbred line 'zheng 58' and wild type benthonic tobacco (Nicotiana benthamiana) seeds are provided by the Qingdao university crop breeding laboratory. Strains: coli competent cells BL21 (DE 3) and DH 5. Alpha. And Agrobacterium (Agrobacterium rhizogenes) competent cells LBA4404 were supplied by Bokang BioCo., ltd (Qingdao). Plasmid: pMD19-T was purchased from Takara, plant expression vector pCAMBIA1300 and prokaryotic expression vector pET28a, supplied by Qingdao university molecular laboratory. Reagent: isopropanol, absolute ethanol, AMP, X-gal, IPTG, kanamycin Kana, rifampin Rif, hygromycin Hyg, cephalosporin Cef, 2000DNA markers, 15000DNA markers, nucleic acid dye, MS medium, 1/2MS medium, LB and YEB medium are all conventional reagents. Instrument: the real-time fluorescence quantitative PCR instrument, the constant temperature incubator, the shaking table, the refrigerated centrifuge, the ultra-clean bench, the spectrophotometer and the nucleic acid concentration measuring instrument (NANODrop 2000) are all conventional instruments.

Primer sequence: the GenBank number of the Zheng 58 corn amino-acylating enzyme-1 gene (ZmACY-1 gene) is: np_001150325.2. The protein sequence is shown as SEQ ID NO.1 in the sequence table. The nucleotide coding sequence is shown as SEQ ID NO.2 in the sequence table. Based on the information such as ZmACY-1 gene sequence, a Primer was designed by using software Primer Premier5 and synthesized by Qingdao qing catalpa biological technology Co., ltd, and the Primer is as follows (Table 1).

TABLE 1 primer sequences

Material cDNA acquisition: RNA extraction was performed according to the instructions provided by Takara Plant RNA Extraction Kit kit; then, the concentration detection is carried out on the extracted RNA by utilizing a spectrophotometer, the purity and the concentration of the RNA are measured, and then, the agarose gel electrophoresis detection is carried out; and then reverse transcription is carried out, wherein the system is as follows: master mix 2.0. Mu.L, RNA 2.0-4.0. Mu.L (< 500 ng), RNase Free water make up 10. Mu.L, PCR protocol: the reaction was carried out at 37℃for 15min, at 85℃for 5s and at 4℃for preservation. The cDNA of the test material thus obtained was used for fluorescent quantitative PCR.

Example 1: cloning of the maize ZmACY-1 Gene

Extraction of RNA: extraction of total RNA (Total RNA) of zheng 58 maize leaf tissue was performed according to the instructions provided by the Plant RNA Extraction Kit kit from Takara corporation.

(II) RNA quality detection: and (3) detecting the concentration of the extracted RNA by using a spectrophotometer, determining the purity and concentration of the RNA, and then detecting by agarose gel electrophoresis.

(III) cDNA template synthesis: a reverse transcription system was prepared on ice, specifically using Master mix 2.0. Mu.L, total RNA 2.0-4.0. Mu.L (< 500 ng), and RNase Free water to bring the reaction system to 10. Mu.L. Adding the reaction solution according to the reaction system, and fully and uniformly mixing. The reaction is carried out in a PCR instrument, and the reaction procedure is as follows: the reaction was carried out at 37℃for 15min, at 85℃for 5s and at 4℃for preservation.

(IV) high-fidelity enzyme amplification ZmACY-1 gene: RNA was extracted from the leaves and reverse transcribed. Using the total cDNA reverse transcribed from the RNA in the step (III) as a reaction template, and carrying out gene amplification by using high-fidelity enzyme (PrimerSTAR Max DNA Polymerase), wherein the amplification ZmACY-1 gene is obtained by the following system (Takara): takara PrimerSTAR Max DNA Polymerase 25. Mu.L, zmACY-1-F1. Mu.L shown in Table 1, zmACY-1-R1. Mu.L shown in Table 1, cDNA 2. Mu.L, and RNase free water were added to the reaction system to 50. Mu.L. The PCR reaction steps are as follows: denaturation at 98℃for 10s; annealing at 58 ℃ for 30s; extending at 72 ℃ for 15s; a cycle number of 35; preserving at 4 ℃.

A single band of approximately 1320bp in size was obtained as shown in FIG. 1. The result shows that the specificity of the primer is better, the amplified fragment is clear, the size of the strip is consistent with the size of the target gene, and further cloning experiments can be carried out.

(V) recovering amplified product and adding A tail

The procedure according to the gel recovery kit (Nanjinouzan Biotechnology Co., ltd., model DC 301-01) was as follows:

1. the 100. Mu. LPCR product was pipetted into a sterilized 1.5mL centrifuge tube using a pipette. 2. Equal volumes of Buffer GDP are added, inverted or vortexed. 3. After the DNA adsorption column was set in the collection tube, the mixture was transferred to the DNA adsorption column, and centrifuged at 10,000Xg for 1min.4. After discarding the filtrate, the column was replaced in the collection tube, 600 μLBuffer GW (absolute ethanol added) was added to the column, and the column was centrifuged at 12,000Xg for 1min.5. And (4) repeating the step 4.6. The filtrate was discarded and the column was returned to the recovery header and centrifuged at 12,000Xg for 2min.7. The column was placed in a sterilized 1.5mL centrifuge tube, 30. Mu. LELUTION Buffer was added to the center of the column, and after standing for 2min, 12,000Xg was centrifuged for 1min. The adsorption column was discarded, and the purified DNA solution was stored in a-20℃refrigerator. 8. The product was added with "A" (deoxyadenosine), 14.5. Mu.L of the PCR product was taken as a template, 2. Mu.L of Taq Buffer, 3. Mu.L of dNTPs and 0.5. Mu.L of Taq enzyme were added, and the reaction was carried out at 72℃for 30 minutes.

Construction of cloning vector pMD19T-ZmACY-1

The construction procedure of the intermediate vector pMD19T-ZmACY-1 is as follows, and DNA purification and recovery are carried out after the amplification product is subjected to the addition reaction of "A".

(1) Ligation of the recovered fragments to the Linear pMD19-T vector

The ligation reaction solution was prepared in a microcentrifuge tube. The pMD19T-ZmACY-1 system is: T-Vector pMD 19. Mu.L; 1 μl of PCR product; DNA Ligase 5. Mu.L; RNase free water was added to the system at 10. Mu.L.

The reaction system was added to the EP tube and thoroughly mixed. Placing into a PCR instrument, setting the temperature at 16 ℃ and reacting for 30min. After the reaction, the reaction solution was added to 100. Mu.L of E.coli competent cells BL21 (DE 3) at a volume ratio of 1:10, and the mixture was rapidly placed on ice for reaction for 40min. Then placing the mixture into a water bath at 42 ℃ for heating treatment for 90 seconds, then rapidly placing the mixture into ice for reaction for 2 minutes, finally adding the reaction liquid into LB liquid medium without antibiotics, and placing the LB liquid medium into an oscillating table for shake culture at 37 ℃ for 60 minutes.

(seventh) screening of cloning vector pMD19T-ZmACY-1

(1) Screening blue and white spots. 30 mu. L X-gal and 3. Mu.L IPTG were added to the LB plates containing Amp in a super clean bench, and the plates were left to stand for 1 hour, then the transformed E.coli was spread on the plates, left to stand for 30 minutes, and then incubated at 37℃overnight upside down.

(2) And (5) screening positive bacteria. White single colonies were selected and bacterial liquid PCR was performed to screen positive recombinant plasmids. The reaction system is as follows: 2X TaqPlusMasterMixII (DyePlus). Mu.L; zmACY-1-F1 mu L; zmACY-1-R1 mu L; 2. Mu.L of Strain solution; RNase free water was added to the system at 50. Mu.L.

Identification of cloning vector pMD19T-ZmACY-1

(1) Plasmid extraction

White single colonies were picked and inoculated into LB medium (containing Amp) and cultured overnight at 37 ℃. The bacterial liquid is sucked into a centrifuge tube, and the bacterial liquid is centrifuged for 1min at the rotating speed of 10,000rpm, so as to collect bacterial bodies. Extracting plasmids according to the steps of the plasmid extraction kit: 1. 2mL of the recombinant E.coli bacterial liquid cultured overnight (12-16 h) was taken and added into a 2mL centrifuge tube, and 10,000Xg was centrifuged for 1min, and the centrifuge tube was back-off on filter paper to drain the residual liquid. 2. 250. Mu.L Buffer P1 (checked before use whether RNase A has been added) was added to the tube with the cell pellet left, and vortexed and mixed. 3. To step 2, 250. Mu.L Buffer P2 was added, and the tube was gently inverted 10 times to lyse the cells well. 4. 350. Mu.L Buffer P3 was added to step 3, and the tube was immediately gently turned upside down 10 times to thoroughly neutralize Buffer P2 and centrifuged at 13,000Xg for 10min.5. FastPure DNA Mini Columns adsorption columns were placed in Collection Tube 2mL Collection tubes. The supernatant from step 4 was carefully transferred to an adsorption column with a pipette and prevented from sucking up to sediment, and centrifuged at 13,000Xg for 1min. The waste liquid in the collecting pipe is discarded, and the adsorption column is put into the collecting pipe again. 6. 600. Mu.L Buffer PW2 (diluted with absolute ethanol) was added to the column and centrifuged at 13,000Xg for 1min. Discarding the waste liquid, and putting the adsorption column back into the collection pipe. 7. And (6) repeating the step 6.8. The adsorption column was placed back into the collection tube. The column was dried by centrifugation at 13,000Xg for 1min to thoroughly remove the residual rinse solution from the column. 9. The column was replaced in a fresh sterilized 1.5mL centrifuge tube. 50. Mu.L of the absorption Buffer was added to the center of the column membrane. The mixture was allowed to stand at room temperature for 2min, and the plasmid was eluted by centrifugation at 13,000Xg for 1min. 10. The adsorption column was discarded and the plasmid was stored at-20℃to prevent DNA degradation.

(2) And (3) enzyme digestion verification: the plasmid was double digested with restriction endonucleases BamH I and Hind III, and the digested pMD19T-ZmACY-1 plasmid system was: 10 XK Buffer 5. Mu.L; bamHI 1. Mu.L; hindIII 1. Mu.L; plasmid 15. Mu.L; RNase free water was added to the system at 50. Mu.L.

The PCR identification of pMD19T-ZmACY-1 plasmid colonies is shown in FIG. 2A, and single bands of approximately 1320bp in size are amplified. The results of the double digestion verification of the correct transformant extracted plasmid for bacterial liquid PCR are shown in FIG. 2B. And (5) the identified positive clone is sent to Qingdao qing Ke catalpa, and the sequencing is carried out by the biological technology limited company. Sequencing results show that the part of the PCR product related to the coding sequence is identical to SEQ ID NO.2, which shows that cloning is successful.

Example 2: bioinformatics analysis

Amino acid sequence alignment was performed on the sequencing results using DNAMAN software, and the base was aligned using MEGA 5.1 softwareAs a result of performing phylogenetic tree analysis. The cloned ZmACY-1 gene is subjected to sequence analysis by using Bioxm software, and the analysis shows that the sequence length of an open reading frame coded by the gene (without counting a stop codon) is 1317bp, and 439 amino acids are coded. Prediction analysis is performed by ProtParam (https:// web. Expasy. Org/protParam /) online software, and the result shows that the molecular formula of the protein coded by the ZmACY-1 gene is C ₂₁₇₃ H ₃₃₆₇ N ₅₉₉ O ₆₂₅ S ₁₄ The relative molecular weight is 48.33kD, the theoretical isoelectric point is 6.02, the total number of positive charge residues (Arg+Lys) is 42, the total number of negative charge residues (Asp+Glu) is 50, the instability coefficient is 46.03, and the protein belongs to an unstable protein. Analysis of the conserved region of the amino acid sequence encoded by the ZmACY-1 gene by NCBI's conserved functional region analysis program CDS (conserved domains) is shown in FIG. 3, indicating that ZmACY-1 belongs to the zinc peptidase superfamily, the M20 aminoacylase-1 subfamily, having an M20-acylase domain and five zinc binding sites. The ZmACY-1 evolution analysis is shown in FIG. 4.

Example 3: construction of prokaryotic expression vector pET28a-ZmACY-1

(one) construction of recombinant bacteria

The recombinant plasmid pMD19T-ZmACY-1 was PCR amplified with high fidelity enzyme using XbaI-ZmACY-1-F and BamHI-ZmACY-1-R as primers shown in Table 1, thereby adding protective bases at both ends of the plasmid. The PCR reaction system is as follows: takara PrimerSTAR Max DNA Polymerase 25 μL; xbaI-ZmACY-1-F1 mu L; bamHI-ZmACY-1-R1 μL; pMD19T-ZmACY-1 2 μL (0.1-10 ng); RNase free water was added to the system at 50. Mu.L.

And (3) recovering target fragments by using glue, and respectively carrying out enzyme digestion on amplified fragments of a prokaryotic expression vector pET28a and a positive clone PMD19T-ACY-1 by using Xba I and BamH I at 37 ℃ for 5 hours, wherein the enzyme digestion reaction system is as follows: 10 XK Buffer 5. Mu.L; xba I1 μL; bamHI 1. Mu.L; plasmid 20. Mu.L; RNase free water was added to the system at 50. Mu.L.

After the enzyme digestion reaction is finished, a product is recovered according to a gel recovery kit, and then the target fragment and the carrier fragment are connected according to the following system, and are placed into a PCR instrument for overnight connection at 16 ℃ to obtain a prokaryotic expression carrier pET28a-ZmACY-1. The connection system is as follows: vector 3. Mu.L; PCR product 12. Mu.L; DNA Ligase 1. Mu.L; 10 XBuffer 2. Mu.L; RNase free water was added to the system at 20. Mu.L.

The recombinant plasmid was transformed into prokaryotic expression host bacterium BL21 (DE 3), then cultured overnight on LB medium containing kanamycin (50. Mu.g/mL), and after screening, the plasmid was extracted for enzyme digestion identification, wherein positive clones were screened and PCR verification results were shown in FIG. 5A. The strain after successful PCR verification is amplified and shaken, plasmids are extracted, the double enzyme digestion identification result of the recombinant plasmids is shown in figure 5B, and the result shows that the pET28a-ZmACY-1 prokaryotic expression vector is successfully constructed. The positive plasmid is sent to catalpa ovata biological technology limited company of Qingdao, and sequenced to obtain the same coding sequence shown in SEQ ID NO. 2.

(II) prokaryotic expression fusion proteins

Prokaryotic expression of ZmACY-1 gene in escherichia coli BL21 (DE 3)

1. Inoculating overnight cultured host strain BL21 (DE 3) into 10mL LB liquid medium containing Amp resistance at a volume ratio of 1:100, shake culturing at 37deg.C for 5 hr at 200rpm in a shake incubator, and measuring OD to OD ₆₀₀ Reaching 0.8.

2. IPTG at a concentration of 0.1M was added to a final concentration of 0.05mmol/L, and the experiment was conducted in an incubator at 28℃for 5 hours with no addition of the inducer IPTG. Then 100. Mu.L of the bacterial liquid was subjected to SDS-PAGE electrophoresis after centrifugation to determine whether the recombinant protein was expressed. 3. Adding 100 μl of BL21 (pET 28 a-ACY-1) recombinant bacteria into 10mL of LB liquid culture medium, performing induced expression according to the method, centrifuging at 10,000rpm for 1min, pouring out supernatant, re-suspending the precipitate with PBS, centrifuging at 10,000rpm for 10min, repeating for three times, placing the collected bacteria into ice bath, performing ultrasonic crushing for 5min, repeating for one time, and centrifuging at 10,000rpm for 20min when the bacteria are clear. 4. mu.L of the supernatant and the pellet suspension were taken, 10. Mu.L of 5X SDS loading buffer was added, respectively, and the mixture was boiled in boiling water at 100℃for 10 minutes, and SDS-PAGE analysis was performed to determine whether the expression product was soluble (in the supernatant) or in the form of inclusion bodies (in the pellet). The protein coded by ZmACY-1 is subjected to IPTG induction, centrifugation, resuspension and ultrasonic crushing centrifugation, SDS-PAGE detection is carried out, the result is shown in a figure 6, the size of the protein is about 48.33kD, and the protein is consistent with the molecular weight of the ZmACY-1 predicted by ProtParam, which shows that pET28a-ZmACY-1 can express the protein in escherichia coli BL21, so that the next salt-tolerant anti-polyethylene glycol analysis can be carried out.

Example 4: salt and polyethylene glycol resistance test of recombinant host bacterium BL21 (pET 28 a-ZmACY-1)

Salt resistance analysis of recombinant host BL21 (pET 28 a-ZmACY-1): three LB liquid culture media containing NaCl with different concentrations (the final concentration of NaCl is 0.4mol/L, 0.6mol/L and 0.8mol/L respectively), BL21 (pET 28 a-ZmACY-1) host bacteria induced by 1mLIPTG and BL21 (pET 28 a) host bacteria of a control group are respectively taken, added into the culture medium and evenly mixed, placed in a shaking incubator for shaking culture at 37 ℃, and OD measurement is carried out every L hours ₆₀₀ Values. 3 replicates were measured for each treatment.

Anti-polyethylene glycol analysis of recombinant host bacterium BL21 (pET 28 a-ZmACY-1): setting three LB liquid culture media containing PEG6000 at different concentrations (5%, 10% and 15% final concentration of PEG 6000), respectively taking 1mL of induced BL21 (pET 28a-ZmACY-1 host bacteria and control BL21 (pET 28 a) host bacteria, adding into the culture media, mixing, placing into an shake incubator, shake culturing at 37deg.C, and measuring OD every l hours ₆₀₀ Values. 3 replicates were measured for each treatment.

The effect of different stress times on the growth of the host bacteria was observed. On the abscissa, the culture time, average OD ₆₀₀ Values are on the ordinate and growth curves are plotted. The results show that compared with the host bacteria transformed with pET28a, the host bacteria transformed with pET28a-ZmACY-1 grow well in the early stage of low-concentration salt stress (figure 7A), but the host bacteria transformed with pET28a-ZmACY-1 grow weaker than the control host bacteria under medium-concentration salt stress (figure 7B) and high-concentration salt stress (figure 7C), and the growth curve of the host bacteria transformed with pET28a-ZmACY-1 cannot continue to grow along with the time lapse under 0.8mol/L of high-concentration salt stress, and the medium-concentration salt stress tolerance and the high-salt stress tolerance of the host bacteria are weakened by high-concentration salt. However, under different concentrations of PEG6000 stress (FIG. 7D-F), pET28a-ZmACY-1 host bacteria grew better than pET28a host bacteria, indicating that overexpression of ZmACY-1 in E.coli BL21 caused host bacteria The method has a certain anti-polyethylene glycol capability, and the OD difference amplitude is larger at a high PEG concentration than at a low PEG concentration, for example, the OD value of the transgenic PEG strain is only slightly increased at a 5% PEG concentration, and the OD value of the transgenic PEG strain is increased by about 50% at a 15% PEG concentration, which shows that the anti-PEG capability of the ZmACY-1 gene on escherichia coli is enhanced along with the increase of the PEG concentration in the PEG concentration range used by the method.

Example 5: zmACY-1 transgenic tobacco

Construction of plant expression vector pCambia1300-ZmACY-1

PMD19-ACY-1 was used as a template, zmACY-1-XbaI-F (the sequence is the same as that of XbaI-ZmACY-1-F shown in Table 1) and ZmACY-1-BamHI-R (the sequence is the same as that of BamHI-ZmACY-1-R shown in Table 1) were used as primers, and PCR amplification was performed with high fidelity enzyme, thereby adding a protecting base to both ends of the plasmid, and recovering the target fragment. The pCambia1300 expression vector and the amplified fragment of the positive clone PMD19T-ZmACY-1 were digested simultaneously with XbaI and BamHI at 37℃for 3h, respectively, in the following manner: 10 XK Buffer 5. Mu.L; xba I1 μL; bamHI 1. Mu.L; plasmid 20. Mu.L; RNase free water was added to the system at 50. Mu.L. The connection system is as follows: T-Vector pCambia1300 (after cleavage) 3. Mu.L; 12. Mu.L of PCR product (after cleavage); DNA Ligase 1. Mu.L; 10 XBuffer 2. Mu.L; RNase free water was added to the system at 20. Mu.L.

The recombinant plasmid was transformed into E.coli DH 5. Alpha., cultured overnight at 37℃in LB medium containing kanamycin (50. Mu.g/mL), and the plasmid was extracted for enzyme digestion identification, PCR identification and enzyme digestion patterns are shown in FIG. 8, whereby it was found that the plant expression vector pCambia1300-ZmACY-1 was successfully constructed. The positive plasmid is sent to catalpa in Qingdao and Qingdao biological technology limited company for sequencing, and the correctness of the vector construction is verified.

Genetic transformation of ZmACY-1 Gene into Bensheng cigarette

Transformation of LBA4404 competent cells

1. The competent cells of the agrobacterium LBA4404 are taken out from an ultralow temperature refrigerator at the temperature of minus 80 ℃ and are inserted into ice for 10min after the competent bacterial liquid is slightly melted. 2. In an ultra-clean bench, 1 μl of expression vector plasmid pCambia1300-ZmACY-1 was added to competent cells, gently stirred and mixed, ice-bathed for 5min, transferred into liquid nitrogen for 5min, transferred into a 37 ℃ water bath for 5min, and then ice-bathed for 2min.3. Adding 600 mu LLB liquid culture medium into ice-bath agrobacteria competence, placing into a shaking incubator, and setting the conditions: the culture is carried out for 2 to 3 hours at 28 ℃ under the rotation speed of 200 rpm. 4. Centrifugation at 10,000rpm for 3min, the supernatant was discarded, and the precipitated cells were resuspended in 100. Mu.LYEB (or LB) liquid medium. 5. The bacterial liquid after the resuspension is smeared on a YEB solid culture medium containing 50mg/L kanamycin and 20mg/L rifampicin, and the bacterial liquid is poured into an incubator to be cultured for 36-48 hours at the temperature of 28 ℃.6. The single colony of the agrobacterium after 36-48h of culture is picked and inoculated into a centrifuge tube filled with YEB (or LB) liquid culture medium (50 mg/L Kan+20 mg/LRif) for overnight culture (> 16 h), and the bacterial liquid is turbid and then is subjected to PCR verification. And (3) preserving bacteria of the bacterial liquid and sterilized 50% glycerol according to a ratio of 1:1, and storing the bacterial liquid and the sterilized glycerol in a refrigerator at the temperature of minus 80 ℃.

Genetic transformation of Bensheng cigarette (leaf disc method)

1. 200 mu L of Agrobacterium LBA4404 strain containing expression vector pCambia1300-ZmACY-1 stored in a-80 ℃ ultra-low temperature refrigerator is taken out and inoculated into 10mLYEB liquid culture medium (50 mg/L Kan+20 mg/LRif) for overnight>16h) Shake culturing and activating strain. 2. The activated bacterial liquid was subjected to continuous shake culture in YEB liquid medium (50 mg/L Kan) at a volume ratio of 1:50, overnight culture, and then subjected to centrifugation (centrifugation at 7,000rpm for 3 min). 3. In an ultra clean bench, the bacterial solution was resuspended in 1/2 medium (agar-free) with 5% sucrose (OD ₆₀₀ Value: 0.6-0.8), immersing the pre-cultured leaf pieces of the raw tobacco in the heavy suspension for 5-10min, airing on filter paper after immersing, and then transferring the leaf pieces to a co-culture medium (MS+3 mg/L6BA+0.2 mg/LNAA+150 mu mol/L acetosyringone) covered by two layers of filter paper for co-culture in the dark for 3 days. 4. Tobacco leaves after 3 days of co-cultivation were transferred to hygromycin and cephalosporin added induced callus medium (MS+3 mg/L6BA+0.2mg/LNAA+25mg/LHyg+250mg/LCef, pH=5.8) for selection culture (25 ℃, 16h of light) with medium replacement every week. 5. After adventitious buds grow out of the callus, cutting off tender buds in an ultra-clean workbench, transferring the tender buds to a bud growth medium (MS+20mg/LHyg+200mg/LCef, pH=5.8) for screening, cutting off plants when the tender buds form 3-5cm seedlings, and transferring the seedlings to a culture medium (1/2MS+15mg/L Hy) g+150mg/LCef, ph=5.6) rooting screening medium. 6. After the screened seedlings grow out of the roots, the tissue culture bottle cap is opened, the sealing film is replaced, and a plurality of cracks are cut through the sealing film by using a surgical knife to exercise the seedlings. After two days, the tissue culture seedlings are gently removed by forceps, and the roots of the tissue culture seedlings are washed by clean water until the root culture medium is washed cleanly and then transplanted into sterilized soil. 7. The transplanted benthonic tobacco is watered thoroughly and put into illumination culture to be cultured at constant temperature of 25 ℃ and needs to be covered with a film for moisturizing in the first three days. After three days, the culture was normal.

(IV) planting of Bensheng tobacco

(1) Seed disinfection and aseptic seeding

Firstly placing seeds on a clean vessel, removing impurities, then transferring the seeds to an EP pipe, washing the seeds once with sterile water, and then soaking the seeds in 75% alcohol for 30-60s. The raw tobacco seeds were then rinsed with 2% NaClO for 10min. Finally, the mixture is washed for 3 to 5 times by sterile water. The sterilized seeds were spotted onto MS medium using a sterile 1mL gun head.

(2) Transplanting of the present raw tobacco

Firstly, sterilizing the nutrient soil for 30min at 120 ℃ before transplanting, and filling the nutrient soil into a flowerpot for planting the natural tobacco after the soil is cooled. The grown and strong benthonic tobacco is moved into the nutrient soil by forceps, and 1 plant is transplanted in each pot. And (5) placing the transplanted raw tobacco in an illumination incubator, and culturing at a constant temperature of 25 ℃. An overview of the infection and screening process of the raw tobacco leaves is shown in figure 9. The infected leaves were screened in the hygromycin-added induced callus screening medium (fig. 9A) to obtain callus (fig. 9B), the callus was replaced with hygromycin-added bud screening medium to screen and obtain buds (fig. 9C), the buds were cut out in the rooting screening medium to continue the screening culture (fig. 9D), the buds were grown into shaped plants and the roots were grown (fig. 9E) to indicate that the screening was completed, the seedlings were trained and moved to soil (fig. 9F).

(V) identification of Bensheng tobacco

Extraction of DNA

Transplanting positive tobacco seedlings subjected to hygromycin screening into nutrient soil for two weeks, and then cutting tobacco leaves for DNA extraction. The method comprises the following steps: 1. 0.5g tobacco leaves were placed in a mortar pre-cooled with liquid nitrogen, ground thoroughly and added continuously with liquid nitrogen, and rapidly transferred into a 2mL EP tube. 2. 700. Mu.L of CTAB (preheated in a water bath at 65 ℃) extraction buffer was added to the EP tube, mixed well and 20. Mu.L of beta-mercaptoethanol was added. The mixture was gently shaken for every 20min in a water bath at 3.65℃for 40 min. 4. After cooling to room temperature, an equal volume of phenol was added: chloroform: the isoamyl alcohol (1:1:1) solution was thoroughly mixed, ice-bath for 5min, at 12,000rpm, and centrifuged for 15min.5. Taking out the supernatant, and repeating the step 4.6. Sucking the supernatant into a new EP tube, adding equal volume of isopropanol, mixing, standing in a refrigerator at-20deg.C for 1 hr, 12,000rpm, and centrifuging for 8min.7. The supernatant was discarded, and the pellet was washed 3 times with 70% absolute ethanol after pre-cooling at 4℃and centrifuged at 12,000rpm for 2min.8. After leaving to dry for several minutes at room temperature, 50. Mu.L of deionized water was added, and after centrifugation, the mixture was stored at-20℃for further use.

And (3) PCR amplification detection: the DNA extracted in the previous step is used as a template to amplify the ZmACY-1 gene, and the Real Time PCR reaction system is as follows: TBGreenPremixExTaqII 12.5. Mu.L; qRT-ZmACY-1-F0.5 mu L; qRT-ZmACY-1-R0.5 mu L, cDNA 2.0 mu L after reverse transcription; RNase free water to 25. Mu.L. The Real Time PCR reaction steps are: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s; annealing at 55-60 ℃ for 30s; extending at 72 ℃ for 30s; cycling was performed 40 times.

Real-time fluorescent quantitative PCR: the expression of ZmACY-1 in this smoke was detected by fluorescent quantitative PCR using a TBGreenPremixExTaqII (TliRNaseHPlus) kit (TaKaRa) as a control. Experiment the relative expression level of ZmACY-1 at the transcription level was determined by using the endogenous gene NbEF1a of the present smoke as an internal reference gene (using the NbEF1a-F and NbEF1a-R shown in Table 1 as primers), and qRT-ZmACY-1-F and qRT-ZmACY-1-R as primers. The real-time fluorescent quantitative PCR reaction system is the same as that described above.

Extracting DNA of leaf blade of screening seedling obtained by hygromycin screening, and making T ₀ Molecular detection of positive transformants. As a result, as shown in FIG. 10A, six T strains were obtained in total ₀ The positive seedlings of the generation ZmACY-1 are positive seedlings which are successfully transformed, and 3 lines which are converted into ZmACY-1 and are respectively named as OE1, OE3 and OE5 are selected as subsequent experimental materials. Extraction of T ₃ Substitution of ZmACY-1 real-time fluorescence quantification of purified seedling and wild type leaf RNA of the present tobacco to detect the relative expression level of ZmACY-1 in each strain, the method is the same as above, and the result is shown in FIG. 10B. From this, zmACY-1 was hardly expressed in wild-type benthonic cigarettes, but was highly expressed in OE1, OE3 and OE5 strains, which indicated that the altered phenotype (trait) in OE1, OE3 and OE5 strains relative to wild-type benthonic cigarettes was caused directly or indirectly by the ZmACY-1 gene.

Identification of the phenotype of ZmACY-1-transformed tobacco

1. Determination and comparison of germination rates of ZmACY-1-transformed tobacco seeds and wild tobacco seeds

Transgenic lines OE1, OE3, OE5 and wild-type tobacco seeds were aseptically sown in ultra-clean bench in culture dishes containing MS medium, one hundred seeds were spotted per plate, and germination rates were measured and compared from the third day (statistical data see FIG. 11A). To compare the vigor of transgenic and wild-type lines (see FIG. 11-B-D), seed from each line was aseptically sown in the same plate, with 25 seeds per line. Therefore, the ZmACY-1 gene can improve the germination rate of tobacco seeds and promote the growth of tobacco.

2. Identification of phenotype of ZmACY-1-transformed tobacco plants and wild tobacco plants

For T ₃ After 15 days of culture of the transgenic lines OE1, OE3, OE5 and wild-type tobacco seedlings in the MS medium, the seedlings were transplanted into the soil, and after 15 days, the plant height of the tobacco plants of one month old (see FIG. 12C for results), the leaf area (see FIG. 12B for results), the stem thickness (see FIG. 12D for results), the root length (see FIG. 12F for results), the leaf number (see FIG. 12A for results), the root area (see FIG. 12G for results), the fresh weight of the aerial parts (see FIG. 12E for results), and the fresh weight of the underground parts (see FIG. 12H for results) were measured.

3. The transgenic strains OE1, OE3 and OE5 and leaf RNA of the same part of the wild tobacco benthonic tobacco are extracted for real-time fluorescence quantitative PCR, and the PCR method is the same as that of the fifth section of the example 5, and the NBEXPA1 gene is amplified by adopting NbEXPA1-F, nbEXPA1-R shown in the table 1 as a primer. The NBEIN2 gene was amplified using the NbEIN2-F, nbEIN2-R primers shown in Table 1. The relative expression amounts of two genes NBEXPA1 (GenBank: NM-001325646.1) and NBEIN2 (GenBank: XM-016579720.1) were measured. The results are shown in FIG. 13.

NBEXPA1 belongs to the family of swollenins and plays an extremely important role in promoting leaf growth. NbEIN2 is an essential positive regulator in the ethylene signal pathway. As can be seen from FIG. 13, the expression levels of NBEXPA1 and NBEIN2 in transgenic benthames were significantly higher than those of the wild type. It is shown that the overexpression of ZmACY-1 gene in the present smoke can cause the plant growth speed to be accelerated by positively regulating the expression of plant growth related genes NBEXPA1 and NBEIN 2. Therefore, zmACY-1 not only responds to the regulation and control of plant hormones, thereby promoting the growth and development process of plants, but also plays an important role in improving the biomass and the yield of the plants.

5. The mid-maturity full pods of tobacco were picked for weighing and fruit length and width measurements (see fig. 14 for results).

As shown in fig. 11, fig. 12 and fig. 14, the overexpression of ZmACY-1 in the present raw tobacco significantly promoted the growth and development of plants, and the germination rate, plant height, root length, stem thickness, leaf area (fifth functional leaf), root area, fresh weight of aerial parts, fresh weight of underground parts, and pods (mature plants) of the transgenic plants were significantly greater than those of the wild plants. ZmACY-1 can promote the growth of the tobacco of the primary tobacco.

Study of the response of (seventh) ZmACY-1 tobacco to salt stress and PEG stress

Influence of salt stress and PEG stress on physiological and biochemical transformation of ZmACY-1. Sup. Th-primary cigarette

1. Salt stress is carried out on young Nicotiana benthamiana seedlings of one month old by irrigating 350mmol/L NaCl, and PEG stress is treated by 20% PEG6000 aqueous solution.

Response of ZmACY-1 transformed Bensheng cigarette to salt stress

For pair T ₃ The results of 350mmol/L NaCl solution treatment on the primary smoke of one month old show that after 10 days of treatment, the wild type and the ZmACY-1 transformed strain gradually lose green and turn yellow, but the ZmACY-1 transformed primary smoke has more obvious green and turn yellow, and is significantly higher than the wild type (figure 15A). In addition, fresh water irrigation was used as a control to measure the tobacco of each strain of the raw tobacco after 10 days of salt stress treatment The fresh weight of the aerial parts (figure 15B) and the chlorophyll content (figure 15C) show that the fresh weight and the chlorophyll content of the aerial parts of the transgenic plant lines after 10 days of water irrigation are obviously higher than those of the wild type, the fresh weight and the chlorophyll content of the aerial parts of each plant line are reduced after 10 days of salt stress, and the fresh weight and the chlorophyll content of the aerial parts of the transgenic plant lines are obviously lower than those of the wild type. This suggests that overexpression of ZmACY-1 in this smoke weakens the salt tolerance of the plant.

Response of ZmACY-1 transformed Bensheng cigarette to drought stress.

The study is directed to T ₃ Drought treatment is carried out on the primary smoke of the first month of age, and clean water irrigation is used as a control. As a result, it was found that the transgenic lines were more serious in wilting degree after drought treatment for 7 days than the wild-type raw tobacco, and that the wild-type raw tobacco was significantly recovered to a higher degree than the ZmACY-1 transgenic lines after rehydration for two days (FIG. 16A). In addition, the present study also measured the fresh weight of the aerial parts of the tobacco of each strain in the untreated and drought conditions, and as a result, it was found that drought stress significantly resulted in a decrease in the fresh weight of the aerial parts of both the wild type and transgenic strain, and that in the untreated condition the fresh weight of the aerial parts of the transgenic strain was significantly higher than that of the wild type, however, the fresh weight of the aerial parts of the transgenic strain was lower than that of the wild type in the drought stress condition (FIG. 16B). The result shows that, unlike the expression of escherichia coli BL21, the overexpression of ZmACY-1 in the raw tobacco weakens the drought resistance of plants.

(1) Malondialdehyde (MDA) content determination

Shearing 0.2g of the leaf of the present cigarette, adding 3mL of 5% trichloroacetic acid (TCA), grinding into slurry, centrifuging at 5,000rmp for 10min, taking the supernatant, adding into a test tube, adding 0.5% thiobarbituric acid (TBA) with equal volume, mixing uniformly, putting into boiling water bath, taking out after 30min, cooling, and measuring the absorbance at 450nm, 532nm and 600nm ^[5] 。

MDA content (nmol/L) =6.45× (A ₅₃₂ -A ₆₀₀ )-0.56×A ₄₅₀

As a result, referring to fig. 17D, it can be seen that the salt stress and drought stress post-conversion ZmACY-1 present cigarette has stronger peroxidase activity than the wild present cigarette.

(2) Determination of relative conductivity

Cutting about 0.2g of the leaf of the cigarette, transferring to a test tube, adding 20mL of deionized water until the leaf is immersed, standing for 4 hours at room temperature, fully shaking, and measuring the leaf conductivity as R by using a conductivity meter ₁ Boiling in boiling water bath for 25min, cooling, shaking, and measuring conductivity R again ₂ ^[6] 。

Relative conductivity=r ₁ /R ₂ ×100％

As a result, referring to fig. 17E, it can be seen that the salt stress and drought stress post-conversion ZmACY-1 present cigarettes were stronger in relative conductivity than the wild present cigarettes.

(3) Determination of protective enzyme Activity

Extracting crude enzyme liquid: shearing 0.2g of the raw tobacco leaves, putting the raw tobacco leaves into a mortar, adding 2mL of precooled 0.1mol/L Tris-HCL buffer solution, grinding, sucking the ground raw tobacco leaves into a 2mL centrifuge tube, flushing the residual substances in the mortar by using 1mL buffer solution, and sucking the residual substances into the centrifuge tube. Centrifuging at 4deg.C at 8,000rpm for 30min, collecting supernatant to obtain crude enzyme solution, packaging, and storing in-20deg.C refrigerator.

(1) Superoxide dismutase (SOD) activity assay

Measuring SOD activity by adopting Nitrogen Blue Tetrazolium (NBT) method, measuring absorbance value of sample at 560nm by enzyme-labeled instrument ^[7] . SOD activity (U/mg protein) = (a) ₀ -A)×V _T ×(0.5A ₀ ×W×V ₁ ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein A is ₀ : absorbance at 560nm for the control tube; a: absorbance of the sample tube at 560 nm; v (V) _T : total volume of enzyme extract (mL); v (V) ₁ : measuring the volume (mL) of enzyme solution added during measurement; w: fresh weight of sample (g).

As a result, referring to fig. 17B, it can be seen that the salt stress and drought stress post-conversion ZmACY-1 present cigarette has stronger peroxidase activity than the wild present cigarette.

(2) Peroxidase (POD) Activity assay

POD activity was measured by guaiacol chromogenic method, and absorbance at 470nm was measured on a crude enzyme solution sample using an ELISA reader, once every 1min (5 times). At each timeMinute change of 0.01 to 1 enzyme activity unit ^[8] 。

POD activity = 10 ⁵ ×△A ₄₇₀ /C·V _S ·t；△A ₄₇₀ : light absorption value changes over the reaction time (t); c: concentration of enzyme solution protein (. Mu.g/. Mu.L); v (V) _S : taking the volume (mL) of the enzyme solution during measurement; t: reaction time (min).

As a result, referring to fig. 17A, it can be seen that the salt stress and drought stress post-conversion ZmACY-1 present cigarette has stronger peroxidase activity than the wild present cigarette.

(3) Catalase (CAT) Activity assay

The guaiacol chromogenic method is adopted to measure, a crude enzyme liquid sample is used for measuring the absorbance value at 470nm by a spectrophotometer, and the absorbance value is measured every 1min (5 times). The change per minute was 0.01 to 1 enzyme activity unit.

3. Catalase (CAT)

mu.L of the enzyme extract was aspirated, and 1.5mL of 100mM PBS and 3.75uL of 30% H were added ₂ O ₂ Mixing and removing bubbles. The absorbance was recorded at 240nm every 10sec, and the enzyme activity was finally expressed as absorbance change. As a result, referring to fig. 17C, it can be seen that the salt stress and drought stress post-conversion ZmACY-1 present cigarette has stronger catalase activity than the wild present cigarette.

In conclusion, under the conditions of salt stress and drought stress, superoxide dismutase (SOD) can convert harmful substances in cells into H ₂ O ₂ And O ₂ Whereas CAT and POD will scavenge these products, thus the three antioxidant enzymes reduce the damage to plants from stress under synergistic action. MDA is the final breakdown product of lipid peroxidation, and the level of MDA reflects the extent of plant damage. To further verify that overexpression of ZmACY-1 in the present raw tobacco weakens the drought and salt tolerance of the plants, the present study measured the content of protective enzyme POD, SOD, CAT in the leaves of each strain, as well as the MDA content and relative conductivity after two days of treatment with clear water (CK), 20% PEG600 and 350mmol/L NaCl. As a result, it was found that drought stress and salt stress all increased the activities of the protective enzymes POD, SOD and CAT of transgenic lines and wild-type raw tobacco And an increase in MDA content and relative conductivity (FIGS. 17A-E). Under salt stress and drought stress, activities of protective enzymes POD, SOD and CAT of the transgenic strain are lower than those of the wild type strain, but MDA content and relative conductivity of the transgenic strain are higher than those of the wild type strain.

Data acquisition 2 ^-△△Ct Relative quantitative analysis method ^[9] And (5) calculating. Data were collated using Microsoft Excel 2007 and plotted for analysis.

It can be seen that the damage to the cigarette produced by converting ZmACY-1 after salt stress and drought stress is more serious.

It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.

Sequence listing

<110> Qingdao university of agriculture

<120> use of aminoacylase-1

<130> C1CNCN220007

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 439

<212> PRT

<213> corn (Zea mays)

<400> 1

Met Pro Pro Pro Leu Arg Cys Leu Leu Leu Ala Phe Val Val Val Leu

1 5 10 15

Ser Gly Phe Pro Arg Leu Ala His Pro Phe Thr Ala Leu Glu Ser Asp

20 25 30

Gln Ile Ala Arg Phe Gln Glu Tyr Leu Arg Ile Arg Thr Ala His Pro

35 40 45

Ser Pro Asp Tyr Ala Gly Ala Ser Ala Phe Leu Leu His Tyr Ala Ala

50 55 60

Ser Leu Gly Leu His Thr Thr Thr Leu His Phe Thr Pro Cys Lys Thr

65 70 75 80

Lys Pro Leu Leu Leu Leu Thr Trp Arg Gly Ser Asp Pro Ser Leu Pro

85 90 95

Ser Val Leu Leu Asn Ser His Met Asp Ser Val Pro Ala Glu Pro Glu

100 105 110

His Trp Ala His Pro Pro Phe Ala Ala His Arg Asp Pro Thr Thr Gly

115 120 125

Arg Ile Tyr Ala Arg Gly Ala Gln Asp Asp Lys Cys Leu Pro Val Gln

130 135 140

Tyr Leu Glu Ala Ile Arg Gly Leu Gln Ala Ala Gly Phe Ala Pro Ala

145 150 155 160

Arg Thr Ile His Ile Ser Leu Val Pro Asp Glu Glu Ile Gly Gly Ala

165 170 175

Asp Gly Phe Asp Lys Phe Ala Arg Ser Glu Glu Phe Arg Ala Leu Asn

180 185 190

Ile Gly Phe Met Leu Asp Glu Gly Gln Ala Ser Pro Thr Asp Val Phe

195 200 205

Arg Val Phe Tyr Ala Asp Arg Leu Val Trp Arg Leu Val Val Lys Ala

210 215 220

Ala Gly Ala Pro Gly His Gly Ser Arg Met Leu Asp Gly Ala Ala Val

225 230 235 240

Asp Asn Leu Met Asp Cys Val Glu Thr Ile Ala Ala Phe Arg Asp Ala

245 250 255

Gln Phe Arg Met Val Lys Ser Gly Glu Lys Gly Pro Gly Glu Val Val

260 265 270

Ser Val Asn Pro Val Tyr Met Lys Ala Gly Ile Pro Ser Pro Thr Gly

275 280 285

Phe Val Met Asn Met Gln Pro Ser Glu Ala Glu Val Gly Phe Asp Leu

290 295 300

Arg Leu Pro Pro Thr Glu Asp Ile Glu Gln Ile Lys Arg Arg Val Glu

305 310 315 320

Glu Glu Trp Ala Pro Ser His Lys Asn Leu Thr Tyr Glu Leu Val Gln

325 330 335

Lys Gly Pro Ala Thr Asp Val Ser Gly Arg Pro Val Ser Thr Ala Thr

340 345 350

Asn Ala Ser Asn Pro Trp Trp Leu Thr Phe Glu Arg Ala Ile Ala Ser

355 360 365

Ala Gly Gly Glu Leu Ser Lys Pro Glu Ile Leu Ser Ser Thr Thr Asp

370 375 380

Ser Arg Phe Ala Arg Gln Leu Gly Ile Pro Ala Leu Gly Phe Ser Pro

385 390 395 400

Met Thr Arg Thr Pro Ile Leu Leu His Asp His Asn Glu Phe Leu Glu

405 410 415

Asp Arg Val Phe Leu Arg Gly Ile Gln Val Tyr Glu His Val Ile Arg

420 425 430

Ala Leu Ser Ser Phe Gln Gly

435

<210> 2

<211> 1320

<212> DNA

<213> corn (Zea mays)

<400> 2

atgccgccgc ctctccgctg tctccttctc gccttcgtcg tcgtcctctc cggcttcccc 60

cgtctcgccc accccttcac ggctctcgag tctgaccaga tcgcccgctt ccaggaatac 120

ctccgcatcc gaactgcgca cccatccccc gactacgccg gcgccagcgc cttcctccta 180

cactacgccg cttcgctcgg tctccacacc accacgctcc acttcacccc gtgcaagacc 240

aagcccctgc tcctcctcac ctggcgaggc tccgatccct ccctcccctc cgtgctcctc 300

aactcccaca tggactccgt ccccgcggag cccgagcact gggcgcaccc tccattcgcc 360

gcgcaccgcg acccgaccac gggccgcatc tacgcgcgcg gcgcacagga cgacaagtgc 420

ctccccgtcc agtacctcga ggcgatccgg ggcctgcagg ccgcggggtt cgctcccgcc 480

cgcaccatcc acatctcgct tgtccccgac gaggagatcg gcggcgcgga tgggttcgac 540

aagttcgccc gatcggagga gttccgcgcc ctcaacatcg ggtttatgct cgacgagggg 600

caggcgtcgc cgacggacgt gttcagagtc ttttacgcgg acaggctggt gtggaggctc 660

gtcgtgaagg cggcgggggc gccagggcat gggtcgagga tgttggacgg cgccgccgtt 720

gacaatttga tggattgcgt ggagaccatc gctgcgttca gggatgcgca gttcaggatg 780

gtgaagtccg gggagaaggg tcctggggag gtggtctcag tcaaccctgt gtacatgaag 840

gccggcatac caagccccac gggtttcgtg atgaacatgc aaccttcaga agcggaggtc 900

ggctttgacc tccgccttcc tccaaccgaa gacatcgagc agatcaagcg gagggtcgaa 960

gaggaatggg caccatctca caaaaacctg acctacgagc tggtgcagaa aggtccggcg 1020

acggatgtgt ccggacgtcc cgtatccaca gcgacgaacg cgtcgaaccc gtggtggctg 1080

acgttcgaga gggccatcgc ctccgcgggt ggggagctgt ctaagcctga gatcctgtct 1140

tcgaccacgg actcacgctt tgcgcggcag ctgggcatcc ctgccctcgg gttttctccg 1200

atgaccagga cgcccatact gctacatgac cataacgagt ttctggaaga cagagtgttc 1260

ctgaggggca tccaagtgta cgaacatgtc atcagagcac taagctcgtt ccaaggctga 1320

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atgccgccgc cgcctctccg ctgt 24

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tcagccttgg aacgagctta gtgc 24

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aagacatcga gcagatcaag c 21

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tcgctgtgga tacgggac 18

<210> 7

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gctctagaat gccgccgccg cctctccgct gt 32

<210> 8

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cgggatcctc agccttggaa cgagcttagt gc 32

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cctcaagaag gttggataca ac 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcttgggctc attaatctgg tc 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ttgtttctct gcttctggat gg 22

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cttaatgcag cagtgtttgt acca 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggcataatag atctggcatt ttcc 24

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tatctaagag catcggtgca gttg 24

Claims (10)

1. Use of an aminoacylase-1 for increasing the anti-polyethylene glycol of a bacterium, wherein the aminoacylase-1 is a protein shown in SEQ ID No.1, and the bacterium is Escherichia coli.

2. The use according to claim 1, characterized in that: the bacterial anti-polyethylene glycol is that the bacteria are grown in a liquid medium containing 1wt% to 20wt% polyethylene glycol.

3. Use according to claim 2, characterized in that: the bacterial anti-polyethylene glycol is that the bacteria are grown in a liquid medium containing 5wt% to 15wt% polyethylene glycol.

4. The use according to claim 1, characterized in that: the nucleic acid sequence for encoding the amino-acylase-1 is shown in SEQ ID NO. 2.

5. The use according to claim 1, characterized in that: the bacterium is escherichia coli BL21.

6. A method for improving polyethylene glycol resistance of bacteria comprises transferring expressible amino-acylase-1 gene into bacteria, wherein the amino-acylase-1 is protein shown as SEQ ID NO.1, and the bacteria are Escherichia coli.

7. The method of claim 6, wherein: the bacterial anti-polyethylene glycol is that the bacteria are grown in a liquid medium containing 1wt% to 20wt% polyethylene glycol.

8. The method of claim 7, wherein: the bacterial anti-polyethylene glycol is that the bacteria are grown in a liquid medium containing 5wt% to 15wt% polyethylene glycol.

9. The method of claim 6, wherein: the nucleic acid sequence for encoding the amino-acylase-1 is shown in SEQ ID NO. 2.

10. The method of claim 6, wherein: the bacterium is escherichia coli BL21.