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WO2022053009A1 - Promoteur recombinant, cassette d'expression génique et son application dans l'élevage de plantes - Google Patents

Promoteur recombinant, cassette d'expression génique et son application dans l'élevage de plantes Download PDF

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Publication number
WO2022053009A1
WO2022053009A1 PCT/CN2021/117586 CN2021117586W WO2022053009A1 WO 2022053009 A1 WO2022053009 A1 WO 2022053009A1 CN 2021117586 W CN2021117586 W CN 2021117586W WO 2022053009 A1 WO2022053009 A1 WO 2022053009A1
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promoter
recombinant
izmhsp70
epsps
expression cassette
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Inventor
李树秀
李晓娇
贾志伟
吕玉平
孙宇
韩雨颖
邓言赛
贺志豪
卢娟
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Longping Biotechnology Hainan Co Ltd
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Longping Biotechnology Hainan Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the invention belongs to the technical field of molecular biology and plant breeding, in particular to a recombinant promoter, a gene expression cassette and its application in plant breeding.
  • hybrid vigor is one of the important measures to increase crop yield and improve product quality.
  • a large number of hybrid seeds must be prepared first, and seed production must first pass the level of emasculation of the female parent.
  • Casting is a very laborious job.
  • Manual emasculation is not only time-consuming and labor-intensive, but also increases the cost of seed production; if the emasculation is not timely and complete, it will also reduce the quality of hybrids and affect the yield increase effect.
  • the use of plant male sterile lines to produce hybrid seeds is one of the most effective methods to reduce the cost of hybrid seed production, ensure hybrid purity, and protect intellectual property rights.
  • the current commercialized promoter for breeding is the 35S+iZmHsp70 promoter, which significantly affects the yield of female parent seed production in the process of seed production, so it still needs to be modified and optimized.
  • the purpose of the present invention is to provide a recombinant gene promoter, which can regulate the formation and expression of plant pollen by applying glyphosate, and produce male sterile lines, which is expected to break the monopoly of foreign-funded enterprises and promote breeding technology in my country. development of.
  • the invention utilizes the particularity of the FMV promoter, and uses the promoter to drive the herbicide gene, so that spraying the herbicide at a specific period can kill pollen, thereby achieving male sterility.
  • One aspect of the present invention provides a recombinant promoter, which consists of an FMV promoter and a maize heat shock 70kDa intron (iZmHSP70) in series.
  • nucleotide sequence of the recombinant promoter is SEQ ID NO: 1.
  • the 5' end and the 3' end of the recombinant promoter are respectively connected with restriction endonuclease enzyme cleavage site sequences. More preferably, the 5' end of the recombinant promoter is connected with a HindIII restriction site sequence, and the 3' end is also connected with an AvrII restriction site sequence. The above restriction sites are added to facilitate the ligation of the corresponding cloning vector.
  • the present invention further provides a gene expression cassette for plant breeding, wherein the gene expression cassette comprises the above-mentioned recombinant promoter, and a chemical resistance gene sequence whose expression is regulated by the recombinant promoter.
  • the chemoresistance gene is selected from the herbicide resistance gene, preferably the glyphosate resistance gene EPSPS.
  • the present invention also provides a recombinant vector, recombinant microorganism, transgenic cell line, callus or plant containing the above-mentioned recombinant promoter.
  • the present invention further provides the application of the above-mentioned recombinant promoter in plant breeding.
  • Yet another aspect of the present invention provides a method of inducing male sterility in a transgenic plant, comprising:
  • a transgenic plant comprising a gene expression cassette as described above is grown and an effective amount of herbicide is applied during the developmental stage of male reproductive tissue of the transgenic plant to induce male sterility in the transgenic plant.
  • the transgenic plant is maize.
  • said developmental stage of said male reproductive tissue is selected from one or more of the V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 stages of maize development a stage.
  • V8 and V10 are preferred.
  • the herbicide is selected from the group consisting of acetyl-CoA carboxylase inhibitors, acetolactate synthase inhibitors, photosystem II inhibitors, protoporphyrinogen oxidase inhibitors, 4- One or more of hydroxyphenylpyruvate dioxygenase inhibitor, 5-enolpyruvylshikimate-3-phosphate synthase inhibitor, glutamine synthase inhibitor or synthetic auxin.
  • the herbicide is glyphosate, and further preferably, the applied amount of the glyphosate is 150-350 ml/mu.
  • the present invention further provides a method for producing hybrid seeds, comprising: 1) applying an effective amount of herbicide during the development period of the male reproductive tissue of a transgenic plant containing the above-mentioned gene expression cassette, thereby inducing induction in the transgenic plant male sterility; 2) fertilizing the transgenic plant with pollen from a plant of another line; 3) harvesting hybrid seed from the transgenic plant.
  • the transgenic plant is maize.
  • said developmental stage of said male reproductive tissue is selected from one or more of the V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 stages of maize development a stage.
  • V8 and V10 are preferred.
  • the herbicide is selected from the group consisting of acetyl-CoA carboxylase inhibitors, acetolactate synthase inhibitors, photosystem II inhibitors, protoporphyrinogen oxidase inhibitors, 4- One or more of hydroxyphenylpyruvate dioxygenase inhibitor, 5-enolpyruvylshikimate-3-phosphate synthase inhibitor, glutamine synthase inhibitor or synthetic auxin.
  • the herbicide is glyphosate, and further preferably, the applied amount of the glyphosate is 150-350 ml/mu.
  • the recombinant promoter provided by the invention utilizes the particularity of the FMV promoter, and uses the promoter to drive the herbicide gene, so that the herbicide is sprayed in a specific period, and the effect of killing pollen can be played, thereby realizing male sterility .
  • the recombinant promoter technology provided by the present invention solves the problem that the commercial transformation event uses the 35S+iZmHsp70 promoter to affect the seed production of the female parent during the seed production process.
  • the recombinant promoter provided by the present invention can not only achieve male sterility seed production in plant breeding, but does not affect the seed production yield compared with 35S+iZmHSP70-EPSPS (Mon87427).
  • Fig. 1 is the construction flow chart of the recombinant cloning vector LP06-T containing pFMV+iZmHSP70-EPSPS nucleotide sequence of the present invention
  • Fig. 2 is the preparation method of the male sterility vector of the present invention: the construction flow chart of the recombinant expression vector LP-PT06 containing the nucleotide sequence of pFMV+iZmHSP70-EPSPS;
  • Figure 3 is the PCR detection diagram of the male sterile vector transformants of the present invention, wherein WT is a wild-type plant, PC is a plasmid control, NC is a water control, 1-15 are 15 positive transformants, of which 1-5 are OsAct- EPSPS transformants, 6-10 are 35S+iZmHSP70-EPSPS transformants and FMV+iZmHSP70-EPSPS transformants.
  • the reagents used in the present invention can be purchased from commercial sources.
  • nucleotide sequence (1406 nucleotides) of the pFMV+iZmHSP70 combination promoter is shown in SEQ ID NO: 1 in the Sequence Listing.
  • the pFMV+iZmHSP70 nucleotide sequence (as shown in SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Company; the synthesized pFMV+iZmHSP70 nucleotide sequence (SEQ ID NO: 1) The 5' end is connected with a HindIII restriction site, and the 3' end is connected with an AvrII restriction site.
  • the synthetic pFMV+iZmHSP70 nucleotide sequence was connected to the cloning vector pEASY-T5 (Transgen, Beijing, China, CAT: CT501-01), and the operation steps were carried out according to the instructions of the Transgen product pEASY-T5 vector to obtain the recombinant cloning vector LP06 -T, its construction process is shown in Figure 1 (wherein Kan represents the kanamycin resistance gene; Amp represents the ampicillin resistance gene; pUC origin represents the replication region sequence of the plasmid pUC, which can guide the double-stranded DNA replication process; LacZ is the LacZ initiation codon; pFMV+iZmHSP70 is the pFMV+iZmHSP70 nucleotide sequence (SEQ ID NO: 1).
  • the recombinant cloning vector LP06-T was transformed into E. coli T1 competent cells (Transgen, Beijing, China; Cat. No: CD501) by heat shock method, and the heat shock conditions were: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant cloning vector LP01-T), water bath at 42°C for 30 seconds; water bath at 37°C for 45 minutes (shaking at 200 rpm), coated with ampicillin (100 mg/L) on LB plates (tryptone 10 g/L, Yeast extract 5g/L, NaCl 10g/L, agar 15g/L, adjust pH to 7.5 with NaOH) and grow overnight.
  • E. coli T1 competent cells Transgen, Beijing, China; Cat. No: CD501
  • the heat shock conditions were: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant cloning vector LP01-T), water bath
  • the synthesized pOsAct1 nucleotide sequence was connected to the cloning vector pEASY-T5 to obtain the recombinant cloning vector LP06CK1-T, and the synthesized p35s+iZmHSP70 nucleotides were The sequence is connected to the cloning vector pEASY-T5 to obtain the recombinant cloning vector LP06CK2-T wherein, pOsAct1 is the pOsAct1 nucleotide sequence (SEQ ID NO: 2), and p35s+iZmHSP70 is the p35s+iZmHSP70 nucleotide sequence (SEQ ID NO: 2) 3).
  • Enzyme digestion and sequencing verified the correct insertion of the pOsAct1 and p35s+iZmHSP70 nucleotide sequences in the recombinant cloning vectors LP01CK1-T and LP01CK2-T.
  • Recombinant cloning vector LP06-T and expression vector LP-BB2 were digested with restriction endonucleases HindIII and AvrII, respectively, and the excised pFMV+iZmHSP70 nucleotide sequence fragment was inserted into Between the HindIII and AvrII sites of the expression vector LP-BB2, it is well known to those skilled in the art to construct the vector using a conventional enzyme digestion method, and it is constructed into a recombinant expression vector LP-PT06, and its construction process is shown in Figure 2 ( Kan: kanamycin gene; RB: right border; pFMV+iZmHSP70: pFMV+iZmHSP70 nucleotide sequence (SEQ ID NO: 1); AtCTP: Arabidopsis chloroplast signal transit peptide (SEQ ID NO: 4); EPSPS : glyphos
  • the recombinant expression vector LP-PT06 was transformed into E. coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant expression vector LP-PT06), 42 °C water bath for 30 seconds ; 37 °C of water baths for 1 hour (shaker shaking at 100rpm rotating speed); then on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L containing 50mg/L kanamycin (Kanamycin) , agar 15g/L, adjust the pH to 7.5 with NaOH) and cultivate it for 12 hours at a temperature of 37°C, pick white colonies, in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g /L, kanamycin 50mg/L, adjusted to pH 7.5 with NaOH) and cultured overnight at 37°C.
  • the nucleotide sequences cut from the recombinant cloning vectors LP06CK1-T and LP06CK2-T were cut by HindIII and AvrII into the expression vector LP-BB2 to obtain the recombinant expression vector LP- PT06CK1 and LP-PT06CK2.
  • Enzyme digestion and sequencing confirmed that the nucleotide sequences of pOsAct1 and p35s+iZmHSP70 between the HindIII and AvrII sites of the recombinant expression vectors LP-PT06CK1 and LP-PT06CK2 were the same.
  • the correctly constructed recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method, and the transformation conditions were as follows: : 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37°C warm water bath for 10 minutes; inoculated with transformed Agrobacterium LBA4404 in a LB test tube at a temperature of 28°C and a rotation speed of 200rpm Cultivated for 2 hours under conditions, spread on LB plates containing 50mg/L rifampicin and 50mg/L kanamycin until positive monoclones were grown, pick out monoclones for culture and extract them.
  • the recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 were digested with restriction endonucleases HindIII and AvrII and verified by restriction enzyme digestion. The results showed that the recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 The structure is completely correct.
  • the company's internal maize inbred line AX808 was planted in the field or in the greenhouse, and the maize at 8-10 days (summer)/10-13 days (autumn) after artificial pollination was used as the source of young embryos.
  • the transgenic plants were harvested after flowering and fruiting.
  • the harvested seeds were sown in the greenhouse, and when the plants grew to the 4-6 leaf stage, expression analysis and detection were carried out using PCR technology.
  • the primers for PCR are:
  • FMV-F TTGGGTTCAATCAACAAGGT (SEQ ID NO: 7)
  • FMV-R CTTCAAATGGGAATGAATGC (SEQ ID NO: 8)
  • EPSPS-F CTACGATTTCGACAGCACCT (SEQ ID NO: 9)
  • EPSPS-R AATCGGATATTCGTCGATCA (SEQ ID NO: 10)
  • the conditions of the PCR reaction were: 30 cycles, each cycle was 30' at 95°C, 30' at 58°C, and 40' at 72°C.
  • EE111-01 was extracted from its genomic DNA, and the copy number of EPSPS gene was detected by TransStart Green fluorescence quantitative PCR method. At the same time, wild-type maize plants were used as control, and the detection and analysis were carried out according to the above method. The experiment was repeated three times, and the average value was taken.
  • Step 11 Take 100 mg of the leaves of the maize plant and the wild-type maize plant that have been transferred into the EPSPS nucleotide sequence, respectively, and grind them into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
  • Step 12 Use Transgen's EasyPure Plant Genomic DNA Kit (containing RNase A) (Transgen, Beijing, China, Cat: EE111-01) to extract the genomic DNA of the above-mentioned sample, and the specific method refers to its product manual;
  • Step 13 measure the genomic DNA concentration of the above-mentioned sample with NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above-mentioned sample to the same concentration value, and the range of the concentration value is 80-100ng/ ⁇ l;
  • Step 15 Use the TransStart Green fluorescence quantitative PCR method to identify the copy number of the sample, take the identified sample with known copy number as the standard, and take the wild-type corn plant sample as the control, each sample is repeated 3 times, and the average value is taken. ; Fluorescence quantitative PCR primer and probe sequences are:
  • EPSPS-qF GTGTCGGAAAACCCTGTCAC (SEQ ID NO: 11)
  • EPSPS-qR CCTTCGTATCGGAGAGTTCG (SEQ ID NO: 12)
  • the following primers are used to detect the nucleotide sequence of 18s for internal reference leveling
  • 18srRNA-R CCTGTCGGCCAAGGCTATATAC (SEQ ID NO: 14)
  • the PCR reaction system is:
  • the experimental results show that the EPSPS nucleotide sequence has been integrated into the genome of the tested maize plants, and the maize plants transformed with the EPSPS nucleotide sequence have obtained transgenic maize plants containing a single copy.
  • Extraction buffer 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO4 12H2O, 0.2g/L KCl, 5.5ml/L Tween-20, pH 7.4;
  • Washing buffer PBST 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO4 12H2O, 0.2g/L KCl, 0.5ml/L Tween-20, pH 7.4;
  • wild-type maize plants and non-transgenic maize plants identified by fluorescence quantitative PCR were used as controls, and detection and analysis were carried out according to the above method.
  • 3 strains (O1, O2, O3) were selected for the nucleotide sequence of OsAct1-EPSPS
  • 3 strains (S1, S2, S3) were selected for the nucleotide sequence of 35S+iZmHSP70-EPSPS
  • 3 strains (S1, S2, S3) were transferred into FMV+
  • the nucleotide sequences of iZmHSP70-EPSPS were selected from 3 lines (F1, F2, F3), and 1 line was identified as non-transgenic (NGM) and 1 line with wild type (CK) by fluorescence quantitative PCR; 3 strains of each strain were selected for testing, and each strain was repeated 6 times.
  • EPSPS protein herbicide resistance protein
  • Table 1 The experimental results of herbicide resistance protein (EPSPS protein) content in transgenic maize leaves are shown in Table 1.
  • the ratios (ng/g) of the average expression of herbicide-resistant protein (EPSPS protein) to the fresh weight of leaves in maize plants transformed with EPSPS nucleotide sequences were measured to be 4642.20, 4753.30, and 4555.42, respectively, which indicated that EPSPS protein High expression level and stability were obtained in maize leaves.
  • EPSPS protein herbicide-resistant protein
  • Table 2 The experimental results of the content of herbicide-resistant protein (EPSPS protein) in transgenic corn pollen are shown in Table 2.
  • the expression levels of +iZmHSP70-EPSPS and 35S+iZmHSP70-EPSPS in pollen are low, and male sterility can be achieved by spraying glyphosate herbicide during the pollen development period.
  • Treatment 1 Composition of 157ml/mu of glyphosate (weed control) in V3 stage;
  • Treatment 2 consisted of: 157ml/mu of glyphosate (weed control) in V3 stage, then 157ml/mu in V8 stage, and then 157ml/mu in V10 stage;
  • Treatment 3 consisted of 157 ml/mu of glyphosate at V3 (weed control), followed by 262 ml/mu at V8), then 262 ml/mu at V10.
  • the last two sprays i.e. V8 and V10) are referred to as sterile sprays.
  • This example illustrates plant tolerance to glyphosate spray and male sterility under glyphosate spray in field trials of transgenic plants tested for yield.
  • OsAct-EPSPS, 35S+iZmHSP70-EPSPS and FMV+iZmHSP70-EPSPS One transformation event per construct was selected and planted in standard plots at 100 plants/plot with corresponding non-transgenic material (no grass Glyphosate spraying, good isolation) for open pollination to prepare F1 hybrid seeds.
  • the spray treatments were:
  • Treatment 1 consisted of 314ml/mu of glyphosate (weed control) in V3 stage;
  • Treatment 2 Consists of 314ml/mu of glyphosate in the V3 stage, then 157ml/mu in the V8 stage, and then 157ml/mu in the V11 stage;
  • Treatment 3 consisted of 314 ml/mu of glyphosate at V3 stage, followed by 262 ml/mu at V8 stage, then 262 ml/mu at V10 stage.
  • Each of the three lines of OsAct-EPSPS, 35S+iZmHSP70-EPSPS and FMV+iZmHSP70-EPSPS showed good phytotolerance as measured by plant height under all three glyphosate treatment regimens.
  • These three transformed lines were fully male fertile when treated with the weed control only glyphosate treatment regimen (Treatment 1), but when treated with glyphosate treatment regimens 2 or 3. The results are shown in Figure 4.

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Abstract

La présente invention concerne un promoteur recombinant. Le promoteur recombinant est composé d'un promoteur de FMV et d'un intron de protéine de choc thermique de maïs de 70 kDa (iZmHSP70) qui sont séquentiellement connectés en série. L'invention concerne également une cassette d'expression génique et son application à la sélection de plantes. Grâce à la spécificité du promoteur FMV, le promoteur est utilisé pour piloter des gènes herbicides, l'herbicide est pulvérisé à une période spécifique, l'effet de destruction du pollen peut être obtenu, et ainsi la stérilité mâle est obtenue. Le promoteur recombinant résout le problème suivant : dans un événement de transformation commerciale, le rendement de la production de semences du parent femelle est affecté dans le processus de production de semences en utilisant un promoteur 35S+iZmHsp70.
PCT/CN2021/117586 2020-09-10 2021-09-10 Promoteur recombinant, cassette d'expression génique et son application dans l'élevage de plantes Ceased WO2022053009A1 (fr)

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CN112080501B (zh) * 2020-09-10 2021-11-23 隆平生物技术(海南)有限公司 重组启动子、基因表达盒及其在植物育种中的应用
CN116606856B (zh) * 2023-07-14 2023-09-15 隆平生物技术(海南)有限公司 一种水稻绿色组织特异性启动子pOsPTHR及其应用
CN116606855B (zh) * 2023-07-14 2023-09-12 隆平生物技术(海南)有限公司 一种水稻绿色组织特异性启动子pOsRBBI3及其应用
CN116676304B (zh) * 2023-07-20 2023-09-26 隆平生物技术(海南)有限公司 转基因玉米事件lp016-1及其检测方法

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