CN102618521A - ATP(Adenosine Triphosphate) -dependent metalloproteinase FtsH (Filamentation temperature-sensitive H - Google Patents

Abstract

The present invention relates to an ATP-dependent metalloprotease FtsH, the amino acid sequence of the enzyme is SEQ ID NO:1. The ATP-dependent metalloprotease FtsH obtained from the thermophilic bacterium Alicyclobacillus hesperidium has ATPase activity, proteolysis activity and molecular chaperone activity, and can be applied to the genetic modification of crops. The ATP-dependent metalloprotease FtsH of the present invention can be applied to the transformation of genetically engineered crops, can better improve crop resistance to environmental pressure, and greatly increase the yield of crops.

Description

An ATP-dependent metalloprotease FtsH

technical field

The invention belongs to the technical field of enzyme proteins, and in particular relates to an ATP-dependent metalloprotease FtsH.

Background technique

Proteins in organisms often lose their function due to various forms of damage, and repairing/removing damaged proteins is a form of protein quality control. Molecular chaperones can assist the refolding of damaged proteins, and some enzymatic repair processes can also reverse the damage of some proteins, and proteins that cannot be repaired are degraded by the proteolytic system to be completely removed. AAA protease (ATPases Associated with a variety of cellular Activities) has both protease and molecular chaperone activities, can mediate the degradation and repair of membrane proteins in bacteria and mitochondria, and form a quality control system for membrane-bound proteins.

FtsH (Filamentation temperature-sensitive H) belongs to the AAA protease family and is an important membrane-bound protease. In vivo, FtsH oligomerizes to form a hexameric ring structure, burying the proteolytic active site in the center of the hexameric complex cavity. The conserved modules of FtsH protein include N-terminal transmembrane domain, AAA structure, zinc ion binding module and so on. FtsH has ATPase activity, proteolytic activity and molecular chaperone activity, participates in the control of protein mass balance, and is also associated with responses to heat shock, hyperosmosis, light stress, low temperature, and disease. Due to the diversity of FtsH functions, it is related to many metabolic activities and developmental processes in cells, which is of great significance. In recent years, some studies have been done on its protease activity, but there are still many problems to be further solved. Moreover, in different species, the sequence of FtsH varies greatly. Therefore, it has been a difficult problem to isolate and determine the sequence of FtsH.

Contents of the invention

The object of the present invention is to provide an ATP-dependent metalloprotease FtsH from the thermophilic bacterium Alicyclobacillus hesperidium, which provides a research object under extreme living conditions to supplement the deficiencies of the prior art.

The invention provides an ATP-dependent metalloprotease FtsH, characterized in that: the amino acid sequence of the enzyme is SEQ ID NO: 1.

The present invention also provides a nucleotide for encoding the above-mentioned ATP-dependent metalloprotease FtsH.

The sequence of the nucleotide is SEQ ID NO:2.

The present invention also provides a recombinant plasmid for expressing the ATP-dependent metalloprotease FtsH of the present invention, the gene sequence carried by the recombinant plasmid is SEQ ID NO:2.

The above-mentioned protease is used to study the stress response of cells, and can be applied to the genetic modification of crops.

The ATP-dependent metalloprotease FtsH obtained from the thermophilic bacterium Alicyclobacillus hesperidium has ATPase activity, proteolysis activity and molecular chaperone activity, and can be applied to the genetic modification of crops. The ATP-dependent metalloprotease FtsH of the present invention can be applied to the transformation of genetically engineered crops, can better improve crop resistance to environmental pressure, and greatly increase the yield of crops.

Detailed ways

The method of the present invention will be further described below in conjunction with examples. However, the examples are for illustration only and are not limited thereto. For the experimental methods that do not indicate specific conditions in the following examples, conventional conditions can usually be used, such as the conditions described in the "Molecular Cloning Experiment Guide" written by J. Sambrook (Sambrook), or according to the manufacturer's suggestion conditional run.

Embodiment 1: ATP-dependent metalloprotease FtsH gene full-length cDNA acquisition of the present invention

In the present invention, firstly, the whole genome is sequenced, spliced and annotated using the next-generation sequencing technology. The genes with the FtsH conserved region were captured from the start codon AUG to the stop codon UGG. The specific steps are:

1. Whole genome sequencing of Alicyclobacillus hesperidium:

Total DNA extraction and library construction: Alicyclobacillus hesperidium cells were obtained by centrifugation, and the total DNA was extracted with reference to the GeneJET Genomic DNA Purification Kit from Fermentas. The sequencing library was constructed according to KAPA NGS Library Preparation Kit. Whole-genome sequencing was performed with the most advanced Solexa paired-end of Illumina HiSeq2000, generating data with 160-fold coverage. Apply Velvet (Sequence assembler for very short reads) splicing method to get 130 contigs. And preliminarily annotated the genome, and found about 1800 protein-coding genes.

2. Prediction of full-length cDNA of ATP-dependent metalloprotease FtsH gene:

Bioinformatics analysis found the protein sequence of the conserved region of ATP-dependent metalloprotease FtsH, such as N-terminal transmembrane domain, AAA structure, and zinc ion binding module, among 1800 protein-coding genes. From the start codon AUG to the stop codon UAA is the predicted cDNA sequence. The full-length cDNA reading frame is 1809bp long, and it is predicted that it encodes a protein containing 602 amino acid residues. The nucleotide sequence is shown in the sequence listing as SEQ ID NO: 1 Described, the nucleotide sequence of its corresponding gene is SEQ ID NO:2.

3. Acquisition of the full-length cDNA of the ATP-dependent metalloprotease FtsH gene:

Extraction of total RNA and synthesis of cDNA: Alicyclobacillus hesperidium cells were obtained by centrifugation, and total RNA was extracted according to the instructions of the Qiagen RNeasy Mini Kit kit. The amplification of the full-length cDNA gene fragments used Invitrogen's M-MLV Reverse transcriptase to synthesize the first strand, and 10 μl of cDNA first strand products were obtained.

cDNA full-length PCR reaction: Design specific primers P1 and P2 according to the sequence of the predicted ATP-dependent metalloprotease FtsH gene fragment (P1: 5′-ATGAACCGTTTTTACCGGAGC-3′SEQ ID NO: 3; P2: 5′-TCAGCCACAGCTTTCCATGATC-3′ SEQ ID NO: 4). Take 2 μl of cDNA for PCR reaction, 50 μl of the system. The reaction conditions were 94°C for 3 min; 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min; and 72°C for 10 min. A fragment of 1809 bp in the reading frame of the ATP-dependent metalloprotease FtsH gene was amplified, and the fragment was purified with an Omega gel cutting kit. According to TransGen's pEASY-Blunt Cloning Kit, the ligation reaction was carried out and E.coli competent cell DH5α was transformed. Take 200 μl of the transformation solution and spread it on the plate, and cultivate it overnight. Ten positive clones were randomly selected for identification, and after activation, they were sent to Invitrogen for sequencing identification.

Example 2: Heterologous expression of Alicyclobacillus hesperidium ATP-dependent metalloprotease FtsH full-length cDNA.

The prokaryotic expression system was used to induce the expression of Alicyclobacillus hesperidium ATP-dependent metalloprotease FtsH recombinant protein, and the specific steps were as follows:

1. Construction and identification of the recombinant plasmid FtsH-pET32a: According to the full-length cDNA sequence of the Alicyclobacillus hesperidium ATP-dependent metalloprotease FtsH gene, restriction endonuclease sites Sac I and Sal I sites were introduced at both ends of the gene to amplify The ATP-dependent metalloprotease FtsH gene of the present invention was subcloned into the prokaryotic expression plasmid pET32a, and sequenced to confirm that it was correctly inserted into the gene carrier.

2. The recombinant plasmid FtsH-pET32a was transformed into Escherichia coli E.coli BL21 (DE3) competent, cultured overnight at 37°C on an LA (containing 100 μg/m1) plate, and screened transformants by blue-white screening. Pick a single colony of the white transformant and inoculate it on an LA plate, and culture overnight at 37°C. Use a toothpick to dip a few colonies for PCR. PCR conditions: 94°C for 10 min; 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min; complete amplification after 72°C for 10 min. 1.0% agarose gel electrophoresis was used to detect PCR amplification products.

3. Induced expression and purification of ATP-dependent metalloprotease FtsH: The verified E. coli BL21 carrying the recombinant plasmid FtsH-pET32a was streaked on an LA plate and cultured at 37°C. Pick a single colony and put it into a liquid LA test tube, and cultivate overnight at 37°C on a shaker. Inoculate 100ml of LA medium at 1% inoculum size, culture on a shaker at 37°C at 210r/min until the bacterial concentration _OD600 reaches 0.6-0.8, add IPTG to a final concentration of 1mM, and culture on a shaker at 37°C for 4 hours to induce the expression of FtsH protein. The supernatant was collected by centrifugation. The supernatant was purified by a nickel ion chelate affinity chromatography column, the protein elution peak was collected, and the protein purification was detected by SDS-PAGE. The eluate containing the target protein is desalted and concentrated to obtain the ATP-dependent metalloprotease FtsH derived from Alicyclobacillus hesperidium.

4. Analysis of ATP-dependent metalloprotease FtsH activity and thermal stability: the purified ATP-dependent metalloprotease FtsH of the present invention was dissolved in 50mM Tris-acetate (pH8.0), and incubated at 70°C for 2 hours; 2 μg of the present invention ATP-dependent metalloprotease FtsH or ATP-dependent metalloprotease FtsH after warm bath were mixed with 1 μg β-casein (Sigma), final concentration 50mM Tris-acetic acid (pH8.0), 5mM magnesium acetate, 12.5μM zinc acetate, 80mM sodium chloride, 100μg/mL BSA, and 1.4mM β-mercaptoethanol solution were mixed, 20μl reaction system was added, 5mMATP was added before the reaction, and the reaction was carried out at 38°C for 2 hours. Add loading buffer (0.25M Tris, 1.92M glycine, 1% SDS, pH8.4-8.9) to terminate the reaction, and keep in ice bath. The samples were electrophoretically separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The ATP-dependent metalloprotease FtsH of the present invention was not added as a blank group. It was found that in the reaction system with the addition of the ATP-dependent metalloprotease FtsH of the present invention, the brightness of the β-casein band was significantly weaker than that of the blank group, and the activity of the protein remained unchanged after being incubated at 70°C.

Induced expression of Alicyclobacillus hesperidium ATP-dependent metalloprotease FtsH recombinant protein with eukaryotic expression system, the specific steps are as follows:

1. Construction and identification of the recombinant plasmid pROK2-FtsH: According to the full-length cDNA sequence of the Alicyclobacillus hesperidium ATP-dependent metalloprotease FtsH gene, restriction endonuclease sites Kpn I and Sac I sites were introduced at both ends of the gene to amplify The ATP-dependent metalloprotease FtsH gene of the present invention is subcloned into the eukaryotic expression plasmid pROK2, and sequenced to determine the correct inserted gene carrier.

2. Freeze-thaw method to transform recombinant plasmid pROK2-FtsH into Agrobacterium tumefaciens LBA4404, culture on YEB (containing 50mg/L kanamycin and 125mg/L rifampicin) plate at 28°C for 2-3d, and select transformants . A single colony of transformed Agrobacterium was picked and inoculated in YEB liquid medium (containing 50 mg/L kanamycin and 125 mg/L rifampicin), shaken at 220 rpm for 16 hours at 28° C., and used for PCR. PCR conditions: 94°C for 5min; 94°C for 30sec, 60°C for 1min, 72°C for 2min, a total of 35 cycles; 72°C for 10min to complete the amplification. 1.0% agarose gel electrophoresis was used to detect PCR amplification products.

3. Genetic transformation of rice: inoculate positive Agrobacterium on YEB solid medium, and culture at 28° C. for 2 days. A single colony was inserted into 5ml of YEB liquid medium, cultured by shaking at 220rpm for 24 hours, centrifuged at 5000rpm for 10 minutes, and the supernatant was discarded. Resuspend in 20ml of AAM medium containing 100μM acetosyringone, shake vigorously, adjust the OD ₆₀₀ of the bacterial solution to about 0.1-1.0, and let stand for 1 hour. Take immature ears 15-20 days after rice pollination, clean and disinfect the seeds after shelling. The immature embryos were picked out and inoculated in the induction medium, and cultured in the dark at 27°C for 10-14 days. Wait for the callus to grow up and subculture, select the embryogenic callus after the third generation, add the above-mentioned Agrobacterium bacteria solution, shake it slightly and rest for 30 minutes, dry the callus on sterile filter paper and inoculate it in co-culture cultured at 25°C for 3 days in the dark. Pick the co-cultured callus into a wide-mouth culture bottle and wash it with sterile water. Finally, sterilized water with 250 mg/L ampicillin was left to stand for one hour and dried. The next day, transfer to selection medium (250mg/L ampicillin, 1mg/L ZT, 500mg/L pipera) to select resistant callus.

4. Cultivation and observation of transgenic rice: the callus was transferred to a new selection medium every two weeks, and it took about three weeks to see tumor-like resistant callus growing from the brown and shriveled callus. The bright yellow colored resistant calli were transferred to differentiation medium for 3-5 days. A part was selected and transferred to the differentiation medium, and sprouts and roots grew after 3 weeks. The seedlings were moved to rooting medium, and the seedlings were used for analysis such as heat tolerance.

5. Analysis of heat resistance of transgenic rice and blank group: plants of transgenic rice group and non-transgenic blank group were pretreated at 42°C for 2 hours at the same time, and then heat-shocked in a light incubator at 50°C for 8 hours, and then the heat-stressed seedlings were moved to Recover growth under normal growth conditions; observe the plant phenotype after 2 weeks of recovery. The result shows that after 2 weeks, the metalloprotease FtsH transgenic rice group blank group that contains the ATP-dependency of Alicyclobacillus hesperidium source of the present invention basically recovers normal growth, and the damage of the blank group is heavier, illustrates that the ATP of the blank group plant than containing Alicyclobacillus hesperidium source of the present invention --dependent metalloproteinase FtsH transgenic rice plants were more susceptible to injury.

6. Analysis of chlorophyll fluorescence characteristics of ATP-dependent metalloprotease FtsH transgenic rice containing Alicyclobacillus hesperidium source of the present invention and blank group under high temperature stress: after 6 weeks of growth of resistant seedlings, carry out high temperature at 48°C and 50°C in a constant temperature light incubator Stress treatment, followed by recovery culture under normal growth conditions. Before heat shock treatment, after heat shock treatment, after 1 day of recovery, and after 2 days of recovery, the chlorophyll fluorescence parameters of the largest unfolded leaf were detected with a portable fluorescence analyzer, including initial fluorescence (F ₀ ), maximum fluorescence (F _m ), Variable fluorescence (F _v ), photochemical efficiency of chloroplasts (F _v /F _m ). Dark adaptation was carried out for 20 minutes before the measurement, and the detection light (<0.05 μmol/m ² /s) was irradiated first, and then the saturated pulse light (12000 μmol/m ² /s) was irradiated. For each transgenic line, 5 resistant plants were selected, and the same number of blank plants were selected for various heat shock treatments. The experimental results showed that after a 2-day recovery period, the F _v / _m of the transgenic rice plants basically returned to normal, while in the blank group plants, the F _v / F _m was still at a low level, indicating that under high temperature stress, the blank group plants suffered more severe damage than transgenic plants containing the ATP-dependent metalloprotease FtsH derived from Alicyclobacillus hesperidium of the present invention.

Claims (6)

1. the metalloprotease FtsH that relies on of an ATP-, it is characterized in that: the aminoacid sequence of said enzyme is SEQ ID NO:1.

2. Nucleotide, the metalloprotease FtsH that the described ATP-of claim 1 that is used to encode relies on.

3. Nucleotide as claimed in claim 2, its sequence are SEQ ID NO:2.

4. a recombinant plasmid is characterized in that, described recombinant plasmid is used to express the metalloprotease FtsH that the described ATP-of claim 1 relies on.

5. recombinant plasmid as claimed in claim 4 is characterized in that, the nucleotides sequence of the gene that described recombinant plasmid carries is classified SEQ ID NO:2 as.

6. the metalloprotease FtsH of the described ATP-dependence of claim 1 is used to study the stress response of cell.