WO2023005160A1 - 一种禾本科植物的遗传转化方法 - Google Patents

一种禾本科植物的遗传转化方法 Download PDF

Info

Publication number
WO2023005160A1
WO2023005160A1 PCT/CN2022/071299 CN2022071299W WO2023005160A1 WO 2023005160 A1 WO2023005160 A1 WO 2023005160A1 CN 2022071299 W CN2022071299 W CN 2022071299W WO 2023005160 A1 WO2023005160 A1 WO 2023005160A1
Authority
WO
WIPO (PCT)
Prior art keywords
genetic transformation
cup
plants
leaf
seedlings
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/071299
Other languages
English (en)
French (fr)
Inventor
蒋立希
朴学成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University
Original Assignee
Zhejiang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University filed Critical Zhejiang University
Publication of WO2023005160A1 publication Critical patent/WO2023005160A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • the invention relates to the field of agricultural biotechnology, in particular to a method for genetic transformation of gramineous plants.
  • Plant genetic transformation is an important technological advancement in modern science, which not only promotes human understanding of plant physiology and development, but also ushers in a new era of crop genetic improvement.
  • efficient transformation and regeneration remains a great challenge for many crops.
  • the method of plant genetic transformation mainly contains following several kinds: (1) based on the plant genetic transformation technology mediated by Agrobacterium, (2) based on the plant genetic transformation technology mediated by particle gun plasmid bombardment, (3) based on Bioactive beads (calcium alginate beads) mediated plant genetic transformation, (4) plant genetic transformation based on pollen and pollen growth channel, (5) plant transformation technology based on silicon carbide filament, (6) based on Plant genetic transformation mediated by electroporation, (7) plant genetic transformation technology mediated by microinjection, (8) genetic transformation method based on regulation of apical meristem development.
  • the CRISPR-Cas system is an RNA-guided adaptive immune response evolved in prokaryotic bacteria and archaea to defend against foreign genetic factors such as viruses, transposons, and plasmids.
  • CRISPR/Cas9-mediated genome editing has been performed in many model plants as well as rice, tobacco, wheat, barley, sorghum bicolor, corn, corn, eggplant, potato, rapeseed, soybean, lettuce, cucumber, citrus, Populus tomentosa, honeysuckle, etc.
  • CRISPR/Cas9-mediated genome editing technology also depends on subsequent genetic transformation technology including tissue culture, and tissue culture is extremely difficult for many species.
  • Plant genetic transformation is the application of recombinant DNA technology or gene editing technology, through cell tissue culture technology or germplasm system transformation technology, to purposefully insert foreign genes or DNA fragments into the recipient plant genome/or to target the recipient genome Editing, the technique of obtaining new plants with modified genomes through meiosis. Whether it is gene editing technology based on CRISPR/Cas9 or other nucleases, or transgenic technology based on DNA recombination, plant genetic transformation (transformation) is one of the indispensable key steps to achieve the purpose of gene editing or transgenic.
  • the current prevailing technology is to infect (or bombard with gene gun DNA plasmid) embryos, hypocotyls, cotyledon explants, apical meristems, etc. Tissues with strong meristematic ability, such as tissues, are then cultured to obtain (transgenic or gene-edited) regenerated shoots.
  • the current method has the following limitations: (1) The tissue culture technique is highly dependent on the species and genotype, and the transformation efficiency of the indica subspecies, barley and wheat in rice is very low; The requirements are very strict, and the configuration of the medium, the pretreatment of plant tissues, dark culture/light culture, and the replacement of culture vessels will be polluted if you are not careful; (3) The construction cost and maintenance cost of the tissue culture space are both high and low. Higher, higher energy consumption; (4) the tissue culture process is more time-consuming, through tissue culture to obtain seedlings and flowering and setting seeds, which is 3-6 months longer than normal seed germination to flowering and setting seeds.
  • the application aims at the above-mentioned deficiencies existing in the prior art, provides a kind of new plant genetic transformation technology, overcomes four kinds of crops genetic transformation technology difficulty such as gramineous food crops (rice, corn, wheat, barley etc.) Low), long period, and dependent on species and genotype disadvantages, save the tissue culture step in the genetic transformation process, create a simple and easy to operate, do not depend on genotype, low cost, high efficiency Gramineae crop genetic transformation method .
  • a method for genetic transformation of gramineous plants comprising the following steps:
  • step 2 2) adding the Agrobacterium tumefaciens bacterium liquid with the recombinant vector for gene editing to the cup-shaped structure in step 1);
  • the gramineous plants are rice, corn, wheat, barley, rye, triticale, sorghum, sugarcane and the like. Since the method for genetic transformation of gramineous plants in the present application is to pull out the first blade after the first blade grows after the seed germinates, a cup-shaped structure is formed in the leaf sheath at this time, and since all gramineous plants have a similar structure, , can be generalized and applied to all grasses.
  • step 1) the first leaf grows when the seed germinates for 3 to 5 days, and then the first leaf is pulled out.
  • the vector used for genetic transformation is a binary vector.
  • the binary vector can be a commonly used plasmid for transgenic Agrobacterium, such as plasmid pTX172.
  • Conventional methods in the prior art can be used for the construction of the recombinant vector and the method for transferring the recombinant vector into Agrobacterium.
  • the amount of bacterial solution can be determined according to different types of plants and culture conditions.
  • the amount of bacterial solution added to the cup-shaped structure in step 1) is 15-30 ⁇ L. More preferably, in step 2), the bacterial liquid is added to the cup-shaped structure in step 1) until it is full. Add as much culture liquid as possible to the cup structure, which can improve the transformation efficiency.
  • the seed germination stage of step (1) is carried out under dark conditions or under light conditions.
  • This application is based on the method of genetic transformation. Since the Agrobacterium liquid for transgenesis is added after the plant seeds germinate and grow the first leaf, during the subsequent plant growth process, the meristems will grow from the top of the shoot tip at the bottom of the cup-shaped structure. Cells in stems and leaves newly grown from tissues can be easily transformed, but not all of them can be genetically modified. Therefore, the plants obtained after continued cultivation may be a kind of chimera. If it is desired to obtain homozygous (homozygote) or heterozygous (heterozygote) with the same genetic background, the selection of populations can be segregated by genetic inheritance of offspring.
  • What this application describes is a kind of genetic transformation technology that uses the anatomical structure characteristics of Gramineae crop seedling stage to directly process the apical meristem in the seedling stage.
  • the characteristics of the anatomical structure of corn, wheat, barley, sorghum, rye, triticale, etc.) crops directly infect the plant apical meristem (SAM) with Agrobacterium liquid, no need to go through the tissue culture stage, and independent of genotype Genetic transformation technology, low cost, high efficiency, simple and easy.
  • Figure 1 shows the sequence construction between the left border and the right border of the vector used for gene editing. It shows the positional relationship between the core elements of the vector, PolII promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence. Arrows indicate the position of the ribozyme cleavage sequence; RZ, Ribozyme terminator, indicate the ribozyme termination sequence.
  • Fig. 2 shows the rice seedlings whose OsPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • A japonica rice variety Nipponbare;
  • B indica rice variety Kasalath. Albino seedlings are indicated by white arrows.
  • Figure 3 shows the status of editing the target region of the rice PDS gene (Nipponbare), where the number after # represents the code of the transformant; a1-a18 represents the allelic change created by gene editing, and A0 represents the original allelic base of Nipponbare Sequence; Ho, means that the two DNA strands are homozygous; Bi, means that both DNA strands are edited and a new allele type appears; He, means that the two DNA strands are heterozygous; minus sign (-) means The number of bases lost, plus sign (+) represents the number of bases increased. The same below.
  • FIG. 4 shows the status of the target region of the rice PDS gene being edited (kasaras).
  • Fig. 5 is the maize seedling (CML322) whose ZmPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the white solid line arrow shows the albino seedlings of the whole plant. Such seedlings gradually die due to the inability to carry out photosynthesis.
  • the white dotted line arrow shows the chimeric plants with white leaves.
  • Fig. 6 shows the status of the edited target region of the maize PDS gene (B73).
  • Fig. 7 shows the edited target region of the maize PDS gene (CML322).
  • Fig. 8 shows the wheat seedlings (Chinese spring) whose TaPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the white solid line arrows show the albino seedlings of the whole plant, and such seedlings gradually die due to the inability to carry out photosynthesis.
  • the 2 plants on the left are chimeric plants with partially white flower leaves.
  • Figure 9 shows the status of the target region of the wheat PDS gene being edited (Chinese Spring).
  • Figure 10 shows the status of editing the target region of the wheat PDS gene (Yangmai 16).
  • Fig. 11 shows the barley seedlings (Zheda 9, ZU9) whose HvPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the black solid line arrows indicate albino seedlings, which gradually die due to the inability to carry out photosynthesis.
  • Figure 12 shows the status of the target region of the barley PDS gene being edited (Golden Hope, GP).
  • Figure 13 shows the edited status of the target region of the barley PDS gene (Zheda No. 9).
  • the left and right seedlings in each figure in Fig. 14 show the state of the seedlings under light culture and dark culture respectively.
  • the dark culture (the seedling on the right) forms a larger cup-shaped space than the light culture (the left seedling) and can carry a larger volume Agrobacterium infection solution.
  • 1 the rice seedlings 5 days after the seeds absorbed water and germinated; 2: gently pull out the first leaf (bud) from the leaf sheath by hand or tools, and after the leaves are pulled out, a cup-shaped space is formed in the leaf sheath; 3: use drops About 5 microliters (left) and 25 microliters (right) of Agrobacterium infection solution were dripped into the "cup-shaped" structural space formed in the leaf sheath after pulling out the first leaf; 4: After injection of Agrobacterium infection solution Within 1-3 days, maintain a certain volume of bacterial liquid in the "cup-shaped” container, so that the Agrobacterium bacterial liquid drips through the "cup bottom” tissue, penetrates into the meristem cells at the top of the shoot tip, and effectively infects SAM; 5 : After 5 days, move to an artificial incubator for cultivation under normal conditions (ie 28° C. under light conditions for 16 hours, and 25° C. under dark conditions for 8 hours).
  • the rice variety is kashalash.
  • the Agrobacterium bacterial liquid penetrates into the "cup bottom” tissue, invades the shoot tip SAM, and effectively infects the SAM.
  • the SAM tissue and the Agrobacterium bacterial fluid were co-cultured under light or dark conditions (the effect of the dark condition is better).
  • the transformed SAMs differentiated into new green shoots and leaves. Collect about 14-21 days of leaves (from the date of germination) to sequence the target gene (hereinafter, PDS gene as an example).
  • results show that, depending on the species and variety, 20-80% of the new leaves of a single plant are extracted The bases of single or double strands of DNA are effectively genetically modified. Subsequently, the single seedling whose genome has been modified is transplanted to a culture medium (such as soil or culture medium) for planting, and after the seedling grows and enters reproductive growth, seeds of the T2 generation are produced.
  • a culture medium such as soil or culture medium
  • Agrobacterium bacterial liquid centrifuge the Agrobacterium bacterial liquid at 4000 rpm, take out the precipitated Agrobacterium cells with a medicine spoon, and dissolve them in 30 ml of 1/2MS culture medium (containing 30 microliters of DMSO with a concentration of 10 mg Acetosyringone (Acetosyringone) per milliliter, carefully resuspend the Agrobacterium cells with a disposable sterilized pipette. The processed bacterial solution is used for the following treatment within 2 hours.
  • 1/2MS culture medium containing 30 microliters of DMSO with a concentration of 10 mg Acetosyringone (Acetosyringone) per milliliter
  • Seedling SAM was co-cultured with Agrobacterium in dark condition. After 3-5 days, move to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.). About 15 days later, select half a leaf from each seedling to extract a small amount of DNA, and then use it for the detection of the target gene sequence; after sequencing, eliminate the target gene that has not been genetically modified (not edited or the target fragment has not been inserted into the plant genome ) negative plants, transplant the positive plants to common media (soil or other culture media) and carefully cultivate them.
  • the seedlings enter reproductive growth through vegetative growth, and the seeds of the T2 generation plants are harvested after the plants mature.
  • T0 generation the generation in which the genomic DNA sequence of the vegetative organ has not been modified
  • T1 generation the generation in which the genomic DNA sequence of the vegetative organ has been modified
  • T2 generation the seeds harvested from the T1 generation plants produce T2 generation plants, and the T2 generation population can be isolated to homozygous mutants.
  • the seedlings of rice, corn, wheat, barley, etc. are treated with the Agrobacterium liquid carrying the target gene target sequence or insert vector.
  • ICCU method genetic transformation method using endogenous cup-shaped structure
  • PDS phytoene desaturase
  • the reason why this gene is taken as an example is because the homozygous mutant of this gene presents the phenomenon of albino seedlings, which is very easy to be observed intuitively.
  • genomic DNA from the leaves of seedlings about 14-21 days after germination, and sequenced them to understand the genetic modification of target genes.
  • Example 1 Editing effect of ICCU method on rice PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target gene OsPDS (LOC_Os03g08570).
  • Figure 1 shows the positional relationship between the core elements of the molecular construction Pol II promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the DNA base sequence transcribed into sgRNA1 is as follows: AGTTGCTTCAGCATGGATAC; the base sequence of sgRNA2 is as follows: CGGGACAACTTCCTACTCAT.
  • the sgRNA1 and sgRNA2 sequences correspond to the positions on LOC_Os03g08570 separated by 50 bases.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope.
  • Seedlings were cultured under dark conditions. After about 3 days, move to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation rice seedlings treated by ICCU method under dark conditions According to the above method, we used ICCU method to treat 60 late rice varieties Nipponbare and 60 early rice varieties Kasalath seedling. Among the 60 Nipponbare rice plants, we found 4 seedlings with white flowers, 5 seedlings with light green leaves, and 51 seedlings with completely normal color. After ICCU treatment, some Nipponbare plants are shown in Figure 2A. Among the 60 Kasaras rice plants, we found 5 seedlings with white flowers, 8 seedlings with light green leaves, and 47 seedlings with completely normal color. After ICCU treatment, some of the Kasaras plants were as follows: Figure 2B.
  • Example 2 The editing effect of the ICCU method on the corn PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: GV3101.
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • the base sequence design of sgRNA1 and sgRNA2 is as follows: AGTTGCTCAACAATGGACAC; the base sequence of sgRNA2 is as follows: TAGACATCCTGCCTTGCAGG; the corresponding positions of sgRNA1 and sgRNA2 sequences on the maize PDS gene are separated by 50 bases.
  • Infection treatment of corn SAM by Agrobacterium bacterium liquid (a) select corn seeds with germination ability, soak the seeds in clear water for 1 hour, and first use 70% alcohol to disinfect the seed epidermis for 1 minute , wash 3 times with clear water, then soak the seeds with 3% H2O2 or 3% sodium hypochlorite solution for 30 minutes, wash the treated seeds 5 times with sterile clear water, then soak them in sterile clear water for 3 hours. (b) Germinate the seeds under dark conditions. After 4 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand or other tools. The pull-out process should not damage the SAM as much as possible, and minimize the residual tissue remaining on the upper layer of the SAM.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 30 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • Seedlings were cultured under dark conditions. After 4 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 maize seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 50 B73 and 50 CML322 seedlings. Among the 50 B73 plants, we found 6 seedlings with white flowers, 9 seedlings with light green leaves, and 35 seedlings with completely normal color. Among the 50 CML322 plants, we found 3 seedlings with white flowers, 8 seedlings with light green leaves, and 39 seedlings with completely normal color. After ICCU treatment, some of the seedling plants of CML322 are shown in Figure 5.
  • Example 3 Editing effect of ICCU method on wheat PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target genes TraesCS4A02G004900.1 (chromosome 4A: 3133866-3141315), TraesCS4B02G300100.1 (chromosome 4B: 586574959-586580910), TraesCS4B02G300100.2 (chromosome 4D: 468262170-468267694).
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • the base sequence design of sgRNA1 and sgRNA2 is as follows: TTGTTTGCCAAGATTTTCCA; the base sequence of sgRNA2 is as follows: GAGGCAAGAGATGTGTTGGG; sgRNA1 and sgRNA2 sequences correspond to the positions on the wheat PDS gene with an interval of 50 bases.
  • Infection treatment of Agrobacterium bacterium liquid to wheat SAM (a) select corn seeds with germination ability, the seeds are absorbed in clear water for 1 hour, and after 1 minute the epidermis of the seeds is sterilized with 70% alcohol, Wash 3 times with clear water, then soak the seeds with 3% H2O2 or 3% sodium hypochlorite solution for 20 minutes, wash the treated seeds 5 times with sterile clear water, then soak them in sterile clear water for 3 hours. (b) Germinate the seeds under dark conditions. After 5 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope.
  • Seedlings were cultured under dark conditions. After 3 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation wheat seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 35 Chinese spring and 35 Yangmai 16 seedlings. Among the 30 Chinese spring plants, we found 3 seedlings with white flowers, 6 seedlings with light green leaves, and 26 seedlings with completely normal color. Among the 35 Yangmai 16 plants, we found 1 seedling with white flowers, 6 seedlings with light green leaves, and 28 seedlings with completely normal color. After ICCU treatment, some of the seedlings of Chinese spring after treatment are shown in Figure 8 Show.
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the base sequence of sgRNA1 is as follows: TTGTTTGCCAAGATTTTCCA; the base sequence of sgRNA2 is as follows: GAGGCAAGAGATGTGTTGGG; the positions of sgRNA1 and sgRNA2 sequences on the barley PDS gene are separated by 50 bases.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette gun, inject about 20 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • Seedlings were cultured under dark conditions. After 5 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation barley seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 35 Golden Hope and 35 Zheda No. 9 seedlings. Among the 35 plants of Golden Hope, we found 4 seedlings with white flowers, 7 seedlings with light green leaves, and 24 seedlings with completely normal color. Among the 35 Zheda No. 9 plants, we found 0 seedlings with white flowers, 4 seedlings with light green leaves, and 31 seedlings with completely normal color. After ICCU treatment, some of the seedling plants of Zheda No. 9 are shown in Figure 11 .
  • Embodiment 5 Comparison of cup-shaped structure and genetic transformation effect formed by ICCU method light culture and dark culture treatment of germinated seeds
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target gene OsPDS (Os03g08570).
  • Figure 1 shows the positional relationship between the core elements of the molecular construction Pol II promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the base sequence of sgRNA1 is as follows: AGTTGCTTCAGCATGGATAC; the base sequence of sgRNA2 is as follows: CGGGACAACTTCCTACTCAT. The positions on Os03g08570 corresponding to the sequences of sgRNA1 and sgRNA2 are separated by 50 bases.
  • Infection treatment of rice SAM by Agrobacterium bacterium liquid (a) select rice seeds with germination ability, soak the seeds in clear water for 1 hour, first treat the seed epidermis with 70% alcohol for 1 minute, and use Clean water 3 times, then use 3% H2O2 or 3% sodium hypochlorite solution to carry out 20 minutes to seed, the seed after treatment is washed 5 times with sterile water, then soak in sterile water for 3 hours. (b) The seeds were germinated under light and dark conditions, respectively. After 5 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand or other tools.
  • the pull-out process should not damage the SAM as much as possible, and minimize the residual tissue remaining on the upper layer of the SAM.
  • "cup-shaped" container structures of different sizes are formed in the leaf sheath (coleoptile).
  • (c) Using a pipette gun, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the darkened "cup-shaped" structure space, inject about 15 microliters (prepared in step (6) The Agrobacterium infection solution prepared in (6) was injected into the light-treated "cup-shaped" structural space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • (d) Continue culturing the seedlings under light or dark conditions respectively.
  • Table 1 The comparison of the final transformation efficiency of germinated seeds under light or dark culture treatment

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

提供了一种禾本科植物的遗传转化方法，包括以下步骤：(1)将禾本科植物种子萌发、长出第一张叶片后，拔出第一张叶片，此时叶鞘内形成一个杯状结构；(2)向步骤(1)杯状结构中加入具有用于基因编辑的重组载体的根癌农杆菌菌液；(3)继续培养，得到至少部分细胞被基因编辑的转基因植株。该方法在苗期活体(营养体)上，利用禾本科作物解剖结构的特点，直接用农杆菌菌液感染植物顶端分生组织(SAM)、不需要经过组培阶段、不依赖基因型的遗传转化技术。

Description

一种禾本科植物的遗传转化方法 技术领域
本发明涉及农业生物技术领域，特别是涉及一种禾本科植物的遗传转化方法。
背景技术
植物遗传转化是现代科学中的一项重要技术进步，不仅促进人类对植物生理与发育过程的认知，而且开创了作物遗传改良的新纪元。然而对于许多农作物而言，有效的转化和再生仍然是一个很大的挑战。
现有技术中，植物遗传转化的方法主要有如下几种：(1)基于农杆菌介导的植物遗传转化技术，(2)基于基因枪质粒轰击介导的植物遗传转化技术，(3)基于生物活性珠(藻酸钙珠)介导的植物遗传转化，(4)基于花粉及花粉生长通道介导的植物遗传转化，(5)基于碳化硅丝介导的植物转化技术，(6)基于电击穿孔介导的植物遗传转化，(7)基于显微注射介导的植物遗传转化技术，(8)基于调控顶端分生组织发育的遗传转化方法。
CRISPR-Cas系统是在原核细菌和古细菌中进化产生的RNA引导的适应性免疫反应，用以抵御诸如病毒、转座子和质粒之类的外来遗传因素的侵害。目前CRISPR/Cas9介导的基因组编辑已经在多个模式植物以及水稻、烟草、小麦、大麦、双色高粱、玉米、玉米、茄子、马铃薯、油菜、大豆、莴苣、黄瓜、柑橘、毛白杨、金银花等物种上取得成功，然而CRISPR/Cas9介导的基因组编辑技术也要依赖后续包括组织培养在内的遗传转化技术，而组织培养对于许多物种而言，难度极大。
总而言之，无论是DNA重组还是基因编辑技术，都迫切需要简便、实用、高效率、低成本的遗传转化技术。随着合成生物学的快速发展，用于精确基因组编辑和基因整合的新型载体系统的研发，有可能带来作物遗传改良革命性的进步，为农业的可持续发展展示了美好的前景。因此，不依赖物种(或基因型)的、高效植物遗传转化技术将是下一阶段作物遗传改良的主攻方向之一。
植物遗传转化是应用重组DNA技术或者基因编辑技术，通过细胞组织培养技术或种质系统转化技术，有目的地将外源基因或DNA片段插入到受体植物基因组中/或对受体基因组进行定点编辑，经过减数分裂获得基因组受到修饰之后的新植株的技术。无论是基于CRISPR/Cas9或其它核酸酶的基因编辑技术，还是基于DNA重组的转基 因技术，植物遗传转化(transformation)都是达到基因编辑或者转基因目的不可或缺的关键步骤之一。
对于水稻、玉米、小麦、大麦等禾本科粮食作物的遗传转化实践，目前通行的技术是通过农杆菌感染(或者用基因枪DNA质粒轰击)胚、下胚轴、子叶外植体、顶端分生组织等分生能力强的组织，然后通过对愈伤组织的培养来获得(转基因或者受到基因编辑)再生苗。目前的方法具有以下的局限性：(1)组培技术高度依赖物种与基因型，水稻中的籼稻亚种、大麦与小麦的转化效率都非常低；(2)组培过程对环境洁净度的要求非常苛刻，培养基的配置、植物组织的前处理、暗培养/光培养、培养器皿的更换等环节，稍有不慎就会受到污染；(3)组培空间的建设成本和维护成本均较高，能耗较高；(4)组培过程比较耗时间，经过组培得到成苗并开花结籽，比正常种子萌发到开花结籽时间上长3-6个月。
发明内容
本申请针对现有技术中存在的上述不足，提供了一种新的植物遗传转化技术，克服禾本科粮食作物(水稻、玉米、小麦、大麦等)等四种作物遗传转化技术难度大(转化效率低)、周期长、且依赖物种种类与基因型缺点，省去遗传转化过程中的组织培养步骤，创建一种简单易操作、不依赖基因型、低成本、高效率的禾本科作物遗传转化方法。
一种禾本科植物的遗传转化方法，包括以下步骤：
1)将禾本科植物种子萌发、长出第一张叶片后，拔出第一张叶片，此时叶鞘内形成一个杯状结构；
2)向步骤1)杯状结构中加入具有用于基因编辑的重组载体的根癌农杆菌菌液；
3)继续培养，得到至少部分细胞被遗传转化的植株。
优选的，所述禾本科植物为水稻、玉米、小麦、大麦、黑麦、小黑麦、高梁、甘蔗等。由于本申请禾本科植物遗传转化方法是通过种子萌发后第一张叶片长出后将第一张叶片拔掉，此时叶鞘内形成一个杯状结构，由于禾本科植物都存在类似的结构，所以，可以推广应用到所有的禾本科植物。
优选的，步骤1)中，种子萌发3～5天时长出第一张叶片，然后拔出第一张叶片。
优选的，步骤2)中，用于由于遗传转化的载体为双元载体。双元载体可以是常用的农杆菌转基因时用的质粒，比如质粒pTX172。重组载体的构建以及重组载体转入农杆菌的方法均可以使用现有技术中常规的方法。
菌液量可以根据不同种类的植株以及培养条件情况确定。优选的，步骤2)中，向步骤1)杯状结构中加入的菌液量为15～30μL。更优选的，步骤2)中，向步骤1)杯状结构中加入菌液到加满。向杯状结构中尽可能多地加入菌液，这样可以提高转化效率。
优选的，步骤(1)种子萌发阶段在黑暗条件下进行或者光照条件下进行。
本申请基于此遗传转化的方法，由于在植株种子萌发长出第一片叶子后才加入转基因用的农杆菌菌液，所以，后续植株生长过程中，从杯状结构底部的茎尖顶端分生组织新生长出的茎、叶中细胞容易被转化，但也并不一定全部都能够受到遗传修饰，所以，可能继续培养后得到的植株是一种嵌合体。如果需要获得遗传背景一致的纯合体(homozygote)或者杂合体(heterozygote)，则可以通过后代遗传分离群体的选择。
本申请描述的是一种利用禾本科作物幼苗期解剖结构特征，直接在幼苗时期处理顶端分生组织的遗传转化技术，是一种在苗期活体(营养体)上，利用禾本科(水稻、玉米、小麦、大麦、高梁、黑麦、小黑麦等)作物解剖结构的特点，直接用农杆菌菌液感染植物顶端分生组织(SAM)、不需要经过组培阶段、不依赖基因型的遗传转化技术，成本低、效率高，简单易行。
附图说明
图1为用于基因编辑的载体左边界与右边界之间的序列构建。示意该载体的核心元件PolII启动子、Cas9核酸蛋白基因、PolyA、核糖酶剪切序列、sgRNA1、sgRNA2，核糖酶终止序列之间的位置关系。箭头所指，示核糖酶剪切序列位置；RZ，Ribozyme terminator，示核糖酶终止序列。
图2为经ICCU法遗传转化处理之后，OsPDS基因受到编辑的水稻幼苗。(A)粳稻品种日本晴(Nipponbare)；(B)籼稻品种卡萨拉斯(Kasalath)。白色箭头所示为白化苗。
图3为水稻PDS基因的目标区域受到编辑的状况(日本晴)，其中，#后面的数字代表转化株代码；a1-a18代表基因编辑创造的等位变化，A0代表日本晴原来的等位基因碱基序列；Ho，代表两条DNA链呈纯合状态；Bi，代表两条DNA链均受到编辑而出现新的等位类型；He，代表两条DNA链呈杂合状态；减号(-)代表失去的碱基数目，加号(+)代表增加的碱基数目。下同。
图4为水稻PDS基因的目标区域受到编辑的状况(卡莎拉斯)。图5为经ICCU法遗传转化处理之后，ZmPDS基因受到编辑的玉米幼苗(CML322)。白 色实心线箭头所示为全株白化苗，这样的苗由于无法进行光合作用而逐渐死去，白色虚线箭头示部分叶片呈白色的嵌合株。
图6为玉米PDS基因的目标区域受到编辑的状况(B73)。
图7为玉米PDS基因的目标区域受到编辑的状况(CML322)。
图8为经ICCU法遗传转化处理之后，TaPDS基因受到编辑的小麦幼苗(中国春)。白色实心线箭头示全株白化苗，这样的苗由于无法进行光合作用而逐渐死去。左边2株为具有部分白花叶片的嵌合体植株。
图9为小麦PDS基因的目标区域受到编辑的状况(中国春)。
图10为小麦PDS基因的目标区域受到编辑的状况(杨麦16)。
图11为经ICCU法遗传转化处理之后，HvPDS基因受到编辑的大麦幼苗(浙大9号，ZU9)。黑色实心线箭头所示为白化苗，这样的苗由于无法进行光合作用逐渐死去。
图12为大麦PDS基因的目标区域受到编辑的状况(金色希望，GP)。
图13为大麦PDS基因的目标区域受到编辑的状况(浙大9号)。图14中各图中左右两株幼苗分别示光照培养与黑暗培养下幼苗的状态，黑暗培养(右侧幼苗)比光照培养(左侧幼苗)形成更大的杯状空间，能承载更大体积的农杆菌感染液。其中，1：种子吸水萌发后5天的水稻幼苗；2：轻轻用手或工具将第一片叶子(芽)拔离叶鞘，叶片拔离之后，叶鞘内形成杯状空间；3：用滴管分别将大约5微升(左)和25微升(右)的农杆菌感染液滴注到拔除第一页之后叶鞘内形成的“杯状”结构空间内；4：注射农杆菌感染液后1-3天内，维持“杯状”容器内一定的菌液容量，使农杆菌菌液滴透“杯底”组织，渗透侵入茎尖顶端分生组织细胞，并对SAM进行有效的感染；5：5天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。水稻品种是kashalash。
具体实施方式
(1)总体方案
禾本科作物(水稻、玉米、小麦、大麦等)种子萌发后约5天左右，待长出第一张叶片(Primary leaf)后，用手或其它工具将其拔出。第一片叶移除之后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间与茎尖顶端分生组织(Shoot Apical Meristem，简称SAM)之间留有数层(第一叶移除之后留下的)残余细胞，形成“杯底”。将大约10-50微升含有双元载体的根癌农杆菌菌液用移液枪或其它实验工具注射到“杯状”容 器内，并使容器内维持接近100％的盈满度。维持“杯状”容器内一定的菌液容量，使农杆菌菌液渗透“杯底”组织，侵入茎尖SAM，并对SAM进行有效的感染。在杯状空间内注入农杆菌菌液之后的约4天时间内，在光明或者暗条件下(黑暗条件效果更佳)，SAM组织和农杆菌菌液共培养。4天后之后，受到遗传转化后的SAM分化出新的绿芽和叶片。采集大约(从萌发之日算起的)14-21天的叶片进行目的基因(以下以PDS基因为例)测序，结果表明，依物种与品种的不同，20-80％单株新生叶片中提取的DNA的单链或者双链的碱基受到有效的遗传修饰。随后，基因组受到修饰的单株幼苗移植到培养介质(如泥土或培养基)种植，幼苗生长并进入生殖生长后，产生T2代的种子。
(2)具体实验步骤(仅作例举，实施例中提到的基因编辑的载体、农杆菌菌种等不是唯一选项)
①前期准备：选择用于遗传转化的载体、农杆菌菌株，例如，基因编辑载体pTX172(addgene公司，质粒编号89259)、农杆菌菌株EHA105，GV3031等。用牙签挑取农杆菌单克隆培养物之后，将牙签置于50毫升LB培养液内(内含30毫克/毫升利福平与50毫克/毫升的卡那霉素)，28度、200转/分摇床内震荡培养，经12-18小时的震荡培养之后，培养液的浓度达到分光光度计600纳米吸收峰读值0.1-0.5(OD600＝0.1-0.5)后，在25℃温度条件下用4000转/分的转速离心农杆菌菌液，用药勺将沉淀下来的农杆菌细胞取出来，并溶解在30毫升1/2MS培养液中(内含30微升溶解于DMSO的、浓度为10毫克/毫升的乙酰丁香酮(Acetosyringone)，用一次性经消毒的吸管小心地再悬浮农杆菌细胞。处理好的菌液在2小时内用于以下处理。
②农杆菌菌液对禾本科萌发种子SAM的侵染处理：
(a)选择具有萌发能力的禾本科作物(水稻、玉米、小麦、大麦等)的种子，将种子在清水中吸涨1个小时，用70％酒精将种子浸泡1分后进行表皮消毒后，用清水清洗3次，然后再用3％H 2O 2或者3％的次氯酸钠溶液对种子进行30分钟浸泡，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡。
(b)将种子置于黑暗条件(26℃)下萌发。5天后待种子萌发，并生长出幼芽之后，用手或其它工具把叶芽拔出。拔出过程中要做到即不损伤顶端分生组织(SAM)，又尽可能地减少留在SAM上层的残余组织。幼芽拔出后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间与SAM组织之间留有数层残余细胞，形成杯状结构的“杯底”。
(c)使用移液枪或注射器，(视不同作物)将10-50微升的(在步骤①中制备好 的)农杆菌感染液注射到经②(b)处理之后形成的“杯状”结构空间内。注射过程可以在体视显微镜或放大镜下操作。
(d)幼苗SAM与农杆菌菌液在黑暗条件下共培养。3-5天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。大约15天后，每株幼苗上选取半张叶片，用以提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰目标基因未受到遗传修饰(未受到编辑或者目标片段未插入植株基因组)的阴性植株，将阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。
(e)幼苗经营养生长进入生殖生长，待植株成熟后收获T2代植物的种子。
注：本文对T0-T2代的定义。T0代：营养器官基因组DNA序列未受到修饰的世代；T1代：营养器官的基因组DNA序列受到修饰的世代；T2代，T1代植株收获的种子产生T2代植株，T2代群体可以分离到纯合的突变体。
通过上述“利用内生杯状结构遗传转化法”(以下简称ICCU法)，用携带具备目的基因靶标序列或插入片段载体的农杆菌菌液处理水稻、玉米、小麦、大麦等幼苗。作为例子，我们以编码八氢番茄红素脱饱和酶(phytoene desaturase，PDS)基因为靶基因，设计gRNA，通过基因编辑手段敲除PDS基因。之所以以这个基因为例子，是因为该基因的纯合突变体呈现白化苗的现象，非常容易被直观地观察到。在表型观察的基础上，我们提取萌发后大约14-21天的幼苗叶片中的基因组DNA，通过测序，了解靶基因受到遗传修饰的情况。
实施例1：ICCU法对水稻PDS基因的编辑效果
1、技术步骤
(1)载体与农杆菌菌株：载体，pTX172；农杆菌菌株：EHA105。
(2)被转化的水稻品种：粳稻品种Nipponbare和籼稻品种Kasalath。
(3)目标基因：OsPDS(LOC_Os03g08570)。
(4)载体的构建：载体的分子构建见图1。图1示意该分子构建的核心元件Pol II启动子、Cas9核酸蛋白基因、PolyA、核糖酶剪切序列、sgRNA1、sgRNA2、核糖酶终止序列之间的位置关系。
(5)sgRNA1与sgRNA2的碱基序列设计：转录成sgRNA1的DNA碱基序列如下：AGTTGCTTCAGCATGGATAC；sgRNA2的碱基序列如下：CGGGACAACTTCCTACTCAT。sgRNA1与sgRNA2序列相对应LOC_Os03g08570上的位置间隔50个碱基。
(6)农杆菌液的培养：用牙签挑取农杆菌单克隆培养物之后，将牙签置于50毫升LB培养液内(内含30毫克/毫升利福平与50毫克/毫升的卡那霉素)，28度、200转/分摇床内震荡培养，经12-18小时的震荡培养之后，培养液的浓度达到分光光度计600纳米吸收峰读值0.1-0.5(OD600＝0.1-0.5)后，在25℃温度条件下用4000转/分的转速离心农杆菌菌液，用药勺将沉淀下来的农杆菌细胞取出来，并溶解在30毫升1/2MS培养液中(内含30微升溶解于DMSO的、浓度为10毫克/毫升的乙酰丁香酮(Acetosyringone)，用一次性经消毒的吸管小心地再悬浮农杆菌细胞。处理好的菌液在2小时内用于以下处理。
(7)农杆菌菌液对水稻SAM的侵染处理：(a)选择具有萌发能力的水稻种子，将种子在清水中吸涨1个小时，用70％酒精将种子浸泡1分后进行表皮消毒后，用清水清洗3次，然后再用3％H 2O 2或者3％的次氯酸钠溶液对种子进行30分钟浸泡，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡12小时。(b)将种子置于黑暗条件下萌发。大约3天后待种子萌发，并生长出第一片叶幼芽之后，用手或其它工具把第一片叶幼芽拔出。拔出过程尽可能做到即不损伤SAM，又尽可能减少留在SAM上层的残余组织。幼芽拔出后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间与SAM组织之间的数层残余细胞，形成杯状结构的“杯底”。(c)使用移液枪，将大约25微升(在步骤(6)中制备好的)农杆菌感染液注射到“杯状”结构空间内。注射过程可以在体视显微镜下操作。(d)幼苗在黑暗条件下培养。大约3天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。15天后，每株剪取半张幼苗叶片，用来提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰目标基因未受到编辑的阴性植株，将一条DNA链或2条DNA都受到编辑的阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。(e)幼苗经营养生长进入生殖生长，待水稻植株成熟后收获T2代水稻种子。
2、处理结果
(1)暗条件下ICCU法处理后T1代水稻幼苗的叶色：根据以上方法，我们一共用ICCU法处理了60株晚稻品种日本晴(Nipponbare)和60株早稻品种卡莎拉斯(Kasalath)的幼苗。在60株日本晴水稻植株中，我们一共发现4株白花苗，5株部分叶片呈淡绿色的苗，以及51株颜色完全正常的苗。ICCU处理之后，部分日本晴的植株如图2A所示。在60株卡莎拉斯水稻植株中，我们一共发现5株白花苗，8株部分叶片呈淡绿色的苗，以及47株颜色完全正常的苗，ICCU处理之后，部分卡莎拉斯的植株如图2B所示。
(2)ICCU法处理后T1代水稻叶片的测序分析：我们一共提取了45株T1代的日本晴植株和43株T1代卡莎拉斯植株幼苗的DNA。测序的引物为：TTGCAGACGCTCTTGCG(正链，5’-3’)，CAAAGCTCACATGCTGACTACT(负链，5’-3’)。经测序分析，在日本晴中，一共有15株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有10株，两条链都被编辑的有5株；两条链都被编辑到的植株中，其中杂合突变的有3株，纯合突变的有2株；目标区域的等位突变归纳于图3。经测序分析，在卡莎拉斯中，一共有21株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有16株，两条链都被编辑的有5株；两条链都被编辑到的植株中，其中杂合突变的有4株，纯合突变的有1株；目标区域的等位突变归纳于图4。
实施例2：ICCU法对玉米PDS基因的编辑效果
1、技术步骤
(1)载体与农杆菌菌株：载体，pTX172；农杆菌菌株：GV3101。
(2)被转化的玉米品种：B73、CML322。
(3)目标基因：Zm00001d027936。
(4)载体的构建：载体的分子构建见图1。图1示意该分子构建的核心元件Pol II启动子，Cas9核酸蛋白基因，PolyA，核糖酶剪切序列，sgRNA1，sgRNA2，核糖酶终止序列之间的位置关系。
(5)sgRNA1与sgRNA2的碱基序列设计：sgRNA1的碱基序列如下：AGTTGCTCAACAATGGACAC；sgRNA2的碱基序列如下：TAGACATCCTGCCTTGCAGG；sgRNA1与sgRNA2序列相对应玉米PDS基因上的位置间隔50个碱基。
(6)农杆菌液的培养：与实施例1，处理水稻的农杆菌液培养过程相同。
(7)农杆菌菌液对玉米SAM的侵染处理：(a)选择具有萌发能力的玉米种子，将种子在清水中吸涨1个小时，先用70％酒精对种子表皮进行1分钟消毒处理，用清水清洗3次，然后再用3％H2O2或者3％的次氯酸钠溶液对种子进行30分钟的浸泡，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡3小时。(b)将种子置于黑暗条件下萌发。4天后待种子萌发，并生长出第一片叶幼芽之后，用手或其它工具把第一片叶幼芽拔出。拔出过程尽可能做到即不损伤SAM，又尽可能减少留在SAM上层的残余组织。幼芽拔出后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间 与SAM组织之间的数层残余细胞，形成杯状结构的“杯底”。(c)使用移液枪，将大约30微升(在步骤(6)中制备好的)农杆菌感染液注射到“杯状”结构空间内。注射过程可以在体视显微镜或放大镜下进行操作。(d)幼苗在黑暗条件下培养。4天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。15天后，每株玉米植株剪半张叶片，用来提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰目标基因未受到编辑的阴性植株，将阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。(e)玉米幼苗经营养生长进入生殖生长之后，收获T2代植物的种子。
2、处理结果
(1)暗条件下ICCU法处理后T1代玉米幼苗的叶色：根据以上方法，我们一共处理了50株B73和50株CML322的幼苗。在50株B73植株中，我们一共发现6株白花苗，9株部分叶片呈淡绿色苗，以及35株颜色完全正常的苗。在50株CML322植株中，我们一共发现3株白花苗，8株部分叶片呈淡绿色苗，以及39株颜色完全正常的苗，ICCU处理之后，部分CML322的幼苗植株如图5所示。
(2)ICCU法处理后T1代玉米叶片的测序分析：我们一共提取了41株T1代B73和38株T1代CML322的幼苗的DNA。测序的引物为：GTGTGGAGCCTAGAGGGGAG(正链，5’-3’)，GGGGCGTTCGCTGTTTCTAA(负链，5’-3’)。经测序分析，在B73品种中，一共有14株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有10株，两条链都被编辑的有4株；两条链都被编辑到的植株中，其中杂合突变的有4株，纯合突变的有0株；目标区域的等位突变归纳于图6。经测序分析，在CML322品种中，一共有9株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有8株，两条链都被编辑的有1株；两条链都被编辑到的植株中，其中杂合突变的有1株，纯合突变的有0株；目标区域的等位突变归纳于图7。
实施例3：ICCU法对小麦PDS基因的编辑效果
1、技术步骤
(1)载体与农杆菌菌株：载体，pTX172；农杆菌菌株：EHA105。
(2)被转化的小麦品种：中国春(CS)、杨麦16。
(3)目标基因：TraesCS4A02G004900.1(染色体4A：3133866-3141315)、TraesCS4B02G300100.1(染色体4B：586574959-586580910)、TraesCS4B02G300100.2 (染色体4D：468262170-468267694)。
(4)载体的构建：载体的分子构建见图1。图1示意该分子构建的核心元件Pol II启动子，Cas9核酸蛋白基因，PolyA，核糖酶剪切序列，sgRNA1，sgRNA2，核糖酶终止序列之间的位置关系。
(5)sgRNA1与sgRNA2的碱基序列设计：sgRNA1的碱基序列如下：TTGTTTGCCAAGATTTTCCA；sgRNA2的碱基序列如下：GAGGCAAGAGATGTGTTGGG；sgRNA1与sgRNA2序列相对应小麦PDS基因上的位置间隔50个碱基。
(6)农杆菌液的培养：与实施例1中农杆菌液培养过程相同。
(7)农杆菌菌液对小麦SAM的侵染处理：(a)选择具有萌发能力的玉米种子，将种子在清水中吸涨1个小时，用70％酒精对种子的表皮消毒1分钟之后，用清水清洗3次，然后再用3％H2O2或者3％的次氯酸钠溶液对种子浸泡20分钟，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡3小时。(b)将种子置于黑暗条件下萌发。5天后待种子萌发，并生长出第一片叶幼芽之后，用手把第一片叶幼芽拔出。幼芽拔出后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间与SAM组织之间的数层残余细胞，形成杯状结构的“杯底”。(c)使用移液枪，将大约25微升(在步骤(6)中制备好的)农杆菌感染液注射到“杯状”结构空间内。注射过程可以在体视显微镜下进行操作。(d)幼苗在黑暗条件下培养。3天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。15天后，每株剪半张叶片，用来提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰TaPDS因未受到编辑的阴性植株，将阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。(e)小麦幼苗经营养生长进入生殖生长之后，收获T2代植物的种子。
2、处理结果
(1)暗条件下ICCU法处理后T1代小麦幼苗的叶色：根据以上方法，我们一共处理了35株中国春和35株杨麦16的幼苗。在30株中国春中，我们一共发现3株白花苗，6株部分叶片呈淡绿色苗，以及26株颜色完全正常的苗。在35株杨麦16中，我们一共发现1株白花苗，6株部分叶片呈淡绿色苗，以及28株颜色完全正常的苗，ICCU处理之后，部分处理后中国春的幼苗植株如图8所示。
(2)ICCU法处理后T1代小麦叶片的测序分析：我们一共提取了30株T1代中国春和30株杨麦16幼苗的DNA。A基因组测序的引物为：A AGCAAGTAAGATCTTTTGC(正链，5’-3’)，CATAAGGAGCACAATTTTAGAATT (负链，5’-3’)，B基因组测序的引物为：AAGCAAGTAAGATCTTTTGC(正链，5’-3’)，GATAACAGTGAATATGAGCTAC(负链，5’-3’)，D基因组测序的引物为：AAGCAAGTAAGATCTTTTGC(正链，5’-3’)，ACGAGCACAATTTTAGAGAT(负链，5’-3’)。经测序分析，在中国春中，一共有10株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有8株，两条链都被编辑的有2株；两条链都被编辑到的植株中，其中杂合突变的有1株，纯合突变的有1株；目标区域的等位突变归纳于图9。经测序分析，在杨麦16中，一共有10株PDS基因对应sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有9株，两条链都被编辑的有1株；两条链都被编辑到的植株中，其中杂合突变的有1株，纯合突变的有0株；目标区域的等位突变归纳于图10。
实施例4：ICCU法对大麦PDS基因的编辑效果
1、技术步骤
(1)载体与农杆菌菌株：载体，pTX172；农杆菌菌株：EHA105。
(2)被转化的大麦品种：金色希望(Golden Promise)(GP)、浙大9号
(3)目标基因：HORVU4Hr1G077450.1
(4)载体的构建：载体的分子构建见图1。图1示意该分子构建的核心元件Pol II启动子，Cas9核酸蛋白基因，PolyA，核糖酶剪切序列，sgRNA1，sgRNA2，核糖酶终止序列之间的位置关系。
(5)sgRNA1与sgRNA2的碱基序列设计：sgRNA1的碱基序列如下：TTGTTTGCCAAGATTTTCCA；sgRNA2的碱基序列如下：GAGGCAAGAGATGTGTTGGG；sgRNA1与sgRNA2序列相对应大麦PDS基因上的位置间隔50个碱基。
(6)农杆菌液的培养：与实施例1中农杆菌液培养过程相同。
(7)农杆菌菌液对小麦SAM的侵染处理：(a)选择具有萌发能力的大麦种子，将种子在清水中吸涨1个小时，先用70％酒精对种子表皮进行1分钟处理，用清水清洗3次，然后再用3％H 2O 2或者3％的次氯酸钠溶液对种子进行20分钟浸泡处理，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡3小时。(b)将种子置于黑暗条件下萌发。5天后待种子萌发，并生长出第一片叶幼芽之后，用手或其它工具把第一片叶幼芽拔出。拔出过程尽可能做到即不损伤SAM，又尽可能减少留在SAM上层的残余组织。幼芽拔出后，叶鞘(coleoptile)内形成一个“杯状”容器结构，杯状空间与 SAM组织之间的数层残余细胞，形成杯状结构的“杯底”。(c)使用移液枪，将大约20微升(在步骤(6)中制备好的)农杆菌感染液注射到“杯状”结构空间内。注射过程可以在体视显微镜或放大镜下进行操作。(d)幼苗在黑暗条件下培养。5天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。15天后，剪半张被检测的幼苗的叶片，用来提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰目标基因未受到编辑的阴性植株，将阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。(e)大麦幼苗经营养生长进入生殖生长之后，收获T2代植物的种子。
2、处理结果
(1)暗条件下ICCU法处理后T1代大麦幼苗的叶色：根据以上方法，我们一共处理了35株金色希望和35株浙大9号幼苗。在35株金色希望中，我们一共发现4株白花苗，7株部分叶片呈淡绿色苗，以及24株颜色完全正常的苗。在35株浙大9号中，我们一共发现0株白花苗，4株部分叶片呈淡绿色苗，以及31株颜色完全正常的苗，ICCU处理之后，部分浙大9号的幼苗植株如图11所示。
(2)ICCU法处理后T1代大麦叶片的测序分析：我们一共提取了30株T1代金色希望和30株浙大9号幼苗的DNA。测序的引物为：GGACAACTTCATACGCAG(正链，5’-3’)，ACGAGCACAATTTTAGAGAT(负链，5’-3’)。经测序分析，在金色希望中，一共有8株sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有6株，两条链都被编辑的有2株；两条链都被编辑到的植株中，其中杂合突变的有2株，纯合突变的有0株；目标区域的等位突变归纳于图12。经测序分析，在浙大9号中，一共有6株sgRNA1与sgRNA2序列之间(或附近)的序列被有效编辑，其中一条链被编辑的有6株，两条链都被编辑的有0株；两条链都被编辑到的植株中，其中杂合突变的有x株，纯合突变的有0株；目标区域的等位突变归纳于图13。
实施例5：ICCU法光培养与暗培养处理萌发种子所形成的杯状结构、遗传转化效果对比
1、技术步骤
(1)载体与农杆菌菌株：载体，pTX172；农杆菌菌株：EHA105。
(2)被转化的水稻品种：水稻品种卡莎拉斯。
(3)目标基因：OsPDS(Os03g08570)。
(4)载体的构建：载体的分子构建见图1。图1示意该分子构建的核心元件Pol II启动子、Cas9核酸蛋白基因、PolyA、核糖酶剪切序列、sgRNA1、sgRNA2、核糖酶终止序列之间的位置关系。
(5)sgRNA1与sgRNA2的碱基序列设计：sgRNA1的碱基序列如下：AGTTGCTTCAGCATGGATAC；sgRNA2的碱基序列如下：CGGGACAACTTCCTACTCAT。sgRNA1与sgRNA2序列相对应Os03g08570上的位置间隔50个碱基。
(6)农杆菌液的培养：与实施例1相同。
(7)农杆菌菌液对水稻SAM的侵染处理：(a)选择具有萌发能力的水稻种子，将种子在清水中吸涨1个小时，先用70％酒精对种子表皮处理1分钟，用清水清洗3次，然后再用3％H2O2或者3％的次氯酸钠溶液对种子进行20分钟，将处理后的种子用无菌清水洗5遍，然后在无菌清水中浸泡3小时。(b)分别将种子置于光照与黑暗条件下萌发。5天后待种子萌发，并生长出第一片叶幼芽之后，用手或其它工具把第一片叶幼芽拔出。拔出过程尽可能做到即不损伤SAM，又尽可能减少留在SAM上层的残余组织。幼芽拔出后，叶鞘(coleoptile)内形成不同大小的“杯状”容器结构。(c)使用移液枪，将大约25微升(在步骤(6)中制备好的)农杆菌感染液注射到经黑暗处理的“杯状”结构空间内、将大约15微升(在步骤(6)中制备好的)农杆菌感染液注射到经光照处理的“杯状”结构空间内。注射过程可以在体视显微镜或放大镜下进行操作。(d)分别继续将幼苗在光照或黑暗条件下培养。3天后，移至人工培养箱的正常条件下培养(即28℃光照条件16个小时，25℃黑暗条件8个小时)。15天后，剪半张被检测的幼苗的叶片，用来提取微量DNA，并后续用于目标基因序列的检测；测序之后，淘汰目标基因未受到编辑的阴性植株，将阳性植株移植到普通的介质(土壤或其它培养介质)上精心培育。(e)幼苗经营养生长进入生殖生长之后，收获T2代植物的种子。
2、处理结果
(1)光处理和黑暗处理形成的“杯状结构”大小比较：根据以上方法，我们分别用光照与黑暗条件处理了50粒种子，我们取典型2粒光照培养与黑暗培养的种子在图14中进行比较。光照或黑暗处理5天后，然后拔除第一片幼叶之后形成的“杯状结构”，每图左侧与右侧苗分别经光照与黑暗处理，5天后，黑暗处理后形成的“杯状结构”明显要比光照处理后形成的“杯状结构”体积要大，分别可以容纳大约30微升与10微升的农杆菌菌液。
表1光照或黑暗培养处理萌动种子最后转化效率的比较
  处理的幼苗数 单链受到编辑株数 双链受到编辑株数 转化率(％)
光处理 40 3 0 7.5％
黑暗处理 40 14 3 42.5％
(2)光处理和黑暗处理遗传转化率的比较：无论是对萌动种子进行光照处理还是黑暗处理，虽然形成的“杯状结构”空间大小具有明显的差异，但是都具有一定的转化效率，黑暗处理最后转化效率42.5％，光照处理最后的转化效率也有7.5％(表1)。

Claims (8)

  1. 一种禾本科植物的遗传转化方法，其特征在于，包括以下步骤：
    1)将禾本科植物种子萌发、长出第一张叶片后，拔出第一张叶片，此时叶鞘内形成一个杯状结构；
    2)向步骤1)杯状结构中加入具有用于遗传转化的载体质粒的根癌农杆菌菌液；
    3)共培养，得到至少部分顶端分生组织细胞基因组被修饰的转化植株。
  2. 如权利要求1所述的遗传转化方法，其特征在于，所述禾本科植物为水稻、玉米、小麦、大麦、高梁、黑麦、小黑麦、狗尾草或甘蔗。
  3. 如权利要求1所述的遗传转化方法，其特征在于，步骤1)中，种子萌发3～5天时长出第一张叶片，然后拔出第一张叶片。
  4. 如权利要求1所述的遗传转化方法，其特征在于，步骤2)中，用于遗传转化的载体为双元载体。
  5. 如权利要求1所述的遗传转化方法，其特征在于，步骤2)中，向步骤1)杯状结构中加入的菌液量为15～30μL。
  6. 如权利要求5所述的遗传转化方法，其特征在于，步骤2)中，向步骤1)杯状结构中加入菌液到加满。
  7. 如权利要求1所述的遗传转化方法，其特征在于，步骤1)种子萌发阶段在黑暗条件下进行或者光照条件下进行。
  8. 如权利要求1所述的遗传转化方法，其特征在于，将步骤3)得到的至少部分细胞被遗传转化的转基因植株培养后获得种子，并通过筛选得到其中杂合或者纯合突变体。
PCT/CN2022/071299 2021-07-28 2022-01-11 一种禾本科植物的遗传转化方法 Ceased WO2023005160A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110861664.7A CN113604497B (zh) 2021-07-28 2021-07-28 一种禾本科植物的遗传转化方法
CN202110861664.7 2021-07-28

Publications (1)

Publication Number Publication Date
WO2023005160A1 true WO2023005160A1 (zh) 2023-02-02

Family

ID=78305934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/071299 Ceased WO2023005160A1 (zh) 2021-07-28 2022-01-11 一种禾本科植物的遗传转化方法

Country Status (2)

Country Link
CN (1) CN113604497B (zh)
WO (1) WO2023005160A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487847A (zh) * 2023-10-31 2024-02-02 中国热带农业科学院橡胶研究所 一种获得橡胶树纯合基因编辑植株的方法
CN121931168A (zh) * 2026-03-26 2026-04-28 中国海洋大学三亚海洋研究院 一种农杆菌介导的条斑紫菜高效稳定遗传转化藻株的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113604497B (zh) * 2021-07-28 2023-03-24 浙江大学 一种禾本科植物的遗传转化方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7494787A (en) * 1986-06-30 1988-01-07 University Of Toledo, The Process for transforming gramineae and the products thereof
US5187073A (en) * 1986-06-30 1993-02-16 The University Of Toledo Process for transforming gramineae and the products thereof
CN1305005A (zh) * 2001-02-26 2001-07-25 山东大学 一种转化大粒种子植物的方法及其应用
WO2014029044A1 (zh) * 2012-08-22 2014-02-27 河北省农林科学院遗传生理研究所 充分微创种子芽生长点的单子叶植物转基因方法
CN113604497A (zh) * 2021-07-28 2021-11-05 浙江大学 一种禾本科植物的遗传转化方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103205455B (zh) * 2012-01-11 2014-12-31 山东省农业科学院作物研究所 利用幼穗拯救获得转基因小麦的方法
CN105420257A (zh) * 2015-12-10 2016-03-23 山东大学 玉米分泌型磷脂酶A2基因ZmsPLA2-2在改变植物抗旱性中的应用
EP3833747A1 (en) * 2018-06-28 2021-06-16 Pioneer Hi-Bred International, Inc. Methods for selecting transformed plants

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7494787A (en) * 1986-06-30 1988-01-07 University Of Toledo, The Process for transforming gramineae and the products thereof
US5187073A (en) * 1986-06-30 1993-02-16 The University Of Toledo Process for transforming gramineae and the products thereof
CN1305005A (zh) * 2001-02-26 2001-07-25 山东大学 一种转化大粒种子植物的方法及其应用
WO2014029044A1 (zh) * 2012-08-22 2014-02-27 河北省农林科学院遗传生理研究所 充分微创种子芽生长点的单子叶植物转基因方法
CN113604497A (zh) * 2021-07-28 2021-11-05 浙江大学 一种禾本科植物的遗传转化方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HE, CONGFEN ET AL.: "Genetic transformation of aloe barbadensis Miller by agrobacterium tumefaciens", JOURNAL OFGENETICS AND GENOMICS, vol. 34, no. 12, 31 December 2007 (2007-12-31), XP022937560, ISSN: 1053-1060, DOI: 10.1016/S1673-8527(07)60120-9 *
LIU FANG, LV WEI-TAO, CUI DE-CAI, ZHAO TAN-FANG: "Transformation of Wheat with Anti-Phospholipase Dγ Gene", LETTERS IN BIOTECHNOLOGY, JUNSHI YIXUE KEXUEYUAN, CN, vol. 17, no. 2, 31 March 2006 (2006-03-31), CN , pages 189 - 191, XP093028925, ISSN: 1009-0002 *
LIU TAO, WU BAOWEI; LI ZHIJIE; LIU ZHENLAN; WANG QIAN; ZHAO HUIXIAN: "Identification and Functional Analysis of miR5062 Target Genes in Wheat (Triticum aestivum)", JOURNAL OF TRITICEAE CROPS, vol. 41, no. 1, 1 January 2021 (2021-01-01), pages 1 - 10, XP093028924, ISSN: 1009-1041, DOI: 10.7606/j.issn.1009-1041.2021.01.01 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487847A (zh) * 2023-10-31 2024-02-02 中国热带农业科学院橡胶研究所 一种获得橡胶树纯合基因编辑植株的方法
CN121931168A (zh) * 2026-03-26 2026-04-28 中国海洋大学三亚海洋研究院 一种农杆菌介导的条斑紫菜高效稳定遗传转化藻株的方法

Also Published As

Publication number Publication date
CN113604497B (zh) 2023-03-24
CN113604497A (zh) 2021-11-05

Similar Documents

Publication Publication Date Title
CN105821073A (zh) 一种通过基因瞬时表达对整株植物定点改造的方法
CN103602657B (zh) Eat1基因的应用及恢复eat1基因缺失导致水稻雄性不育的方法
WO2022135246A1 (zh) 一种控制大豆-根瘤菌匹配性的r基因及其蛋白质和应用
CN113604497B (zh) 一种禾本科植物的遗传转化方法
JP2000510325A (ja) 植物性材料の繁殖および／または選択方法
CN105602984A (zh) 一种快速高效的农杆菌介导的小麦茎尖遗传转化方法
CN107557385A (zh) 一种菠萝体细胞胚发生受体类激酶基因AcSERK1的遗传转化方法及其应用
CN113564196A (zh) Lsa-MIR408基因及其在调控生菜产量及种子大小中的应用
CN104004783B (zh) 一种提高棉花转基因效率的方法
CN104513831B (zh) 一种促进植物生长的方法
JP4463456B2 (ja) 葉柄移植片を使用する綿の高効率アグロバクテリア菌仲介形質転換
CN121065205A (zh) 一种通过表达GmSWEET3c培育耐盐植物的方法
CN109735538A (zh) 一种提高森林草莓叶片再生效率的载体及其制备方法和应用
CN1769463A (zh) 通过转基因聚合betA、NHX1、PPase基因提高玉米、小麦耐盐耐旱性的方法
CN112931227B (zh) 一种杜仲各部位全株诱导植物再生及构建转基因植株再生体系的方法
CN118126150A (zh) GmSIG1基因在调控大豆株型中的应用
CN118638821A (zh) 水稻cdpk20基因及其在提高植物抗非生物胁迫中的用途
CN118389569A (zh) 梅花毛状根遗传转化体系的构建方法及应用
CN104726489A (zh) 农杆菌介导的活体幼胚转化获得转基因棉花的方法
CN102229946B (zh) 一种改良的农杆菌介导的小麦幼穗转化方法及其应用
CN102703497B (zh) 水稻矮杆株系创制的方法及其用途、恢复矮杆性状的方法
CN115125252A (zh) CsSH1基因在提高黄瓜苗期耐徒长中的应用和方法
CN121183029B (zh) 抗虫转基因玉米am11外源插入片段的旁侧序列及其应用
CN120519474B (zh) 甘蓝感病基因bpm6及其在甘蓝抗病育种中的应用
CN118086370B (zh) 转录因子OsbZIP62在水稻种子活力负向调控中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22847781

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22847781

Country of ref document: EP

Kind code of ref document: A1