Method for improving transformation efficiency of nano magnetic bead mediated corn pollen

Abstract

The invention discloses a method for improving the transformation efficiency of nano magnetic bead mediated maize pollen, which comprises the following steps: constructing Ac / Ds transposon elements of maize into plasmids, constructing complete expression cassettes of a herbicide selection marker gene and a target gene between 3 'Ds and 5' Ds of maize transposon, then cutting down fragments from 3 'Ds to 5' Ds by utilizing transposase AcTpase, and screening the fragments from 3 'Ds to 5' Ds to obtain the nano magnetic bead mediated maize pollen. The exogenous gene is integrated into the corn genome by using the transposon characteristics. According to the method, the average stable genetic efficiency reaches 9.6% through herbicide screening and identification, and compared with the original 1.41%, the transformation efficiency of the method is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, in particular to a method for improving the efficiency of nanomagnetic bead-mediated maize pollen transformation. BACKGROUND

[0002] The new method of nanomagnetic bead-mediated maize pollen transformation independent of genotype can efficiently deliver exogenous DNA into maize pollen through the opened pollen germination pore, and then deliver it into the maize embryo through the natural pollination process. The delivery efficiency of exogenous DNA can be as high as 55% through fluorescence labeling observation and GUS staining (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 is usually as follows: first, it is integrated into the maize genome by some means to obtain stable inheritance; second, it is degraded by intracellular nucleases; third, it is gradually diluted and finally degraded by intracellular nucleases as the cells rapidly divide after fertilization. Compared with the high delivery rate of exogenous DNA, the efficiency of stable integration of exogenous DNA into the maize genome is only 1.41% (herbicide selection results), so greatly improving the stable inheritance efficiency of this method has great significance and application value for maize molecular breeding and maize gene function identification. SUMMARY

[0003] The technical problem to be solved by the present application is how to improve the efficiency of exogenous fragment integration into the maize genome by the nanomagnetic bead-mediated pollen transformation method. To solve the above technical problem, the present application provides a transposon integration system, which comprises a transposon element and a transposase. The transposon element is mainly Ac and Ds, and the transposase is mainly AcTpase. The present application also provides a vector containing an expression cassette of the coding gene of the above 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 nanomagnetic bead-mediated maize pollen transformation, comprising the following steps: The application discloses a vector containing a corn Ac / Ds transposon system, and a construction method, which comprises the following steps: constructing a complete expression frame of an herbicide screening marker gene and a target gene between a corn transposon 3'Ds and 5'Ds, cutting the fragment from 3'Ds to 5'Ds by using a transposase AcTpase, and finally integrating the exogenous gene into the corn genome by using the transposon characteristics, so as to greatly improve the efficiency of integration of the exogenous DNA into the corn genome in a nano-magnetic bead-mediated transformation method.

[0006] The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the target gene is Cas9 The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the target gene is bar The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the target gene is Cas9 The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the target gene is bar The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the target gene is

[0007] The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein the vector construction method comprises the following steps: (1) amplifying a NOS terminator fragment and constructing a pk-tNOS vector; (2) amplifying a CaMV 35S promoter-bar-CaMV poly(A) signal fragment and constructing a pk-tNOS-bar vector; (3) constructing a pk-AcTpase-3Ds-bar vector; (4) amplifying a 5'Ds fragment and constructing a pk-AcTpase-3Ds-bar-5Ds vector; (5) amplifying a Ubi-Cas9 fragment and constructing a pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector.

[0008] The application discloses a method for improving the efficiency of nano-magnetic bead-mediated corn pollen transformation, wherein: The step (1) specifically comprises the following method: taking a pCAMBIA3301 vector as a template, performing PCR amplification by using primers S-tNOS-N-16KrF and S-tNOS-N-16KrR, performing electrophoresis separation on the product, recovering a target gene DNA fragment, and recombining the recovered product into a pMD18-T vector by using a seamless cloning technology. EcoThe RV enzyme cut pBM16K linearized vector is constructed as a 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 method: using the CPB vector as a template, PCR amplification is performed with primers bar-p35S-F and bar-M13R2, the product is separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the RV enzyme cut pk-tNOS linearized vector by seamless cloning technology to construct a pk-tNOS-bar vector. Nde I, Nhe The RV enzyme cut pk-tNOS linearized vector is constructed as a 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: the 35sAct-AcTpase-3'Ds fragment is synthesized into the pk-tNOS-bar vector at the 35sAct-AcTpase-3'Ds fragment and the RV enzyme cut site. Sma I and Spe The RV enzyme cut pk-tNOS linearized vector is constructed as a 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 (4) specifically includes the following method: using the synthesized PUC-SP-5Ds vector as a template, PCR amplification is performed with primers 5Ds-LB-QF and 5Ds-M13R-HR, the product is separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the RV enzyme cut pk-AcTpase-3Ds-bar linearized vector by seamless cloning technology to construct a pk-AcTpase-3Ds-bar-5Ds vector. Nhe The RV enzyme cut pk-tNOS linearized vector is constructed as a 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 (5) specifically includes the following method: using the CPB vector as a template (the CPB vector is derived from the Xie Chuanxiao team of the Chinese Academy of Agricultural Sciences Crop Science Institute, and contains a Cas9 expression frame), PCR amplification is performed with primers Ubi-F and Cas9-NLS-R, the product is separated by electrophoresis, the target gene DNA fragment is recovered, and the recovered product is recombined into the RV enzyme cut pk-AcTpase-3Ds-bar-5Ds linearized vector by seamless cloning technology. Hin dIII、 Spe The RV enzyme cut pk-tNOS linearized vector is constructed as a 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;

[0009] The recombinant product is transformed into E. coli competent cells, a single colony is picked for plasmid extraction, and then the plasmid is usedEco RI digestion verification, the correct plasmid was sequenced, and the sequence was verified by sequencing, thus the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector construction was completed, and the strain containing the plasmid was stored in a low-temperature refrigerator at-80 DEG C for subsequent test.

[0010] Application of transposase AcTpase in improving nanometer magnetic bead mediated maize pollen transformation efficiency.

[0011] The maize Ac / Ds transposition system of the application is applied in improving nanometer magnetic bead mediated maize pollen transformation efficiency.

[0012] The pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector of the application is applied in improving nanometer magnetic bead mediated maize pollen transformation efficiency.

[0013] The transformation method of the application is different from the prior art in that: Compared with the high delivery rate of exogenous DNA, the efficiency of stably integrating exogenous DNA into the maize genome in the prior art is only 1.41% (herbicide screening result), and the stable genetic efficiency of the nanometer magnetic bead mediated maize pollen transformation method is greatly improved, which has great significance and application value for maize molecular breeding and maize gene function identification.

[0014] The application improves the nanometer magnetic bead mediated maize pollen transformation method by modifying the plasmid vector, constructing the maize Ac / Ds transposon element into the plasmid, and constructing the herbicide selection marker gene bar and the gene editing enzyme gene Cas9 into a complete expression frame between the maize transposon 3'Ds and 5'Ds.

[0015] The method for improving nanometer magnetic bead mediated maize pollen transformation efficiency of the application will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0016] Figure 1 The figure is a flowchart for constructing the pk-tNOS vector in the method of the application; Figure 2 The figure is a flowchart for constructing the pk-tNOS-bar vector in the method of the application; Figure 3The flowchart for constructing the pk-AcTpase-3Ds-bar-5Ds vector in the method of the application is shown in the figure; Figure 4 The flowchart for constructing the pk-AcTpase-3Ds-bar-5Ds vector in the method of the application is shown in the figure; Figure 5 The flowchart for constructing the pk-AcTpase-3Ds-bar-5Ds vector in the method of the application is shown in the figure; Figure 6 The sequencing result of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector in the method of the application is shown in the figure; Figure 7 The schematic diagram of the pk-tNOS vector in the method of the application is shown in the figure; Figure 8 The schematic diagram of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector in the method of the application is shown in the figure; Figure 9 The herbicide-resistant transgenic lines in the HZ178 inbred line background screened in the method of the application are shown in the figure; Figure 10 The herbicide-resistant transgenic lines in the Zheng 58 inbred line background screened in the method of the application are shown in the figure; Figure 11 The PCR detection of the target gene in the HZ178 transgenic line in the method of the application is shown in the figure; Cas9 The figure shows the results; wherein, the numbers 1-132 represent different transgenic lines, HZ178 is the wild-type non-transgenic control, and water is the negative control; Figure 12 The PCR detection of the target gene in the Zheng 58 transgenic line in the method of the application is shown in the figure; Cas9 The figure shows the results; wherein, the numbers 1-40 represent different transgenic lines, Z58 is the wild-type non-transgenic control, and water is the negative control. DETAILED DESCRIPTION

[0017] Example 1

[0018] The application provides a method for improving the efficiency of integration of exogenous DNA into the corn genome in a nano-magnetic bead-mediated transformation method. The Ac / Ds transposon element of corn is constructed into a plasmid, the complete expression frame of the herbicide selection marker gene and the target gene is constructed between the 3'Ds and 5'Ds of the corn transposon, then the fragment from 3'Ds to 5'Ds is cut off by the transposase AcTpase, and finally the exogenous gene is integrated into the corn genome by using the transposon characteristics, so as to greatly improve the efficiency of integration of exogenous DNA into the corn genome in the nano-magnetic bead-mediated transformation method.

[0019] AcTpase protein sequence: MTPPVGNNPPSGSAIRLAKLMSTTRAPSTRKTNSVFSAYAQGLKRKAEASSSRIQNVRARARGHGCGRTSPSSSTAEAERHFIQSVSSSNANGTATDPSQDDMAIVHEPQPQPQPQPEPQPQPQPEPEEEAPQKRAKKCTSDVWQHFTKKEIEVEVDGKKYVQVWGHCNFPNCKAKYRAEGHHGTSGFRNHLRTSHSLVKGQLCLKSEKDHGKDINLIEPYKYDEVVSLKKLHLAIIMHEYPFNIVEHEYFVEFVKSLRPHFPIKSRVTARKYIMDLYLEEKEKLYGKLKDVQSRFSTTMDMWTSCQNKSYMCVTIHWIDDDWCLQKRIVGFFHVEGRHTGQRLSQTFTAIMVKWNIEKKLFALSLDNASANEVAVHDIIEDLQDTDSNLVCDGAFFHVRCACHILNLVAKDGLAVIAGTIEKIKAIVLAVKSSPLQWEELMKCASECDLDKSKGISYDVSTRWNSTYLMLRDALYYKPALIRLKTSDPRRYDAICPKAEEWKMALTLFKCLKKFFDLTELLSGTQYSTANLFYKGFCEIKDLIDQWCVHEKFVIRRMAVAMSEKFEKYWKVSNIALAVACFLDPRYKKILIEFYMKKFHGDSYKVHVDDFVRVIRKLYQFYSSCSPSAPKTKTTTNDSMDDTLMENEDDEFQNYLHELKDYDQVESNELDKYMSEPLLKHSGQFDILSWWRGRVAEYPILTQIARDVLAIQVSTVASESAFSAGGRVVDPYRNRLGSEIVDALICTKDWVAASRKGATYFPTMIGDLEVLDSVIAAATNHENHMDEDEDAIEFSKNNEDVASGSSP

[0020] DNA sequence encoding the 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: TTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC Third step: construction of pk-AcTpase-3Ds-bar vector As shown in Figure 1, the 35sAct-AcTpase-3'Ds fragment was synthesized by Nanjing Kings River Biotech Co., Ltd. to pk-tNOS-bar vector (pUC57-tNOS-bar) between the restriction sites of I and I, and the pk-AcTpase-3Ds-bar vector was constructed. Figure 3 Sma Spe

[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] Fourth step: amplifying the 5' Ds fragment and constructing the pk-AcTpase-3Ds-bar-5Ds vector

[0030] As Figure 4As shown, the PUC-SP-5Ds vector synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. was used as a template, and primers 5Ds-LB-QF and 5Ds-M13R-HR were used for PCR amplification, and the size of the amplification product 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 (digested by Nhe I) by seamless cloning technology to construct 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.

[0031] 5Ds-LB-QF: CACAATATATCCTGCCAGCTAGCCTGCAGAGCTTCTTTAGGCTAACCAC

[0032] 5Ds-M13R-HR: GGCAGACGCGATATCCCGCTAGCTCTAGACCAGGGATGAAAGTAGGATG

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

[0034] Fifth step: amplifying the Ubi-Cas9 fragment and constructing the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector

[0035] As shown, the CPB vector (the CPB vector is derived from the Xie Chuanxiao team of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and contains Figure 5 an expression frame) was used as a template, and primers Ubi-F and Cas9-NLS-R were used for PCR amplification, and the size of the amplification product was 6307 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-5Ds linearized vector (digested by Cas9 dIII、 Hin I) by seamless cloning technology. The recombinant product was transformed into E. coli competent cells, and the plasmid was extracted after single colonies were picked and verified by plasmid digestion using Spe RI. The correct plasmid was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequence was correct after sequencing verification, and thus the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds vector was constructed (the sequencing results are shown in Eco Figure 6 ​The strain containing the plasmid was stored in a low-temperature refrigerator at -80°C for subsequent experiments. The nucleotide sequences of primers Ubi-F and Cas9-NLS-R are shown as 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. The specific method and steps of corn transformation and screening mediated by nanomagnetic beads are as follows: patent ZL202111418727.8.

[0039] 1. Transformation method

[0040] (1) Prepare plasmid DNA for transfection (pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds plasmid); (2) Combine nano magnetic beads MNP with plasmid DNA under room temperature conditions to form an MNP-DNA complex; (3) Collect fresh pollen at the corn flowering stage, and store in an ice box under water-proof conditions, and quickly bring back to the room; (4) Mix the corn pollen transformation solution with sieved corn pollen, and perform pore opening pretreatment under low temperature conditions; (5) Add the MNP-DNA complex obtained in step (2) to the pore opening pretreatment solution of step (4), mix gently, and place on a low-temperature pre-cooled magnetic plate for transfection; (6) After transfection is completed, the pollen suspension is taken to the field with an ice box, and pollination is directly performed on the corn ear; In steps (4) and (5), the low temperature is 6-10°C.

[0041] 2. Screening method

[0042] Sow the harvested corn seeds in the field, and at the three-leaf stage of the plants, spray 200 mg / L of glufosinate herbicide for screening.

[0043] III. Results: 1. Herbicide screening to obtain resistant transgenic lines In the background of the inbred line HZ178, 3300 corn seeds transformed by nano magnetic bead mediated pollen transformation of plasmid pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds were sown in the field in Hainan in winter, and 2300 plants were emerged (emergence rate 69.6%). At the three-leaf stage of the plants, 200 mg / L of glufosinate herbicide was sprayed, and after glufosinate screening, 206 resistant transgenic lines were obtained (transformation efficiency 8.9%), as shown in Table 1. Figure 9

[0044] In the background of the inbred line Zheng 58, 1900 corn seeds transformed by nano magnetic bead mediated pollen transformation of plasmid pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds were sown in the field in Beijing in summer, and 470 plants were emerged (emergence rate 24.7%). At the three-leaf stage of the plants, 200 mg / L of glufosinate herbicide was sprayed, and after glufosinate screening, 60 resistant transgenic lines were obtained (transformation efficiency 12.7%), as shown in Table 2. Figure 10

[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 above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art are intended to fall within the scope of the present application defined by the claims.

Claims

1. A nucleotide sequence encoding the transposase AcTpase, as shown in SEQ ID NO:

1.

2. A method for improving the efficiency of maize pollen conversion mediated by magnetic nanobeads, characterized in that: The vector containing the maize Ac / Ds transposon system is constructed using the following steps: the complete expression frames of the herbicide selection marker gene and the target gene are constructed between the 3'Ds and 5'Ds of the maize transposon, and then the 3'Ds to 5'Ds fragment is cut out using the transposon enzyme AcTpase. The foreign gene is then integrated into the maize genome using the transposon properties.

3. The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to claim 2, characterized in that: 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.

4. The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to claim 3, characterized in that: The carrier construction method 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.

5. The method for improving the efficiency of maize pollen conversion mediated by nanomagnetic beads according to claim 4, 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.

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

7. The application of the maize Ac / Ds transposable system as described in claim 2 in improving the efficiency of maize pollen conversion mediated by nanomagnetic beads.

8. The application of the pk-AcTpase-3Ds-Ubi-Cas9-bar-5Ds carrier according to any one of claims 3 to 5 in improving the efficiency of maize pollen conversion mediated by magnetic nanobeads.

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