CN117887683A - Heat-resistant regulatory gene BrAK of flowering cabbage and application thereof - Google Patents
Heat-resistant regulatory gene BrAK of flowering cabbage and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention belongs to the field of plant genetic engineering, and particularly relates to a heat-resistant regulation gene BrAK of a vegetable heart and application thereof, wherein the heat-resistant regulation gene of the vegetable heart is an aspartokinase gene BrAK. The nucleotide sequence of BrAK is shown as SEQ ID NO. 1. The BrAK gene disclosed by the invention has very important effect in regulating and controlling the heat resistance of the vegetable center, can effectively regulate the tolerance of the vegetable center to high temperature, can be applied to regulating and controlling the heat resistance of the vegetable center, lays a theoretical foundation for researching the mechanism of the plant to respond to high temperature signals, provides gene resources for cultivating new varieties of heat-resistant vegetable center, and has good potential application value.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a heat-resistant vegetable heart regulatory gene BrAK and application thereof.
Background
The vegetable core (Brassica campestris L. Ssp. Chinensis var. Utilis TSEN ET LEE), also called as a vegetable bolt, is a first and second annual herb plant of Brassica genus of Brassicaceae, is selected and cultivated and domesticated for a long time from Chinese cabbage subspecies and Chinese cabbage subspecies, and is a special product in the south China. The vegetable heart is cool, the growth and development of the vegetable heart are affected in the high-temperature weather with the temperature of above 30 ℃ and the proper temperature of 15-25 ℃ is suitable, so that the vegetable is thin and inferior in quality, and the single plant weight and commodity yield are greatly reduced. Particularly in the south China, the high-temperature duration time in summer is long, the extremely high-temperature frequency exceeding 40 ℃ appears, and the high temperature becomes one of important factors for limiting the normal growth of the vegetable cores in the south China.
At present, most of research on the aspect of high temperature stress resistance of the flowering cabbage is focused on the aspect of economic character indexes or physiological and biochemical changes of the flowering cabbage, but related research on molecular mechanisms of the flowering cabbage responding to the high temperature stress is less, and the functions of most high temperature response genes are not clear. Therefore, the heat-resistant regulation mechanism of the cabbage heart is analyzed, and the heat-resistant gene is mined, so that a molecular basis can be provided for cultivating new heat-resistant cabbage heart varieties.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a heart heat-resistant regulatory gene BrAK and application thereof.
The aim of the invention is achieved by the following technical scheme: the heat-resistant regulation gene of the cabbage is an aspartokinase gene BrAK, and the nucleotide sequence of the aspartokinase gene BrAK is shown as SEQ ID NO. 1.
Further, the protein encoded by aspartokinase gene BrAK has the amino acid sequence shown in SEQ ID NO. 2.
The gene BrAK is applied to the aspect of regulating and controlling the heat resistance of the cabbage heart.
Further, the gene BrAK affects the heat resistance of the heart by regulating heat resistant gene expression and ROS signaling.
The invention also provides a heat-resistant cabbage heart overexpression system, which contains the cabbage heart heat-resistant regulation gene aspartokinase gene BrAK.
The invention also provides a construction method of the heat-resistant cabbage heart overexpression system, which uses a homologous recombination method to construct the CDS full-length sequence of the aspartokinase gene BrAK into the pEarley gate 101 vector, thereby obtaining a BrAK gene overexpression system, namely a vector pEarley gate 101-BrAK3; the pEarley gate 101-BrAK3 over-expression vector was transferred to Agrobacterium GV3101 to obtain BrAK gene over-expression plants.
The invention also provides a construction method of the heat-resistant cabbage heart silencing system, which uses a homologous recombination method to construct a segment with the length of 90bp of CDS non-conservation section of BrAK genes and a complementary segment thereof into a pK7GWIWG (I) vector, so as to obtain a BrAK3 gene silencing vector pK7GWIWG (I) -BrAK3; the pK7GWIWG (I) -BrAK3 silencing vector was transferred to Agrobacterium GV3101 to obtain BrAK gene silencing plants.
Further, the nucleotide sequence of the CDS non-conservation region 90bp long fragment of BrAK gene is shown as SEQ ID NO. 3.
The invention has the following advantages:
(1) The cabbage heart high-temperature response gene BrAK provided by the invention has the advantages that under the high-temperature condition, the cabbage heart material over-expressed by the BrAK gene is more resistant to high temperature, the BrHsfA, brHsp and BrMn-SOD gene expression and the antioxidant enzyme SOD activity of the over-expressed plants are obviously higher than those of blank control plants, and the relative conductivity and the H 2O2 content are obviously reduced compared with those of the blank control plants. The BrAK gene silencing is opposite, after high temperature stress, the BrHsfA, brHsp, brMn-SOD gene expression level and SOD activity and chlorophyll content of BrAK gene silencing plants are obviously reduced compared with those of blank control plants, and the relative conductivity and H 2O2 content are obviously increased.
(3) The BrAK gene disclosed by the invention has very important effect in regulating and controlling the heat resistance of the vegetable heart, can effectively regulate the tolerance of the vegetable heart to high temperature, lays a theoretical foundation for researching the mechanism of plant response to high temperature signals, provides gene resources for cultivating new varieties of heat-resistant vegetable heart, and has good potential application value.
Drawings
FIG. 1 shows a plasmid map of pK7GWIWG (I).
FIG. 2 is a diagram showing transcriptional expression analysis of BrAK gene on heat-resistant material CX1-7 and heat-sensitive material CX 7-3.
FIG. 3 is a diagram of a BrAK gene cis-acting element analysis.
FIG. 4 is a pEarley gate 101 plasmid map.
FIG. 5 is a graph showing the overexpression of BrAK gene and the expression of BrAK gene in leaf of control cabbage.
FIG. 6 is a graph showing the overexpression of BrAK gene and the expression of heat-resistant marker gene and ROS signal response gene before and after high temperature stress in leaf of control cabbage.
FIG. 7 is a graph showing phenotype changes before and after high temperature stress of BrCK gene overexpression and blank control flowering cabbage plants.
FIG. 8 is a graph showing the results of measurement of chlorophyll content, relative conductivity, H 2O2 content and SOD activity in BrAK gene-overexpressing plants and mock cabbage leaves.
FIG. 9 is a graph showing BrAK gene silencing and BrAK gene expression in control heart leaves.
FIG. 10 is a graph showing the expression of heat-resistant marker genes before and after high temperature stress and ROS signal response genes in BrAK gene silencing and in control heart leaves.
FIG. 11 is a graph showing the change in phenotype of BrAK gene-silenced plants and mock heart plants before and after high temperature stress.
FIG. 12 is a graph showing the results of chlorophyll content, relative conductivity, H 2O2 content, and SOD activity measurements in BrAK gene-silenced plants and mock cabbage leaves.
Detailed Description
The invention will be further described with reference to examples, but the scope of the invention is not limited to the following:
Example 1: transcriptional expression detection of BrAK gene in heat-resistant and heat-sensitive cabbage materials
The gene of the flowering cabbage BrAK is discovered by the inventor through analysis of the data of the flowering cabbage high temperature stress transcriptome. The heat-resistant materials CX1-7 and the heat-sensitive materials CX7-3 (CX 1-7 and CX7-3 are disclosed in a patent publication CN113348992A and are stored in the institute of vegetables of the academy of agricultural sciences of Hainan province) used in the transcriptome database are subjected to high-temperature stress identification in a seedling stage and natural high-temperature identification in a field. Transcriptome sequencing was done by Beijing Baimai Biotechnology Co., ltd, using a high temperature stress treatment temperature of 37℃and a treatment time of 6 hours, specific treatment reference "(Pang Jiangjiang et al, transcriptome analysis of cabbage leaves in response to high temperature stress, transcription factor screening [ J/OL ]. Molecular plant breeding: 1-23[2023-05-24]. The transcriptional expression level of the flowering cabbage BrAK gene before and after high temperature stress is shown in figure 2.
It can be found that the transcriptional expression level of the flowering cabbage BrAK gene in the thermosensitive material CX7-3 and the heat-resistant material CX1-7 is lower before high temperature stress (0 h); after 6h of high temperature stress, the transcriptional expression level of BrAK gene in CX1-7 is rapidly increased to 15.58 times that before high temperature stress, and the expression level in CX7-3 is only 2.30 times that before high temperature stress; in addition, the amount of BrAK gene expressed in CX1-7 after 6h of high temperature stress is 22.02 times that in CX 7-3. The above results indicate that the heart BrAK gene is a potential high Wen Xiangying regulatory gene.
Example 2: cis-acting element analysis
The BrAK gene was analyzed for cis-acting element discovery using an online tool PlantCAR (http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html /), and the results are shown in FIG. 3. As can be seen from FIG. 3, brAK genes contain 11 high temperature stress response elements CAAT-box.
Example 3: brAK3 gene over-expression system construction and heat resistance analysis
The full length CDS sequence of BrAK gene was constructed into pEarley gate 101 vector (available from Shanghai ze leaf Biotechnology Co., ltd.) using homologous recombination method, the vector size was 12454bp, the insertion position of BrAK3 gene was specifically ccdB gene between attR1 and attR2 after plasmid 35S promoter, thus obtaining BrAK gene overexpression vector pEarley gate 101-BrAK3, and the vector map and insertion position are shown in FIG. 4.
The length of the full-length CDS sequence of BrAK gene is 681bp, as shown in SEQ ID NO.1, the specific sequence is:
5'-ATGCGGCCAGCTAGCGAAGGGAACATTCCTGTAAGGGTTAAGAACTCTTACAA TCCCACTGCACCAGGAACTCTCATCACTAGATCAAGAGACATGAGCAAGGCTGTACTGACCAGCATCGTTCTGAAACGTAATGTTACCATGTTGGACATCACTAGCAACCGTATGCTCGGTCAATATGGTTTCCTTTCCAAGGTGCTCTCCACATTTGAAAAGATGGGCATATCTGTAGATGTTGTTGCAACCAGCGAAGTTAGCGTTTCCTTGACATTGGATCCTTCAAAGTTCTGCAGCAAAGAGTTAATTCAACAGGAGATTAATCACATGGTAGAGGAACTGGAGAAGATTGCTTCGGTAAACCTGCTTCAGCACAGATCAATTATCTCACTCATTGGAAATTTTCAGAGATCATCATCTTTCATATTAGAGAAGGGCTTCCGAGTTCTTAGAACCAATGGAATCAATGTCCAGATGATCTCTCAGGGTGCATCTAAGGTAAACATCTCGCTGATAGTGAATGATGATGAGGCAGAGCATTGTGTGAAGGCTCTCCATTCAGCCTTCTTCGAGACAGGAACCTCCAAAGCTGTTCCTCAGATAGGGCGTCTAGCTACTACACCTCTGCCTTGTCGTGAAAACCTCTTGGTGCAACCTGACCTGCGAGATCTACAAGTAGTTTGA-3'
the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and specifically comprises the following steps:
MRPASEGNIPVRVKNSYNPTAPGTLITRSRDMSKAVLTSIVLKRNVTMLDITSNRML
GQYGFLSKVLSTFEKMGISVDVVATSEVSVSLTLDPSKFCSKELIQQEINHMVEELEK
IASVNLLQHRSIISLIGNFQRSSSFILEKGFRVLRTNGINVQMISQGASKVNISLIVNDDEAEHCVKALHSAFFETGTSKAVPQIGRLATTPLPCRENLLVQPDLRDLQVV
Referring to the method of the predecessor on the vegetable core, specifically referring to the construction methods (1) - (7) of the transient over-expression/gene silencing vegetable core leaf gene expression model in the invention patent with publication number CN114934054A, namely the vegetable core latex protein gene BraMLP applied to downy mildew resistance, the pEarley gate 101-BrAK3 over-expression vector is transferred to agrobacterium GV3101, and after BrAK gene over-expression plants are obtained, brAK3 gene amplification primers are designed for positive identification. The experimental material used in this section was a thermosensitive material CX7-3.
Treating BrAK gene over-expression and blank control plants with 37 ℃ for 0h and 6h, photographing, recording the phenotype change of the plants before and after treatment, collecting heart leaves, and analyzing physiological indexes and related gene expression. The gene expression analysis method comprises the following steps: cDNA synthesis and purification were accomplished using TRIzol kit (Invitrogen TRIzol) extract RNA, superScript IV first strand cDNA Synthesis System kit (Invitroge), and related gene expression analysis was performed. The cDNA product is diluted 3 times and then used as a template for PCR amplification to carry out qRT-PCR amplification, wherein the reagent used for the qRT-PCR amplification is Premix Ex TaqTM kit (TAKARA), and the real-time fluorescent quantitative PCR instrument is BioRad CFX96 TM (BioRad Bere, USA). The qRT-PCR amplification reaction was 5. Mu.L 2X SYBR Premix Ex TaqTM, 0.5. Mu.L forward primer (10. Mu. Mol/L), 0.5. Mu.L reverse primer (10. Mu. Mol/L), 4. Mu.L cDNA template (3-fold dilution) and 10. Mu.L sterile distilled water. qRT-PCR reaction procedure: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s, annealing at 60℃for 20s, extension at 72℃for 20s,40 cycles. Each reaction was repeated 3 times. The gene expression was analyzed by 2 -△△Ct method and mapped, and BrCK I gene was used as an internal reference gene (see Chinese patent publication No. CN115927296A for nucleotide sequence).
Wherein the expression amplification primers of BrCK I gene are:
F:5’-GTCTAACTTCCTCGCCACCT-3’SEQ ID NO.4
R:5’-GATTCGACGACAAGCCAGAC-3’SEQ ID NO.5
The expression amplification primers of BrAK gene are:
F:5’-GGCAGATGGGTTCAGATTCG-3’SEQ ID NO.6
R:5’-AAGAGGCGTGCTCATACTCA-3’SEQ ID NO.7
the expression amplification primers of BrHsfA gene are:
F:5’-GACCATTGCTCCAAGACACC-3’SEQ ID NO.8
R:5’-GGAAGAGACGGTGACCTACG-3’SEQ ID NO.9
The expression amplification primers of BrHsp gene are:
F:5’-CTGCAACAGTCTCACCCTCT-3’SEQ ID NO.10
R:5’-CTGAGAGCAAGTGGTGGAAC-3’SEQ ID NO.11
the expression amplification primers of BrMn-SOD gene are:
F:5’-AGATGAGTGCTGAAGGTGCT-3’SEQ ID NO.12
R:5’-CTGATTGGCGGTTGTGTCAA-3’SEQ ID NO.13
The physiological index measuring method comprises the following steps: the chlorophyll content is measured by adopting an SPDA method, the relative conductivity is measured by adopting a conductivity meter method, the H 2O2 content is measured by adopting a spectrophotometry method, the SOD activity is measured by adopting a nitrogen blue tetrazolium method, and the above index measurement is repeated for 3 times.
The positive identification result of BrAK gene over-expression plants is shown in figure 5, and after 6 hours of high temperature stress, the BrAK gene expression quantity of BrAK gene over-expression plants is obviously higher than that of blank control plants, which indicates that BrAK gene over-expression system construction is successful.
As shown in FIG. 6, the expression levels of the heat-resistant marker genes BrHsfA, brHsp and the ROS scavenger enzyme gene BrMn-SOD are remarkably increased after high temperature stress compared with the blank control plant, and the expression level in the BrAK gene over-expressed plant is remarkably higher than that of the blank control plant, which indicates that the BrAK3 gene can influence the heat resistance of the vegetable core by regulating and controlling high temperature and ROS signal transduction.
The results of phenotypic observation and physiological and biochemical index measurement are shown in fig. 7 and 8. After high temperature stress, the phenotype of the blank control plant is obviously changed, the leaf color is shallow, and the phenotype of the BrCK gene over-expression plant is not obviously changed. From the aspect of physiological and biochemical indexes, after 6 hours of high-temperature stress, the relative conductivity and H 2O2 content of BrAK gene over-expression plants are reduced, SOD activity is increased, chlorophyll content is not changed obviously, and heat resistance is obviously enhanced compared with that of blank control plants.
Example 4: brAK3 Gene silencing System construction and Heat resistance analysis
The BrAK gene silencing system was constructed by the following steps and results were obtained:
(1) The plasmid used for the BrAK gene silencing system was pK7GWIWG (I) (available from Shanghai ze leaf Biotechnology Co., ltd.) and was 13599bp in size, and the plasmid map was shown in FIG. 1. The CDS non-conservation segment 90bp (CDS 592-681 bp) length fragment of BrCK gene and the complementary fragment thereof are substituted for the plasmid ccdB gene by a gene recombination exchange mode, wherein the specific position of the ccdB gene is between attR1 and attR2 after a 35S promoter, so that BrAK gene silencing vector pK7GWIWG2 (I) -BrAK3 is obtained.
The CDS non-conservation section of BrCK gene used in the invention has a length of 90bp (592-681), as shown in SEQ ID NO.3, and the specific sequence is:
5‘-CCTCAGATAGGGCGTCTAGCTACTACACCTCTGCCTTGTCGTGAAAACCTCTT GGTGCAACCTGACCTGCGAGATCTACAAGTAGTTTGA-3’
BrCK3 Gene-silenced plants were obtained in the same manner as in example 3. After obtaining the silencing plants, primers (primer sequences identical to example 3) were designed for positive identification. The experimental materials used in this section were heat-resistant materials CX1-7.
Treating BrAK gene silencing plants and blank control plants thereof with 37 ℃ for 0h and 6h, photographing, recording the phenotype changes of the plants before and after treatment, collecting leaves of flowering cabbage seedlings, and analyzing physiological indexes and related gene expression. The gene expression analysis and the physiological index measurement method were the same as in example 3.
The results of the gene detection are shown in FIGS. 9 and 10. The expression level of BrAK gene in the silenced cabbage leaves is obviously lower than that of a control plant, which indicates that the construction of a silencing system of BrAK gene is successful. Compared with the control plants, the expression of the heat-resistant marker genes BrHsfA and BrHsp and the ROS scavenger enzyme gene BrMn-SOD is inhibited, which shows that BrAK gene can influence the heat resistance of the vegetable heart by regulating high temperature and ROS signal transduction.
The results of phenotypic observation and physiological and biochemical index measurement are shown in fig. 11 and 12. Under high temperature stress, compared with a control plant, the heat resistance of the BrAK gene silencing plant is reduced, the color of the leaf is light, the green content of the leaf is reduced, the relative conductivity and the H 2O2 content are increased, the SOD activity is reduced, and the heat resistance is obviously reduced compared with a blank control.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.
Claims (8)
1. The heart heat-resistant regulation gene is characterized by being an aspartokinase gene BrAK, and the nucleotide sequence of the aspartokinase gene BrAK is shown as SEQ ID NO. 1.
2. The heart-resistant regulatory gene of claim 1, wherein the protein encoded by aspartokinase gene BrAK has the amino acid sequence shown in SEQ ID No. 2.
3. Use of the gene BrAK according to claim 1 or 2 for regulating heat resistance of a heart of a vegetable.
4. The use of claim 3, wherein said gene BrAK affects the heat resistance of the heart of the dish by regulating heat resistant gene expression and ROS signaling.
5. A heat-resistant flowering cabbage overexpression system, which is characterized by comprising the flowering cabbage heat-resistant regulatory gene aspartokinase gene BrAK as claimed in claim 1 or 2.
6. The method for constructing a heat-resistant flowering-core overexpression system according to claim 5, wherein the full-length CDS sequence of aspartokinase gene BrAK is constructed into pEarley gate101 vector by using homologous recombination method, thereby obtaining BrAK3 gene overexpression system, namely vector pEarley gate101-BrAK 3; the pEarley gate101-BrAK3 over-expression vector was transferred to Agrobacterium GV3101 to obtain BrAK gene over-expression plants.
7. A construction method of a heat-resistant cabbage silencing system is characterized in that a homologous recombination method is used, a CDS non-conservation segment of BrAK genes with a length of 90bp and a complementary segment thereof are constructed to a pK7GWIWG (I) vector, so as to obtain a BrAK3 gene silencing vector pK7GWIWG (I) -BrAK3; the pK7GWIWG (I) -BrAK3 silencing vector was transferred to Agrobacterium GV3101 to obtain BrAK gene silencing plants.
8. The method of claim 7, wherein the nucleotide sequence of the 90bp long fragment of the CDS non-conserved region of BrAK gene is shown in SEQ ID No. 3.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410067587.1A CN117887683A (en) | 2023-07-26 | 2023-07-26 | Heat-resistant regulatory gene BrAK of flowering cabbage and application thereof |
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| CN202410067587.1A CN117887683A (en) | 2023-07-26 | 2023-07-26 | Heat-resistant regulatory gene BrAK of flowering cabbage and application thereof |
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| US5243039A (en) * | 1989-04-10 | 1993-09-07 | Regents Of The University Of Minnesota | Bacillus MGA3 aspartokinase II gene |
| US20130167266A1 (en) * | 2010-07-08 | 2013-06-27 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | Plant heat-resistance gene jaz5a and use thereof |
| CN113348992A (en) * | 2021-06-08 | 2021-09-07 | 海南省农业科学院蔬菜研究所 | Evaluation method for heat resistance of cabbage |
| CN114934054A (en) * | 2022-05-25 | 2022-08-23 | 广东省农业科学院设施农业研究所 | Application of cabbage heart latex protein gene BraMLP1 in resisting downy mildew |
| CN115927296A (en) * | 2022-09-13 | 2023-04-07 | 海南省农业科学院蔬菜研究所 | Cabbage heart high-temperature stress reference gene and screening method and application thereof |
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| US20140317782A1 (en) * | 2011-08-05 | 2014-10-23 | Shanghai Insititute For Biological Sciences, Chinse Academy Of Sciences | High temperature resistant plant gene and use thereof |
| CN114941008B (en) * | 2022-05-25 | 2023-03-24 | 广东省农业科学院设施农业研究所 | Application of flowering cabbage LRR receptor protein kinase gene BraEFR in downy mildew resistance |
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- 2023-07-26 CN CN202410067587.1A patent/CN117887683A/en active Pending
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| CN113348992A (en) * | 2021-06-08 | 2021-09-07 | 海南省农业科学院蔬菜研究所 | Evaluation method for heat resistance of cabbage |
| CN114934054A (en) * | 2022-05-25 | 2022-08-23 | 广东省农业科学院设施农业研究所 | Application of cabbage heart latex protein gene BraMLP1 in resisting downy mildew |
| CN115927296A (en) * | 2022-09-13 | 2023-04-07 | 海南省农业科学院蔬菜研究所 | Cabbage heart high-temperature stress reference gene and screening method and application thereof |
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| CN116732053A (en) | 2023-09-12 |
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