CN117304286A - Application of rice OsLAT5 mutant gene in cultivation of bipyridine herbicide resistant rice variety - Google Patents
Application of rice OsLAT5 mutant gene in cultivation of bipyridine herbicide resistant rice variety Download PDFInfo
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Abstract
The invention discloses application of a rice OsLAT5 mutant gene in cultivation of a bipyridine herbicide resistant rice variety. The invention designs an OsLAT5 gene target sequence for targeting to endow rice bipyridine herbicide resistance, and discovers that the OsLAT5 gene point editing amino acid loci Pro-44, phe-45 and Val-51 which can endow rice bipyridine herbicide resistance can be targeted, and the locus edited rice can not only retain excellent resistance to bipyridine herbicide, but also can not influence the agronomic characters and quality of the rice by using a gene editing technology. The resistant variety can also obtain bipyridine herbicide resistance after sexual or asexual reproduction. The OsLAT5 mutant gene can be applied to rice bipyridine herbicide resistant breeding.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of a rice OsLAT5 mutant gene in cultivation of bipyridine herbicide resistant rice varieties.
Background
Weed control is a key for ensuring high and stable yield of rice, and particularly the importance of the rice is more prominent under the background that the simplified planting of the rice becomes a main pushing technology in production. The weeds in the paddy fields are various, various herbicides are used for preventing and controlling on a large scale, and the problems of weed resistance, soil pollution, pesticide residues and the like are easily caused. The biocidal herbicide has high efficiency, small dosage and simple application mode, and can greatly reduce the dosage of the herbicide and reduce the labor cost, so that the biocidal herbicide is widely applied. However, the lack of selectivity of the biocidal herbicide between weeds and rice makes the cultivation of resistant varieties of the biocidal herbicide of great significance.
Bipyridylium herbicide belongs to a biocidal herbicide and only comprises two important varieties of paraquat and diquat. Early studies (chinese patent CN111574605 a) have demonstrated that rice OsLAT5 develops resistance to rice by mediating bipyridine herbicide transport, and that gene editing technology is used to inactivate OsLAT5 transporter and prevent bipyridine herbicide from reaching the rice target site, thus obtaining resistant rice varieties, however, inactivation of its OsLAT5 gene affects rice yield and quality, so that it is necessary to provide other mutation strategies for rice OsLAT5 to ensure that rice has bipyridine herbicide resistance while not affecting rice growth, rice yield and quality.
Disclosure of Invention
The invention aims to overcome the defects and defects of affected growth and reduced yield of herbicide resistant varieties and reduced yield caused by an OsLAT5 mutation method in the prior art, provides an OsLAT5 mutation strategy, discovers that an OsLAT5 gene point editing amino acid locus Pro-44, phe-45 and Val-51 for targeting on the bipyridine herbicide resistance of rice through carrying out saturation mutation and site-directed mutation on the amino acid locus of the specific identification bipyridine herbicide of the OsLAT5, and utilizes the gene editing technology to maintain excellent resistance of the rice on the bipyridine herbicide after editing the locus point, does not influence the agronomic characters and quality of the rice, can more efficiently and more targetedly create the bipyridine herbicide resistant rice germplasm, and can carry out sexual or asexual reproduction on the bipyridine herbicide resistant rice on other rice.
The first object of the present invention is to provide a rice OsLAT5 mutant protein and an OsLAT5 mutant gene.
Another object of the invention is to provide the application of the rice OsLAT5 mutant protein and OsLAT5 mutant gene in cultivating bipyridine herbicide resistant rice germplasm resources.
The above object of the present invention is achieved by the following technical solutions:
the invention carries out multi-species homologous protein search on rice OsLAT5 protein sequence (SEQ ID NO. 1), compares protein sequences of 27 species with OsLAT5 by utilizing MEGA software, searches conserved amino acid sites of the OsLAT5 protein, designs 116 target sequences for saturation mutation aiming at the conserved amino acid sites, and carries out saturation mutation; the molecular docking predicts that the medicine and protein binding site finds out that partial conserved amino acid sites (Pro-44, phe-45 and Val-51) belong to important sites for the combination of OsLAT5 and bipyridine herbicides, a yeast system and a plant system verify that the mutation of the amino acid sites generates resistance to the bipyridine herbicides, the obtained resistant variety does not influence the agronomic characters of rice, and in addition, the bipyridine herbicide resistance can be obtained after sexual or asexual reproduction.
Therefore, the invention firstly provides a rice OsLAT5 mutant protein, the amino acid sequence of which is obtained after mutation of any one or more sites of 44 th, 45 th or 51 th amino acids of a wild OsLAT5 protein shown in SEQ ID NO. 1.
Further, the mutation is a glutamic acid substitution mutation.
The invention also provides a rice OsLAT5 mutant gene for encoding the rice OsLAT5 mutant protein.
The invention also claims the following applications for the OsLAT5 mutant gene and OsLAT5 mutant protein:
the application of any one of the above rice OsLAT5 mutant proteins or rice OsLAT5 mutant genes in cultivation of bipyridine herbicide resistant rice varieties.
Specifically, the bipyridine herbicide resistant rice variety is obtained by mutating any one or more sites of 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein with the sequence shown as SEQ ID NO.1 in rice.
Further, a gene editing system for editing any one or more sites of 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein shown in SEQ ID NO.1 at fixed point is constructed for the OsLAT5 gene, and rice plants are transformed to obtain rice mutant strains with any one or more sites of 44 th, 45 th or 51 th amino acids of the wild OsLAT5 protein shown in SEQ ID NO.1 in the rice sequences, namely the bipyridine herbicide resistant rice varieties.
Further, the gene editing is by gene saturation mutation or gene site-directed editing.
Further, the gene saturation mutation target sequence is one or more of SEQ ID NO. 2 to SEQ ID NO. 117.
The invention also provides application of the reagent for mutating any one or more sites of 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein shown in SEQ ID NO.1 in cultivation of bipyridine herbicide resistant rice varieties.
Further, the bipyridyl herbicides include, but are not limited to, paraquat and diquat, and further include other compounds having a bipyridyl structural backbone.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses application of a rice OsLAT5 mutant gene in cultivation of a bipyridine herbicide resistant rice variety. The invention discovers that the gene locus editing amino acid loci Pro-44, phe-45 and Val-51 of the OsLAT5 which can be targeted to endow rice with bipyridine herbicide resistance can not only keep excellent resistance to the bipyridine herbicide but also can not influence the agronomic characters and quality of the rice after the locus editing by using a gene editing technology. The resistant variety can also obtain bipyridine herbicide resistance after sexual or asexual reproduction. The OsLAT5 mutant gene can be applied to rice bipyridine herbicide resistant breeding.
Drawings
FIG. 1 shows the results of OsLAT5 and 27 species homologous proteins.
FIG. 2 shows the results of molecular docking of OsLAT5 protein and bipyridyl herbicide. Wherein A: the structural model predicts the region where OsLAT5 interacts with paraquat. B: the structural model predicts the region where OsLAT5 interacts with diquat.
FIG. 3 shows colony growth of yeast heterologously expressing mutant OsLAT5 protein in bipyridine herbicides.
FIG. 4 shows the tolerance of wild rice and OsLAT5 gene site-directed mutant to bipyridyl herbicide during seed germination.
FIG. 5 shows tolerance of wild rice and OsLAT5 gene site-directed mutant after spraying bipyridine herbicide in seedling stage.
FIG. 6 shows the tolerance of the conventional rice variety Guiyu No.11 and OsLAT5 gene site-directed mutant hybrid offspring to bipyridine herbicides.
FIG. 7 is a comparison of agronomic traits of fixed point mutants of the flower No.11 and OsLAT5 genes in conventional rice varieties.
FIG. 8 shows the agronomic characteristics of the fixed point mutants of OsLAT5 gene and Guiyu No.11 of conventional rice variety.
FIG. 9 is a graph showing the results of fixed-point editing of plants on ZH-44 rice.
FIG. 10 is a graph showing the results of target editing of saturated mutant plants of ZH-45 and ZH-51.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Coli DH 5. Alpha. And Agrobacterium EHA105 are common strains in the examples and commercially available. The primers used in the examples were synthesized by Shenzhen Dacrogene, inc., and sequencing was performed by Shenzhen Dacrogene, inc.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
EXAMPLE 1 construction of OsLAT5 saturated mutant plants
(1) OsLAT5 amino acid conservation site prediction
Multi-species homologous protein searches were performed on OsLAT5 using the website (https:// blast.ncbi.nlm.nih.gov/blast.cgiprogram = blastp & page_type = BlastSearch & link_loc = blastthome), with sequence alignment of homologous proteins of 27 species with OsLAT5 using MEGA software, amino acid up-taken is a highly conserved site, as shown in fig. 1. The target site and primer design can be performed later with reference to this result.
(2) Design of saturation mutation target and primer
Inputting a gene OsLAT5 sequence in a targetDesign applet of software CRISPR-GE, selecting a corresponding PAM type, and screening target sequences according to information such as an exon sequence, an amino acid conserved site and the like of the OsLAT5 to obtain 116 target sequences meeting requirements; the 20bp target sequence and PAM locus are input into the primerDesign applet of the software CRISPR-GE, and the corresponding OsU a, osU b and OsU3 promoters are selected, so that the specific primers #U-R and #gR-R for constructing the expression cassette are obtained (overlay PCR method).
Target sequence:
Target-1：5’-CCAATGGAGGATTGTGTTGG-3’(SEQ ID NO.2)；
Target-2：5’-GGCGTCCCAAAGGTTTCCAT-3’(SEQ ID NO.3)；
Target-3：5’-GATGATGGAAACCTTTGGGA-3’(SEQ ID NO.4)；
Target-4：5’-AGGAAAATGAGTGGGATGAT-3’(SEQ ID NO.5)；
Target-5：5’-GAATATGAGGAAAATGAGTG-3’(SEQ ID NO.6)；
Target-6：5’-CCCCCAGAAACTTCATAGAA-3’(SEQ ID NO.7)；
Target-7：5’-CTATGAAGTTTCTGGGGGTC-3’(SEQ ID NO.8)；
Target-8：5’-TCAATCCCAAACGGACCCCC-3’(SEQ ID NO.9)；
Target-9：5’-ATTGAGGATAGTGTCAAGGC-3’(SEQ ID NO.10)；
Target-10：5’-TTGACACTATCCTCAATCCC-3’(SEQ ID NO.11)；
Target-11：5’-AGGATAGTGTCAAGGCTGCT-3’(SEQ ID NO.12)；
Target-12：5’-ATTGCTAGGAGTGGGCCAGC-3’(SEQ ID NO.13)；
Target-13：5’-AGTGCAAACAGCAGAAATCC-3’(SEQ ID NO.14)；
Target-14：5’-TCCATATGAGTGCAAACAGC-3’(SEQ ID NO.15)；
Target-15：5’-CCGGAAGCCCTGATTACTGC-3’(SEQ ID NO.16)；
Target-16：5’-TGCAGTAATCAGGGCTTCCG-3’(SEQ ID NO.17)；
Target-17：5’-GAGATGGGCACTATGTTTCC-3’(SEQ ID NO.18)；
Target-18：5’-GGAAACATAGTGCCCATCTC-3’(SEQ ID NO.19)；
Target-19：5’-CCTGAGAATGGTGGTTACGT-3’(SEQ ID NO.20)；
Target-20：5’-ACGACGTAACCACCATTCTC-3’(SEQ ID NO.21)；
Target-21：5’-GGCTGAAGAGACCCAGACGA-3’(SEQ ID NO.22)；
Target-22：5’-AAAACCCCAGAATGGCCCAA-3’(SEQ ID NO.23)；
Target-23：5’-TTCAGCAAGGCTGGGCAAAG-3’(SEQ ID NO.24)；
Target-24：5’-TCAGCCACTTTGCCCAGCCT-3’(SEQ ID NO.25)；
Target-25：5’-TGGGCAAAGTGGCTGAGTGG-3’(SEQ ID NO.26)；
Target-26：5’-GTGTCATAGATAATGCTCTC-3’(SEQ ID NO.27)；
Target-27：5’-AGGAAGAGGACTGGATAGAG-3’(SEQ ID NO.28)；
Target-28：5’-CATAGTCGAGGAAGAGGACT-3’(SEQ ID NO.29)；
Target-29：5’-GGACTTAACATAGTCGAGGA-3’(SEQ ID NO.30)；
Target-30：5’-TCTCCCAAGGACCTTGGCGG-3’(SEQ ID NO.31)；
Target-31：5’-GCAACTGTGAGGATAAGCAC-3’(SEQ ID NO.32)；
Target-32：5’-CTCACAGTTGCACTTACTTA-3’(SEQ ID NO.33)；
Target-33：5’-ATGAACTACAGAGGGTTGAC-3’(SEQ ID NO.34)；
Target-34：5’-TGTCAACCCTCTGTAGTTCA-3’(SEQ ID NO.35)；
Target-35：5’-TAGAGAGAACACGCCAAGAA-3’(SEQ ID NO.36)；
Target-36：5’-AACAAAAAACGGGAGTAGAG-3’(SEQ ID NO.37)；
Target-37：5’-GGATTAATAGCTATTCCCCG-3’(SEQ ID NO.38)；
Target-38：5’-CCATCTTGAGGGTTCGATTC-3’(SEQ ID NO.39)；
Target-39：5’-GGTTCCAAAACAGTGTGTTT-3’(SEQ ID NO.40)；
Target-40：5’-GAACCTCAATTATTGGGACT-3’(SEQ ID NO.41)；
Target-41：5’-TCAATTATTGGGACTCAATC-3’(SEQ ID NO.41)；
Target-42：5’-AATCAGTACCCTTGCTGGAG-3’(SEQ ID NO.43)；
Target-43：5’-GGAGAGGTTGAGAATCCAAA-3’(SEQ ID NO.44)；
Target-44：5’-GAATCCAAAGAGAACACTCC-3’(SEQ ID NO.45)；
Target-45：5’-CATAAGAAAGTGCCCTTGGG-3’(SEQ ID NO.46)；
Target-46：5’-CCACCACTAAAACTAGAGCA-3’(SEQ ID NO.47)；
Target-47：5’-TAGAGGTATCCCCCCACCAC-3’(SEQ ID NO.48)；
Target-48：5’-ACAGGTGATCAGAGGGTAGA-3’(SEQ ID NO.49)；
Target-49：5’-TGTACAGCAGCAGTTCCAGT-3’(SEQ ID NO.50)；
Target-50：5’-TCCAGAACTCCCGAACAACT-3’(SEQ ID NO.51)；
Target-51：5’-TGAGAAATATCCATCCGTCC-3’(SEQ ID NO.52)；
Target-52：5’-GAAACCACCAAGAATTCTCG-3’(SEQ ID NO.53)；
Target-53：5’-AGTGCAACCAGAAACCACCA-3’(SEQ ID NO.54)；
Target-54：5’-TTGAAGCCACGAGTGCAACC-3’(SEQ ID NO.55)；
Target-55：5’-GCTTCAAGCAGCTGCTGCAC-3’(SEQ ID NO.56)；
Target-56：5’-CTGTCCAACATGGGCAATTT-3’(SEQ ID NO.57)；
Target-57：5’-ACGAAATTGCCCATGTTGGA-3’(SEQ ID NO.58)；
Target-58：5’-GGTAAGAATCACTGCTCATT-3’(SEQ ID NO.59)；
Target-59：5’-GAGAAGCTGGTAAGAATCAC-3’(SEQ ID NO.60)；
Target-60：5’-GCCATCCCGAGAAGCTGGTA-3’(SEQ ID NO.61)；
Target-61：5’-TCTGGAAGCATTCCACGCTC-3’(SEQ ID NO.62)；
Target-62：5’-TTGGCGAAAAACTCTGGAAG-3’(SEQ ID NO.63)；
Target-63：5’-TAGCGAGATCTCTTGGCGAA-3’(SEQ ID NO.64)；
Target-64：5’-CGCTATGGAACCCCACTTAT-3’(SEQ ID NO.65)；
Target-65：5’-AACATGATGCCAATAAGTGG-3’(SEQ ID NO.66)；
Target-66：5’-CGGAGAACATGATGCCAATA-3’(SEQ ID NO.67)；
Target-67：5’-CAGGACCACACCAAACGCGG-3’(SEQ ID NO.68)；
Target-68：5’-CCAGGACAGCAGGACCACAC-3’(SEQ ID NO.69)；
Target-69：5’-GGAAGCTCATCCAGGACAGC-3’(SEQ ID NO.70)；
Target-70：5’-CCAGGAGATCATCGCTGCGG-3’(SEQ ID NO.71)；
Target-71：5’-CTGCGGAGAACTACCTGTAC-3’(SEQ ID NO.72)；
Target-72：5’-CATACCGAAGCAGTACAGGT-3’(SEQ ID NO.73)；
Target-73：5’-TTCCAGGATCATACCGAAGC-3’(SEQ ID NO.74)；
Target-74：5’-GATGAAGGCGATGAATTCCA-3’(SEQ ID NO.75)；
Target-75：5’-CTGAGGGTGGTCCACCCAAA-3’(SEQ ID NO.76)；
Target-76：5’-CCACTGGGCACCATCGGCGC-3’(SEQ ID NO.77)；
Target-77：5’-GGTAGGTGGGATGATCATCA-3’(SEQ ID NO.78)；
Target-78：5’-GACGATCAGAATGGTAGGTG-3’(SEQ ID NO.79)；
Target-79：5’-GCATCATCACCACGACGATC-3’(SEQ ID NO.80)；
Target-80：5’-TTATGGAGAACTTCAGCCAC-3’(SEQ ID NO.81)；
Target-81：5’-CTCATTGACACTGCTGTACT-3’(SEQ ID NO.82)；
Target-82：5’-TTCGCCCTCATTGACACTGC-3’(SEQ ID NO.83)；
Target-83：5’-ATGGCCTCCCTTACGCTCTT-3’(SEQ ID NO.84)；
Target-84：5’-GGACGCCATGGCCTCCCTTA-3’(SEQ ID NO.85)；
Target-85：5’-CCTCCAAGAGCTGGAATGCT-3’(SEQ ID NO.86)；
Target-86：5’-GGAGACCACCTCCAAGAGCT-3’(SEQ ID NO.87)；
Target-87：5’-GCACCGCCAAGGTCCTTGGG-3’(SEQ ID NO.88)；
Target-88：5’-GCCAACTATTGTCAACCCTC-3’(SEQ ID NO.89)；
Target-89：5’-CCATTTCAAGCCATCTTGAG-3’(SEQ ID NO.90)；
Target-90：5’-CATTCCCCAAGTCCATTTCA-3’(SEQ ID NO.91)；
Target-91：5’-AAGAATTCTCGCAACGTCTG-3’(SEQ ID NO.92)；
Target-92：5’-AAACGCCTCCCGACCTTACA-3’(SEQ ID NO.93)；
Target-93：5’-CACCATCACCTTGAAGGACG-3’(SEQ ID NO.94)；
Target-94：5’-AACCAGCATTGCCATGATGC-3’(SEQ ID NO.95)；
Target-95：5’-CCCAACCAGCATTGCCATGA-3’(SEQ ID NO.96)；
Target-96：5’-AGCACGAACCCAACCAGCAT-3’(SEQ ID NO.97)；
Target-97：5’-CACCAGAGCCGGCTGCAGCA-3’(SEQ ID NO.98)；
Target-98：5’-TCTCCACGTACACCAGAGCC-3’(SEQ ID NO.99)；
Target-99：5’-TGGCAGTTCTGCGCTTATGG-3’(SEQ ID NO.100)；
target-100:5'-CTTCCTCAACGTTCGAGTAC-3' (SEQ ID NO. 101); target-101:5'-GTGCTGTCTTCCTCAACGTT-3' (SEQ ID NO. 102); target-102:5'-GCACAATCCCACTTGTGTGC-3' (SEQ ID NO. 103); target-103:5'-GGTCAGCACACAAGTGGGAT-3' (SEQ ID NO. 104); target-104:5'-CAACACAATCCTCCATTGGA-3' (SEQ ID NO. 105); target-105:5'-CACTATCCTCAATCCCAAAC-3' (SEQ ID NO. 106); target-106:5'-AAGGCTGCTGGCCCACTCCT-3' (SEQ ID NO. 107); target-107:5'-TCCAGCAATTGCTAGGAGTG-3' (SEQ ID NO. 108); target-108:5'-CATATGGAGTGTCCCGGAAG-3' (SEQ ID NO. 109); target-109:5'-CCCAAGGGCTGAAGAGACCC-3' (SEQ ID NO. 110); target-110:5'-CTTCAGCCCTTGGGCCATTC-3' (SEQ ID NO. 111); target-111:5'-ATGACACCACTCAGCCACTT-3' (SEQ ID NO. 112); target-112:5'-GAGAACACGCCAAGAAAGAC-3' (SEQ ID NO. 113); target-113:5'-ATCCCATAACAAAAAACGGG-3' (SEQ ID NO. 114); target-114:5'-CCATTTCAAGCCATCTTGAG-3' (SEQ ID NO. 115); target-115:5'-TCCAGCAAGGGTACTGATTG-3' (SEQ ID NO. 116); target-116:5'-ATCCCCCCACCACTAAAACT-3' (SEQ ID NO. 117). First round amplification universal primers:
U-F：5’-CTCCGTTTTACCTGTGGAATCG-3’；
gR-R：5’-CGGAGGAAAATTCCATCCAC-3’。
the first round amplified specific primers (lower case sticky ends):
LAT5-gRT1:5’-CCAATGGAGGATTGTGTTGGgtttcagagctagaaat-3’；
LAT5-OsU6aT1:5’-CCAACACAATCCTCCATTGGCggcagccaagccagca-3’；LAT5-gRT2：5’-GCGTCCCAAAGGTTTCCATgtttcagagctagaaat-3’；
LAT5-OsU6aT2：5’-ATGGAAACCTTTGGGACGCCggcagccaagccagca-3’；LAT5-gRT3：5’-ATGATGGAAACCTTTGGGAgtttcagagctagaaat-3’；
LAT5-OsU6aT3：5’-TCCCAAAGGTTTCCATCATCggcagccaagccagca-3’；LAT5-gRT4：5’-AGGAAAATGAGTGGGATGATgtttcagagctagaaat-3’
LAT5-OsU6aT4：5’-ATCATCCCACTCATTTTCCTCggcagccaagccagca-3’；
LAT5-gRT5：5’-AATATGAGGAAAATGAGTGgtttcagagctagaaat-3’；LAT5-OsU6aT5：5’-CACTCATTTTCCTCATATTCggcagccaagccagca-3’；
LAT5-gRT6：5’-CCCCCAGAAACTTCATAGAAgtttcagagctagaaat-3’；
LAT5-OsU6aT6：5’-TTCTATGAAGTTTCTGGGGGCggcagccaagccagca-3’；LAT5-gRT7：5’-CTATGAAGTTTCTGGGGGTCgtttcagagctagaaat-3’；
LAT5-OsU6aT7：5’-GACCCCCAGAAACTTCATAGCggcagccaagccagca-3’；LAT5-gRT8：5’-TCAATCCCAAACGGACCCCCgtttcagagctagaaat-3’
LAT5-OsU6aT8：5’-GGGGGTCCGTTTGGGATTGACggcagccaagccagca-3’；LAT5-gRT9：5’-ATTGAGGATAGTGTCAAGGCgtttcagagctagaaat-3’；
LAT5-OsU6aT9：5’-GCCTTGACACTATCCTCAATCggcagccaagccagca-3’；LAT5-gRT10：5’-TTGACACTATCCTCAATCCCgtttcagagctagaaat-3’；
LAT5-OsU6aT10：5’-GGGATTGAGGATAGTGTCAACggcagccaagccagca-3’；LAT5-gRT11：5’-AGGATAGTGTCAAGGCTGCTgtttcagagctagaaat-3’；
LAT5-OsU6aT11：5’-AGCAGCCTTGACACTATCCTCggcagccaagccagca-3’；LAT5-gRT12：5’-ATTGCTAGGAGTGGGCCAGCgtttcagagctagaaat-3’；
LAT5-OsU6aT12：5’-GCTGGCCCACTCCTAGCAATCggcagccaagccagca-3’；LAT5-gRT13：5’-AGTGCAAACAGCAGAAATCCgtttcagagctagaaat-3’；
LAT5-OsU6aT13：5’-GGATTTCTGCTGTTTGCACTCggcagccaagccagca-3’；LAT5-gRT14：5’-TCCATATGAGTGCAAACAGCgtttcagagctagaaat-3’；
LAT5-OsU6aT14：5’-GCTGTTTGCACTCATATGGACggcagccaagccagca-3’；LAT5-gRT15：5’-CCGGAAGCCCTGATTACTGCgtttcagagctagaaat-3’；
LAT5-OsU6aT15：5’-GCAGTAATCAGGGCTTCCGGCggcagccaagccagca-3’；LAT5-gRT16：5’-TGCAGTAATCAGGGCTTCCGgtttcagagctagaaat-3’；
LAT5-OsU6aT16：5’-CGGAAGCCCTGATTACTGCACggcagccaagccagca-3’；LAT5-gRT17：5’-AGATGGGCACTATGTTTCCgtttcagagctagaaat-3’；
LAT5-OsU6aT17：5’-GGAAACATAGTGCCCATCTCggcagccaagccagca-3’；LAT5-gRT18：5’-GAAACATAGTGCCCATCTCgtttcagagctagaaat-3’；
LAT5-OsU6aT18：5’-GAGATGGGCACTATGTTTCCggcagccaagccagca-3’；
LAT5-gRT19：5’-CCTGAGAATGGTGGTTACGTgtttcagagctagaaat-3’；
LAT5-OsU6aT19：5’-ACGTAACCACCATTCTCAGGCggcagccaagccagca-3’；
LAT5-gRT20：5’-ACGACGTAACCACCATTCTCgtttcagagctagaaat-3’；
LAT5-OsU6aT20：5’-GAGAATGGTGGTTACGTCGTCggcagccaagccagca-3’；
LAT5-gRT21:5’-GCTGAAGAGACCCAGACGAgtttcagagctagaaat-3’；
LAT5-OsU6aT21：5’-TCGTCTGGGTCTCTTCAGCCggcagccaagccagca-3’；
LAT5-gRT22：5’-AAAACCCCAGAATGGCCCAAgtttcagagctagaaat-3’；
LAT5-OsU6aT22：5’-TTGGGCCATTCTGGGGTTTTCggcagccaagccagca-3’；
LAT5-gRT23：5’-TTCAGCAAGGCTGGGCAAAGgtttcagagctagaaat-3’；
LAT5-OsU6aT23：5’-CTTTGCCCAGCCTTGCTGAACggcagccaagccagca-3’；
LAT5-gRT24：5’-TCAGCCACTTTGCCCAGCCTgtttcagagctagaaat-3’；
LAT5-OsU6aT24：5’-AGGCTGGGCAAAGTGGCTGACggcagccaagccagca-3’；
LAT5-gRT25：5’-TGGGCAAAGTGGCTGAGTGGgtttcagagctagaaat-3’；
LAT5-OsU6aT25：5’-CCACTCAGCCACTTTGCCCACggcagccaagccagca-3’；
LAT5-gRT26：5’-TGTCATAGATAATGCTCTCgtttcagagctagaaat-3’；
LAT5-OsU6aT26：5’-GAGAGCATTATCTATGACACggcagccaagccagca-3’；
LAT5-gRT27：5’-AGGAAGAGGACTGGATAGAGgtttcagagctagaaat-3’；
LAT5-OsU6bT27：5’-CTCTATCCAGTCCTCTTCCTCaacacaagcggcagc-3’；
LAT5-gRT28：5’-CATAGTCGAGGAAGAGGACTgtttcagagctagaaat-3’；
LAT5-OsU6bT28：5’-AGTCCTCTTCCTCGACTATGCaacacaagcggcagc-3’；
LAT5-gRT29：5’-GACTTAACATAGTCGAGGAgtttcagagctagaaat-3’；
LAT5-OsU6bT29：5’-TCCTCGACTATGTTAAGTCCaacacaagcggcagc-3’；
LAT5-gRT30：5’-TCTCCCAAGGACCTTGGCGGgtttcagagctagaaat-3’；
LAT5-OsU6bT30：5’-CCGCCAAGGTCCTTGGGAGACaacacaagcggcagc-3’；
LAT5-gRT31：5’-CAACTGTGAGGATAAGCACgtttcagagctagaaat-3’；
LAT5-OsU6bT31：5’-GTGCTTATCCTCACAGTTGCaacacaagcggcagc-3’；
LAT5-gRT32：5’-CTCACAGTTGCACTTACTTAgtttcagagctagaaat-3’；
LAT5-OsU6bT32：5’-TAAGTAAGTGCAACTGTGAGCaacacaagcggcagc-3’；
LAT5-gRT33：5’-ATGAACTACAGAGGGTTGACgtttcagagctagaaat-3’；
LAT5-OsU6bT33：5’-GTCAACCCTCTGTAGTTCATCaacacaagcggcagc-3’；
LAT5-gRT34：5’-TGTCAACCCTCTGTAGTTCAgtttcagagctagaaat-3’；
LAT5-OsU6bT34：TGAACTACAGAGGGTTGACACaacacaagcggcagc-3’；
LAT5-gRT35：5’-TAGAGAGAACACGCCAAGAAgtttcagagctagaaat-3’；
LAT5-OsU6bT35：5’-TTCTTGGCGTGTTCTCTCTACaacacaagcggcagc-3’；
LAT5-gRT36：5’-AACAAAAAACGGGAGTAGAGgtttcagagctagaaat-3’；
LAT5-OsU6bT36：5’-CTCTACTCCCGTTTTTTGTTCaacacaagcggcagc-3’；
LAT5-gRT37：5’-GATTAATAGCTATTCCCCGgtttcagagctagaaat-3’；
LAT5-OsU6bT37：5’-CGGGGAATAGCTATTAATCCaacacaagcggcagc-3’；
LAT5-gRT38：5’-CCATCTTGAGGGTTCGATTCgtttcagagctagaaat-3’；
LAT5-OsU6bT38：5’-GAATCGAACCCTCAAGATGGCaacacaagcggcagc-3’；
LAT5-gRT39：5’-GTTCCAAAACAGTGTGTTTgtttcagagctagaaat-3’；
LAT5-OsU6bT39：5’-AAACACACTGTTTTGGAACCaacacaagcggcagc-3’；
LAT5-gRT40：5’-AACCTCAATTATTGGGACTgtttcagagctagaaat-3’；
LAT5-OsU6bT40：5’-AGTCCCAATAATTGAGGTTCaacacaagcggcagc-3’；
LAT5-gRT41：5’-TCAATTATTGGGACTCAATCgtttcagagctagaaat-3’；
LAT5-OsU6bT41：5’-GATTGAGTCCCAATAATTGACaacacaagcggcagc-3’；
LAT5-gRT42：5’-AATCAGTACCCTTGCTGGAGgtttcagagctagaaat-3’；
LAT5-OsU6bT42：5’-CTCCAGCAAGGGTACTGATTCaacacaagcggcagc-3’；
LAT5-gRT43：5’-GAGAGGTTGAGAATCCAAAgtttcagagctagaaat-3’；
LAT5-OsU6bT43：5’-TTTGGATTCTCAACCTCTCCaacacaagcggcagc-3’；
LAT5-gRT44：5’-AATCCAAAGAGAACACTCCgtttcagagctagaaat-3’；
LAT5-OsU6bT44：5’-GGAGTGTTCTCTTTGGATTCaacacaagcggcagc-3’；
LAT5-gRT45：5’-CATAAGAAAGTGCCCTTGGGgtttcagagctagaaat-3’；
LAT5-OsU6bT45：5’-CCCAAGGGCACTTTCTTATGCaacacaagcggcagc-3’；
LAT5-gRT46：5’-CCACCACTAAAACTAGAGCAgtttcagagctagaaat-3’；
LAT5-OsU6bT46：5’-TGCTCTAGTTTTAGTGGTGGCaacacaagcggcagc-3’；
LAT5-gRT47：5’-TAGAGGTATCCCCCCACCACgtttcagagctagaaat-3’；
LAT5-OsU6bT47：5’-GTGGTGGGGGGATACCTCTACaacacaagcggcagc-3’；
LAT5-gRT48：5’-ACAGGTGATCAGAGGGTAGAgtttcagagctagaaat-3’；
LAT5-OsU6bT48：5’-TCTACCCTCTGATCACCTGTCaacacaagcggcagc-3’；
LAT5-gRT49：5’-TGTACAGCAGCAGTTCCAGTgtttcagagctagaaat-3’；
LAT5-OsU6bT49：5’-ACTGGAACTGCTGCTGTACACaacacaagcggcagc-3’；
LAT5-gRT50：5’-TCCAGAACTCCCGAACAACTgtttcagagctagaaat-3’；
LAT5-OsU6bT50：5’-AGTTGTTCGGGAGTTCTGGACaacacaagcggcagc-3’；
LAT5-gRT51：5’-TGAGAAATATCCATCCGTCCgtttcagagctagaaat-3’；
LAT5-OsU6bT51：5’-GGACGGATGGATATTTCTCACaacacaagcggcagc-3’；
LAT5-gRT52：5’-AAACCACCAAGAATTCTCGgtttcagagctagaaat-3’；
LAT5-OsU6bT52：5’-CGAGAATTCTTGGTGGTTTCaacacaagcggcagc-3’；
LAT5-gRT53：5’-GTGCAACCAGAAACCACCAgtttcagagctagaaat-3’；
LAT5-OsU3T53：5’-TGGTGGTTTCTGGTTGCACTgccacggatcatctgc-3’；
LAT5-gRT54：5’-TTGAAGCCACGAGTGCAACCgtttcagagctagaaat-3’；
LAT5-OsU3T54：5’-GGTTGCACTCGTGGCTTCAATgccacggatcatctgc-3’；
LAT5-gRT55：5’-GCTTCAAGCAGCTGCTGCACgtttcagagctagaaat-3’；
LAT5-OsU3T55：5’-GTGCAGCAGCTGCTTGAAGCTgccacggatcatctgc-3’；
LAT5-gRT56：5’-CTGTCCAACATGGGCAATTTgtttcagagctagaaat-3’；
LAT5-OsU3T56：5’-AAATTGCCCATGTTGGACAGTgccacggatcatctgc-3’；
LAT5-gRT57：5’-CGAAATTGCCCATGTTGGAgtttcagagctagaaat-3’；
LAT5-OsU3T57：5’-TCCAACATGGGCAATTTCGTgccacggatcatctgc-3’；
LAT5-gRT58：5’-GGTAAGAATCACTGCTCATTgtttcagagctagaaat-3’；
LAT5-OsU3T58：5’-AATGAGCAGTGATTCTTACCTgccacggatcatctgc-3’；
LAT5-gRT59：5’-GAGAAGCTGGTAAGAATCACgtttcagagctagaaat-3’；
LAT5-OsU3T59：5’-GTGATTCTTACCAGCTTCTCTgccacggatcatctgc-3’；
LAT5-gRT60：5’-GCCATCCCGAGAAGCTGGTAgtttcagagctagaaat-3’；
LAT5-OsU3T60：5’-TACCAGCTTCTCGGGATGGCTgccacggatcatctgc-3’；
LAT5-gRT61：5’-TCTGGAAGCATTCCACGCTCgtttcagagctagaaat-3’；
LAT5-OsU3T61：5’-GAGCGTGGAATGCTTCCAGATgccacggatcatctgc-3’；
LAT5-gRT62：5’-TTGGCGAAAAACTCTGGAAGgtttcagagctagaaat-3’；
LAT5-OsU3T62：5’-CTTCCAGAGTTTTTCGCCAATgccacggatcatctgc-3’；
LAT5-gRT63：5’-TAGCGAGATCTCTTGGCGAAgtttcagagctagaaat-3’；
LAT5-OsU3T63：5’-TTCGCCAAGAGATCTCGCTATgccacggatcatctgc-3’；
LAT5-gRT64：5’-CGCTATGGAACCCCACTTATgtttcagagctagaaat-3’；
LAT5-OsU3T64：5’-ATAAGTGGGGTTCCATAGCGTgccacggatcatctgc-3’；
LAT5-gRT65：5’-ACATGATGCCAATAAGTGGgtttcagagctagaaat-3’；
LAT5-OsU3T65：5’-CCACTTATTGGCATCATGTTgccacggatcatctgc-3’；
LAT5-gRT66：5’-CGGAGAACATGATGCCAATAgtttcagagctagaaat-3’；
LAT5-OsU3T66：5’-TATTGGCATCATGTTCTCCGTgccacggatcatctgc-3’；
LAT5-gRT67：5’-CAGGACCACACCAAACGCGGgtttcagagctagaaat-3’；
LAT5-OsU3T67：5’-CCGCGTTTGGTGTGGTCCTGTgccacggatcatctgc-3’；
LAT5-gRT68：5’-CCAGGACAGCAGGACCACACgtttcagagctagaaat-3’；
LAT5-OsU3T68：5’-GTGTGGTCCTGCTGTCCTGGTgccacggatcatctgc-3’；
LAT5-gRT69：5’-GGAAGCTCATCCAGGACAGCgtttcagagctagaaat-3’；
LAT5-OsU3T69：5’-GCTGTCCTGGATGAGCTTCCTgccacggatcatctgc-3’；
LAT5-gRT70：5’-CCAGGAGATCATCGCTGCGGgtttcagagctagaaat-3’；
LAT5-OsU3T70：5’-CCGCAGCGATGATCTCCTGGTgccacggatcatctgc-3’；
LAT5-gRT71：5’-CTGCGGAGAACTACCTGTACgtttcagagctagaaat-3’；
LAT5-OsU3T71：5’-GTACAGGTAGTTCTCCGCAGTgccacggatcatctgc-3’；
LAT5-gRT72：5’-CATACCGAAGCAGTACAGGTgtttcagagctagaaat-3’；
LAT5-OsU3T72：5’-ACCTGTACTGCTTCGGTATGTgccacggatcatctgc-3’；
LAT5-gRT73：5’-TTCCAGGATCATACCGAAGCgtttcagagctagaaat-3’；
LAT5-OsU3T73：5’-GCTTCGGTATGATCCTGGAATgccacggatcatctgc-3’；
LAT5-gRT74：5’-GATGAAGGCGATGAATTCCAgtttcagagctagaaat-3’；
LAT5-OsU3T74：5’-TGGAATTCATCGCCTTCATCTgccacggatcatctgc-3’；
LAT5-gRT75：5’-CTGAGGGTGGTCCACCCAAAgtttcagagctagaaat-3’；
LAT5-OsU3T75：5’-TTTGGGTGGACCACCCTCAGTgccacggatcatctgc-3’；
LAT5-gRT76：5’-CCACTGGGCACCATCGGCGCgtttcagagctagaaat-3’；
LAT5-OsU3T76：5’-GCGCCGATGGTGCCCAGTGGTgccacggatcatctgc-3’；
LAT5-gRT77：5’-GGTAGGTGGGATGATCATCAgtttcagagctagaaat-3’；
LAT5-OsU3T77：5’-TGATGATCATCCCACCTACCTgccacggatcatctgc-3’；
LAT5-gRT78：5’-GACGATCAGAATGGTAGGTGgtttcagagctagaaat-3’；
LAT5-OsU3T78：5’-CACCTACCATTCTGATCGTCTgccacggatcatctgc-3’；
LAT5-gRT79：5’-CATCATCACCACGACGATCgtttcagagctagaaat-3’；
LAT5-OsU6aT79：5’-GATCGTCGTGGTGATGATGCggcagccaagccagca-3’；
LAT5-gRT80：5’-TTATGGAGAACTTCAGCCACgtttcagagctagaaat-3’；
LAT5-OsU6bT80：5’-GTGGCTGAAGTTCTCCATAACaacacaagcggcagc-3’；
LAT5-gRT81：5’-CTCATTGACACTGCTGTACTgtttcagagctagaaat-3’；
LAT5-OsU6aT81：5’-AGTACAGCAGTGTCAATGAGCggcagccaagccagca-3’；
LAT5-gRT82：5’-TTCGCCCTCATTGACACTGCgttttagagctagaaat-3’；
LAT5-OsU6bT82：5’-GCAGTGTCAATGAGGGCGAACaacacaagcggcagc-3’；
LAT5-gRT83：5’-TGGCCTCCCTTACGCTCTTgttttagagctagaaat-3’；
LAT5-OsU3T83：5’-AAGAGCGTAAGGGAGGCCATgccacggatcatctgc-3’；
LAT5-gRT84：5’-GACGCCATGGCCTCCCTTAgttttagagctagaaat-3’；
LAT5-OsU6aT84：5’-TAAGGGAGGCCATGGCGTCCggcagccaagccagca-3’；
LAT5-gRT85：5’-CCTCCAAGAGCTGGAATGCTgttttagagctagaaat-3’；
LAT5-OsU6bT85：5’-AGCATTCCAGCTCTTGGAGGCaacacaagcggcagc-3’；
LAT5-gRT86：5’-GGAGACCACCTCCAAGAGCTgttttagagctagaaat-3’；
LAT5-OsU3T86：5’-AGCTCTTGGAGGTGGTCTCCTgccacggatcatctgc-3’；
LAT5-gRT87：5’-CACCGCCAAGGTCCTTGGGgttttagagctagaaat-3’；
LAT5-OsU6aT87：5’-CCCAAGGACCTTGGCGGTGCggcagccaagccagca-3’；
LAT5-gRT88：5’-CCAACTATTGTCAACCCTCgttttagagctagaaat-3’；
LAT5-OsU6bT88：5’-GAGGGTTGACAATAGTTGGCaacacaagcggcagc-3’；
LAT5-gRT89：5’-CCATTTCAAGCCATCTTGAGgttttagagctagaaat-3’；
LAT5-OsU3T89：5’-CTCAAGATGGCTTGAAATGGTgccacggatcatctgc-3’；
LAT5-gRT90：5’-CATTCCCCAAGTCCATTTCAgttttagagctagaaat-3’；
LAT5-OsU6aT90：5’-TGAAATGGACTTGGGGAATGCggcagccaagccagca-3’；
LAT5-gRT91：5’-AAGAATTCTCGCAACGTCTGgttttagagctagaaat-3’；
LAT5-OsU6bT91：5’-CAGACGTTGCGAGAATTCTTCaacacaagcggcagc-3’；
LAT5-gRT92：5’-AACGCCTCCCGACCTTACAgttttagagctagaaat-3’；
LAT5-OsU3T92：5’-TGTAAGGTCGGGAGGCGTTTgccacggatcatctgc-3’；
LAT5-gRT93：5’-CACCATCACCTTGAAGGACGgttttagagctagaaat-3’；
LAT5-OsU6aT93：5’-CGTCCTTCAAGGTGATGGTGCggcagccaagccagca-3’；LAT5-gRT94：5’-AACCAGCATTGCCATGATGCgttttagagctagaaat-3’；
LAT5-OsU6bT94：5’-GCATCATGGCAATGCTGGTTCaacacaagcggcagc-3’；LAT5-gRT95：5’-CCCAACCAGCATTGCCATGAgttttagagctagaaat-3’；
LAT5-OsU3T95：5’-TCATGGCAATGCTGGTTGGGTgccacggatcatctgc-3’；
LAT5-gRT96：5’-AGCACGAACCCAACCAGCATgttttagagctagaaat-3’；
LAT5-OsU6aT96：5’-ATGCTGGTTGGGTTCGTGCTCggcagccaagccagca-3’；LAT5-gRT97：5’-CACCAGAGCCGGCTGCAGCAgttttagagctagaaat-3’；
LAT5-OsU6bT97：5’-TGCTGCAGCCGGCTCTGGTGCaacacaagcggcagc-3’；LAT5-gRT98：5’-TCTCCACGTACACCAGAGCCgttttagagctagaaat-3’；
LAT5-OsU3T98：5’-GGCTCTGGTGTACGTGGAGATgccacggatcatctgc-3’。LAT5-gRT99：5’-TGGCAGTTCTGCGCTTATGGgttttagagctagaaat-3’；
LAT5-OsU6aT99：5’-CCATAAGCGCAGAACTGCCACggcagccaagccagca-3’；LAT5-gRT100：5’-CTTCCTCAACGTTCGAGTACgttttagagctagaaat-3’；
LAT5-OsU6bT100：5’-GTACTCGAACGTTGAGGAAGCaacacaagcggcagc-3’；LAT5-gRT101：5’-GTGCTGTCTTCCTCAACGTTgttttagagctagaaat-3’；
LAT5-OsU3T101：5’-AACGTTGAGGAAGACAGCACTgccacggatcatctgc-3’；LAT5-gRT102：5’-CACAATCCCACTTGTGTGCgttttagagctagaaat-3’；
LAT5-OsU6aT102：5’-GCACACAAGTGGGATTGTGCggcagccaagccagca-3’；LAT5-gRT103：5’-GTCAGCACACAAGTGGGATgttttagagctagaaat-3’；
LAT5-OsU6bT103：5’-ATCCCACTTGTGTGCTGACCaacacaagcggcagc-3’；LAT5-gRT104：5’-CAACACAATCCTCCATTGGAgttttagagctagaaat-3’；
LAT5-OsU3T104：5’-TCCAATGGAGGATTGTGTTGTgccacggatcatctgc-3’；LAT5-gRT105：5’-CACTATCCTCAATCCCAAACgttttagagctagaaat-3’；
LAT5-OsU6aT105：5’-GTTTGGGATTGAGGATAGTGCggcagccaagccagca-3’；LAT5-gRT106：5’-AAGGCTGCTGGCCCACTCCTgttttagagctagaaat-3’；
LAT5OsU6bT106：5’-AGGAGTGGGCCAGCAGCCTTCaacacaagcggcagc-3’；LAT5-gRT107：5’-TCCAGCAATTGCTAGGAGTGgttttagagctagaaat-3’；
LAT5-OsU3T107：5’-CACTCCTAGCAATTGCTGGATgccacggatcatctgc-3’；LAT5-gRT108：5’-CATATGGAGTGTCCCGGAAGgttttagagctagaaat-3’；
LAT5-OsU6aT108：5’-CTTCCGGGACACTCCATATGCggcagccaagccagca-3’；
LAT5-gRT109：5’-CCCAAGGGCTGAAGAGACCCgttttagagctagaaat-3’；
LAT5-OsU6bT109：5’-GGGTCTCTTCAGCCCTTGGGCaacacaagcggcagc-3’；
LAT5-gRT110：5’-CTTCAGCCCTTGGGCCATTCgttttagagctagaaat-3’；
LAT5-OsU3T110：5’-GAATGGCCCAAGGGCTGAAGTgccacggatcatctgc-3’；
LAT5-gRT111：5’-ATGACACCACTCAGCCACTTgttttagagctagaaat-3’；
LAT5OsU6aT111：5’-AAGTGGCTGAGTGGTGTCATCggcagccaagccagca-3’；
LAT5-gRT112：5’-AGAACACGCCAAGAAAGACgttttagagctagaaat-3’；
LAT5-OsU6bT112：5’-GTCTTTCTTGGCGTGTTCTCaacacaagcggcagc-3’；
LAT5-gRT113：5’-TCCCATAACAAAAAACGGGgttttagagctagaaat-3’；
LAT5-OsU3T113：5’-CCCGTTTTTTGTTATGGGATgccacggatcatctgc-3’；
LAT5-gRT114：5’-CCATTTCAAGCCATCTTGAGgttttagagctagaaat-3’；
LAT5OsU6aT114：5’-CTCAAGATGGCTTGAAATGGCggcagccaagccagca-3’；
LAT5-gRT115：5’-TCCAGCAAGGGTACTGATTGCgttttagagctagaaat-3’；
LAT5OsU6bT115：5’-GCAATCAGTACCCTTGCTGGACaacacaagcggcagc-3’；
LAT5-gRT116：5’-TCCCCCCACCACTAAAACTgttttagagctagaaat-3’；
LAT5-OsU3T116：5’-AGTTTTAGTGGTGGGGGGATgccacggatcatctgc-3’；
second round amplification universal primers:
Pps-R：5’-TTCAGAGGTCTCTACCGACTAGTCACGCGTATGGAATCGG
CAGCAAA-3’；
Pgs-2：5’-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3’；
Pps-2：5’-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3’；
Pgs-3：5’-AGCGTGGGTCTCGTCTTCACTCCATCCACTCCAAGCTC-3’；
Pps-3：5’-TTCAGAGGTCTCTAAGACTTTGGAATCGGCAGCAAAGG-3’；
Pgs-L：5’-AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGC-3’。
(3) Construction of sgRNA expression cassettes
1) First round PCR, amplifying promoter and gRNA respectively
The following promoter amplification systems were formulated: 2 XPimeStar Max 7.5. Mu.L, U-F0.5. Mu.L, # U-R0.5. Mu.L, promoter template 50ng, ddH 2 O is added to 15 mu L, and the amplification reaction is carried out in a PCR instrument under the following reaction conditions: 98 ℃, 1min,1 cycle; 98 ℃,20 s,58 ℃,20 s,72 ℃,10 s,32 cycles; 72℃for 5min,1 cycle. Obtaining a promoter sequence with a sticky end; the following gRNA amplification system was formulated: 2 XPimeStar Max 7.5. Mu.L, gR-R0.5. Mu.L, #gR-F0.5. Mu.L, 50ng promoter template, ddH2O were made up to 15. Mu.L, and amplification was performed in a PCR apparatus at 58℃to give a gRNA sequence with a sticky end. After amplification, electrophoresis was performed to determine if the band was correct.
2) Second round of PCR, ligation of promoter and corresponding gRNA
According to the brightness of the first round of amplified bands, the promoter and the corresponding gRNA are mixed in equal proportion, and 9 volumes of ddH are added 2 O is used as a second round of amplification template for standby; mixing Pps-R and Pgs-2 in equal proportion, named PT1R, mixing Pps-2 and Pgs-3 in equal proportion, named PT2R, mixing Pps-3 and Pgs-L in equal proportion, named PT3L, and using as a second round amplification primer for standby; the following amplification systems were formulated: 2 XPrimeStar Max 15. Mu.L, PT1R, PT R or PT3L0.5. Mu.L, promoter+gRNA 1. Mu.L, ddH2O make up 30. Mu.L, and amplification reaction was performed in a PCR apparatus under the following conditions: 98 ℃, 1min,1 cycle; 98 ℃,20 s,58 ℃,20 s,72 ℃, 25s,32 cycles; 72℃for 5min,1 cycle. An expression cassette sequence with cohesive ends was obtained.
3) The PCR products were electrophoretically detected and used with the kit E.Z.N.Gel Extraction Kit (OMEGA) purification
4) Edge trimming connection construction knockout carrier
Two expression cassettes with target sites were constructed on one knockout vector, a total of 39 knockout vectors were predicted, designated in turn as V1-V39, and the specific combinations of target sites were as follows:
V1：Target1+Target39+Target78
V2：Target2+Target40+Target77
V3：Target3+Target38+Target76
V4：Target4+Target41+Target75
V5：Target5+Target37+Target74
V6：Target6+Target42+Target73
V7：Target7+Target36+Target72
V8：Target8+Target43+Target71
V9：Target9+Target35+Target70
V10：Target10+Target44+Target69
V11：Target11+Target34+Target68
V12：Target12+Target45+Target67
V13：Target13+Target33+Target66
V14：Target14+Target46+Target65
V15：Target15+Target32+Target64
V16：Target16+Target47+Target63
V17：Target17+Target31+Target62
V18：Target18+Target48+Target61
V19：Target19+Target30+Target60
V20：Target20+Target49+Target59
V21：Target21+Target29+Target58
V22：Target22+Target50+Target57
V23：Target23+Target28+Target56
V24：Target24+Target51+Target55
V25：Target25+Target27+Target54
V26：Target26+Target52+Target53
V27：Target79+Target80
V28：Target81+Target82+Target83
V29：Target84+Target85+Target86
V30：Target87+Target88+Target89
V31：Target90+Target91+Target92
V32：Target93+Target94+Target95
V33：Target96+Target97+Target98
V34：Target99+Target100+Target101
V35：Target102+Target103+Target104
V36：Target105+Target106+Target107
V37：Target108+Target109+Target110
V38：Target111+Target112+Target113
V39：Target114+Target115+Target116
the following edge trimming and connecting system is prepared: 10×CutSmart Buffer 1.5. Mu.L; 10×ATP 1.5. Mu.L; t4 DNA Ligase 0.3. Mu.L; 150NG of the complete knockout carrier FRENY-ABE/CBE-NG; the second round of PCR purified products were 25ng each; bsaI-HF-v2 0.5. Mu.L; ddH2O was made up to 15. Mu.L and the edge ligation was performed in a PCR instrument by the following procedure: 37 ℃,5min,1 cycle; 37 ℃,5min, 10 ℃, 2min,20 ℃,5min, 16 cycles; 37℃for 5min,1 cycle. Coli DH5 alpha is transformed by the connection product, the resistance LB plate is cultivated for 12 hours (containing 50mg/L kanamycin), positive strains are selected for sequencing, and 33 recombinant vectors containing target sequences and sg-RNA with correct sequencing are obtained.
(3) And (3) delivering the recombinant vector obtained in the step (4) to a wild rice of Zhonghua 11 transferred by Wuhan Aidi crystal company, and taking 2 vectors as 1 transformation event in a transformation combination.
2. Identification of mutant rice by amplification and determination of editing conditions
Extracting DNA from the transgenic plants (T0 generation) transplanted in the step (3), designing specific primers aiming at DNA fragments within 220bp of the target site, and amplifying the DNA fragments containing the target site. And (3) purifying the PCR product obtained by amplification, sending the purified PCR product to An Nuo Youda company for high-throughput sequencing, comparing the sequencing result with a wild plant sequence, and screening out successfully edited single-point mutation plants. The ratio of the coefficient quantity of the edited rice plants to the number of the unedited rice plants is about 1:1 after sequencing comparison.
Example 2 molecular docking of OsLAT5 with bipyridyl herbicides
1. A three-dimensional model of OsLAT5 was obtained by deep learning prediction using trRosetta (http:// yanglab. Nankai. Edu. Cn/trRosetta /). The formulas for paraquat and diquat are available from PubCHem (https:// PubChem. Ncbi. Lm. Nih. Gov /).
2. Paraquat and diquat molecules are molecular-butted with OsLAT5 active sites by DOCK 6.9 software. The binding pocket of OsLAT5 is set to x= -6.6,16.5, y= -4.3,15.1, z= -34.7, -16.1. After the coordinates of the docking pocket are determined, docking is carried out in the LeDock, then high-score docking is selected from the output result, and further analysis and three-dimensional graph preparation are carried out by using PyMOL 1.7.
The results indicate that the region where OsLAT5 interacts with paraquat is predicted by the structural model to include amino acid residues Pro-44, phe-45 and Phe-180 (FIG. 2A), which result in hydrophobic interactions with paraquat. Diquat is located in a cavity consisting of residues Phe-37, ser-41, pro-44, phe-45, phe-180, tyr-252, ser-298 and Asn-302, where Ser-41, ser-298 and Asn-302 form hydrogen bond interactions with diquat and the other residues form hydrophobic interactions with diquat (FIG. 2B).
Example 3 sensitivity of Yeast Strain heterologously expressing mutant OsLAT5 proteins to bipyridine herbicides
1. A plurality of amino acid residues of OsLAT5 interacting with paraquat or diquat in molecular docking and Ser-50, val-51 and Phe-451 amino acid sites edited by saturation mutation are mutated into alanine or glutamic acid, and the sensitivity and the transport capacity of yeast expressing the OsLAT5 before and after mutation on the paraquat or diquat are evaluated, so that the specific binding amino acid site of the OsLAT5 and bipyridyl herbicide in the sites is identified.
2. Construction of site-directed mutagenesis Yeast expression vector pYES-dest52-OsLAT5 and acquisition of Yeast Strain
According to the gene sequence to be mutated and the target site, inPrimers were designed on Primer Design Program (www.agilent.com.cn/store/prime design program. Jsp.
Yeast expression vectors were transferred into yeast strains BY4741 (wild type) and Δagp2 (polyamine-deficient) using a yeast transformation kit (coolaber, SK 2400), and after successful transfer BY extraction of yeast plasmid sequencing verification, a sensitivity experiment was performed.
3. Sensitivity test of Yeast Strain in solid Medium
Suspending single colony yeast in 1mL SC-galactose culture medium to induce expression for 16-20h, measuring yeast liquid OD at the moment 600 The values were then diluted uniformly to 0.4 with SC-galactose medium and diluted 10, 50 and 100 times with sterile water. Yeast suspensions of different concentrations were inoculated at 3. Mu.L on SC-galactose solid medium containing 3mM paraquat and 1.5mM diquat, cultured upside down in a 30℃incubator for 5-6d, and photographed for recording.
The results show that substitution of glutamic acid for Pro-44, phe-45 and Val-51 reduced the activity of OsLAT5 in absorbing paraquat, whereas yeast with Pro-44-Glu only showed tolerance to diquat (FIG. 3), demonstrating that the method of finding the amino acid site of OsLAT 5-specific recognition bipyridyl herbicide by homology alignment and molecular docking was correct.
Example 4 sensitivity test of Rice at germination stage to bipyridine herbicides
The Zhonghua No.11 is used as a material, a rice gene precise editing system is used for carrying out site-directed mutagenesis on the 44 th amino acid site of the OsLAT5 gene and screening positive plants, and simultaneously, saturated mutant plants V8 and V10 containing the 45 th and 51 th amino acid site target sequences are also subjected to 45 th and 51 th amino acid site editing positive plant screening, and rice plants with 44 th, 45 th and 51 th amino acid site mutations are named as ZH-44, ZH-45 and ZH-51 respectively.
The growth condition of three types of mutant rice of OsLAT5 in bipyridine herbicides is sensitively observed through the form of tissue culture seedlings. MS culture medium is prepared: adding 15g of sucrose and 2.215g M&S basal medium with vitamins into 500mL of secondary water, regulating the pH value to be 5.7 by NaOH, sub-packaging 80mL of each tissue culture bottle, adding 0.28g of Phytagel for high-temperature steam sterilization, adding bipyridine herbicide when the temperature is reduced to about 60 ℃ to ensure that the final concentration is 0.05 and 0.1 mu M, mixing uniformly, cooling and solidifying, and thus the product can be used.
Resuscitating the seeds to be planted for 3-4d at 49 ℃, and sterilizing the seeds before planting the tissue culture seedlings: firstly, preparing 75% alcohol with sterilized water, and cleaning for 3min each time for three times; then washing twice with 30% sodium hypochlorite water solution (1-2 drops of Tween 20 are added dropwise to make the disinfectant better adhere to the surface of the seeds) for 20min once for 3min; the impurities attached to the surfaces of the seeds can be removed completely by rapid shaking during cleaning, so that the possibility of subsequent pollution is reduced; finally, the plants are planted after being cleaned by sterile water. The pre-culture period (about 5 d) was incubated at 28℃in the absence of light, and after emergence of seedlings, the culture was continued for 10d after transfer to normal conditions (light/dark=14 h/10h,28 ℃). Photographing and recording the growth conditions after different treatments.
The results show that the growth of all lines of rice was not significantly different without application. Further studies have found that root sprout length of OsLAT5 mutant seedlings is not affected at all under stress with bipyridyl herbicide at any concentration, while wild type under co-treatment is inhibited (fig. 4). The results show that the rice mutated by the OsLAT5 gene at the 3 amino acid sites has very good tolerance to bipyridine herbicides in the germination period.
Example 5 test of resistance of seedling stage Rice to spray bipyridine-based herbicide
Every 2 seedlings are planted in one flowerpot, 4 seedlings in total are treated in 2 pots, and after the seedlings grow to 30-35d, spraying experiments are carried out, and 200mg/L herbicide spraying liquid is prepared by 0.01 per mill of Silwet L-77. After spraying, the liquid drops on the surface of the leaf can be moved back to the incubator for culture after the liquid drops are volatilized, the damage condition of the plant is observed every day, and the record is photographed before spraying and after spraying for 8 days respectively.
As shown in FIG. 5, the wild rice was completely dead and withered after 8d of treatment with bipyridyl herbicide, while the mutant was also yellow and curled, but the growth was still relatively normal, which indicated that the rice after mutation of OsLAT5 gene at the 3 amino acid position exhibited very good tolerance to bipyridyl herbicide during seedling stage.
Example 6 determination of resistance of conventional Rice variety Guiyu No.11 and OsLAT5 Gene mutant hybrid offspring to bipyridyl herbicides
When the rice ears are completely or partially extracted, the three mutants of OsLAT5 can bloom in the same day and serve as male parent plants; the conventional rice variety Guiyu No.11 has been completely or partially extracted from the rice ears, but has plants with more grains which have not yet flowering as female parent. Shearing off the branches of the female parent plant which are completely bloomed on the upper part of the rice spike and the branches which are not developed on the lower part of the rice spike by using scissors, and then shearing off the glume which is not transparent in the middle and has 1/2-2/3 of the upper part of the grain with anther, wherein the standard is that the anther in the grain is sheared off and female organs in the grain are not damaged. The treated ears were then sprayed with a spray can filled with 70% medicinal alcohol to remove the male bag. Artificial pollination is performed in noon on the day or in noon on the next day. The rice ears with large pollen amount are gently sheared at the ears and necks by scissors in the full-bloom stage of the male parent rice ears, the rice ears are gently placed downwards into a kraft hybridization bag of a female parent plant prepared in advance, and the pollen of the male parent is fully fallen onto the rice ears of the female parent plant by shaking off, so that the aim of pollination is fulfilled. F1 was obtained by identifying the mutation at the OsLAT5 gene locus as in example 1, selecting homozygous mutants, and carrying out 3 successive generations of selfing, each generation identifying the mutation at the target locus. The homozygous hybrid lines obtained for ZH-44, ZH-45 and ZH-51 were GY-44, GY-45 and GY-51, respectively, and the bipyridyl herbicide resistance of the plants was identified as in example 4, using Guiyu No.11 as a control.
As a result, it was found that the growth states of the GY-44, GY-45 and GY-51 mutant rice and Guiyu No.11 were comparable without applying bipyridine herbicides; under the treatment of 200mg/L paraquat or diquat, the osmanthus plant No.11 withers and dies 8 days. The rice plant lines GY-44, GY-45 and GY-51 mutant all have no obvious phytotoxicity, and have the same growth situation as the plant line without herbicide spraying (figure 6). The result shows that the OsLAT5 gene confers the resistance of rice adult plants to bipyridine herbicides after mutation at the 3 amino acid sites, and bipyridine herbicide resistant germplasm resources can be created through a hybridization mode.
EXAMPLE 7 barrel cultivation and agronomic trait phenotyping of different Rice lines
All rice lines were subjected to a barrel cultivation experiment and agronomic phenotypic analysis, ZH-44, ZH-45 and ZH-51 rice lines were controlled by medium flower No.11, GY-44, GY-45 and GY-51 rice lines were controlled by Guiyu No.11, and the experiment was performed in two environments. Firstly, the rice is cultivated in a plant climate chamber in the seedling stage, and after the seedlings are strong (about 30-35 d), the rice is moved to a school farm for carrying out. The planting pond sludge is used as barrel hilling in the farm, and the planting pond sludge is used for 10 times in a single plant. Additional fertilizer or foliar fertilizer is periodically supplemented in the rice fertilizer-requiring period. The rice plant height, the tiller number of a single plant, the fruiting rate, the thousand grain weight and other relevant phenotypes are investigated during harvesting.
The results are shown in fig. 7 and 8, and there was no significant difference in agronomic traits related to yield between the site-directed mutant strain and wild type compared to the reduction in plant height and thousand kernel weight caused by the normal OsLAT5 mutant strain. The result shows that the OsLAT5 gene has no influence on rice growth, rice yield and rice quality after the mutation of the 3 amino acid sites. FIG. 9 is a graph showing the results of fixed-point editing of plants on ZH-44 rice, showing that the amino acid at position 44 of OsLAT5 protein is mutated from proline (Pro) (CCG) to arginine (Arg) (CGG). FIG. 10 is a graph showing the editing result of the plant targets of saturated ZH-45 and ZH-51 mutant, showing that OsLAT5 protein has amino acid 45 mutated from phenylalanine (Phe) (TTT) to proline (Pro) (CCC) and amino acid 51 mutated from valine (Val) (GTC) to alanine (Ala) (GCC).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A mutant protein of rice OsLAT5 is characterized in that the mutant protein is obtained by mutating any one or more sites of 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein shown in SEQ ID NO. 1.
2. The rice OsLAT5 mutant protein according to claim 1, wherein the mutation is an arginine, proline or alanine substitution mutation.
3. A rice OsLAT5 mutant gene encoding the rice OsLAT5 mutant protein according to claim 1 or 2.
4. Use of the rice OsLAT5 mutant protein according to claim 1 or 2 or the rice OsLAT5 mutant gene according to claim 3 for breeding bipyridyl herbicide-resistant rice varieties.
5. The use according to claim 4, wherein the bipyridine herbicide resistant rice variety is obtained by mutating any one or more of the 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein with the sequence shown as SEQ ID NO.1 in rice.
6. The application of a reagent for mutating any one or more sites of 44 th, 45 th or 51 th amino acids of wild OsLAT5 protein shown in SEQ ID NO.1 in cultivating bipyridine herbicide resistant rice varieties.
7. The use according to claim 5, wherein a gene editing system for site-directed editing of any one or more sites of 44 th, 45 th or 51 th amino acids of wild-type OsLAT5 protein shown in SEQ ID No.1 is constructed for the OsLAT5 gene, and transformed into rice plants to obtain rice mutant strains in which any one or more sites of 44 th, 45 th or 51 th amino acids of wild-type OsLAT5 protein shown in SEQ ID No.1 in the rice sequences are mutated, namely bipyridine herbicide resistant rice varieties.
8. The use according to claim 7, wherein the gene editing is by gene saturation mutagenesis or gene site-directed editing.
9. The use according to claim 8, wherein the gene saturation mutagenesis target sequence is one or more of SEQ ID No. 2 to SEQ ID No. 117.
10. The use according to any one of claims 4 to 9, wherein the bipyridylium herbicide is paraquat and/or diquat.
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| CN202311015585.XA CN117304286A (en) | 2023-08-11 | 2023-08-11 | Application of rice OsLAT5 mutant gene in cultivation of bipyridine herbicide resistant rice variety |
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| CN202311015585.XA CN117304286A (en) | 2023-08-11 | 2023-08-11 | Application of rice OsLAT5 mutant gene in cultivation of bipyridine herbicide resistant rice variety |
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