CN114134173B - Expression vector and application thereof in genetic transformation of plants - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering and transgenosis, and particularly relates to an expression vector and application thereof in plant genetic transformation. The invention constructs a new expression vector, and the vector is used as an auxiliary vector for crop genetic transformation, so that the transformation efficiency of plants, especially maize, wheat and other plant species which depend on genotypes and are difficult to transform and the quantity of high-quality transformed seedlings can be improved.

Description

Expression vector and application thereof in genetic transformation of plants

Technical Field

The invention belongs to the technical field of genetic engineering and transgenosis, and particularly relates to an expression vector and application thereof in crop genetic transformation.

Background

The plant genetic transformation technology is a key element of the research and development of transgenic plant products. Agrobacterium-mediated methods have become the most widely used genetic transformation technique in plant transgenic research today.

Corn and wheat are monocotyledonous plants, are not natural hosts of agrobacterium, and agrobacterium-mediated genetic transformation has the problem of low transformation efficiency. Many factors influence the transformation efficiency of these species, such as Agrobacterium strains, acceptor materials, medium composition and screening means. Among them, the receptor genotype has a great influence on agrobacterium-mediated transformation efficiency of plant species such as corn, wheat, etc. For example, the recipient materials commonly used in maize are inbred line A188, hybrid HiII (A188×B73), etc., which are significantly more efficient than other materials, because most maize inbred lines induce Type I callus that is difficult to transform, while recipient materials such as A188 can produce Type II callus. Type ii calli are embryogenic calli, have the ability to differentiate embryoids and can be long-term subcultured. The problem of receptor genotype dependence has limited to a certain extent the development of transgenic technologies for maize, wheat and other species.

Baby bottom (Bbm) and Wuschel2 (Wus) are key regulatory factors in plant stem cell development. Bbm encodes an AP2/ERF transcription factor that plays an important role in maintaining stem cells in an undifferentiated state; wus2 encodes a homeodomain protein that confers stem cell characteristics to surrounding cells. Several research units have applied Bbm and Wus genes to maize genetic transformation methods to increase transformation efficiency from 2% single digit to 25% and above (transformation efficiencies for different receptors remain different) and further break through the limitations of genotypes (Lowe K, et al, morphogenic regulators Baby boom and Wuschel improve monocot transformation.plant Cell,2016,28:1998-2015;Mookkan M,et al, selectable marker independent transformation of recalcitrant maize inbred B and srghum P898012 mediated by morphogenic regulators BABY BOOM and wuschel2.plant Cell Rep,2017, 36:1477-1491).

Although the technical data disclosed above using Bbm and Wus successfully improved maize transformation efficiency, the resulting transformed plants often exhibited malformations that were not commercially viable. There is therefore a need to further optimize the way Bbm and Wus2 are used in assisting maize genetic transformation.

In order to solve the problems, a plurality of Bbm and Wus2 expression auxiliary vectors are constructed, transformation tests are carried out on some maize and wheat backbone inbred lines, so that the application modes of Bbm and Wus2 in auxiliary maize and other plant genetic transformation are optimized, the plant genetic transformation efficiency is improved, and the development of transgenic crops is assisted.

Disclosure of Invention

The invention aims to provide a method for improving the genetic transformation efficiency of plants.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a protein combination, which is characterized by comprising three proteins of Bbm, wus2 and Barnase;

wherein the Barnase protein is expressed by an expression cassette comprising a nucleic acid molecule encoding the Barnase protein linked to a Photosystem II 10kDa promoter;

the amino acid sequence of the Barnase protein used in the embodiment of the invention is shown as SEQ ID NO.1, the sequence of the Photosystem II 10kDa promoter is shown as SEQ ID NO.2, and the nucleic acid molecule sequence for encoding the Barnase protein is shown as SEQ ID NO. 3; the amino acid sequence of Bbm protein is shown as SEQ ID NO.4, and the amino acid sequence of Wus protein is shown as SEQ ID NO. 5.

In some embodiments, the above-described expression cassette further comprises a nos terminator; the nos terminator is a commonly used transcription terminator, and the nos terminator sequence used in the examples of the present invention is shown in SEQ ID NO. 6.

In some embodiments, the Bbm protein is expressed from an expression cassette comprising a pZmPLTP promoter linked to a nucleic acid molecule encoding a Bbm protein linked to a tT28 terminator and the Wus2 protein is expressed from an expression cassette comprising a pZmAXIG1 promoter linked to a nucleic acid molecule encoding a Wus2 protein linked to a tIN-1 terminator.

The pZmPLTP promoter sequence used in the embodiment of the invention is shown in SEQ ID NO.7, the nucleic acid molecule encoding Bbm protein is derived from a maize Bbm gene, the sequence is shown in SEQ ID NO.8, the tT28 terminator sequence is shown in SEQ ID NO.9, the pZmAXIG1 promoter sequence is shown in SEQ ID NO.10, the nucleic acid molecule encoding Wus2 protein is derived from a maize Wus2 gene, the sequence is shown in SEQ ID NO.11, and the tIN2-1 terminator sequence is shown in SEQ ID NO. 12.

The invention also provides an expression vector, which is characterized by comprising the following expression cassettes:

(1) The Photosystem II 10kDa promoter is linked to a nos terminator to which is linked a nucleic acid molecule encoding a Barnase protein;

(2) The pZmPLTP promoter is linked to a nucleic acid molecule encoding a Bbm protein linked to the tT28 terminator;

(3) The pZmAXIG1 promoter is linked to a nucleic acid molecule encoding a Wus protein linked to a tIN-1 terminator.

The expression vector used in the examples of the present invention is shown in FIG. 1.

The invention also provides a host cell, which is characterized in that the host cell comprises the expression vector;

in some embodiments, the host cell is an agrobacterium cell.

The invention also provides a plant genetic transformation method, which is characterized in that the plant is transformed by the expression vector and another expression vector or the host cell and another host cell together to obtain a regenerated plant.

The additional expression vectors in the examples of the present invention contain HTP and GFP gene expression cassettes, or expression cassettes for editing the wall gene (two pZmU6 promoter linked to sgRNA expression cassette, and pzmebi promoter linked to Cas9 gene linked to NOS terminator) and selectable marker expression cassettes (35S promoter linked to Bar gene linked to poly a terminator).

The invention also provides any one of the protein combinations, the expression vector, the host cell and the application of the method in improving the genetic transformation efficiency of plants.

In some embodiments the plant is selected from any one of wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, cotton, soybean, canola, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard, cabbage, broccoli, cabbage, cucumber, pumpkin, wax gourd, bitter gourd, luffa, melon, watermelon, melon, tomato, eggplant, chilli, bean, cowpea, green bean, leek, green onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, yellow flower, grape, strawberry, beet, sugarcane, tobacco, alfalfa, pasture, turf grass, tea, and cassava.

The invention has the advantages and beneficial effects as follows: according to the invention, the influence of the mixed transformation of the expression auxiliary vectors of Bbm and Wus2 and the target gene vector on the transformation efficiency and the high-quality transformation efficiency is tested, and the result shows that the transformation efficiency and the high-quality transformation efficiency of species such as corn and wheat can be obviously improved by adding the Bacillus amyloliquefaciens Barnase gene driven by the Photosystem II 10kDa promoter into the independent expression auxiliary vectors of Bbm and Wus, the advantages of the Bbm and Wus genes can be furthest exerted, the negative influence can be reduced, and the limitation of genotypes can be broken through. The transgenic plant obtained by the transformation system has no abnormal phenotype and adverse agronomic characters, has effects on various difficultly transformed species such as corn, wheat and the like, can be applied to genetic transformation of all plant species, and has very important application value.

Drawings

FIG. 1 pWMDR003 vector.

FIG. 2 is a schematic representation of the T-DNA region of each vector.

FIG. 3 is a photograph of each transformation stage. a-C: a callus induction stage; d: a callus differentiation stage; e: abnormal phenotype plants.

FIG. 4 shows an electrophoresis pattern of the mixed-transformed genotyping detection PCR amplification products. The upper row bar gene was amplified and the lower row Bbm gene was amplified.

FIG. 5 shows an electrophoretogram of PCR amplification products using genotyping isolation after lethal gene conversion. The upper row bar gene was amplified and the lower row Bbm gene was amplified.

FIG. 6 pWMV024 vector.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be construed according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are incorporated by reference in their entirety.

As used herein, "maize" is any maize plant and includes all plant varieties that can be bred with maize, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, whole plant cells in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. Unless otherwise indicated, nucleic acids are written in the 5 'to 3' direction from left to right; the amino acid sequence is written in the amino to carboxyl direction from left to right. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may be referred to by commonly accepted single letter codes. The numerical range includes the numbers defining the range.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present application. Examples follow conventional experimental conditions, such as the molecular cloning laboratory manual of Sambrook et al (Sambrook J & Russell D W, molecular cloning: a laboratory manual, 2001), or conditions recommended by the manufacturer's instructions, unless otherwise indicated. Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.

Examples

Example 1 expression Bbm and Wus2 improves maize transformation efficiency

Constitutive expression of maize morphogenic transcription factors Bbm and Wus2 in maize can greatly increase transformation efficiency, but also induce phenotypic abnormalities and sterility in maize plants, making commercial use difficult. pZmPLP is a phosphotransferase protein gene promoter, and is specifically expressed in callus and embryo, and pZmAxig1 is a promoter with auxin induction property, and the pZmPLP and pZmAxig1 promoters are used for driving the space-time specific expression of Bbm and Wus2, so that negative effects (Lowe K, et al, rapid gene "independent" Zea mays L. (size) transformation via direct somatic emulogenesis.In Vitro Cell Dev Biol Plant,2018, 54:240-252) caused by Bbm and Wus2 genes can be overcome to a certain extent. The invention firstly tests the transformation auxiliary effect of pZmPLP and pZmAxig1 promoters Bbm and Wus on some common maize backbone inbred lines in China. The expression vector used in the test was pWMDR001, and the schematic of the T-DNA region is shown in FIG. 2. The vector T-DNA region comprises six expression cassettes, two of which promote transformation: the pZmAXIG1 promoter is connected with a ZmWus2 gene and tIN-1 terminator, and the pZmPLTP promoter is connected with a ZmBbM gene and tT28 terminator; three expression cassettes for editing the wall gene: the pZmU6 promoter is connected with two sgRNA expression cassettes, and the pZmUBI promoter is connected with the Cas9 gene and connected with the NOS terminator; screening marker expression cassette: the 35S promoter is linked to the bar gene and to the PolyA terminator.

The pWMDR001 vector was transformed into 7 backbone inbred lines Zheng 58, X923-1, IL3, IL4, beijing 724, KN5585 and B104 using conventional maize transformation methods (Agrobacterium-infected immature embryos). The specific transformation steps are as follows:

1) Streaking an AGL1 or EHA105 agrobacterium strain containing a glufosinate resistance gene on an activation culture medium, and culturing in the dark at 28 ℃ for 24 hours;

2) 6-15 days after pollination of the maize inbred line, immersing young embryo obtained by stripping maize ears when the maize young embryo grows to 0.5-2.0mm in a suspension culture medium, suspending and immersing for 10-30min, removing liquid after young embryo collection, performing heat shock for 5min, adding into an infection culture medium in AGL1 or EHA105 agrobacterium containing glufosinate resistance genes for infection for 5min, and blowing air into the agrobacterium infection liquid during the period; the specific operation method of blowing air is as follows: using a disposable dropper or a pipette loaded with 200 mu L or 1000 mu L of gun heads, blowing and beating agrobacterium immersed in the explant for 3-5 times at intervals of about 10 seconds to generate bubbles;

3) Transferring the young embryo onto a co-culture medium, and culturing in darkness at 23 ℃ for 24-48h;

4) Transferring the young embryo onto a rest culture medium, and culturing in the dark at 26-34 ℃ for 1-2 weeks;

5) Transferring the young embryo to a selection medium for culture, wherein the selection medium contains glufosinate-induced resistant callus; transferring the resistant callus to a differentiation medium, culturing at 25 ℃ and 5000lx under illumination for 3 weeks, and differentiating to form regenerated plantlets;

6) Rooting the regenerated plantlets on a rooting culture medium, hardening off and transplanting to obtain transgenic corn;

the medium formulation used in the transformation procedure was as follows:

agrobacterium activation medium: d-glucose 20g/L+MES 19.5g/L+NaH ₂ PO ₄ 0.06 g/L+NH ₄ Cl 1g/L+MgSO4·7H ₂ O 0.3g/L+KCl 0.15g/L+CaCl ₂ ·2H ₂ O 0.0132 g/L+FeSO ₄ ·7H ₂ O0.0025 g/L+15 g/L agar;

suspension medium: 1/2 MS+sucrose 68.5 g/L+glucose 36 g/L+L-proline 0.115g/L; and

infection medium: 1/2 MS+sucrose 68.5 g/L+glucose 36 g/L+L-proline 0.115 g/L+acetosyringone 200 mM+cysteine 200mg/L; and

co-culture medium: 1/2 MS+sucrose 20 g/L+glucose 10 g/L+proline 0.115 g/L+thiamine hydrochloride 0.5mg/L+AgNO3 20mM+L-cysteine 200mg/L+2, 4-D0.5 mg/L+picloram 2.2 mg/L+acetosyringone 200mM; and

rest medium: 2 XSS+30 g/L of sucrose+1.38 g/L of proline+0.5 mg/L of thiamine hydrochloride+20 mM of AgNO 3+0.5 g/L of hydrolyzed casein+2, 4-D0.5 mg/L+2.2 mg/L of picloram+200 mg/L of timentin; and

screening the culture medium: MS+sucrose 30g/L+6-BA 0.1 mg/L+KT1mg/L+timentin 200 mg/L+glufosinate 10mg/L; and

seedling strengthening culture medium: MS+sucrose 30g/L+6-BA 0.1 mg/L+KT1mg/L+timentin 200 mg/L+glufosinate 10mg/L

Rooting medium: MS+sucrose 20g/L+MES 0.5g/L+IBA 0.2mg/L.

The Agrobacterium infection was found 3 days after transformationEmbryogenic projections due to the expression of Bbm and Wus2 (FIG. 3A) were observed on the calli, and after 7 days, distinct clustered embryoids (FIG. 3B) and abundant embryogenic projections (FIG. 3C) were observed, which were able to successfully differentiate into shoots on the medium (FIG. 3D). The statistical result of the transformation efficiency shows that the transformation efficiency of different inbred lines is between 6.0% and 24.0%, and the average transformation efficiency can reach 14.0% (Table 1). If the Bbm and Wus2 genes are not used, the transformation efficiency of KN5585 and B104 is only 10.0%, and the transformation seedlings cannot be obtained by other inbred lines (Zheng 58, X923-1, IL3, IL4 and Beijing 724) at all, and the transformation efficiency is 0. These results show that the expression of Bbm and Wus2 is indeed able to increase the transformation efficiency of multiple maize inbred lines. However, at all T's obtained ₀ The transformed plants contain Bbm and Wus genes. Since these two genes are not valuable after successful transformation and their expression can cause plants to exhibit different degrees of abnormalities, such as tassel portion to appear as female (FIG. 3E), the vector is not suitable for commercial use.

TABLE 1 transformation efficiency of pWMDR001 vector in maize backbone inbred and conventional recipient materials

Example 2 improved high quality conversion efficiency by mixing conversion separations Bbm and Wus2

Although the expression Bbm and Wus2 can obviously improve the genetic transformation efficiency of corn, various unfavorable agronomic characters such as development disorder still exist in the obtained offspring corn plants, and the popularization and application of the technology are limited. In order to solve the above problems, the present invention first attempted to independently construct Bbm and Wus2 expression auxiliary vectors (pWMDR 002, see FIG. 2 for schematic T-DNA region) and wall gene editing vectors (p 193412, see FIG. 2 for schematic T-DNA region), and obtain transgenic plants free of Bbm and Wus2 genes by means of a mixed transformation strategy. However, since it is possible to include both pWMDR002 and p193412 in2 vectors in one transgenic plant at the time of mixed transformation, the elimination of plants containing the Bbm and Wus2 genes would result in the loss of many successfully transformed and edited plants. It is uncertain how many plants can be obtained that contain only editing elements and that grow normally.

Specific testing work was conducted with Zheng 58 as the receptor. The results show that after mixed transformation at T ₀ The generation was able to obtain transgenic plants which did not contain the Bbm and Wus2 genes but contained the gene editing elements (see FIG. 4; samples 3, 9). These transgenic plants, which do not contain the Bbm and Wus genes, grew normally, and sequencing results showed that the wax gene was successfully edited, and the editing type was also rich, being a good quality transformant (no Bbm and Wus2 genes, grew normally). However, the efficiency of the high quality transformed seedlings obtained by this method is still low, between 0.4% and 6.8%, and on average 3.2% (Table 2), and such efficiency is still not suitable for large-scale, high throughput production of maize transformed seedlings.

TABLE 2 effect of pWMDR002 and p193412 vector on Mixed rotation Zheng 58

Example 3 use of lethal Gene isolation Bbm and Wus2 to promote high quality transformation efficiency

In order to further increase the high quality transformation efficiency, the present invention contemplates that lethal genes may be used such that calli containing the Bbm and Wus2 genes cannot differentiate into shoots. However, it is uncertain which lethal gene is used and in what manner the best effect can be obtained. The invention first uses Barnase gene for testing. Barnase encodes an extracellular ribonuclease, which is strongly toxic and can cause cell death. The invention uses four promoters of Phs-II 10 and Ubiquitin, actin, nos to drive Barnase genes to test effects.

The expression vectors used in the test are pWMDR003, pWMDR004, pWMDR005 and pWMDR006, and the schematic diagram of the T-DNA region is shown in FIG. 2. The series of vector T-DNA regions comprise three expression cassettes, two that promote transformation, respectively: the pZmAXIG1 promoter is linked to the ZmWus2 gene linked to the tIN-1 terminator and the pPhs-II 10 (or Ubiquitin, or action, or Nos) promoter is linked to the Barnase gene linked to the NOS terminator.

The transformation efficiency was tested for pWMDR003/pWMDR004/pWMDR005/pWMDR006 and p193412 vector mixed transformation Zheng 58, and it was found that superior transformation efficiency was obtained using the pPhs-II 10 promoter, which was significantly higher than using other promoters (Table 3). The pPhs-II 10 promoter drives barnase lethal gene, can lead embryoid bodies to be successfully induced, and then, the transformed seedlings containing the element are dead in the process of growing into seedlings, so that only the transformed seedlings without Bbm and Wus genes are reserved. Other tested promoter combinations can not achieve efficient transformation and Bbm and Wus gene knockout at the same time, and the high-quality transformation efficiency is low.

TABLE 3 high-quality transformation efficiency test of different lethal Gene vectors on Zheng 58

The test result of a large amount of transformation again shows that after pWMDR003 and p193412 carrier are mixed and transformed into Zheng 58, the separation rate of high-quality transformed seedlings is obviously increased (as shown in figure 5; samples 1, 4, 5, 6, 8, 9 and 10), and the average separation rate can reach 89.2%. The conversion efficiency of the high quality reaches 16.4% on average, and is improved by more than 5 times compared with 3.2% in the embodiment 2. Using this method to transform other inbred lines (B104, beijing 724, waxy IN 12), very high quality transformation efficiencies could be obtained as well (Table 4).

TABLE 4 Mixed transfer Effect of pWMDR003 and p193412 vectors

Example 4 test results in wheat

Transformation of wheat is also difficult and is greatly affected by genotype. The invention further tests the application condition of the technical scheme in wheat. The vectors used for the test include the above-described pWMDR002 and pWMDR003, and a test vector pWMV024 (containing HTP and GFP gene expression cassettes, vector diagram is shown in FIG. 6).

The specific steps of wheat transformation are as follows:

1) Streaking an AGL1 or EHA105 agrobacterium strain containing hygromycin resistance gene on an activation medium, and culturing in the dark at 28 ℃ for 24 hours;

2) Cutting wheat ears with young embryo length of 0.5-2.0mm 10-14 days after pollination, peeling to obtain young embryo, immersing in suspension culture medium for 10-30min, removing liquid after young embryo collection, heat-shocking for 5min, and adding into infection culture medium containing hygromycin resistance gene EHA105 Agrobacterium for 5min;

3) Transferring the young embryo onto a co-culture medium, and culturing in darkness at 23 ℃ for 24-48h;

4) Transferring the young embryo onto a rest culture medium, and culturing in the dark at 26-34 ℃ for 1-2 weeks;

5) Transferring the young embryo to a selection medium for culture, wherein the selection medium contains hygromycin to induce resistant callus; transferring the resistant callus to a differentiation medium, culturing at 25 ℃ and 5000lx under illumination for 3 weeks, and differentiating to form regenerated plantlets;

6) And hardening seedlings after the regenerated seedlings root on a rooting culture medium, and transplanting to obtain the transgenic wheat.

The medium formulation used in the transformation procedure was as follows:

suspension medium: MS+glucose 10g/L+Silwet L-77 0.5mL/L; and

infection medium: MS+glucose 10g/L+Silwet L-77 0.5 mL/L+acetosyringone 200 mM+cysteine 200mg/L; and

co-culture medium: 1/2 MS+sucrose 20 g/L+glucose 10 g/L+proline 0.115 g/L+thiamine hydrochloride 0.5mg/L+AgNO3 20mM+L-cysteine 200mg/L+2, 4-D0.5 mg/L+picloram 2.2 mg/L+acetosyringone 200mM; and

rest medium: 2 XSS+30 g/L of sucrose+1.38 g/L of proline+0.5 mg/L of thiamine hydrochloride+20 mM of AgNO 3+0.5 g/L of hydrolyzed casein+2, 4-D0.5 mg/L+2.2 mg/L of picloram+200 mg/L of timentin; and

screening the culture medium: MS+sucrose 30g/L+6-BA 0.1 mg/L+KT1mg/L+timentin 200 mg/L+hygromycin 50mg/L; and

seedling strengthening culture medium: MS+sucrose 30g/L+6-BA 0.1 mg/L+KT1mg/L+timentin 200 mg/L+hygromycin 50mg/L; and

rooting medium: MS+sucrose 20g/L+MES 0.5g/L+IBA 0.2mg/L.

The results show that the transformation efficiency of high quality seedlings of different wheat varieties (Yangmai and Fielder) transformed with the combination of the pWMDR003 and pWMV024 vectors is significantly higher than that of the combination of the pWMDR002 and pWMV024 vectors (tables 5 and 6), indicating that the technical scheme has effect in various different species.

TABLE 5 Mixed transformation of pWMDR002 and pWMV024 vectors

TABLE 6 Mixed transformation of pWMDR003 and pWMV024 vectors

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Oryza Glutinosa (Jiangsu) Limited, university of agriculture in China, academy of agricultural sciences in Jilin province

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Arg Ile Ala Ser Gln Leu Gly Asp Gly Gly Ala Leu Ala Ala Ala Tyr

485 490 495

Gly Ala His Tyr His Gly Ala Ala Trp Pro Thr Ile Ala Phe Gln Pro

500 505 510

Gly Ala Ala Ser Thr Gly Leu Tyr His Pro Tyr Ala Gln Gln Pro Met

515 520 525

Arg Gly Gly Gly Trp Cys Lys Gln Glu Gln Asp His Ala Val Ile Ala

530 535 540

Ala Ala His Ser Leu Gln Asp Leu His His Leu Asn Leu Gly Ala Ala

545 550 555 560

Gly Ala His Asp Phe Phe Ser Ala Gly Gln Gln Ala Ala Ala Ala Ala

565 570 575

Met His Gly Leu Gly Ser Ile Asp Ser Ala Ser Leu Glu His Ser Thr

580 585 590

Gly Ser Asn Ser Val Val Tyr Asn Gly Gly Val Gly Asp Ser Asn Gly

595 600 605

Ala Ser Ala Val Gly Gly Ser Gly Gly Gly Tyr Met Met Pro Met Ser

610 615 620

Ala Ala Gly Ala Thr Thr Thr Ser Ala Met Val Ser His Glu Gln Val

625 630 635 640

His Ala Arg Ala Tyr Asp Glu Ala Lys Gln Ala Ala Gln Met Gly Tyr

645 650 655

Glu Ser Tyr Leu Val Asn Ala Glu Asn Asn Gly Gly Gly Arg Met Ser

660 665 670

Ala Trp Gly Thr Val Val Ser Ala Ala Ala Ala Ala Ala Ala Ser Ser

675 680 685

Asn Asp Asn Met Ala Ala Asp Val Gly His Gly Gly Ala Gln Leu Phe

690 695 700

Ser Val Trp Asn Asp Thr

705 710

<210> 5

<211> 302

<212> PRT

<213> Zea mays

<400> 5

Met Ala Ala Asn Ala Gly Gly Gly Gly Ala Gly Gly Gly Ser Gly Ser

1 5 10 15

Gly Ser Val Ala Ala Pro Ala Val Cys Arg Pro Ser Gly Ser Arg Trp

20 25 30

Thr Pro Thr Pro Glu Gln Ile Arg Met Leu Lys Glu Leu Tyr Tyr Gly

35 40 45

Cys Gly Ile Arg Ser Pro Ser Ser Glu Gln Ile Gln Arg Ile Thr Ala

50 55 60

Met Leu Arg Gln His Gly Lys Ile Glu Gly Lys Asn Val Phe Tyr Trp

65 70 75 80

Phe Gln Asn His Lys Ala Arg Glu Arg Gln Lys Arg Arg Leu Thr Ser

85 90 95

Leu Asp Val Asn Val Pro Ala Ala Gly Ala Ala Asp Ala Thr Thr Ser

100 105 110

Gln Leu Gly Val Leu Ser Leu Ser Ser Pro Pro Pro Ser Gly Ala Ala

115 120 125

Pro Pro Ser Pro Thr Leu Gly Phe Tyr Ala Ala Gly Asn Gly Gly Gly

130 135 140

Ser Ala Val Leu Leu Asp Thr Ser Ser Asp Trp Gly Ser Ser Gly Ala

145 150 155 160

Ala Met Ala Thr Glu Thr Cys Phe Leu Gln Asp Tyr Met Gly Val Thr

165 170 175

Asp Thr Gly Ser Ser Ser Gln Trp Pro Arg Phe Ser Ser Ser Asp Thr

180 185 190

Ile Met Ala Ala Ala Ala Ala Arg Ala Ala Thr Thr Arg Ala Pro Glu

195 200 205

Thr Leu Pro Leu Phe Pro Thr Cys Gly Asp Asp Gly Gly Ser Gly Ser

210 215 220

Ser Ser Tyr Leu Pro Phe Trp Gly Ala Ala Ser Thr Thr Ala Gly Ala

225 230 235 240

Thr Ser Ser Val Ala Ile Gln Gln Gln His Gln Leu Gln Glu Gln Tyr

245 250 255

Ser Phe Tyr Ser Asn Ser Asn Ser Thr Gln Leu Ala Gly Thr Gly Asn

260 265 270

Gln Asp Val Ser Ala Thr Ala Ala Ala Ala Ala Ala Leu Glu Leu Ser

275 280 285

Leu Ser Ser Trp Cys Ser Pro Tyr Pro Ala Ala Gly Ser Met

290 295 300

<210> 6

<211> 270

<212> DNA

<213> Escherichia coli

<400> 6

gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 60

cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 120

catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 180

catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 240

ggtgtcatct atgttactag atcgggaatt 270

<210> 7

<211> 1062

<212> DNA

<213> Zea mays

<400> 7

ccctagctcc ctgcggctgt tacgcggtcc cccatcaatc ttctgttctt gcggttgtag 60

cctgtgtaac agtgctagag tatgtatgat aaataggttt taagtctgct tacatgacat 120

tttttattgt ggaagagaca tataaaaatt agagagagtg gttctcatgc aacggcggac 180

ggcccggtgc taaaagagct tcaagacaaa ataatgaaac aggaagagag tagatttatc 240

taagagccaa ctttattata tgaatgtgtt tattgttggc tttagatgat atggtaagga 300

gttagagcta ataatagata ggctctatta ttattattat taattaaact cgctctaagg 360

aggaaagtgg gaggaaggga cgaggacgaa gactactgga agcatcgtgc atggatgatg 420

gatgtggtgt ctcttaatgt aggtggccgg aggatgtacg tgttaattgc gcgataagca 480

ctcagatcca accgcaaact acctccacac tgacacactg atagagagaa agagagacct 540

ccgacgactg ccgccgcaga tgagccacgt acgtatacga cgtctgccgg ccggctcagg 600

ctgccgccat caccctgctc gaaagtcgcg ttaggcggcg ccagctacat aggagtatct 660

agtctagcca gttagtatac tactactgcg ctgatgatga attaactctg catagatact 720

gtacttgcct ccctccaaca cccaaccacc tcctgctcgg ctcttaataa cttggacacg 780

gatcgatgcc atccaaggaa gaacacgacg acgacgacgg aacatccacc atgcaagctt 840

gcatccatac gccgatacgc gtgcatccat ccatccacca ttatttccat tttccaccga 900

tcacacgtac acaggcctat ttaaggagcg acatcccact gcaactctcc tcaccactca 960

tcaccagcta gctctagcaa agcacttgcc atctaccgac cgccgcattc caaacagccc 1020

gacgagctag cagagcggca ggcacctccc tcctcaagga ac 1062

<210> 8

<211> 2133

<212> DNA

<213> Zea mays

<400> 8

atggccactg tgaacaactg gctcgctttc tccctctccc cgcaggagct gccgccctcc 60

cagacgacgg actccacact catctcggcc gccaccgccg accatgtctc cggcgatgtc 120

tgcttcaaca tcccccaaga ttggagcatg aggggatcag agctttcggc gctcgtcgcg 180

gagccgaagc tggaggactt cctcggcggc atctccttct ccgagcagca tcacaaggcc 240

aactgcaaca tgatacccag cactagcagc acagtttgct acgcgagctc aggtgctagc 300

accggctacc atcaccagct gtaccaccag cccaccagct cagcgctcca cttcgcggac 360

tccgtaatgg tggcttcctc ggccggtgtc cacgacggcg gtgccatgct cagcgcggcc 420

gccgctaacg gtgtcgctgg cgctgccagt gccaacggcg gcggcatcgg gctgtccatg 480

attaagaact ggctgcggag ccaaccggcg cccatgcagc cgagggtggc ggcggctgag 540

ggcgcgcagg ggctctcttt gtccatgaac atggcgggga cgacccaagg cgctgctggc 600

atgccacttc tcgctggaga gcgcgcacgg gcgcccgaga gtgtatcgac gtcagcacag 660

ggtggagccg tcgtcgtcac ggcgccgaag gaggatagcg gtggcagcgg tgttgccggc 720

gctctagtag ccgtgagcac ggacacgggt ggcagcggcg gcgcgtcggc tgacaacacg 780

gcaaggaaga cggtggacac gttcgggcag cgcacgtcga tttaccgtgg cgtgacaagg 840

catagatgga ctgggagata tgaggcacat ctttgggata acagttgcag aagggaaggg 900

caaactcgta agggtcgtca agtctattta ggtggctatg ataaagagga gaaagctgct 960

agggcttatg atcttgctgc tctgaagtac tggggtgcca caacaacaac aaattttcca 1020

gtgagtaact acgaaaagga gctcgaggac atgaagcaca tgacaaggca ggagtttgta 1080

gcgtctctga gaaggaagag cagtggtttc tccagaggtg catccattta caggggagtg 1140

actaggcatc accaacatgg aagatggcaa gcacggattg gacgagttgc agggaacaag 1200

gatctttact tgggcacctt cagcacccag gaggaggcag cggaggcgta cgacatcgcg 1260

gcgatcaagt tccgcggcct caacgccgtc accaacttcg acatgagccg ctacgacgtg 1320

aagagcatcc tggacagcag cgccctcccc atcggcagcg ccgccaagcg cctcaaggag 1380

gccgaggccg cagcgtccgc gcagcaccac cacgccggcg tggtgagcta cgacgtcggc 1440

cgcatcgcct cgcagctcgg cgacggcgga gccctggcgg cggcgtacgg cgcgcactac 1500

cacggcgccg cctggccgac catcgcgttc cagccgggcg ccgccagcac aggcctgtac 1560

cacccgtacg cgcagcagcc aatgcgcggc ggcgggtggt gcaagcagga gcaggaccac 1620

gcggtgatcg cggccgcgca cagcctgcag gacctccacc acctgaacct gggcgcggcc 1680

ggcgcgcacg actttttctc ggcagggcag caggccgccg ccgctgcgat gcacggcctg 1740

ggtagcatcg acagtgcgtc gctcgagcac agcaccggct ccaactccgt cgtctacaac 1800

ggcggggtcg gcgacagcaa cggcgccagc gccgtcggcg gcagtggcgg tggctacatg 1860

atgccgatga gcgctgccgg agcaaccact acatcggcaa tggtgagcca cgagcaggtg 1920

catgcacggg cctacgacga agccaagcag gctgctcaga tggggtacga gagctacctg 1980

gtgaacgcgg agaacaatgg tggcggaagg atgtctgcat gggggactgt cgtgtctgca 2040

gccgcggcgg cagcagcaag cagcaacgac aacatggccg ccgacgtcgg gcatggcggc 2100

gcgcagctct tcagtgtctg gaacgacact taa 2133

<210> 9

<211> 880

<212> PRT

<213> Oryza sativa

<400> 9

Thr Gly Ala Cys Ala Thr Cys Thr Thr Ala Thr Ala Gly Thr Cys Thr

1 5 10 15

Gly Cys Ala Ala Cys Cys Thr Cys Thr Cys Gly Thr Gly Thr Cys Thr

20 25 30

Gly Ala Ala Thr Thr Cys Cys Thr Ala Thr Cys Thr Thr Thr Ala Thr

35 40 45

Cys Ala Ala Gly Thr Gly Thr Thr Ala Thr Thr Gly Cys Thr Thr Cys

50 55 60

Cys Ala Cys Gly Ala Cys Thr Ala Thr Ala Gly Gly Ala Cys Ala Gly

65 70 75 80

Cys Thr Thr Thr Cys Gly Thr Cys Gly Ala Ala Ala Gly Gly Thr Thr

85 90 95

Thr Thr Gly Cys Thr Cys Ala Thr Gly Thr Gly Ala Thr Cys Thr Cys

100 105 110

Gly Ala Ala Gly Gly Ala Thr Thr Cys Ala Thr Cys Thr Ala Gly Thr

115 120 125

Cys Thr Gly Ala Thr Thr Thr Thr Thr Cys Gly Thr Gly Ala Cys Thr

130 135 140

Thr Gly Thr Ala Thr Cys Gly Gly Thr Thr Thr Thr Ala Thr Thr Gly

145 150 155 160

Gly Ala Thr Thr Cys Ala Thr Cys Cys Ala Ala Cys Ala Thr Ala Thr

165 170 175

Ala Thr Cys Ala Ala Thr Ala Ala Ala Ala Ala Ala Thr Gly Ala Gly

180 185 190

Thr Thr Gly Thr Gly Thr Thr Thr Cys Cys Thr Thr Thr Cys Thr Thr

195 200 205

Cys Cys Thr Ala Gly Thr Thr Cys Ala Gly Thr Thr Ala Ala Ala Ala

210 215 220

Thr Thr Ala Thr Thr Thr Cys Cys Cys Thr Cys Cys Thr Gly Cys Gly

225 230 235 240

Cys Thr Thr Gly Thr Gly Cys Thr Gly Thr Ala Ala Thr Thr Gly Thr

245 250 255

Cys Thr Gly Thr Gly Thr Ala Cys Cys Thr Gly Thr Thr Gly Thr Thr

260 265 270

Thr Gly Thr Gly Ala Cys Thr Gly Thr Gly Thr Thr Ala Gly Thr Thr

275 280 285

Cys Cys Cys Thr Thr Gly Gly Ala Thr Ala Thr Gly Ala Thr Thr Thr

290 295 300

Cys Gly Thr Ala Thr Thr Thr Gly Ala Thr Ala Thr Gly Thr Ala Cys

305 310 315 320

Ala Thr Gly Gly Ala Gly Ala Thr Ala Gly Cys Thr Thr Ala Gly Cys

325 330 335

Thr Thr Cys Ala Thr Thr Ala Thr Thr Gly Gly Ala Gly Thr Ala Thr

340 345 350

Gly Ala Ala Gly Thr Thr Ala Gly Thr Ala Thr Gly Ala Cys Ala Thr

355 360 365

Ala Gly Thr Cys Ala Cys Thr Cys Thr Cys Cys Thr Gly Gly Ala Ala

370 375 380

Ala Ala Thr Thr Gly Ala Cys Ala Cys Thr Gly Cys Ala Ala Ala Cys

385 390 395 400

Cys Ala Thr Ala Thr Thr Thr Thr Thr Ala Thr Thr Cys Thr Gly Ala

405 410 415

Ala Cys Cys Ala Cys Ala Ala Ala Thr Cys Cys Thr Ala Gly Thr Cys

420 425 430

Ala Gly Thr Cys Cys Gly Cys Thr Gly Gly Cys Ala Thr Ala Thr Gly

435 440 445

Cys Cys Gly Thr Cys Cys Gly Thr Thr Thr Gly Cys Thr Gly Ala Ala

450 455 460

Thr Cys Cys Ala Gly Ala Ala Cys Gly Thr Gly Gly Gly Thr Thr Thr

465 470 475 480

Gly Gly Ala Gly Ala Thr Gly Thr Ala Cys Gly Gly Cys Thr Gly Ala

485 490 495

Gly Ala Thr Gly Cys Cys Thr Cys Thr Ala Thr Gly Cys Gly Ala Ala

500 505 510

Gly Gly Gly Gly Ala Thr Thr Thr Cys Gly Thr Gly Gly Thr Gly Ala

515 520 525

Ala Ala Cys Gly Ala Gly Ala Thr Gly Gly Gly Ala Gly Thr Ala Gly

530 535 540

Ala Gly Cys Ala Ala Cys Gly Cys Cys Cys Gly Thr Gly Gly Ala Ala

545 550 555 560

Gly Ala Thr Gly Cys Thr Thr Cys Ala Ala Ala Cys Thr Thr Cys Cys

565 570 575

Ala Cys Ala Cys Thr Thr Thr Thr Gly Ala Gly Cys Ala Ala Cys Gly

580 585 590

Ala Thr Cys Gly Gly Cys Ala Gly Thr Ala Gly Thr Ala Ala Gly Gly

595 600 605

Thr Ala Gly Ala Cys Gly Ala Thr Thr Thr Cys Ala Ala Gly Ala Thr

610 615 620

Cys Ala Ala Ala Gly Cys Ala Thr Ala Thr Gly Ala Ala Gly Ala Thr

625 630 635 640

Ala Ala Ala Cys Ala Ala Cys Ala Thr Cys Ala Ala Cys Ala Ala Cys

645 650 655

Ala Ala Ala Ala Thr Thr Thr Gly Thr Thr Gly Gly Gly Gly Thr Thr

660 665 670

Cys Thr Ala Thr Ala Gly Ala Gly Ala Gly Ala Ala Ala Cys Ala Gly

675 680 685

Ala Gly Cys Thr Ala Cys Ala Thr Ala Cys Ala Thr Ala Cys Ala Cys

690 695 700

Thr Gly Thr Thr Thr Thr Gly Thr Ala Thr Cys Thr Ala Cys Cys Ala

705 710 715 720

Thr Cys Thr Gly Ala Gly Ala Thr Gly Ala Thr Gly Ala Ala Ala Ala

725 730 735

Gly Ala Thr Gly Ala Ala Ala Ala Ala Cys Thr Ala Ala Ala Gly Ala

740 745 750

Ala Thr Gly Cys Cys Cys Cys Gly Gly Cys Gly Cys Cys Ala Ala Cys

755 760 765

Gly Cys Cys Ala Gly Gly Ala Cys Ala Cys Gly Cys Cys Gly Cys Gly

770 775 780

Cys Gly Cys Gly Cys Gly Thr Cys Ala Cys Cys Cys Gly Ala Gly Cys

785 790 795 800

Cys Ala Thr Cys Thr Cys Thr Thr Gly Ala Cys Cys Cys Ala Gly Cys

805 810 815

Cys Gly Gly Cys Gly Cys Thr Gly Thr Ala Thr Ala Thr Thr Thr Ala

820 825 830

Cys Ala Cys Ala Cys Gly Thr Thr Gly Cys Ala Gly Cys Ala Thr Cys

835 840 845

Gly Ala Thr Cys Ala Cys Cys Ala Cys Cys Thr Gly Thr Thr Cys Gly

850 855 860

Ala Thr Cys Gly Cys Gly Thr Cys Gly Cys Cys Gly Thr Cys Ala Cys

865 870 875 880

<210> 10

<211> 1096

<212> DNA

<213> Zea mays

<400> 10

aggcgaccca tcgctgcttt gtctacatca tgttcttcat catcctcccc aggcgacgcg 60

tgctgctgtt cttattcaga ctaccgttcg agtgactgca tggcgtacat ctttctgcat 120

cgactttgta cggctacatc gaacatatac acgagatgtc tcgtgtgaat agagtcacta 180

atgccttaag catcggttac tccgtagggt acattctgtt cttcttattt gtgcatattt 240

ttattgttgt ttactgatta tacgagtagt tatacataca tgcacataca tatcatcaca 300

tatatcacaa tatttttcta aattaaatta aaactaaaaa tgactaaatt tctaacacca 360

acgacattgt aatgttttct ccaacaactt tacctattct acattgttct atttcgaatt 420

tcactctata aacaacatag tctacaatgg aaaacagtgc tttgtacgac tatatacgcg 480

atgtgtggct acaacataag acaatatagt cgtttgaaga ttgaacctat atatcggtac 540

ggttaatccg tctatgtacg tgggcatgac gaacacccgt gataacgaag gattaacgtg 600

cacaatcata aatccaaagt aggagcggtg catgatgaga atcgctctca gtactcgaca 660

taatgaacct tacgaggtac aacaggcagg caggcaggga ccaggggccg cctttatttc 720

aggctcgctg gccccacggg cgtgctgcgt gcacgaaggg cactacccca acctctcacc 780

gaaaaccgcg ctggatcggc aaatcaaacg aggtggtgcc ccgtgcccac tctccacgtc 840

cacggcacca tccctctgca gccgctcacc agccatgccg tgtcgcggaa cggcacaacc 900

acccccaacc cactcacgaa accccgtccc ggccgtgccc gtgtcggtcc gcgctcggca 960

acgaggcggc ccgcgctgct gagtcccctg gacacccgac accctgtcgg ccctttgttt 1020

attcatcccg aaatctcatc tgcccccacg gccgactgcg ctgcgccgcc cggatatata 1080

tacccatcgt tatcga 1096

<210> 11

<211> 1096

<212> DNA

<213> Zea mays

<400> 11

aggcgaccca tcgctgcttt gtctacatca tgttcttcat catcctcccc aggcgacgcg 60

tgctgctgtt cttattcaga ctaccgttcg agtgactgca tggcgtacat ctttctgcat 120

cgactttgta cggctacatc gaacatatac acgagatgtc tcgtgtgaat agagtcacta 180

atgccttaag catcggttac tccgtagggt acattctgtt cttcttattt gtgcatattt 240

ttattgttgt ttactgatta tacgagtagt tatacataca tgcacataca tatcatcaca 300

tatatcacaa tatttttcta aattaaatta aaactaaaaa tgactaaatt tctaacacca 360

acgacattgt aatgttttct ccaacaactt tacctattct acattgttct atttcgaatt 420

tcactctata aacaacatag tctacaatgg aaaacagtgc tttgtacgac tatatacgcg 480

atgtgtggct acaacataag acaatatagt cgtttgaaga ttgaacctat atatcggtac 540

ggttaatccg tctatgtacg tgggcatgac gaacacccgt gataacgaag gattaacgtg 600

cacaatcata aatccaaagt aggagcggtg catgatgaga atcgctctca gtactcgaca 660

taatgaacct tacgaggtac aacaggcagg caggcaggga ccaggggccg cctttatttc 720

aggctcgctg gccccacggg cgtgctgcgt gcacgaaggg cactacccca acctctcacc 780

gaaaaccgcg ctggatcggc aaatcaaacg aggtggtgcc ccgtgcccac tctccacgtc 840

cacggcacca tccctctgca gccgctcacc agccatgccg tgtcgcggaa cggcacaacc 900

acccccaacc cactcacgaa accccgtccc ggccgtgccc gtgtcggtcc gcgctcggca 960

acgaggcggc ccgcgctgct gagtcccctg gacacccgac accctgtcgg ccctttgttt 1020

attcatcccg aaatctcatc tgcccccacg gccgactgcg ctgcgccgcc cggatatata 1080

tacccatcgt tatcga 1096

<210> 12

<211> 816

<212> DNA

<213> Zea mays

<400> 12

agcaacgcga gctgccactg ctcttcactg gtaccgttaa cagatcaatt cgacaaagca 60

gcattagtcc gttgatcggt ggaagaccac tcgtcagtgt tgagttgaat gtttgatcaa 120

taaaatacgg caatgctgta agggttgttt tttatgccat tgataataca ctgtactgtt 180

cagttgttga actctatttc ttagccatgc caagtgcttt tcttattttg aataacatta 240

cagcaaaaag ttgaaagaca aaaaaaaaaa cccccgaaca gagtgctttg ggtcccaagc 300

ttctttagac tgtgttcggc gttcccccta aatttctccc cctatatctc actcacttgt 360

cacatcagcg ttctctttcc ccctatatct ccacgctcta cagcagttcc acctatatca 420

aacctctata ccccaccaca acaatattat atactttcat cttcaactaa ctcatgtacc 480

ttccaatttt tttctactaa taattattta cgtgcacaga aacttagcaa ggagagagag 540

agcggggtga cccaccttgc tagttggata ttacctcttc tcttcaaagt atccttgaac 600

gctcaccggt tatcaaatct ctacactata gctctgtagt cttgctagat agttagttct 660

ttagctctcg gtgaccaagc ttggcgcgat caagcttatc gataccgtcg acctcgaagc 720

ttggtcaccc ggtccgggcc tagaaggcca gcttcaagtt tgtacaaaaa agcaggctcc 780

ggccagaatg gcccggaccg ggttaccgaa ttctta 816

Claims (7)

1. A combination of proteins, wherein the combination of proteins comprises three proteins, bbm, wus2 and Barnase;

wherein the Barnase protein is expressed by an expression cassette comprising a PhotosystemII 10kDa promoter linked to a nucleic acid molecule encoding the Barnase protein.

2. The protein combination of claim 1, wherein the expression cassette further comprises a nos terminator.

3. The combination of proteins of claim 1, wherein the Bbm protein is expressed from an expression cassette comprising a pZmPLTP promoter linked to a nucleic acid molecule encoding a Bbm protein linked to a tT28 terminator and the Wus2 protein is expressed from an expression cassette comprising a pZmAXIG1 promoter linked to a nucleic acid molecule encoding a Wus2 protein linked to a tIN-1 terminator.

4. An expression vector comprising the following expression cassette:

(1) The Photosystem II 10kDa promoter is linked to a nucleic acid molecule encoding a Barnase protein linked to a nos terminator;

(2) The pZmPLTP promoter is linked to a nucleic acid molecule encoding a Bbm protein linked to the tT28 terminator;

(3) The pZmAXIG1 promoter is linked to a nucleic acid molecule encoding a Wus protein linked to a tIN-1 terminator.

5. A host cell comprising the expression vector of claim 4.

6. A method of genetic transformation, characterized in that maize or wheat is transformed with the expression vector of claim 4 together with an additional expression vector or the host cell of claim 5 together with an additional host cell to obtain a regenerated maize or wheat plant;

wherein the additional expression vector contains an HTP gene expression cassette and a GFP gene expression cassette, or contains an expression cassette for editing the wax gene and a bar gene expression cassette;

wherein the additional host cell is a host cell containing an HTP gene expression cassette and a GFP gene expression cassette, or containing an expression cassette for editing the wall gene and a bar gene expression cassette.

7. Use of any one of the protein combinations of claims 1-3, the expression vector of claim 4, the host cell of claim 5, the method of claim 6 for increasing the genetic transformation efficiency of maize or wheat.