A method to improve the efficiency of maize pollen conversion mediated by magnetic nanobeads

Abstract

This invention discloses a method for improving the efficiency of maize pollen transformation mediated by magnetic nanobeads, comprising the following steps: constructing maize Ac / Ds transposon elements into plasmids; constructing complete expression frames of herbicide selection marker genes and target genes between the 3'Ds and 5'Ds of the maize transposon; then using the transposase AcTpase to cut out the fragment from 3'Ds to 5'Ds; and utilizing the transposon characteristics to integrate the exogenous gene into the maize genome. The method of this invention achieves an average stable genetic efficiency of 9.6% through herbicide screening and identification, significantly improving the transformation efficiency compared to the previous 1.41%.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and in particular to a method for improving the efficiency of maize pollen conversion mediated by magnetic nanobeads. Background Technology

[0002] A novel method for maize pollen transformation mediated by genotype-independent magnetic nanobeads enables the efficient delivery of exogenous DNA into maize pollen through open pollen germination pores, followed by natural pollination and delivery into the maize embryo. Fluorescent labeling and GUS staining observation show that the delivery efficiency of exogenous DNA can reach up to 55% (see invention patent ZL202111418727.8 and Wang Z. et al. J. Integr. Plant Biol. 2022, 64: 1145–1156). The fate of exogenous DNA delivered into the maize embryo typically falls into three categories: first, it integrates into the maize genome and achieves stable inheritance; second, it is degraded by intracellular nucleases; third, with rapid cell division after fertilization, the exogenous DNA is gradually diluted and eventually degraded by intracellular nucleases. Compared to the high delivery rate of exogenous DNA, the efficiency of exogenous DNA being stably integrated into the maize genome is only 1.41% (herbicide screening results). Therefore, significantly improving the stable genetic efficiency of this method is of great significance and application value for maize molecular breeding and the identification of maize gene functions. Summary of the Invention

[0003] The technical problem this invention aims to solve is how to improve the efficiency of exogenous fragment integration into the maize genome using a nanoparticle-mediated pollen transformation method. To address this problem, this invention provides a transposon integration system comprising transposon elements and transposases. The transposon elements are primarily Ac and Ds, and the transposase is primarily AcTpase. This invention also provides a vector containing an expression cassette of the gene encoding the aforementioned transposase, a DNA molecule of the Ac element, and a DNA molecule of the Ds element.

[0004] A nucleotide sequence encoding the transposase AcTpase is shown in SEQ ID NO:1.

[0005] A method for improving the efficiency of maize pollen conversion mediated by magnetic nanobeads includes the following steps: The vector containing the maize Ac / Ds transposon system is constructed using the following steps: the complete expression frames of the herbicide screening marker gene and the target gene are constructed between the 3'Ds and 5'Ds of the maize transposon; the 3'Ds to 5'Ds fragment is then cut out using the transposase AcTpase; and finally, the exogenous gene is integrated into the maize genome using the transposon properties, thereby significantly improving the efficiency of exogenous DNA integration into the maize genome in the nanomagnetic bead-mediated transformation method.

[0006] The method for improving the efficiency of maize pollen transformation mediated by magnetic nanobeads according to the present invention, wherein the target gene is... Cas9 The herbicide selection marker gene is a glufosinate resistance gene. bar Using pk-tNOS as the backbone, a novel coronavirus containing pk-tNOS was constructed. Cas9 and glufosinate resistance gene bar The Ac / Ds plant expression vector pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds.

[0007] The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to the present invention includes the following steps in the carrier construction method: (1) Amplify the NOS terminator fragment and construct the pk-tNOS vector; (2) Amplify the CaMV 35S promoter-bar-CaMV poly(A) signal fragment and construct the pk-tNOS-bar vector; (3) Construct the pk-AcTpase-3Ds-bar vector; (4) Amplify the 5'Ds fragment and construct the pk-AcTpase-3Ds-bar-5Ds vector; (5) Amplify the Ubi-Cas9 fragment and construct the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector.

[0008] The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to the present invention, wherein: Step (1) specifically includes the following methods: using the pCAMBIA3301 vector as a template, PCR amplification is performed using primers S-tNOS-N-16KrF and S-tNOS-N-16KrR. The products are separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the target gene using seamless cloning technology. EcoThe pBM16K linearized vector digested by RV enzyme was constructed as the pk-tNOS vector; the nucleotide sequences of primers S-tNOS-N-16KrF and S-tNOS-N-16KrR are shown in SEQ ID NO:2 and SEQ ID NO:3. Step (2) specifically includes the following methods: using the CPB vector as a template, PCR amplification is performed using primers bar-p35S-F and bar-M13R2. The products are separated by electrophoresis, and the target gene DNA fragment is recovered. The recovered product is then recombined into the target gene using seamless cloning technology. Nde I, Nhe The pk-tNOS linearized vector digested with enzyme I was constructed as the pk-tNOS-bar vector; the nucleotide sequences of primers bar-p35S-F and bar-M13R2 are shown in SEQ ID NO:4 and SEQ ID NO:5. Step (3) specifically includes the following method: synthesizing the 35sAct-AcTpase-3'Ds fragment into the pk-tNOS-bar vector. Sma I and Spe Between the I restriction sites, a pk-AcTpase-3Ds-bar vector was constructed; Step (4) specifically includes the following methods: using the synthesized PUC-SP-5Ds vector as a template, PCR amplification is performed using primers 5Ds-LB-QF and 5Ds-M13R-HR. The products are separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the target gene using seamless cloning technology. Nhe The pk-AcTpase-3Ds-bar linearized vector digested with enzyme I was constructed as the pk-AcTpase-3Ds-bar-5Ds vector; the nucleotide sequences of primers 5Ds-LB-QF and 5Ds-M13R-HR are shown in SEQ ID NO:6 and SEQ ID NO:7. Step (5) specifically includes the following methods: using the CPB vector as a template (the CPB vector originated from Xie Chuanxiao's team at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and contains...) Cas9 The expression cassette was amplified by PCR using primers Ubi-F and Cas9-NLS-R. The products were separated by electrophoresis, and the target gene DNA fragment was recovered. The recovered product was then recombined into the expression cassette using seamless cloning technology. Hin dIII、 Spe The pk-AcTpase-3Ds-bar-5Ds linearized vector digested with enzyme I; the nucleotide sequences of primers Ubi-F and Cas9-NLS-R are shown in SEQ ID NO:8 and SEQ ID NO:9.

[0009] The recombinant product was transformed into competent E. coli cells, and plasmids were extracted from single clones for use.Eco RI performed plasmid digestion verification. After successful digestion verification, the plasmid was sequenced, and the sequence was verified to be correct. Thus, the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector was constructed. The strain containing the plasmid was stored in a -80℃ freezer for subsequent experiments.

[0010] Application of transposase AcTpase in improving the efficiency of maize pollen conversion mediated by magnetic nanobeads.

[0011] The application of the maize Ac / Ds transposable system described in this invention in improving the efficiency of maize pollen conversion mediated by nanomagnetic beads.

[0012] The application of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds carrier described in this invention in improving the efficiency of maize pollen conversion mediated by magnetic nanobeads.

[0013] The conversion method of the present invention differs from the prior art in that: Compared to the high delivery rate of exogenous DNA, the efficiency of stable integration of exogenous DNA into the maize genome in existing technologies is only 1.41% (herbicide screening results). This invention significantly improves the stable genetic efficiency of the nanomagnetic bead-mediated maize pollen transformation method, which has great significance and application value for maize molecular breeding and the identification of maize gene functions.

[0014] This invention modifies plasmid vectors to construct maize Ac / Ds transposon elements into plasmids, thereby enabling the inclusion of herbicide selection marker genes. bar and gene editing enzyme gene Cas9 The complete expression frame was constructed between the 3'Ds and 5'Ds of the maize transposon. The transposase AcTpase was then used to cut out the fragment from 3'Ds to 5'Ds. By utilizing the transposon properties, the exogenous gene was integrated into the maize genome, thereby significantly improving the efficiency of exogenous DNA integration into the maize genome in the nanomagnetic bead-mediated transformation method. The average stable genetic efficiency reached 9.6% through herbicide screening, which is significantly improved compared to the original 1.41%.

[0015] The method for improving the efficiency of corn pollen conversion mediated by nanomagnetic beads according to the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the process for constructing the pk-tNOS vector in the method of the present invention; Figure 2 This is a schematic diagram of the process for constructing the pk-tNOS-bar vector in the method of the present invention; Figure 3This is a schematic diagram of the process for constructing the pk-AcTpase-3Ds-bar vector in the method of the present invention; Figure 4 This is a schematic diagram of the process for constructing the pk-AcTpase-3Ds-bar-5Ds vector in the method of the present invention; Figure 5 This is a schematic diagram of the process for constructing the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector in the method of the present invention; Figure 6 This is a sequencing result diagram of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector used in the method of this invention; Figure 7 This is a schematic diagram of the pk-tNOS carrier in the method of the present invention; Figure 8 This is a schematic diagram of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds carrier used in the method of this invention; Figure 9 The herbicide-resistant transgenic lines selected from the HZ178 inbred line background in the method of this invention; Figure 10 This refers to the herbicide-resistant transgenic lines selected from the background of the Zheng 58 inbred line in the method of this invention; Figure 11 This invention relates to the method for PCR detection of the target gene in the HZ178 transgenic line. Cas9 Results diagram; where numbers 1-132 represent different transgenic lines, HZ178 is the wild-type non-transgenic control, and water is the negative control; Figure 12 This invention relates to the method for PCR detection of the target gene in the Zheng 58 transgenic line. Cas9 Results diagram; where numbers 1-40 represent different transgenic lines, Z58 is the wild-type non-transgenic control, and water is the negative control. Detailed Implementation

[0017] Example 1

[0018] This invention provides a method for improving the efficiency of exogenous DNA integration into the maize genome in nanobead-mediated transformation. The method involves constructing maize Ac / Ds transposon elements into a plasmid, constructing the complete expression frames of herbicide selection marker genes and target genes between the 3'Ds and 5'Ds of the maize transposon, and then using the transposase AcTpase to cut out the fragment from 3'Ds to 5'Ds. Finally, utilizing the transposon properties, the exogenous gene is integrated into the maize genome, thereby significantly improving the efficiency of exogenous DNA integration into the maize genome in nanobead-mediated transformation.

[0019] AcTpase Protein Sequence: MTPPVGNNPPSGSAIRLAKLMSTTRAPSTRKTNSVFSAYAQGLKRKAEASSSRIQNVRARARGHGCGRTSPSSSTAEAERHFIQSVSSSNANGTATDPSQDDMAIVHEPQPQPQPQPEPQPQPQPEPEEEAPQKRAKKCTSDVWQHFTKKEIEVEVDGKKYVQVWGHCNFPNCKAKYRAEGHHGTSGFRNHLRTSHSLVKGQLCLKSEKDHGKDINLIEPYKYDEVVSLKKLHLAIIMHEYPFNIVEHEYFVEFVKSLRPHFPIKSRVTARKYIMDLYLEEKEKLYGKLKDVQSRFSTTMDMWTSCQNKSYMCVTIHWIDDDWCLQKRIVGFFHVEGRHTGQRLSQTFTAIMVKWNIEKKLFALSLDNASANEVAVHDIIEDLQDTDSNLVCDGAFFHVRCACHILNLVAKDGLAVIAGTIEKIKAIVLAVKSSPLQWEELMKCASECDLDKSKGISYDVSTRWNSTYLMLRDALYYKPALIRLKTSDPRRYDAICPKAEEWKMALTLFKCLKKFFDLTELLSGTQYSTANLFYKGFCEIKDLIDQWCVHEKFVIRRMAVAMSEKFEKYWKVSNIALAVACFLDPRYKKILIEFYMKKFHGDSYKVHVDDFVRVIRKLYQFYSSCSPSAPKTKTTTNDSMDDTLMENEDDEFQNYLHELKDYDQVESNELDKYMSEPLLKHSGQFDILSWWRGRVAEYPILTQIARDVLAIQVSTVASESAFSAGGRVVDPYRNRLGSEIVDALICTKDWVAASRKGATYFPTMIGDLEVLDSVIAAATNHENHMDEDEDAIEFSKNNEDVASGSSP

[0020] DNA Sequence Encoding AcTpase Protein (SEQ ID NO:1): 3' terminal sequence: GCAATGTGTCTTATGTTTGTTGACAGATGAGCCTTGGTTGTAATAGTTTATGCATGCTAAGTGATCCAGATGTGAGCAAGTGATTATGAATATGTGTTTTAAACTTTATATTGTGTCATGTGTGCTAGTAGACTTATATGGCTTCTTATGTTAGCCAAGAGCCCAAGACTTATCACTTATGTGCTACATTAAACTATGTGTGCTCCAGATTTATATGGATTTTATCTATGTTTAATTAAGACTTGTGTTTACAATTTTTTATATTTGTTTTTA 3'Ds sequence: AGTTTTGAATATATGTTTTCATGTGTGATTTTACCGAACAAAAATACCGGTTCCCGTCCGATTTCGACTTTAACCCGACCGGATCGTATCGGTTTTCGATTACCGTATTTATCCCGTTCGTTTTCGTTACCGGTATATCCCGTTTTCGTTTCCGTCCCGCAAGTTAAATATGAAAATGAAAACGGTAGAGGTATTTTACCGACCGTTACCGACCGTTTTCATCCCTA 5'Ds sequence: Example 2 I. Carrier Construction: Using the pk-tNOS storage vector in our laboratory as a backbone vector, a culturing medium containing pk-tNOS was constructed. Cas9 and glufosinate resistance gene bar The Ac / Ds plant expression vector pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds.

[0021] Step 1: Amplify the NOS terminator fragment and construct the pk-tNOS vector

[0022] like Figure 1 As shown, using the pCAMBIA3301 vector as a template (pCAMBIA3301 is a publicly available vector), PCR amplification was performed using primers S-tNOS-N-16KrF and S-tNOS-N-16KrR. The amplified product was 325 bp in size. The PCR product was separated by 1% agarose gel electrophoresis, and the target gene DNA fragment was recovered. The recovered product was recombined into the pBM16K linearized vector (using seamless cloning technology) Eco The pk-tNOS vector was constructed by RV digestion. The pBM16K vector was purchased from Beijing Bomeide Gene Technology Co., Ltd. The nucleotide sequences of primers S-tNOS-N-16KrF and S-tNOS-N-16KrR are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0023] S-tNOS-N-16KrF sequence: CCTGATGCTTGATATCCCGGGACTAGTGTTTAAACGAGCTCGATCGTTCAAACATTTGG

[0024] S-tNOS-N-16KrR sequence: CAGGCAGACGCGATATCCCGCTAGCCATATGGATCTAGTAACATAGATGAC NOS terminator sequence: GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTCATCTATGTTACTAGATC Step 2: Amplify the CaMV 35S promoter-bar-CaMV poly(A) signal fragment and construct the pk-tNOS-bar vector. like Figure 2 As shown, using the CPB vector as a template (CPB vector originated from Xie Chuanxiao's team at the Institute of Crop Science, Chinese Academy of Agricultural Sciences), PCR amplification was performed using primers bar-p35S-F and bar-M13R2, and the amplified product was 1209 bp in size. The PCR product was separated by 1% agarose gel electrophoresis, and the target gene DNA fragment was recovered. The recovered product was recombined into the pk-tNOS linearized vector (via seamless cloning technology) Nde I, Nhe The vector was constructed by digestion with enzyme I (bar-p35S-F and bar-M13R2). The nucleotide sequences of primers bar-p35S-F and bar-M13R2 are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0025] bar-p35S-F sequence: CATCTATGTTACTAGATCCATATGGAGCTCTGAGACTTTTCAACAAAGGG

[0026] bar-M13R2 sequence: CAGGCAGACGCGATATCCCGCTAGCTGGCAGGATATATTGTGGTGTAAAC

[0027] CaMV 35S promoter sequence: TGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACA bar Sequence: ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATTTGA CaMV poly(A) signal sequence: TTTCCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC Step 3: Constructing the pk-AcTpase-3Ds-bar vector like Figure 3 As shown, the 35sAct-AcTpase-3'Ds fragment was synthesized by Nanjing Genscript Biotech Co., Ltd. into the pk-tNOS-bar vector ( Sma I and Spe (between the I restriction sites), and constructed as the pk-AcTpase-3Ds-bar vector.

[0028] 35sAct promoter sequence: ATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACGCTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTTGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGGGTACCCGGACCTCTAGTGATTTCGTCAGGCTTAGATGTGCTAGATCTTTCTTTCTTCTTTTTGTGGGTAGAATTTGAATCCCTCAGCATTGTTCATCGGTAGTTTTTCTTTTCATGATTTGTGACAAATGCAGCCTCGTGCGGAGCTTTTTTGTAGGTAGAAGTGATCAACC The AcTpase protein sequence, the DNA sequence encoding the AcTpase protein, the 3' terminal sequence, and the 3'Ds sequence are the same as in Example 1.

[0029] Step 4: Amplify the 5'Ds fragment and construct the pk-AcTpase-3Ds-bar-5Ds vector

[0030] As Figure 4As shown, using the PUC-SP-5Ds vector synthesized by Sangon Biotech (Shanghai) Co., Ltd. as a template, PCR amplification was performed using primers 5Ds-LB-QF and 5Ds-M13R-HR, and the amplified product size was 1848 bp. The PCR product was separated by 1% agarose gel electrophoresis, and the target gene DNA fragment was recovered. The recovered product was recombined into the pk-AcTpase-3Ds-bar linearized vector (via seamless cloning technology) Nhe The vector pk-AcTpase-3Ds-bar-5Ds was constructed by digestion with enzyme I. The nucleotide sequences of primers 5Ds-LB-QF and 5Ds-M13R-HR are shown in SEQ ID NO:6 and SEQ ID NO:7.

[0031] 5Ds-LB-QF: CACAATATATCCTGCCAGCTAGCCTGCAGAGCTTCTTTAGGCTAACCAC

[0032] 5Ds-M13R-HR:GGCAGACGCGATATCCCGCTAGCTCTAGACCAGGGATGAAAGTAGGATG

[0033] The 5'Ds sequence is the same as in Example 1.

[0034] Step 5: Amplify the Ubi-Cas9 fragment and construct the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector.

[0035] like Figure 5 As shown, the CPB vector was used as a template (the CPB vector originated from Xie Chuanxiao's team at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and contains...). Cas9 The expression cassette was amplified by PCR using primers Ubi-F and Cas9-NLS-R, and the amplified product was 6307 bp in size. The PCR product was separated by 1% agarose gel electrophoresis, and the target gene DNA fragment was recovered. The recovered product was recombined into the pk-AcTpase-3Ds-bar-5Ds linearized vector (via seamless cloning technology) Hin dIII、 Spe (I enzyme digestion). The recombinant product was transformed into E. coli competent cells, and plasmids were extracted from single clones and then used. Eco RI performed plasmid digestion verification. After successful digestion, the plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing confirmed the sequence was correct. The pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector construction was thus completed (sequencing results are available in [link to sequencing results]). Figure ⑥The strain containing the plasmid was stored at -80°C for subsequent experiments. The nucleotide sequences of primers Ubi-F and Cas9-NLS-R are shown in SEQ ID NO:8 and SEQ ID NO:9.

[0036] Ubi-F: AATATATCCTGTCAAAGCTTTGCAGTGCAGCGTGACCCGG

[0037] Cas9-NLS-R: ATCGAGCTCGTTTAAACACTAGTTTACTTTTTCTTTTTTG

[0038] Ubi promoter sequence: 3xFLAG-SV40 NLS- Cas9 -NLS (underlined is Cas9 sequence): GACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC GACAAGAAGTACAGCATCGGCCTGGACAT CGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCA ACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACC CGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACG AGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGA AAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCG GGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGA CCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTG AGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGAT TGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCA AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCC AAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGC CTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTG AGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAA GAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGA GGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCA TTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGC ATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCAT CACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCG ATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTG ACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGA CCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGA GGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACA ATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTT TAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCA GCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAG CCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAAT GAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGC AGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGG CTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGAC CAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGC GGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGC GAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT GGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCA AGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGAC GCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCT TCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATC GAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCAT GCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGA ACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCC TATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGAT CACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAA AGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCC GGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGA GAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGA TCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGC CCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCA CCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA II. Specific methods and steps for maize conversion and screening mediated by nanomagnetic beads are as described in patent ZL202111418727.8.

[0039] 1. Conversion Method

[0040] (1) Preparation of plasmid DNA for transfection (pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds plasmid). (2) The magnetic nanoparticles MNP bind to plasmid DNA at room temperature to form an MNP-DNA complex; (3) Collect fresh pollen from corn during its peak flowering period, store it in an ice box while it is still wet, and then quickly bring it back indoors; (4) Mix the corn pollen conversion solution with the sieved corn pollen and perform a pre-treatment with opening holes under low temperature conditions. (5) Add the MNP-DNA complex obtained in step (2) to the well pretreatment solution in step (4), mix gently, and place on a pre-cooled magnetic plate for transfection. (6) After the transfection is completed, the pollen suspension is taken to the field in an ice box and directly pollinated on the female ears of corn; In steps (4) and (5), the low temperature is 6~10℃.

[0041] 2. Screening Method

[0042] The harvested corn seeds were sown in the field, and at the three-leaf stage of the plants, they were sprayed with 200 mg / L glufosinate herbicide for screening.

[0043] III. Results: 1. Herbicide screening to obtain resistant transgenic lines In the background of inbred line HZ178, 3300 maize seeds were transformed into plasmid pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds via nano-magnetic bead-mediated pollen transformation in Beijing during the summer. In the winter, these transformed seeds were sown in Hainan, resulting in 2300 seedlings (emergence rate 69.6%). At the three-leaf stage, 200 mg / L glufosinate herbicide was sprayed. After glufosinate screening, 206 resistant transgenic lines were obtained (transformation efficiency 8.9%). Figure 9 As shown.

[0044] In the inbred line Zheng 58, 1900 maize seeds were transformed into plasmid pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds via nano-magnetic bead-mediated pollen transformation in Beijing during the summer. In the winter, these transformed seeds were sown in Hainan, resulting in 470 seedlings (emergence rate 24.7%). At the three-leaf stage, 200 mg / L glufosinate herbicide was sprayed. After glufosinate screening, 60 resistant transgenic lines were obtained (transformation efficiency 12.7%). Figure 10 As shown.

[0045] After screening for herbicides, the efficiency of resistant lines was statistically analyzed, and the average transformation efficiency of the two inbred lines was 9.6%.

[0046] 2. Target gene in herbicide-resistant transgenic lines Cas9 Detection

[0047] Leaf DNA extraction methods, such as patent ZL202111418727.8, involve PCR amplification of the target gene. Cas9 The information is as follows: (1) Primer sequence (5'-3'), amplification product size is 477bp Cas9 JC-F: ATGCCAAACTGCAGCTGAGC Cas9 JC-R:GCAGCTCTCCCAGGTGGATC (2) PCR reaction system (recommended)

[0048] Recommended Mixes: Nanjing Novizan Biotechnology Co., Ltd. 2×Taq Plus Master Mix II (Dye Plus), P213; 2×Rapid Taq Master Mix, P222.

[0049] (3) P213—PCR reaction procedure (recommended)

[0050] P222—PCR Reaction Procedure (Recommended)

[0051] (4) Sequencing: The sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0052] The sequencing primers were Cas9 JC-F / Cas9 JC-R, and unidirectional sequencing was performed.

[0053] Among 206 herbicide-resistant lines of HZ178, [the following was observed / tested]: Cas9 Genes were verified by PCR detection and sequencing, yielding 132 genes with [specific characteristics]. Cas9 Genetic lines (transgenic lines) Cas9 The detection rate of the target gene was 64%, such as Figure 11 As shown.

[0054] Among 60 herbicide-resistant lines of Zheng 58, [the following was observed]: Cas9 The genes were verified by PCR detection and sequencing, and 40 genes with [specific characteristics] were obtained. Cas9 Genetic lines (transgenic lines) Cas9 The target gene detection rate was 66.6%, such as...Figure 12 As shown.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the efficiency of maize pollen conversion mediated by magnetic nanobeads, characterized in that: Includes the following steps: Step 1: Prepare the plasmid DNA for transfection, namely pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds plasmid; The plasmid contains a vector of the maize Ac / Ds transposon system. The construction method specifically includes the following steps: constructing the complete expression frames of the herbicide screening marker gene and the target gene between the 3'Ds and 5'Ds of the maize transposon, then using the transposase AcTpase to cut out the fragment from 3'Ds to 5'Ds, and using the transposon characteristics to integrate the foreign gene into the maize genome; The nucleotide sequence encoding the transposase AcTpase is shown in SEQ ID NO:1; The target gene is Cas9 The herbicide selection marker gene is a glufosinate resistance gene. bar Using pk-tNOS as the backbone, a novel coronavirus containing pk-tNOS was constructed. Cas9 and glufosinate resistance gene bar Ac / Ds plant expression vector pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds; The method for constructing the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector includes the following steps: (1) Amplify the NOS terminator fragment and construct the pk-tNOS vector; (2) Amplify the CaMV 35S promoter-bar-CaMV poly(A) signal fragment and construct the pk-tNOS-bar vector; (3) Construct the pk-AcTpase-3Ds-bar vector; (4) Amplify the 5'Ds fragment and construct the pk-AcTpase-3Ds-bar-5Ds vector; (5) Amplify the Ubi-Cas9 fragment and construct the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector; The method for constructing the pk-tNOS vector includes the following steps: PCR amplification was performed using pCAMBIA3301 vector as template, the target gene DNA fragment was recovered, and the recovered product was recombined into pBM16K linearized vector to construct pk-tNOS vector; Step 2: The magnetic nanoparticles MNP bind to plasmid DNA at room temperature to form an MNP-DNA complex; the obtained MNP-DNA complex is added to the well pretreatment solution for transfection.

2. The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to claim 1, characterized in that: Step (1) specifically includes the following methods: using the pCAMBIA3301 vector as a template, PCR amplification is performed using primers S-tNOS-N-16KrF and S-tNOS-N-16KrR. The products are separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the target gene using seamless cloning technology. Eco The pBM16K linearized vector digested with RV enzyme was constructed into the pk-tNOS vector. The nucleotide sequences of primers S-tNOS-N-16KrF and S-tNOS-N-16KrR are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. Step (2) specifically includes the following methods: using the CPB vector as a template, PCR amplification is performed using primers bar-p35S-F and bar-M13R2. The products are separated by electrophoresis, and the target gene DNA fragment is recovered. The recovered product is then recombined into the target gene using seamless cloning technology. Nde I, Nhe The pk-tNOS linearized vector digested with enzyme I was constructed as the pk-tNOS-bar vector; the nucleotide sequences of primers bar-p35S-F and bar-M13R2 are shown in SEQ ID NO:4 and SEQ ID NO:

5. Step (3) specifically includes the following method: synthesizing the 35sAct-AcTpase-3'Ds fragment into the pk-tNOS-bar vector. Sma I and Spe Between the I restriction sites, a pk-AcTpase-3Ds-bar vector was constructed; Step (4) specifically includes the following methods: using the synthesized PUC-SP-5Ds vector as a template, PCR amplification is performed using primers 5Ds-LB-QF and 5Ds-M13R-HR. The products are separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the target gene using seamless cloning technology. Nhe The pk-AcTpase-3Ds-bar linearized vector digested with enzyme I was constructed as the pk-AcTpase-3Ds-bar-5Ds vector; the nucleotide sequences of primers 5Ds-LB-QF and 5Ds-M13R-HR are shown in SEQ ID NO:6 and SEQ ID NO:

7. Step (5) specifically includes the following methods: using the CPB vector as a template, PCR amplification is performed using primers Ubi-F and Cas9-NLS-R. The products are separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the target gene using seamless cloning technology. Hin dIII、 Spe pk-AcTpase-3Ds-bar-5Ds linearized vector digested with enzyme I; The nucleotide sequences of primers Ubi-F and Cas9-NLS-R are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively. The recombinant product was transformed into competent E. coli cells, and plasmids were extracted from single clones for use. Eco RI performed plasmid digestion verification. After successful digestion verification, the plasmid was sequenced, and the sequence was verified to be correct. Thus, the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector was constructed. The strain containing the plasmid was stored in a -80℃ freezer for subsequent experiments.

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