TWI686477B - Cloning vector, kit, and method for specifically inducing mutagenesis in chloroplast genes, and transgenic plant cells and agrobacterium generated by the same - Google Patents

Abstract

The present application provides a method for generating chloroplast gene modified plant cells, comprising: constructing a pair of nuclear gene modifying vectors; transforming the pair of nuclear gene modifying vectors into plant cells via Agrobacterium; and selecting and culturing the plant cells. The nuclear gene modifying vectors comprises a promoter, a recombinant gene conferred the chloroplast transit peptide and TALEN protein domain, a terminator as well as right border and left border of T-DNA regions. Thus, by using nuclear transformation to mutate chloroplast gene, the problems such as low transformation efficiency, long selection time, labor-intensive and high cost encountering in chloroplast transformation would be avoided.

Description

Transplantation vector, set and method for specifically causing plant chloroplast gene variation, and transgenic plant cells and Agrobacterium produced using the same

The invention relates to a plant gene transfer vector and method, in particular to a plant cell nuclear gene transfer vector and method, which can be used to cause chloroplast gene mutation.

Traditionally, to silence, modify and edit plant chloroplast genes, it is necessary to construct a chloroplast expression vector containing the gene sequence to be modified first, and then use the gene gun bombardment method to introduce the expression vector into the chloroplast and replace it by homologous recombination. The original gene sequence (Bock, 2015). However, the technical threshold of the gene gun bombardment method is high, which requires trained, experienced technicians and expensive instruments to operate and execute, and the cost of the gene gun consumables is high, and the efficiency of chloroplast transformation is low. In addition, since a plant cell contains tens to hundreds of chloroplasts, and a chloroplast also contains tens or hundreds of genomes, the time required for the screening process by the gene gun bombardment method is lengthy.

In addition to the gene gun bombardment method, PEG (polyethylene glycol) vector method can also be used for chloroplast gene transformation. It has also been reported that expression vectors can be successfully introduced into chloroplasts of a few plants via protoplasts (Bock, 2015). Although this method does not require expensive equipment, it requires the preparation of protoplasts, and the protoplasts are fragile and difficult to handle, and plants need to be regenerated from the protoplasts after transplantation. Although the cost of consumables is low, the threshold of protoplast regeneration technology is high, and the time required for the regeneration process is very long. Most plants have not yet established this technology, and currently only a few plants are feasible. In addition, the PEG-mediated method may also produce polyploid cells, low colonization efficiency, and lengthy screening process.

All in all, the existing chloroplast gene transfer technology still has many shortcomings and problems to be overcome and improved, including high threshold, low transfer efficiency, lengthy screening process, and a lot of manpower and material resources. In addition, because the chloroplast genome is small and the gene density is high, the foreign genes are generally inserted into the genome by homologous recombination. Compared with the method of randomly inserting the nuclear genome into the nuclear genome, the efficiency of chloroplast transformation is low. a lot of.

In view of the above problems of the prior art, the object of the present invention is to provide a nuclear gene transfer vector, set and method to cause chloroplast gene mutation, which can effectively improve the traditional chloroplast gene transfer efficiency, low screening time, and cost Human and material issues.

According to an object of the present invention, a nuclear gene transfer vector is proposed, which includes: a promoter; a chloroplast signaling protein gene, which is located downstream of the promoter; and a recombinant TALEN protein domain, which includes SEQ ID NO: 21 or SEQ ID NO: 22 The sequence shown; and the first and second border regions, such as the left and right border regions of T-DNA. The promoter, the chloroplast signaling protein gene, and the recombinant TALEN protein domain are sequentially located between the first border region and the second border region.

Preferably, the promoter may be the rbcS promoter.

Preferably, the recombinant TALEN protein domain may include DNA sequences encoding DNA recognition proteins and endonuclease. More preferably, the endonuclease can be Fok I, and the DNA recognition protein can recognize a fragment of the chloroplast rpoB gene. Most preferably, the DNA sequence encoding the DNA recognition protein may include the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

Preferably, the transfection vector may further include a screening gene, and the screening gene may be a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. More preferably, the screening gene may include a kanamycin (kanamycin) resistance gene or a methionine sulfoximine (MSO) herbicide resistance gene. Most preferably, the kanamycin resistance gene includes nptII or the methionine sulfonimide herbicide resistance gene includes bar .

Preferably, the transfer vector may further include a sequence encoding a marker. More preferably, the marker may comprise a molecule selected from the group consisting of luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescent quenchers, lipids, colored molecules, radioisotopes, scintillators, biotin, avidin, streptavidin , Protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tag, myc tag, HA tag, and detectable markers in the group consisting of enzymes.

Preferably, the transfer vector may comprise the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

Preferably, the transfection vector may further include a 3-terminal non-transcribed region of the rbcS gene, which is located downstream of the recombinant TALEN protein domain.

According to another object of the present invention, a kit for specifically causing plant chloroplast gene variation is proposed, which includes: a pair of the aforementioned nuclear gene transfer vectors. Preferably, the kit may include a first transgenic vector and a second transgenic vector. The first transfer vector includes: a first promoter; a first chloroplast information protein gene, which is located downstream of the first promoter; a first recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 21; and a first border Region and the second boundary region, in which the first promoter, the first chloroplast signaling protein gene and the first recombinant TALEN protein domain are sequentially located between the first boundary region and the second boundary region. The second transfer vector includes: a second promoter; a second chloroplast information protein gene, which is located downstream of the second promoter; a second recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 22; and a third border Region and the fourth border region, in which the second promoter, the second chloroplast signaling protein gene and the second recombinant TALEN protein domain are sequentially located between the third border region and the fourth border region.

According to yet another object of the present invention, a method for generating nuclear gene transplantation of plant cells and causing chloroplast gene mutations is provided, comprising: constructing a pair of nuclear gene transfection vectors as described above; transfecting the pair of nuclear genes through Agrobacterium The vector is incorporated into the plant cell; and the plant cell is selected and cultured.

Preferably, the incorporation may include homology-directed repair mechanisms or non-homologous end-joint-directed repair mechanisms.

Preferably, the method may further include transferring the nuclear gene transfer vector to Agrobacterium.

Preferably, the method may further include analyzing the plant cell and confirming that the nuclear gene transfer vector is incorporated into the plant cell.

According to another object of the present invention, a nuclear gene transgenic plant cell is proposed, which is constructed by the aforementioned method.

According to another object of the present invention, an Agrobacterium is proposed, which includes the aforementioned nuclear gene transfer vector.

In summary, the method of causing chloroplast gene mutation by expressing recombinant TALEN protein in the cell nucleus can at least solve the aforementioned problem of traditional chloroplast gene transfer and provide at least the following advantages:

(1) Improve the efficiency of transformation and solve the problem of high threshold of traditional chloroplast gene transformation technology.

(2) Due to the use of nuclear gene transfer, the screening time is shorter.

(3) The cost of using the Agrobacterium-mediated method for nuclear gene transfer is low, so there is no need for expensive equipment and preparation of protoplasts.

The above and other objects, features, and advantages of the invention claimed in this case will become apparent after referring to the following detailed description and preferred embodiments and accompanying drawings.

The detailed description of each specific example of the present invention is as follows. Other features of the present invention will be clearly presented through the detailed description and patent application scope in the following specific examples.

Without further elaboration, Xian believes that those with ordinary knowledge in the technical field to which the present invention belongs can use the present invention to the fullest extent based on the foregoing description. Therefore, it can be understood that the following description is only for illustrative purposes and does not limit the remaining disclosure in any way.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs.

Unless otherwise stated, all percentages, parts, ratios, etc. are by weight.

As used herein, the term "produced from" is synonymous with "comprising". As used herein, the terms "includes", "includes", "comprises", "has, having", "contains", or any other variants of It is intended to cover non-exclusive inclusions. For example, a composition, process, method, product, or device containing a plurality of elements listed in the list is not necessarily limited to those elements listed on the list, but may include the composition not specifically listed, but Other elements inherent in the process, method, product or device. The term "comprising" is generally used to mean including, which means that one or several other features or components are allowed to exist.

The purpose of the present application is to provide a nuclear gene transfer vector which can be transferred to the plant cell nucleus through Agrobacterium or other methods known to those of ordinary skill in the technical field of the present invention and transcribed by the cell nucleus, and can be transferred to the cytoplasm Translation and synthesis of recombinant TALEN protein. The recombinant TALEN protein can be guided into the chloroplast matrix through the transmission channel (Toc-Tic translocon) of the chloroplast membrane through the guidance of the N-terminal signaling protein, and then the recombinant TALEN protein can specifically bind to the specific location of the target chloroplast DNA. The recombinant TALEN protein is then endonuclease (such as Fok I) to cause the double-stranded chloroplast DNA to break, and then start the repair mechanism of chloroplast DNA, including homologous-oriented repair mechanism (homologous recombination, HR) or different Non-homologous end joining (NHEJ). When the repair mechanism of non-homologous recombination of chloroplast DNA is used, the target gene is often mutated, which will in turn cause the target gene to lose activity.

In one embodiment, as shown in FIG. 1, a nuclear gene transfer vector is provided, including: a promoter; a chloroplast signaling protein gene (TP), which is located downstream of the promoter; a recombinant TALEN protein domain (TALEN), It is located downstream of the chloroplast signaling protein gene; and T-DNA left border region (LB-T border) and T-DNA right border region (RB-T border), in which the promoter, chloroplast signaling protein gene and recombinant TALEN protein domain are in order Located between the left border area of T-DNA and the right border area of T-DNA.

In one embodiment, the nuclear gene transfer vector may have the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the nuclear gene transfer vector is named pTpTalen-L or pTpTalen-R.

It is worth noting that those with ordinary knowledge in the technical field of the present invention can design the sequence of the recombinant TALEN protein domain according to the region to be recognized or cleaved by the desired TALEN protein. For other elements in the transfection vector, such as promoters, selection markers, terminators, etc., those with ordinary knowledge in the technical field to which the present invention belongs may choose suitable elements to replace according to experimental needs.

In one embodiment, a method for producing nuclear gene transgenic plant cells is provided, comprising: constructing a pair of the aforementioned nuclear gene transfection vectors; incorporating the pair of nuclear gene transduction vectors into plant cells through Agrobacterium; and Screen and cultivate plant cells.

In yet another embodiment, through the aforementioned method, a nuclear gene transgenic plant cell or a nuclear gene transgenic plant can be provided, or an Agrobacterium comprising the aforementioned nuclear gene transgenic vector can be provided.

The terms "plant", "plant cell" and "recombinant plant cell" referred to herein may be used interchangeably with each other herein, which may refer to plant cells of dicotyledonous plants, monocotyledonous plants, gymnosperms, etc., such as tobacco, Arabidopsis, potato, soybean, tomato, orchid, rice, corn, wheat, barley, sorghum, sugarcane, etc. One or several vectors described in this case can be introduced into the plant cell or the vector to be inserted into the gene to be expressed by genetic engineering methods. As those of ordinary knowledge in the technical field to which the present invention is understood, the above term means not only a specific cell, but also a progeny or a potential progeny of the cell. Because of genetic mutations or environmental influences, there may be changes in subsequent generations, so the descendants of these cells will not be substantially the same as the cells of the parent, but they are still included in the scope of the term used herein.

The term "chloroplast signaling protein gene" as used herein refers to a nucleotide sequence that can be synthesized by transcription of the nucleus and translation of the cytoplasm. This information protein will appear at one end of the protein, such as the N-terminus. This information protein contains the guiding information needed to transport the protein to the chloroplast. Like the "address tag", this information protein can direct the protein to the surface of the chloroplast. On the surface of the chloroplast, there is a special transmission channel (Toc-Tic translocon), which can recognize this "address label". After recognition, the receiver will transfer the protein containing the correct "address label" into the chloroplast matrix. After entering the chloroplast matrix, this extra piece of signal protein will be excised.

The "screening gene" referred to herein is used to confirm whether the aforementioned vector has successfully transformed into the host. The aforementioned screening gene may be, but not limited to, a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. In a feasible embodiment, the aforementioned screening gene may be a drug resistance screening gene. For example, the aforementioned drug resistance screening gene may be a kanamycin or neomycin resistance gene. In this feasible embodiment, a plant cell (for example, tobacco or Arabidopsis) that has been transformed with the aforementioned vector can develop resistance to kanamycin or neomycin, and can survive in the presence of kanamycin. Or neomycin environment.

The term "non-drug resistance screening gene" as used herein refers to a gene that has not been confirmed by the resistance to antibiotics. The aforementioned non-drug resistance screening genes are, for example but not limited to, Phosphhinothricin N-acetyltransferase gene ( bar or pat ), which can be used as a screening gene to include the herbicide-containing components glufosinate or methionine sulfonamide (Methionine sulfoximine; MSO) and other compounds are acetylated to remove their toxicity, so herbicides such as glufosinate and other compounds can be used to screen transgenic plants. In addition, mutations or excessive performance of the 5‐enolpyruvoyl‐shikimate‐3‐phosphate synthetase (EPSPS) gene may not be inhibited by herbicides containing Glyphosate; N‐(phosphonomethyl)glycine), so it can also be used as a screening gene Utilizing herbicides such as glufosinate to screen transgenic plants.

In a preferred embodiment, the aforementioned non-drug resistance screening genes are nutritional deficiency screening genes (eg, cytosine deaminase ( codA )), sugar metabolism genes (eg, phosphomannose isomerase (manA) ; xylose isomerase ( xylA )), Plant hormone related genes (eg, isopentenyltransferase (ipt) ), heavy metal resistance genes (eg, cadmium resistance genes), D-amino acid racemase (eg, alanine racemase) genes, heat shock proteins (heat shock) protein) gene or reporter gene (eg, β-glucuronidase ( GUS ); luciferase ( LUC ) ; fluorescent proteins ( FPs) ).

The terms "incorporated", "incorporated", or "sending nucleic acid into a cell" as used herein mean using any method to make the extracellular nucleic acid (such as a carrier) with or without accompanying substances (such as another A vector or oligonucleotide) into the host cell. Terms such as "cell incorporation" or "incorporated cells" refer to nucleic acids that are delivered outside the cell in the plant cell or its progeny cells, so the plant cell contains the nucleic acid outside the cell. The nucleic acid is fed into the cell, so that the nucleic acid can be combined with the chromosome in the form of fragments or replicated in the form of extrachromosomal elements, or the nucleic acid disrupts the expression of endogenous genes in the cell.

In one embodiment, there are 兩 ways to force the vector containing the foreign gene into Agrobacterium, namely electroporation and three-parent hybridization. Electroporation is the method of electroporation of Agrobacterium to produce a temporary through hole for the carrier to enter Agrobacterium. The three-parent hybridization method is to cultivate E. coli containing special plastids (referred to as helper strains) together with E. coli containing exogenous genes, and three strains of Agrobacterium containing helper plastids 類, a carrier containing exogenous genes Will enter the Agrobacterium containing helper plastids. In one embodiment, the Agrobacterium containing the foreign gene vector is further used to deliver the foreign gene into a target cell, such as a plant cell.

The indefinite articles "a" and "an" before the element or component in this article are intended to illustrate without limitation the number of instances (ie the number of occurrences) of the element or component. Therefore, "a" or "an" should be understood as including one or at least one, and the singular form of the element or component also includes the plural unless the number obviously refers to the singular.

The materials, methods, and examples herein are illustrative only, and unless specifically stated otherwise, are not intended to limit the invention. Although methods or materials similar or equivalent to those described herein can be used to practice or test the present invention, those described herein are more suitable methods and materials.

Construction of transcription-like activator nuclease ( Transcription activator-like effector nuclease; TALEN )

TALEN is formed by the DNA recognition binding domain and endonuclease active region of artificial recombination transcription activator-like effector (TALE). TALE is a protein secreted by the plant Xanthomonas bacteria. This protein contains a nuclear entry signal (NLS) and transcription activator, which can help the TALE protein enter the nucleus from the cytoplasm and then specifically bind to the promoter of the host gene. , Which in turn activates transcription.

The middle area of TALE is a tandem repeating area composed of multiple tandem repeating units. Each tandem repeating unit generally contains 34 amino acids, of which the amino acids at positions 12 and 13 have high variability and are called repeatable variable District (repeat-variable diresidue, RVD). The RVD in each tandem repeat unit can identify different bases of DNA, and there is a simple one-to-one correspondence. For example, if RVD is NI (Asn-Ile) can recognize A, HD (His-Asp) can recognize C, NG (Asn-Gly) can recognize T, and NN (Asn-Asn) can recognize G/A.

On the other hand, the active region of the endonuclease can be Fok I endonuclease, and Fok I endonuclease needs to form a binary body to cut DNA. Therefore, it is currently possible to synthesize a pair of TALENs to match any desired DNA sequence and specifically use it to cut its double-stranded DNA (Cermak et al., 2011).

In one embodiment, the user can design the recombinant TALEN protein domain according to his target gene, so that the repeated variable region in the recombinant TALEN protein domain can recognize the target nucleic acid sequence. For example, the recombinant TALEN protein domain may include the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, wherein the repeating variable region is represented by "NNNNNN", that is, N may be selected from A, T, C And any one of G, so that the repeated variable regions shown are NI (Asn-Ile) recognizable A, HD (His-Asp) recognizable C, and NG (Asn-Gly) recognizable T, Or the NN (Asn-Asn) of G or A can be recognized. In one embodiment, a pair of nuclear gene transfer vectors are designed. As shown in FIG. 1, Talen-L and Talen-R encoded in pTpTalen-L and pTpTalen-R are a pair of TALEN genes. In one embodiment, the sequences of Talen-L and Talen-R in pTpTalen-L and pTpTalen-R are designed so that the translated protein can recognize the chloroplast rpoB gene sequence of Arabidopsis or tobacco.

design TALEN Target sequence to act on chloroplast gene

In this example, taking the tobacco chloroplast rpoB gene as an example, Talen-L and Talen-R are designed according to their sequences. The rpoB gene is located in the operational genome (Operon) of rpoB-rpoCl-rpoC2 of chloroplast DNA (Little et al., 1988; Inada et al., 1996; Hajdukiewicz et al., 1997). According to previous research, if the rpoB gene is removed from the chloroplast DNA of tobacco, although RNA synthetase encoded by the nucleus still maintains part of the chloroplast function, it will not cause plant death, but it will promote tobacco to form a white appearance. Growth and development are also slower than normal tobacco plants (Allison et al., 1996). Because it is easier to observe, the rpoB gene was selected as the target gene for chloroplast gene editing of TALENs.

First, download the rpoB gene sequences (Accession numbers NC_000932.1 and NC_001879.2, respectively) from Arabidopsis and Tobacco from the NCBI database, and perform sequence alignment to extract a sequence with high common retention and use TAL Effector Nucleotide Targeter 2.0 (https://talent.cac.corne-ll.edu/node/add/talen) The goal of TALENs for web design. In one embodiment, the target DNA sequences identified by Talen-L and Talen-R are 5'-TCCAAAAATTGAA-3' (SEQ ID NO: 11) and 5'-TAATTGAAATTCA-3' (SEQ ID NO: 12).

Constructing nuclear reorganization TALEN Gene expression vector

Based on the aforementioned target DNA sequence, a pair of target DNA sequences SEQ ID NO: 11 and SEQ ID NO: 12 located in the chloroplast rpoB gene were synthesized and constructed using the Golden Gate TALEN assembly kit (Cermak et al., 2011). TALEN gene, so that the RVD sequences of the repeated variable regions that are translated are N'-HD-HD-NI-NI-NI-NI-NI-NG-NG-NN-NI-NI-C' and N'-NI-NI-NG-NG-NN-NI-NI-NI-NG-NG-HD-NI-C', which can specifically recognize and bind to the above target DNA sequences SEQ ID NO: 11 and SEQ ID NO: 12 . In one embodiment, according to the instructions of the Golden Gate TALEN assembly kit, the construction of the aforementioned RVD sequence needs to be divided into the front section and the rear section, which are named pTALEN-L1, pTALEN-L2, pTALEN-R1, and pTALEN-R2, and The RVD sequences are N'-HD-HD-NI-NI-NI-NI-NI-C', N'-NI-NG-NG-NG-NN-NI-C', N'-NI-NI-NG-NG- NN-NI-C' and N'-NI-NI-NG-NG-HD-C'.

Next, the nuclear expression vector of the recombinant TALEN gene was constructed using the aforementioned pTALEN-L1, pTALEN-L2, pTALEN-R1, and pTALEN-R2. In short, the synthesized TALEN gene fragment was recombined into the plant expression vector pImpactVector 4-1, and the final composition of the gene expression cassette was rbcS chloroplast signaling protein and HA tag or Flag tag were recombined at the N-terminus of TALEN, and then rbcS Gene promoter to regulate the performance of recombinant TALEN. Then, the gene expression cassettes were recombined into the Agrobacterium double-coupled vector pBinPlus (containing the nptII gene as a selection marker) or pZGB9R (containing the bar gene as a selection marker), and pTpTalen-R and pTpTalen-L nuclear expression vectors were obtained respectively ,As shown in Figure 1. The experimental steps are described in detail below:

Construction of pTALEN-L1 and pTALEN-R1 vectors:

Using primers to pFUSA-M6-R (AGCGGCGCCGGTCTCGATAGCC; SEQ ID NO: 13) and pFUS-A-Del-F (AGCAAGCGGGTCTCACGGTGCAGCGGCTGTTG; SEQ ID NO: 14) PCR amplification using pFUS-A vector as a template, resulting in 2,573 bp DNA fragment. The PCR amplified fragment of pFUS-A was cut with restriction enzyme BsaI, and then separated by 1% agar electrophoresis, and a 2,560 bp DNA fragment was purified. Reuse primer pairs pTC14-F3 (TCTGATGTTACATTGCACAAGA; SEQ ID NO: 15) and pTC14-R3 (TGAACGCTCTCCTGAGTAGG; SEQ ID NO: 16) with Module plasmid vectors (pTALEN-L1: pHD1, pHD1, pHD2, pNI3, pNI4, pNI5, pNI6; pTALEN-R1: pNI1, pNI2, pNG3, pNG4, pNN5, pNI6) PCR amplification and quantification for each template. After mixing the PCR amplified fragments of Module plasmid, it is cut with restriction enzyme BsaI, and then separated by 2% agarose electrophoresis, and 79～100 bp DNA fragments are purified. The PCR product of Module plasmid was cut by BsaI DNA fragment and the PCR product of pFUS-A was cut by BsaI DNA fragment, and then the adhesion reaction was carried out. After transforming E. coli, and then screening with 100mg/L Spectinomycin, 3,087 bp pTALEN-L1 and 3,087 bp pTALEN-R1 vectors were obtained.

Construction of pTALEN-L2 and pTALEN-R2 vectors:

PFUS-B5 was cut with restriction enzyme BsaI, then separated by 1% agar electrophoresis, and 2,557 bp DNA fragments were purified. Using primer pairs pTC14-F3 (TCTGATGTTACATTGCACAAGA; SEQ ID NO: 15) and pTC14-R3 (TGAACGCTCTCCTGAGTAGG; SEQ ID NO: 16) with Module plasmid vectors (pTALENL2: pNI1, pNG2, pNG3, pNN4, pNI5; pTALEN-R2: pNI1 , PNI2, pNG3, pNG4, pHD5) are the templates for PCR amplification. After mixing the amplified PCR fragments of the Module plasmid, it is cut with the restriction enzyme BsaI, and then separated by 2% agar electrophoresis, and 79 to 100 bp DNA fragments are purified. The PCR product of Module plasmid was cleaved by BsaI and the fragment of pFUS-B5 cleaved by BsaI to carry out the adhesion reaction. After transforming E.coli and then screening with 100 mg/L Spectinomycin, 3,007 bp pTALEN-L2 and 3,007 bp pTALEN-R2 vectors were obtained.

Construction of pTALEN-L carrier:

The pCS2TAL3-DD, pTALEN-L1 and pTALEN-L2 vectors were cut with the restriction enzyme Esp3I, and then separated by 1% agar electrophoresis, and 5,309 bp, 616 bp and 536 bp DNA fragments were respectively purified. The pLR-NI vector was then cut with restriction enzyme Esp3I, separated by 2% agar electrophoresis, and 89 bp DNA fragments were purified. The fragments of pCS2TAL3-DD, TALEN-L1, TALEN-L2, and pLR-NI cleaved by Esp3I were subjected to bonding reaction. After transforming Escherichia coli, it was screened with 100 mg/L Ampicillin to obtain 6,550 bp pTALEN-L vector.

Construction of pTALEN-R carrier:

The pCS2TAL3-RR, pTALEN-R1 and pTALEN-R2 vectors were cut with restriction enzyme Esp3I, and then separated by 1% agar electrophoresis, and 5,339 bp, 616 bp and 536 bp DNA fragments were purified respectively. The pLR-NI vector was then cut with restriction enzyme Esp3I, separated by 2% agar electrophoresis, and 89 bp DNA fragments were purified. The fragments of pCS2TAL3-DD, TALEN-L1, TALEN-L2, and pLR-NI cleaved by Esp3I were subjected to bonding reaction. After transforming Escherichia coli, it was screened with 100 mg/L Ampicillin to obtain 6,580 bp pTALEN-R vector.

Construction of plant tissue-specific expression vectors: the use of the rbcS (Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit) gene promoter from Chrysanthemum spp. (Outchkourov et al., 2003) to regulate gene expression, which can Initiate the expression of downstream genes in tissues that engage in photosynthesis. Therefore, constructing a vector containing the rbcS promoter includes pTpTalen-L and pTpTalen-R and other transition vectors in the process of constructing these two vectors.

Construction of pIV4-TL vector:

Using primer pairs TAL-BamHI-F (CATGGCTCCAAAGAAGGATCCTAAGGTA; SEQ ID NO: 17) and TAL-NsiI-R (GCACCCGTCAGTGCATTGC; SEQ ID NO: 18), using pTALEN-L as a template for PCR amplification, and then use the restriction enzyme Nsi I and BamH I cut the PCR fragment and separated it with 1% agarose electrophoresis. Take 460 bp DNA fragment for purification. At the same time, pTALEN-L and ImpactVector ^TM 1.4 were cut with restriction enzymes Not I/ Nsi I and Not I/ BamH I, respectively, and separated by 1% agar electrophoresis, and 2,233 bp and 4,906 bp DNA fragments were purified respectively. Then the DNA fragments of ImpactVector ^TM 1.4 (4,906 bp), pTALEN-L (2,233 bp) and pTALEN-L PCR product (460 bp) after restriction enzyme cleavage were subjected to the binding reaction. Screened with 100 mg/L Ampicillin, a 7,598 bp pIV4-TL vector was obtained.

Construction of pIV4-TR carrier:

Use primer pairs TAL-BamHI-F (CATGGCTCCAAAGAAGGATCCTAAGGTA; SEQ ID NO: 17) and TAL-NsiI-R (GCACCCGTCAGTGCATTGC; SEQ ID NO: 18), using pTALEN-R as a template for PCR amplification and then use the restriction enzyme Nsi I and BamH I cut the PCR amplified fragments and separated them by 1% agarose electrophoresis, and took 490 bp DNA fragments for purification. At the same time, pTALEN-R and ImpactVector ^TM 1.4 were cut with restriction enzymes Not I/ Nsi I and Not I/ BamH I, respectively, and separated by 1% agar electrophoresis, and 2,233 bp and 4,906 bp DNA fragments were purified respectively. Restriction enzyme cleaved ImpactVector ^TM 1.4 (4,906 bp), pTALEN-R (2,233 bp) and pTALEN-R PCR product (490 bp) DNA fragments were subjected to a binding reaction, after transforming E. coli, and then through 100 Screened with mg/L Ampicillin and obtained 7,628 bp pIV4-TR vector.

Construction of pZGB-IV4 carrier

Using primer pairs AscI-IV-F (TGGCGCGCCAGACAAACACCCCTTGTTATAC; SEQ ID NO: 19) and H3-PacI-IV-R (CATAAGCTTTTAATTAATGCGTCCACTATAGAC; SEQ ID NO: 20) using ImpactVector ^TM 1.4 as a template for PCR amplification and then using restriction enzymes HindIII and AscI cut the PCR amplified fragments and separated them by 1% agarose electrophoresis. The 2,293 bp DNA fragments were purified. At the same time, pZGB9R was cleaved with restriction enzymes Hind III and Asc I, and separated by 1% agarose electrophoresis, and 8,479 bp DNA fragments were purified. Then, the restriction enzyme cleaved pZGB9R (8,479 bp) and ImpactVector ^TM 1.4 PCR product (2,293 bp) DNA fragments were subjected to a binding reaction, after transforming Escherichia coli, and then screened with 100 mg/L Kanamycin to obtain 10,747 bp. pZGB-IV4 vector.

Construction of pTpTalen-L vector:

PIV4-TL and pZGB-IV4 were cut with restriction enzymes Asc I and Pac I, respectively, and separated by 1% agar electrophoresis, and 4,953 bp and 8,478 bp DNA fragments were purified respectively. Then pZGB-IV4 (8,478 bp) and pIV4-TL (4,953 bp) DNA fragments cleaved by restriction enzymes were subjected to a binding reaction. After transforming E. coli, they were screened with 100 mg/L Kanamycin to obtain pTpTalen of 13,435 bp. -L carrier. The repeat variable region sequence in the recombinant TALEN protein domain of the pTpTalen-L vector includes the nucleotide sequence shown in SEQ ID NO: 3.

Construction of pTpTalen-R vector:

The pIV4-TR and pBinPLUS were cut with restriction enzymes Asc I and Pac I, respectively, and separated by 1% agarose electrophoresis, and 4,983 bp and 12,328 bp DNA fragments were purified. Then, the restriction enzyme cleaved pBinPLUS (12,328 bp) and pIV4-TR (4,983 bp) DNA fragments were subjected to a binding reaction, and after transformation into E. coli, and then screened with 100 mg/L Kanamycin, the 17,311 bp pTpTalen-R was obtained. Carrier. The repeat variable region sequence in the recombinant TALEN protein domain of the pTpTalen-R vector includes the nucleotide sequence shown in SEQ ID NO: 4

Expression vector transfer

Transplant the expression vector to tobacco:

In short, the nuclear recombinant TALEN expression vectors constructed as described above were transferred into Agrobacterium by electroporation, respectively, and screened with appropriate antibiotics (kanamycin or hygromycin) to obtain transformed Agrobacterium containing different vectors. The pTpTalen-L and pTpTalen-R-transformed Agrobacterium culture solution was mixed according to the same bacterial solution concentration ratio, and then put into tobacco leaf disc tissue for co-transplantation (Chang et al., 2007).

In detail, take 2 μL of Agrobacterium solution (GV3101) from the frozen bacteria tube and add it to a test tube containing 10 ml LB (containing 100 mg/L Rifomycin and 100 mg/L Gentamycin) at 28 °C, 150 rpm Shake culture for 1 day. Extract 200 μL from the bacterial solution cultured for 1 day and add it to a test tube containing 4 mL of LB (containing 100 mg/L Rifomycin and 100 mg/L Gentamycin), and incubate at 28 °C with shaking at 150 rpm for 4 hours. Pour 1.5 mL into a microcentrifuge tube, spin at 6,000 rpm, 4 °C, centrifuge for 3 minutes, remove the supernatant, and repeat this step until all bacterial cells settle in the microcentrifuge tube. Add 200 μL of sterile 10% glycerol pre-chilled on ice, re-dissolve the bacteria after precipitation, and then centrifuge at 6,000 rpm at 4 °C for 3 minutes. Pour the supernatant into the waste bottle and repeat This step once. Add 100 μL of sterile 10% glycerol pre-chilled on ice, re-dissolve the bacteria and place on ice for 5 minutes. Add 1.5 μg plastid. Place the shock tube on ice before shock. The bacterial solution was then transferred to an electric shock tube and shocked at 2,500 volts and 100 ohms (BTX ECM399 Electroporation Generator, USA). Transfer the bacterial solution to a new microcentrifuge tube, add 1 mL of LB, and incubate at 28 °C with shaking at 150 rpm for 1 hour. Centrifuge the bacterial solution at 5,000 rpm for 1 minute, remove about 950 μL of the supernatant, and mix the remaining supernatant with the bacterial cells, and move to LB solid medium to spread evenly. Incubate at room temperature and observe the appearance of transformed Agrobacterium colonies on the culture plate in about 2 to 3 days.

Screening of transgenic tobacco tissues and plant regeneration:

In short, the plant tissues of the two expression vectors are co-transformed, and combined with two different drugs (kanamycin, methionine sulfoximine (MSO)) for screening according to the screening marker gene carried on the expression vector. Colonization turn differentiate into tobacco plant tissue by regeneration process by the self-pollinated to give seed transfected colonization tobacco (T ₁ progeny). The T ₁ progeny can be transformed into T ₂ progeny by self-pollination. The detailed tobacco nuclear gene transfer process is as follows:

Take a platinum inoculation ring to take a single colony of Agrobacterium containing recombinant plastids, mix in 5 mL of LB containing antibiotics (containing 150 μg/L Rifampicin and 150 μg/L Kanamycin), and culture at 28 °C with shaking at 150 rpm 18 to 24 hours. The next day, the bacteria solution was enlarged and cultured to 50 mL of LB culture medium containing appropriate antibiotics (150 μg/L Rifampicin and 150 μg/L Kanamycin), 1 mL of 500 mM MES, and 5 μL of 0.2M Acetosyringone, shaking culture 18～ 24 hours. The cultured bacterial solution was centrifuged at 1,700 xg at room temperature for 10 minutes, and the supernatant was removed. Add 5 mL of infiltration buffer (10 mM MES 1 mL, 10 mM MgCl ₂ ), and shake back to the lysate evenly. Measure the OD _{600 of the} bacterial solution and adjust the bacterial solution to OD ₆₀₀ of 1. Mix the bacterial solution with 0.2 M Acetosyringone solution (1000:1 ratio), and shake at 150 rpm for 3 to 4 hours. Then the microtubule bundle tissue of tobacco leaf back was gently scraped with a sterile needle. Aspirate 0.1-0.3 mL of bacterial solution with a syringe, hold the injection syringe to place its exit against the wound on the back of the leaf, and gently press the leaf surface with the fingertip of the other hand. Begin to slowly inject the bacterial liquid into the tobacco leaf, and it can be seen that the liquid diffuses in the leaf and fills the interior. Mark the injection area after injection.

Put the injected tobacco plants in a carton in the dark for 2 days, and then culture for 3 days under light. Cut the tissues within the injection area of the leaves, sterilize with 1.2-2% bleach, shake for 10 minutes, and complete the sterilization. Rear. Use a scalpel to cut the wound around the blade, cut the blade into the appropriate size, with the back of the leaf facing up, place it in Tobacco shooting medium (TSM) medium containing the appropriate antibiotic, seal it with a breathable tape, and place it in a 25℃ plant growth box After cultivating for 16 hours of sunshine, callus, stems and leaves are grown, then hair root culture is carried out, and finally transferred to the soil.

Analysis of transgenic plants

a. PCR molecular detection:

According to the foregoing embodiment, many tobacco transgenic lines were obtained through cultivation, and six transgenic lines M0, M11, M14, M17, M29, and M42 were selected for PCR amplification and amplification, thereby detecting foreign genes (TpTalen-L and TpTalen- R) Whether it is inserted into tobacco DNA at the same time.

In one embodiment, Plant genomic DNA mini kit (Geneaid, Taiwan) is used to extract DNA from plant leaf tissue. First, weigh 0.1 g of plant leaf tissue and grind it into powder with liquid nitrogen. Add 400 μL GP1 buffer and 5 μL RNase A, mix and move to a 1.5 mL microcentrifuge tube, and heat at 60 °C for 10 minutes. At this time, preheat the Elution buffer (200 μL /sample) for use. Add 100 μL of GP2 buffer and mix well, then place on ice for 3 minutes. Transfer the plant tissue fluid to the Filter column and centrifuge at 1,000 xg for 1 minute. Move the filtrate to a new 1.5 mL microcentrifuge tube, add 1.5 times the volume of GP3 buffer, and shake for 5 seconds. Transfer 700 μL of plant tissue mixture to the GD column and centrifuge at 14,000 xg for 2 minutes. Remove the filtrate and repeat this step until the plant tissue mixture is separated. Add 400 μL W1 buffer to the GD column and centrifuge at 14,000 xg for 30 seconds, then remove the filtrate. Add 600 μL Wash buffer to the GD column and centrifuge at 14,000 xg for 30 seconds, then remove the filtrate. Centrifuge at 14,000 xg for 3 minutes, remove the column and place in a new 1.5 mL microcentrifuge tube. Add 50～100 μL of Elution buffer to the column membrane, let it stand for 5 minutes, centrifuge at 14,000 xg for 30 seconds to filter the DNA solution from the column, and collect it in a 1.5 ml microcentrifuge tube and place it at -20 Store at °C.

Next, PCR was performed on the control group (WT) and the DNA (150 ng) of the transferred tobacco with a pair of primers Check-F (SEQ ID NO: 5; AATTCACTCATTGGATTCATAGAAG) and Check-R (SEQ ID NO: 6; CTGTAGCCGAGCGTGCGTAG). To confirm whether the foreign genes (TpTalen-L and TpTalen-R) are inserted into tobacco DNA at the same time. The three-stage condition of PCR reaction temperature and time is that the first stage reacts at 95 °C for 2 minutes. The second stage is divided into three parts, first react at 95 °C for 30 seconds for Denaturation, then at 61 °C for 30 seconds for Annealing, then at 72 °C for 45 seconds for Elongation. This stage is repeated about 45 times. In the third stage, the reaction was carried out at 72 °C for 5 minutes. Then use 2.2% agar and perform electrophoretic separation at 100 volts for 40 minutes.

The electrophoresis results are shown in Part B of Figure 2. In the electrophoresis results of these six different transgenic lines, PCR products of 452 bp and 482 bp can be simultaneously detected, that is, the Talen-L and Talen-R genes are simultaneously expressed in 6 Among the transgenic lines, Talen-L and Talen-R genes were not detected in WT. This means that the Talen-L and Talen-R genes have been successfully transformed into tobacco.

b. Observe the external traits of the transformed plants:

Observe the difference in appearance and growth between different transgenic plant lines and the control group. The results are shown in Figure 3, the appearance and growth of the transgenic plants are diverse. Some lines of transgenic plants do not have any appearance traits. The leaves of some lines of transgenic plants are spotted or partially whitened (such as M29), or the entire leaves are white (yellow) (such as M11, M17), while some of the transformed plants It is an all-albino (light yellow) plant (such as M0). In addition, the growth and development of transgenic plants are also delayed to varying degrees. As reported in previous studies, when the rpoB gene of the tobacco chloroplast changes, it will cause the plant to produce albino appearance (Allison et al., 1996). Therefore, it is speculated that the difference in the degree of albinism of the transgenic plants may be related to the amount of recombinant TALEN protein expressed in the leaves of the transgenic plants, which caused the chloroplast rpoB gene to be damaged to varying degrees and caused the leaves to produce these different degrees of appearance traits.

c. High-resolution dissociation analysis of chloroplast rpoB gene mutations:

To investigate whether the generation of plant albino traits is related to the variation of chloroplast rpoB gene. Therefore, the albino leaf tissues of two different transgenic lines (M11 and M17) were selected. After DNA extraction, a pair of primers (SEQ ID NO: 7; GGATTTAATCAGATACAATTTGAAG) and (SEQ ID NO: 8; TTCCAAATTAATCCCGCGGATAC) were used for quantitative PCR (Q -PCR, Fast Evagreen ^® qPCR Master Mixer, Biotium) to amplify and amplify the recombinant TELEN protein in the vicinity of the rpoB gene DNA sequence (the expected PCR product size is 229 bp), and the PCR product is subjected to high resolution dissociation analysis (high After resolution melting (HRM) analysis, compare with the control group (normal tobacco) to observe whether the DNA sequence has changed.

In detail, containing a total reaction volume of 20 μL of ^{2X Fast Evagreen ® qPCR Master Mixer (} Biotium), 2.5 mM MgCl 2, a mixture of 0.2 μM of the preceding primer and 20 ng transfected colonization tobacco DNA is Q-PCR, the expected product is 229 bp. Both Q-PCR and HRM are operated using Roche Light Cycler ^® Nano. First, use 95 °C for 180 seconds, followed by a two-step cycle step, each cycle of 95 °C denature 10 seconds and 60 °C annexing/extension 50 seconds, after 45 cycles, and then 95°C for 60 seconds to completely unwind the double-stranded DNA, followed by treatment at 40°C for 60 seconds to form a heterozygous structure, and finally gradually heated from 65°C to 95° at 0.05 per second C Perform HRM analysis.

The results are shown in Figure 4. Compared with the control group, the white leaf tissues (M11-W and M17-W) of the transgenic plants have obvious melting curve differences, which means that the rpoB genes in these two transgenic lines have Nucleotide variation occurs.

d. Sequencing analysis of chloroplast rpoB gene:

In order to investigate whether the performance of the recombinant TALEN protein in the transgenic plants finally caused the mutation of the chloroplast rpoB sequence, the leaf tissues of the control group (WT) and 4 different transgenic lines (M0, M11, M17 and M29) were extracted, among which M11 and The DNA of the M17 strain plants is extracted from the leaf tissues of white (M11-W and M17-W) and green (M11-G and M17-G), respectively. Use a pair of primers (SEQ ID NO: 9; AGCAGAAGTCTGTTTCTAGGGATGT) and (SEQ ID NO: 10; TAGCTGATGATAGAACTAGAATA) to amplify and amplify the sequence around the recombinant TALEN protein acting on the rpoB gene by PCR and sequence the PCR products. The sequence results were compared using CLC Genomics Workbench 9.5.2 software.

The results shown in Figure 5 show that the mutation of A to T is apparent in the rpoB- 295 position of the white or yellow leaf tissues (M0, M11-W, M17-W, M29) of the transgenic lines M0, M11, M17 and M29. The situation, as indicated by the arrow. However, the rpoB gene sequence of green leaf tissues (M11-G, M17-G) in M11 and M17 was the same as that in the control group. This means that the transformation of tobacco through pTpTalen-L and pTpTalen-R can mutate specific chloroplast genes, such as the mutation of the chloroplast rpoB gene, which in turn leads to changes in plant appearance.

The above is only exemplary and not restrictive. Those with ordinary knowledge in the technical field of the invention to which the invention belongs can design the repeating variable region in the recombinant TALEN protein domain according to the needs and the targeted nucleotide sequence, so that the recombinant TALEN protein can specifically identify the target nucleic acid sequence. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

References: Allison, LA, Simon, LD and Maliga, P. Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. The European Molecular Biology Organization Journal 15, 2802-2809, 1996. Bock, R. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annual Review of Plant Biology , 66 , 211-241, 2015. Cermak, T., Doyle, EL, Christian, M., Wang, L., Zhang, Y ., Schmidt, C., Baller, JA, Somia, NV, Bogdanove, AJ and Voytas, DF Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research 39, e28, 2011. Chang , CC, Huang, PS, Lin, HR, & Lu, CH. Transactivation of protein expression by rice HSP101 in planta and using Hsp101 as a selection marker for transformation. Plant and cell physiology , 48 (8), 1098-1107, 2007 Hajdukiewicz, PT, Allison, LA and Maliga, P. The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct grou ps of genes in tobacco plastids. The European Molecular Biology Organization Journal 16, 4041-4048, 1997. Inada, H., Kusumi, K., Nishimura, M. and Iba, K. Specific expression of the chloroplast gene for RNA polymerase ( rpoB ) at an early stage of leaf development in rice. Plant and Cell Physiology 37, 229-232, 1996. Little, MC and Hallick, RB Chloroplast rpoA , rpoB , and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription. The Journal of Biological Chemistry 263, 14302-14307, 1988. Outchkourov, NS, Peters, J., de Jong, J., Rademakers, W. and Jongsma, MA The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 6, 1003-1012, 2003.

no

FIG. 1 is a map of a nuclear gene transfer vector according to an embodiment of the invention claimed in this case. Among them, part A and part B are pTpTalen-L and pTpTalen-R, respectively, which are both Agrobacterium double-coupled vectors expressed by nuclear genes, and are expression vectors with leaf tissue specificity. The skeleton of pTpTalen-L is from pZGB9R, with KanR ( npt II ) gene as a selection marker; and the skeleton of pTpTalen-R is from pBinPlus, which has bar gene as a selection marker. The meaning of each abbreviation is: LB-T Border and RB-T Border are the left and right borders of Agrobacterium T-DNA, respectively. CaMV 35S is the promoter of Califlower mosaic virus 35S transcript. 35S polyA is the 3'UTR of Califlower mosaic virus 35S transcript. RbcS promoter is the promoter of rbcS gene. RbcS terminator is the 3'UTR of rbcS gene. KanR ( npt II ) is a selection marker gene for kanamycin resistance, and bar is a selection marker gene for herbicide resistance (Methionine sulfoximine, MSO). TP is the sequence of chloroplast signaling protein. Flag is the Flag tag, and HA is the HA tag.

Fig. 2 is an example of the invention claimed in this case, using PCR to confirm whether the genes after co-transfection of pTpTalen-L and pTpTalen-R are embedded in the tobacco genome. (A) The PCR primer pairs Check-F (SEQ ID NO: 5; AATTCACTCATTGGATTCATAGAAG) and Check-R (SEQ ID NO: 6; CTGTAGCCGAGCGTGCGTAG) are located on the pTpTalen-L and pTpTalen-R vectors. (B) 6 co-transgenic tobacco lines (M0, M11, M14, M17, M39, M42), control group (Wt is untransformed tobacco), pTpTalen-L (Lp) and pTpTalen-Rp (Rp) vectors The DNA of such products is amplified by PCR using specific primer pairs (Check-F and Check-R), and then the PCR products are separated by 2.2% agar electrophoresis. In the figure, M is a 100 bp DNA marker. The arrows indicate the expected PCR product sizes of 482 bp and 452 bp, respectively.

FIG. 3 is a plant phenotype of a tobacco line transfected with nuclear genes according to an embodiment of the present invention. As shown in the figure, the four different transgenic lines transfected with nuclear genes exhibited the appearance of leaf albino or yellowing to varying degrees. WT is the control group (untransformed tobacco). M0 is an all-albino plant line. M11 is two different individuals that grow on medium or soil. M17 is two different individuals that grow on medium or soil, respectively. M29 is two different individuals growing in the soil.

4 is a high-resolution dissociation (High Resolution Melting, HRM) analysis of tobacco chloroplast rpoB gene mutation according to an embodiment of the present invention. DNA was extracted from the leaf tissue of the WT control group (untransformed tobacco) and the white leaf tissue of two different transgenic lines (M11 and M17), followed by a pair of primers (SEQ ID NO: 7; GGATTTAATCAGATACAATTTGAAG) and (SEQ ID NO: 8; TTCCAAATTAATCCCGCGGATAC) Q-PCR (Fast Evagreen ^® qPCR Master Mix, Biotium) to amplify the DNA (229 bp) near the area of action of the recombinant TELEN protein, and the PCR products are subjected to high-resolution dissociation analysis (HRM, LightCycler ^® Nano, Roche). The results showed that the white leaf tissues (M11-W and M17-W) of the transformed plants had a significant difference in melting curve from the WT control group, so it was speculated that there was a nucleotide variation in the rpoB gene.

FIG. 5 shows that the recombinant TALEN protein causes variation in the chloroplast rpoB gene sequence of tobacco leaves according to an embodiment of the present invention. Extract the leaf tissues of the control group (WT) and 4 different transgenic lines (M0, M11, M17 and M29), where the DNA of the plants of the M11 and M17 lines are extracted from white (M11-W and M17-W) and green respectively (M11-G and M17-G) Leaf tissue. Use a pair of primers (SEQ ID NO: 9; AGCAGAAGTCTGTTTCTAGGGATGT) and (SEQ ID NO: 10; TAGCTGATGATAGAACTAGAATA) to amplify the amplified DNA using PCR, and sequence the PCR products, using the CLC Genomics Workbench 9.5.2 The software performs multiple sequence alignments. The results showed that in the transgenic lines M0, M11, M17 and M29, the white or yellowed leaf tissues (M0-W; M11-W, M17-W, M29) had a mutation from A to T in the rpoB- 295 position. . However, the rpoB gene sequence in the green leaf tissues (M11-G, M17-G) of M11 and M17 was the same as the control group. The arrows show the nucleotides that have mutated.

In the following detailed description, in order to explain the present invention, many specific details are provided in order to thoroughly understand the disclosed embodiments. However, it is obvious that one or more embodiments can be implemented without the specific details. In other cases, in order to simplify the drawings, the conventional structure and flow will be shown in a schematic manner.

Claims (20)

A nuclear gene transfer vector, including: a promoter; a chloroplast information protein gene, which is located downstream of the promoter; a recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22; And a first border region and a second border region, wherein the promoter, the chloroplast signal protein gene and the recombinant TALEN protein domain are sequentially located between the first border region and the second border region; wherein the recombinant TALEN The protein domain includes a DNA sequence encoding a DNA recognition protein; wherein the DNA recognition protein recognizes a fragment of the chloroplast rpoB gene. The transfer vector according to claim 1, wherein the promoter is an rbcS promoter. The transfer vector according to claim 1, wherein the recombinant TALEN protein domain includes a DNA sequence of an endonuclease. The transfer vector according to claim 3, wherein the endonuclease is Fok I. The transfer vector according to claim 1, wherein the DNA sequence encoding the DNA recognition protein has the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. The transfer vector according to claim 1, further comprising a selection gene. The transfer vector according to claim 6, wherein the screening gene is a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. The transfer vector according to claim 6, wherein the screening gene comprises a kanamycin (kanamycin) resistance gene or a methionine sulfoximine (MSO) herbicide resistance gene. The transfer vector according to claim 8, wherein the kanamycin resistance gene includes nptII or the methionine sulfonimide herbicide resistance gene includes bar . The transfer vector as described in claim 1, further comprising a sequence encoding a marker. The transfection carrier according to claim 10, wherein the marker comprises a luminescent molecule, a chemiluminescent molecule, a fluorescent dye, a fluorescent quencher, a lipid, a colored molecule, a radioactive isotope, a scintillant, biotin, an anti-cancer Detectable labels in the group consisting of biotin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tags, myc tags, HA tags, and enzymes Thing. The transfer vector according to claim 1, which comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The transfection vector according to claim 1, further comprising a 3-terminal non-transcribed region of the rbcS gene, which is located downstream of the recombinant TALEN protein domain. A kit for specific variation of plant chloroplast genes, including: a first transgenic vector, including: a first promoter; a first chloroplast signaling protein gene, which is located downstream of the first promoter; a first recombination TALEN protein domain, including the sequence shown in SEQ ID NO: 21; and a first border region and a second border region, wherein the first promoter, the first chloroplast signaling protein gene and the first recombinant TALEN protein The domain is located between the first border region and the second border region in sequence; and a second transfection vector, including: a second promoter; a second chloroplast signaling protein gene, which is located in the second promoter gene Downstream; a second recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 22; and a third border region and a fourth border region, wherein the second promoter, the second chloroplast signaling protein gene and The second recombinant TALEN protein domain is sequentially located between the third border region and the fourth border region; wherein the first recombinant TALEN protein domain and the second recombinant TALEN protein domain include a DNA sequence encoding a DNA recognition protein; The DNA recognition protein recognizes a fragment of the rpoB gene of the chloroplast. A method for producing nuclear gene transgenic plant cells, comprising: constructing a pair of nuclear gene transfection vectors as described in claim 1; incorporating the nuclear gene transduction vectors into a plant cell through an Agrobacterium; and screening and culturing The plant cell. The method of claim 15, wherein the incorporation includes a homology-directed repair mechanism or a non-homologous end-joint-directed repair mechanism. The method according to claim 15, further comprising transferring the nuclear gene transfer vector to the Agrobacterium. The method of claim 15, further comprising analyzing the plant cell and confirming that the nuclear gene transfer vector is incorporated into the plant cell. A nuclear gene transgenic plant cell is constructed by the method described in claim 15. An Agrobacterium comprising the nuclear gene transfer vector as described in claim 1.

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