CN111793626A - Oligo DNA group of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes - Google Patents

Oligo DNA group of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes Download PDF

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CN111793626A
CN111793626A CN202010745749.4A CN202010745749A CN111793626A CN 111793626 A CN111793626 A CN 111793626A CN 202010745749 A CN202010745749 A CN 202010745749A CN 111793626 A CN111793626 A CN 111793626A
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徐杰
林小丽
贺浩华
姜志树
范寒雨
刘嘉龙
马莹莹
朱昌兰
边建民
傅军如
彭小松
贺晓鹏
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Abstract

The invention provides two sgRNAs for knocking out rice OsMAP65-3.1(LOC _ Os05g47970) genes. Two sgRNA sequences based on CRISPR/Cas9 are designed aiming at the rice OsMAP65-3.1 gene, a DNA fragment containing the sgRNA sequences is connected into a vector carrying CRISPR/Cas9, and rice is transformed through agrobacterium so as to knock out the rice OsMAP65-3.1 gene. Wherein, the nucleotide sequence of the sgRNA action site is shown as SEQ ID NO.1 and SEQ ID NO. 2. According to the invention, the rice endogenous gene OsMAP65-3.1 is edited by a CRISPR-CAS9 technology, and two OsMAP65-3.1-1 and OsMAP65-3.1-2 knockout mutants are obtained. The sgRNA prepared by the method can efficiently, quickly and accurately target the OsMAP65-3.1 gene of rice, and has certain significance in basic research (mitosis of rice) and production practice (high-yield breeding and stress-resistant breeding of rice).

Description

Oligo DNA group of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes
Technical Field
The invention belongs to the field of plant genetic engineering. Specifically, the invention relates to two oligo DNA groups for site-directed knockout of sgRNA of rice OsMAP65-3.1 gene based on CRISPR-CAS9 technology and application thereof.
Background
Cytokinesis in plant cells is caused by the formation of a membrane, an antiparallel microtubule array of mitotic cells in somatic cells, reflecting one of the most prominent features of the dynamic microtubule network. The MAP65 protein can form a dimer, which promotes the binding of the membrane-forming body to the antiparallel microtubules of other microtubule arrays during cell division, and thus, can bridge overlapping microtubules in the membrane-forming body. During plant development, MAP65 family member MAP65-3 is expressed in all dividing cells, expression is under dual regulation of transcriptional and post-transcriptional mechanisms, and MAP65-3 plays a key role in mitosis and cytokinesis in all plant organs. In addition, the MAP65-3 gene is expressed at an early stage in nematode induced large cell formation. Deletion of MAP65-3 alone severely affected cytokinesis and mitosis, and in mutant cells lacking MAP65-3, binding of antiparallel microtubules in the central spindle and the membrane-forming bodies was largely abolished, the membrane-forming body microtubule array was disorganized, the length was increased, and the membrane-forming bodies were more interstitially spaced and loosely arranged in vascular bundles compared to wild-type cells.
With the initial discovery of about 65-Kd MAP65 protein in tobacco, the Arabidopsis genome was predicted to encode 9 homologous proteins with 28-79% amino acid homology to tobacco protein, and MAP65-3 protein has cytokinesis among the 9 MAP65 proteins in Arabidopsis. In Arabidopsis, MAP65-3 is not only a substrate for MAP kinase, but also acts on the MAP65 subtype. Thus, when cell plate assembly is complete, the microtubule stabilizing function of microtubule-associated proteins must be down-regulated in order to allow normal de-aggregation of the membrane-forming body microtubules. Microtubule-associated proteins modify all arrays or one specific array, mainly in arabidopsis thaliana. Some of these proteins are either remotely associated with microtubule-associated proteins in other neighbourhood organisms or specific to flowering plants. Unfortunately, little progress has been made in identifying proteins associated with microtubules in rice and other monocots.
Among proteins interacting with microtubules, rice has more genes encoding members of the MAP65 family than Arabidopsis, and 11 rice genes have been identified as encoding members of the MAP65/Ase1p family, one of which is OsMAP65-3.1, and are involved in encoding members of the MAP65-3 family. The cytokinesis is the basis for maintaining normal growth and development of individuals and is an important process for ensuring continuous and stable animal and higher plant species, and the rice is used as an important food crop in China, so that the research on the function of the OsMAP65-3.1 gene in the rice has important significance for the normal growth and the yield guarantee of the rice, and the function and the action mechanism of the mutant obtained by knocking out the OsMAP65-3.1 gene. The invention obtains transgenic plants by designing sgRNA to construct a gene editing vector, a transgenic technology and a CRISPR-CAS9 technology, obtains plants with OsMAP65-3.1 gene editing (knocking out) and sequence editing conditions by sequencing analysis.
Disclosure of Invention
The invention aims to provide a method for knocking out a rice gene OsMAP65-3.1, and provides sgRNA for knocking out rice OsMAP65-3.1 based on a CRISPR-CAS9 technology and a vector for knocking out a rice OsMAP65-3.1 gene.
A method for targeted knockout of rice OsMAP65-3.1 by using CRISPR/Cas9 technology is characterized by comprising the following steps:
a) the OsMAP65-3.1 gene coding region 74 th to 80 th nucleic acid sequences were selected as target sequences for the CRISPR/Cas9 system (SEQ ID NO. 1): GCGAGACGACGGTCCACGTC, respectively; and selecting the nucleic acid sequence from 156 th to 163 th of the coding region of the OsMAP65-3.1 gene as the target sequence for the CRISPR/Cas9 system (SEQ ID No. 2): GGAGCTGAAGTCCCTTGAAA are provided.
Four oligo DNAs were designed based on the target sequence:
OsMAP65-3.1-1-F1(SEQ ID NO.3)：tgttGCGAGACGACGGTCCACGTC
OsMAP65-3.1-1-R1(SEQ ID NO.4)：aaacGACGTGGACCGTCGTCTCGC
OsMAP65-3.1-2-F1(SEQ ID NO.5)：gtgtGGAGCTGAAGTCCCTTGAAA
OsMAP65-3.1-2-R1(SEQ ID NO.6)：aaacTTTCAAGGGACTTCAGCTCC
b) mixing an oligo DNA group OsMAP65-3.1-1-F1 and OsMAP65-3.1-1-R1, OsMAP65-3.1-2-F1 and OsMAP65-3.1-2-R1, annealing to form double chains, connecting the double chains with linearized PENTR4, and constructing plasmids pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-2 containing rice OsMAP65-3.1 gene target sequences through Gateway recombination;
c) infecting the callus of rice with Agrobacterium tumefaciens EHA105 containing pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-2 plasmids, screening with hygromycin, regenerating to obtain transgenic rice plants, and performing transgenic identification with HPT II gene specific primers;
d) amplifying sequences near a target sequence by using SQ-OsMAP65-3.1-1-F (SEQ ID NO.7), SQ-OsMAP65-3.1-1-R (SEQ ID NO.8), SQ-OsMAP65-3.1-2-F (SEQ ID NO.9) and SQ-OsMAP65-3.1-2-R (SEQ ID NO.10), sequencing amplified genome fragments, identifying the editing condition of OsMAP65-3.1 and screening knockout plants;
SQ-OsMAP65-3.1-1-F(SEQ ID NO.7)：GTTGGTCTCTCGCTGATTCAGATCG
SQ-OsMAP65-3.1-1-R(SEQ ID NO.8)：ATGATTACAGTCACAGATGGGCTC
SQ-OsMAP65-3.1-2-F(SEQ ID NO.9)：CAGTCTAATCAGAAAGCATGTGGAT
SQ-OsMAP65-3.1-2-R(SEQ ID NO.10)：CCCTTTCCATTGAGTTCCTCAACTA
after the gene editing vectors pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-2 transform rice, the rice OsMAP65-3.1 gene can be efficiently, quickly and specifically subjected to targeted editing and knockout, the gene can be directly used as a research material to discuss the functions and action mechanisms of the OsMAP65-3.1 gene, and a foundation is laid for better utilizing the OsMAP65-3.1 gene for genetic improvement in production practice.
Drawings
FIG. 1 shows the structure of OsMAP65-3.1 gene editing vector pUbi-Cas9-OsMAP 65-3.1-1.
FIG. 2 shows the structure of OsMAP65-3.1 gene editing vector pUbi-Cas9-OsMAP 65-3.1-2.
FIG. 3 pUbi-Cas9-OsMAP65-3.1-1 transgene identification map.
FIG. 4 pUbi-Cas9-OsMAP65-3.1-2 transgene identification map.
FIG. 5 PCR products from the region near the target site of OsMAP65-3.1-1 transgenic positive plant.
FIG. 6 PCR products from the region near the target site of OsMAP65-3.1-2 transgenic positive plant.
FIG. 7 Gene editing of transgenic line No. 2OsMAP 65-3.1-1.
FIG. 8 Gene editing of transgenic line No.7 OsMAP 65-3.1-1.
FIG. 9 Gene editing of transgenic line No. 11 OsMAP 65-3.1-1.
FIG. 10 Gene editing of transgenic line No.3, OsMAP 65-3.1-2.
FIG. 11 Gene editing of transgenic line No.13 OsMAP 65-3.1-2.
FIG. 12 Gene editing of transgenic line No. 18 OsMAP 65-3.1-2.
Detailed Description
The invention is further described below with reference to specific examples. These descriptions are not intended to limit the present invention further, and the technical means used in the following examples are conventional means well known to those skilled in the art, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design and Synthesis of rice OsMAP65-3.1 Gene sgRNA sequence
The coding region sequence of the rice OsMAP65-3.1 gene is shown in SEQ ID NO. 13.
e) The CRISPR/Cas9 editing target sequences of the present example are all 20bp in length, one of them is located at bases 74 to 80 of the first exon of the OsMAP65-3.1 coding region, and the editing target sequence is SEQ ID No. 1: GCGAGACGACGGTCCACGTC, and the other located in the 156 th to 163 th nucleic acid sequences of the coding region of the OsMAP65-3.1 gene, the edited target sequence being (SEQ ID NO. 2): GGAGCTGAAGTCCCTTGAAA are provided. The sequence is specific on rice genome and has extremely low off-target probability.
Four oligo DNAs were synthesized from the target sequence:
OsMAP65-3.1-1-F1(SEQ ID NO.3)：tgttGCGAGACGACGGTCCACGTC
OsMAP65-3.1-1-R1(SEQ ID NO.4)：aaacGACGTGGACCGTCGTCTCGC
OsMAP65-3.1-2-F1(SEQ ID NO.5)：gtgtGGAGCTGAAGTCCCTTGAAA
OsMAP65-3.1-2-R1(SEQ ID NO.6)：aaacTTTCAAGGGACTTCAGCTCC
example 2 construction of OsMAP65-3.1 editing vector pUbi-Cas9-OsMAP65-3.1-1, pUbi-Cas9-OsMAP65-3.1-2
Adding water into the synthesized OsMAP65-3.1-1-F1, OsMAP65-3.1-1-R1, OsMAP65-3.1-2-F1 and OsMAP65-3.1-2-R1 for dissolving to 10 mu M, and putting the mixture into a PCR instrument for annealing to form a double chain with a short strip joint. Carrying out linearization on a PENTR4 vector of OsMAP65-3.1-1 by BtgZ I, carrying out linearization on PENTR4 vector of OsMAP65-3.1-2 by Bsa I, cutting and recovering gel, recombining with a target fragment, transforming a ligation product into escherichia coli by a heat shock method, selecting a single colony to an LB liquid culture medium for culturing for 12 hours, carrying out restriction enzyme digestion detection and PCR detection after extracting a plasmid, recombining with Gateway and pUbi-Cas9, and sequencing; the other is the same. Selecting bacteria with correct sequencing to extract plasmid DNA, and obtaining editing vectors pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-1 containing rice OsMAP65-3.1 gene target sequences, wherein vector diagrams are shown in figure 1 and figure 2.
Example 3 pUbi-Cas9-OsMAP65-3.1-1, pUbi-Cas9-OsMAP65-3.1-2 vector Agrobacterium transformation
The plasmid pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-2 are transformed into agrobacterium by a freeze-thaw method, 10 mul of plasmid DNA is added into 200 mul of agrobacterium EHA105 competence, ice bath is carried out for 30min, liquid nitrogen is frozen for 3min, water bath at 37 ℃ is carried out for 5min, 1ml of YEB culture medium is added, and shaking culture is carried out for 3-4h at 28 ℃. The cells were centrifuged at 6000rpm at room temperature for 1min, the supernatant was discarded, 200. mu.l of YEB medium was added to resuspend the cells, and they were plated on YEB solid medium (+ rifampicin) and cultured at 28 ℃ for 3 days. Selecting single colony for identification.
Example 4 transformation of Rice
In the implementation, Nipponbare is used as a receptor for agrobacterium transformation. Selecting about 300 Nipponbare seeds, removing shells, soaking for 1 minute by using 75% ethanol, pouring off the 75% ethanol, sterilizing for 30 minutes by using a sodium hypochlorite solution, washing for 6 times by using sterile water, sucking water by using sterile gauze, then planting the seeds on an NB culture medium containing 2,4-D (2mg/L) for 2 weeks in a dark place at 26 ℃, and selecting the calli which grow vigorously to be used as a transformed receptor. The rice calli were infected with an engineered bacterial solution prepared from EHA105 strain containing the editing vectors (pUbi-Cas9-OsMAP65-3.1-1, pUbi-Cas9-OsMAP65-3.1-2), co-cultured in the dark at 25 ℃ for 3 days, and then cultured in a selection medium containing 50mg/L Hygromycin for about 14 days (light intensity 13200LX, temperature 32 ℃). Transferring the pre-differentiated callus to a differentiation medium, and culturing the pre-differentiated callus under the illumination condition (the illumination intensity is 13200LX, and the temperature is 32 ℃) for about one month to obtain a resistant transgenic plant. Using 1/2MS culture medium to take root and strengthen seedling to obtain T0 generation plant, transplanting into field to plant.
Example 5 analysis of OsMAP65-3.1 Gene editing
DNA is extracted from T0 generation plant leaves, primers HPT-F and HPT-R are designed according to hygromycin gene sequence, and positive transgenic plants are determined, and are shown in figure 3 and figure 4. Then, positive transgenic plants were amplified with the primers SQ-OsMAP65-3.1-1-F and SQ-OsMAP65-3.1-1-R (FIG. 3), and the sequences near the target sites of the OsMAP65-3.1-1 gene (FIGS. 4-7 are the sequencing results of partially edited plants) and the sequences near the target sites of the OsMAP65-3.1-2 gene (FIGS. 4-7 are the sequencing results of partially edited plants) were analyzed by sequencing with the primers SQ-OsMAP65-3.1-2-F and SQ-OsMAP65-3.1-2-R, and the statistics of the overall results are shown in Table 1 and Table 2. The hygromycin gene and the sequence amplification primer sequence near the target site are as follows:
SQ-OsMAP65-3.1-1-F(SEQ ID NO.7)：GTTGGTCTCTCGCTGATTCAGATCG
SQ-OsMAP65-3.1-1-R(SEQ ID NO.8)：ATGATTACAGTCACAGATGGGCTC
SQ-OsMAP65-3.1-2-F(SEQ ID NO.9)：CAGTCTAATCAGAAAGCATGTGGAT
SQ-OsMAP65-3.1-2-R(SEQ ID NO.10)：CCCTTTCCATTGAGTTCCTCAACTA
HPT-F(SEQ ID NO.11)：ACGGTGTCGTCCATCACAGTTTGCC
HPT-R(SEQ ID NO.12)：TTCCGGAAGTGCTTGACATTGGGGA
TABLE 1 analysis of OsMAP65-3.1-1 Gene editing
Figure BDA0002608283960000071
TABLE 2 analysis of OsMAP65-3.1-2 Gene editing
Figure BDA0002608283960000081
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> university of agriculture in Jiangxi
<120> oligo DNA group of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes
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tacaggagga aggtggacca ggcgaaccgc tcccgcgccc agctgcggca ggcgatcgct 180
caatatgaag ctgaactcgc cgccatctgc tccgccattg gcgagacgac ggtccacgtc 240
aggcagtcta atcagaaagc atgtggatta cgggatgagc tcggtgcaat attaccatac 300
ctagaagaga tgaagaggaa gaaggttgaa agatggaacc agtttcttga tgtcgtagga 360
aggataaaga agatctcatc tgagataagg ccagcaaatt ttgacccttt taaagtgtct 420
gtggatcaat ctgatctatc attaagaaag cttgaagagt taagggtgga gctgaagtcc 480
cttgaaaagg agaagggtga gagggtaaag caagttatgg aatatttgaa gactttacat 540
tctttatgtg tagtacttgg tgtcgacttc aagaaaacaa tatctgaaat acaccctagt 600
cttgatgaag ctgaagggcc aaggaatataagcaacacta caattgagat gctagcatgg 660
gcgattcaga gacttcgtga aacaaaaatg cagaggatgc agaagcttca agatcttgca 720
tctaccttgc tagaactatg gaatctaatg gatacgccat ttgaagagca gcaggcatac 780
cagaatataa catgtaatat tgctgcttca gaggctgaat taacagaaca gaacaccctt 840
tccattgagt tcctcaacta tgtggaagct gaggtattaa ggcttgaaca gcacaaagca 900
agcaaaatga aggagcttgt tctcaagaag aaaacagaac tagaagaaca tcgacgacgg 960
gcacatttag ttggagagga aggttatgca actcagttca caattgaggc cattgaagca 1020
ggggctattg atccctcttt gttgctcgag caaattgagg cttatatttc aacagtgaaa 1080
gaggaagctt ttagcaggaa ggacattctc gagcgagttg agaagtggct aaatgcacgt 1140
gaggaagagg cttggttgga agattacaac aaagatgaca accggtataa tgctgggaga 1200
ggtgcacaca ttatgcttaa gagggctgaa aaagcgcgtg ttctggttag caagattcca 1260
ggaatggtag atgttcttga aacaaaaacc agagcttggg aaactgaaag aggcaatgag 1320
ttcacatatg atggcgtccg acttatattg atgcttgaag aatacatggt tgttcgtcaa 1380
gagaaagagc aggaaaggaa gaggcaaagg gatcaaaaga agcttcaaga tcaacgcaaa 1440
gctgagcagg aggcacttta tggatcaaag ccaagctctt caaaatctca cagcactaag 1500
aaggtaccca gaaactccac acctggagtt caacctccta aatcagagat acttcattca 1560
aagactattc gtgcaaccaa gaaaacggaa gatatcaaca ctccttcccc tggtcataaa 1620
ggtttagaca ctgttggtct ccctatcagg aagttatttc catcttccaa ctcgagcact 1680
cttcttgaga tggagacacc ccggaagccc ttctctcaga ttacacctgg aaacatatcc 1740
tcagctcctg tgcgtccaat ctccactggt ggtactgaag agaacaggac tcccaagaca 1800
tttgcaccag ttcctacgac tccgatgaca gtgagccctc atatgcaaat ggcagtgacg 1860
cctgttctta ccgcaaaagc tgtttctgta ctctcttatg acgaaccgga gctgacttca 1920
caagaggaca ccgagtactc atttgaagag aagcgtcttg cagtctatct tgccgcacaa 1980
gtggcttga 1989

Claims (4)

1. The oligo DNA group of sgRNA of two fixed-point knockout rice OsMAP65-3.1 genes is characterized in that: the DNA sequences of the target sites of the two sgRNAs of the OsMAP65-3.1 gene are shown as SEQ ID NO.1 and SEQ ID NO.2, one oligo DNA group is OsMAP65-3.1-1-F1 and OsMAP65-3.1-1-R1, the other oligo DNA group is OsMAP65-3.1-2-F1 and OsMAP65-3.1-2-R1, the nucleotide sequences of OsMAP65-3.1-1-F1 are shown as SEQ ID NO.3, the nucleotide sequences of OsMAP65-3.1-1-R1 are shown as SEQ ID NO.4, the nucleotide sequences of OsMAP65-3.1-2-F1 are shown as SEQ ID NO.5, and the nucleotide sequences of OsMAP65-3.1-2-R1 are shown as SEQ ID NO. 6.
2. The use of the oligo DNA set of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes according to claim 1 in rice OsMAP65-3.1 gene editing breeding.
3. A carrier for site-directed knockout of sgRNA of rice OsMAP65-3.1 gene is characterized in that the carrier is pUbi-Cas9-OsMAP65-3.1-1 and pUbi-Cas9-OsMAP65-3.1-2, oligo DNA groups OsMAP65-3.1-1-F1 and OsMAP65-3.1-1-R1 are mixed, OsMAP65-3.1-2-F1 and OsMAP65-3.1-2-R1 are mixed and annealed to form double chains respectively, then the double chains are connected with linearized PENTR4, pUbi-OsMAP 9-OsMAP 465-1 and pUbi-OsMAP 9-OsMAP 24-3.1-OsMAP 633.1 gene target sequences are constructed through Gateway recombination to obtain pUbi-OsMAP 9-OsMAP 5-OsMAP-3.1-OsMAP plasmid containing rice OsMAP65-3.1 gene target sequences, and the nucleotide sequences of pUbi-OsMAP 9-OsMAP 24-OsMAP-3.1-OsMAP-593.1 gene target sequences are shown in SEQ ID No. 3-593-SEQ ID No. 3-NO. 7, and nucleotide sequences are shown in SEQ ID No, the nucleotide sequence of OsMAP65-3.1-2-F1 is shown as SEQ ID NO.5, and the nucleotide sequence of OsMAP65-3.1-2-R1 is shown as SEQ ID NO. 6.
4. A primer group for identifying the sgRNA target region editing condition of an OsMAP65-3.1 gene, which is characterized in that: comprises two primer pairs, wherein the DNA sequence of one primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8, and the DNA sequence of the other primer pair is shown as SEQ ID NO.9 and SEQ ID NO. 10.
CN202010745749.4A 2020-07-29 2020-07-29 Oligo DNA group of sgRNA of two site-directed knockout rice OsMAP65-3.1 genes Pending CN111793626A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105907773A (en) * 2015-11-27 2016-08-31 南京农业大学 Soybean gene for regulation and control of seed oil synthesis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105907773A (en) * 2015-11-27 2016-08-31 南京农业大学 Soybean gene for regulation and control of seed oil synthesis

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOHN GARDINER: "The evolution and diversification of plant microtubule-associated proteins", 《PLANT J》 *
KAWAHARA,Y.等: "Oryza sativa Japonica Group cultivar Nipponbare chromosome 5, IRGSP-1.0", 《GENBANK DATABASE》 *
WEN XU等: "Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice", 《BMC PLANT BIOL》 *
杨方方: "水稻OsMAP65家族T-DNA插入突变的筛选及表型鉴定", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 *
林小丽等: "基于CRISPR/Cas9技术对水稻细胞质分裂关键基因OsMAP65-3s的编辑", 《核农学报》 *

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Application publication date: 20201020