CN109082438B

CN109082438B - Channel protein with anion transport activity and genome modification thereof for regulating and controlling crop grain size

Info

Publication number: CN109082438B
Application number: CN201810880297.3A
Authority: CN
Inventors: 李乐攻; 任志杰; 李嘉熙
Original assignee: Capital Normal University
Current assignee: Capital Normal University
Priority date: 2018-08-03
Filing date: 2018-08-03
Publication date: 2021-03-30
Anticipated expiration: 2038-08-03
Also published as: CN109082438A

Abstract

The invention relates to a method for obtaining a recombinant or edited DNA fragment suitable for adjusting the size of rice grains, which comprises the following steps: transforming DNA fragments of rice or crops by a genetic engineering technical means to knock out, knock down or silence the TCBG1 gene and homologous genes with similar functions in the rice or the crops; wherein the CDS sequence of the TCBG1 gene is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO. 2. In some embodiments of the invention, the storage capacity of the grain cells is successfully changed by directly regulating the osmotic potential or turgor pressure of the cells, so that the size of the crop grains is increased. The method undoubtedly provides a foundation for the yield increase of crops and has wide application prospect.

Description

Channel protein with anion transport activity and genome modification thereof for regulating and controlling crop grain size

Technical Field

The invention relates to the technical field of genes, in particular to a gene with anion transport activity for regulating and controlling grain size.

Background

Rice is an annual gramineous food crop, and rice is eaten by nearly half of the world population. Increasing the yield of rice or other gramineous crops has been a hot issue of interest to researchers, and the grain type is a trait directly related to the yield, and thus has become an intuitive point of research, wherein the phenotype mainly includes grain length, grain width, grain thickness and the like, which affect the weight of grains, and the phenotype is determined by the number of cells, the shape and the size of the cells.

In recent years, a plurality of genes determining the size of grains are obtained by Quantitative Trait Loci (QTLs) and the like, and mainly relate to physiological processes in several aspects, namely, first, organic matter assimilation-related enzyme genes including starch synthesis-related enzymes and sucrose metabolism-related enzymes such as GBSS, OsCIN1, GIF1/OsCIN2 and the like (Tsai CY, 1974, Smith AM et al, 1997, Guoyu Zhang et al 2011, ayahiko shomura et al, 2008, tatsouro Hirose et al, 2008, tatao Wang et al, 2008). The second class is sugar transporters and their regulatory genes such as SWEET4c, SUT family, OsNF-YB1, etc. (DavideSosso et al, 2015, Ai-NingBai et al, 2015). The third class is cell division and hormone related genes such as GS5, GW2, GW8qSW5/GW5, BG1, etc. (Yibo Li et al, 2011, Xian-Jun Song et al, 2007, Shaokui Wang et al, 2012, ayahiko shomura et al, 2008). Among these reported division-regulating genes affecting grain type, some influence grain size by altering cell number, such as GS3, GW2, GW5, GS5, GW8, qGL3, TGW6, GW6a, and BG1 etc. (Song et al, 2007,2015; Weng et al, 2008; Mao et al, 2010; Li et al, 2011; Wang et al, 2012; Zhang et al, 2012; Ishimaru et al, 2013; Liu et al, 2015); there are also a small fraction of cells whose numbers do not change much and whose size changes, such as PGL1, GL7, and GS2/GL2, etc. (Heang and Sassa, 2012; Che et al, 2015; Duan et al, 2015; Hu et al, 2015; Wang et al, 2015). Among them, most of them affect the grain size by regulating cell division or cell cycle with hormones (Ashikari et al, 1999,2005; Hong et al, 2003; Tanabe et al, 2005; Ishimaru et al, 2013) (Yibo Li et al, 2011, Xian-Jun Song et al, 2007, Shaokui Wang et al, 2012, Liu et al, 2015) regardless of the number or size of cells.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a method for obtaining a recombinant or edited DNA fragment suitable for adjusting the size of rice grains, which comprises the following steps: transforming a DNA fragment of rice by a genetic engineering technical means, so that the TCBG1 gene and homologous genes with similar functions in the rice or crops are knocked out, knocked down or silenced; wherein the CDS sequence of the TCBG1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The method as described above, wherein the genetic engineering technology means CRISPR/Cas 9.

The method as described above, wherein the CRISPR/Cas9 target sequence is one or more of: CGCCGTCGCCGCCGCACGAGAGG, respectively; CCTCGGCCGCCTCGGCGACCTCC, respectively; CCGCCATGGACCCCATCTGCGGG, respectively; CCCAGCTGCCTCAACACCTGCC, respectively; GCGGGCATGGTGTCGATCGTGGG, respectively; CAAGGCCAGACTTGGGATCCAGG are provided.

The method as described above, wherein the CDS sequence of the recombinant DNA fragment is shown as SEQ ID NO.3 and the amino acid sequence is shown as SEQ ID NO. 4.

The method as described above, wherein the genetic engineering means is TALEN technology.

The method as described above, wherein the TALEN recognition region sequence is one or more of: GGCCGTCACCACCGCCTTCCTCGGCCGCCTCGGCGACCTCCAGCTCGCCGCCGGC, respectively; CGCCGTCCTCACCGGCCTCTGCGCCGCCATGGACCCCATCTGCGGGCAGGCGC, respectively; CGCCTCCGCCCTGGCCCTGGCGCTCCACGTCCCCCTCACCATGTGGATGGCCAGG are provided.

The method as described above, wherein the genetic engineering technique is RNAi.

The method as described above, wherein the RNAi target sequence is represented by SEQ ID NO. 5.

A recombinant DNA fragment as described in any one of the above and functionally similar homologous genes thereof.

A plant genome comprising a recombinant DNA segment as described above and functionally similar homologous genes thereof.

A plant cell comprising a recombinant DNA segment as described above.

The recombinant DNA fragment, the plant genome or the plant cell are applied to the aspect of adjusting the grain size of rice or crops.

A method of adjusting the size of a rice or crop kernel comprising: treating the DNA fragment of the rice by a genetic engineering technical means, so that the DNA fragment of the rice cannot be transcribed after being edited, and the TCBG1 gene is silenced; wherein the CDS sequence of the TCBG1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Use of a recombinant DNA segment as described above, a plant genome as described above or a plant cell as described above for modulating cell turgor or osmotic potential.

A method of modulating the turgor or osmotic potential of rice or crop cells comprising: treating the DNA fragment of the rice by a genetic engineering technical means, so that the translated DNA fragment of the rice is silenced by TCBG1 gene; wherein the CDS sequence of the TCBG1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Cell turgor pressure or osmotic potential is an important factor for promoting cell growth and elongation. Cell turgor or osmotic potential can maintain cell size and shape. The cellular water content, sugars, organic acids, various inorganic ion contents, etc. maintain the turgor pressure or osmotic potential of the cells. Such as: aquaporins (Maurel C, et al 2015), glycotransporter proteins, amino acid and other organic transporters (Zifarelli G, Pusch M, 2010; Zhang et al, 2016), inorganic ion channels (Beauzamy et al 2014) on cell membranes directly affect the turgor or osmotic potential of cells. However, the efficiency of dry matter accumulation and grain size are increased by regulating the osmotic potential or cell turgor of crop grain-associated cells, and no report exists so far on no matter the mechanism or the example.

In some embodiments of the invention, the storage capacity of the grain cells is successfully changed by directly regulating the osmotic potential or turgor pressure of the cells, so that the size of the crop grains is increased. The method undoubtedly provides a foundation for the yield increase of crops and has wide application prospect.

Drawings

Preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, in which:

FIG. 1 is the CDS sequence of TCBG1 gene;

FIG. 2 is the amino acid sequence of the TCBG1 gene;

FIG. 3 is an expression pattern analysis of TCBG 1; shown as PRO in the figure_TCBG1-GUS staining of different tissues in GUS transgenic rice; wherein a-d is spikelet shell, and the scale is 0.2 mm; e and f are anthers, scale 1 mm; g is pistil, and the standard ruler is 0.5 mm; h is seed, and the standard ruler is 1 mm;

FIG. 4 is a schematic diagram of a transgenic rice TCBG1 mutant plant induced by Cas 9; panel A is a diagram illustrating the sgRNA target sequence (except double-underlined) Cas9 and the corresponding PAMs [ motif sequence, triple nucleotide NGG sequence ] (double-underlined) TCBG1 gene, underlined as an NcoI restriction site; FIG. B shows the result of analysis of DNA samples of the extracted individual transgenic rice seedlings by detecting sgRNA in transgenic rice plants by PCR/RE, wherein the sgRNA is a mutation induced by Cas 9; the red dash in panel C indicates a deletion, and the right-hand number indicates the type of mutation and the number of nucleotides involved; FIG. D shows the Sanger sequencing results of allelic clones of plants No.1, 2 and 7;

FIG. 5 is a schematic diagram of the induction of transgenic rice TCBG1 mutant plants by transcription activator-like effector nuclease (TALEN) technology; FIG. a shows the position of the selected target sequence in TCBG1, in which the bases in the double underlined parts represent the left target sequence and the right target sequence, respectively; b, the short line indicates the deletion, the right-hand number indicates the type of mutation and the number of nucleotides involved;

FIG. 6 shows the silencing of rice TCBG1 gene by RNAi (RNA interference) technique; panel A shows the positions of selected RNAi target sequences in the TCBG1 gene; FIG. B is a recombinant vector map for constructing RNAi transgenic rice; and the picture C shows that the RT-RCR (fluorescent quantitative PCR) method identifies the transgenic plants.

FIG. 7 is a comparison of tcbg1 and WT brown rice kernels; panel A compares the size and morphology of tcbg1 and WT kernels, scale 1 cm; panel B shows statistics of 500 grain coverage for tcbg1 and WT kernels; panel C shows the thousand kernel weight statistics for tcbg1 and WT brown rice kernels;

FIG. 8 is a comparison of tcbg1 and WT grain morphology; panel A and C are grain length comparisons and statistics for WT, tcbg1-1, and tcbg 1-2; panel B and D are grain width comparisons and statistics for WT, tcbg1-1, and tcbg 1-2; panel E is a comparison of grain thickness and statistics for WT, tcbg1-1 and tcbg 1-2; FIGS. F and H are the comparison and statistics of the grain length of the brown rice of WT, tcbg1-1 and tcbg 1-2; graphs G and I are comparisons of grain widths of whole grain rice WT, tcbg1-1 and tcbg1-2 and statistical results; FIG. J shows the comparison and statistics of the grain thickness of the brown rice grains of WT, tcbg1-1 and tcbg 1-2; wherein the scales of both graphs A, B, F and G are 1 cm;

FIG. 9 is a comparison of tcbg1 and WT grouting speeds; panels a and B are the results comparing dry and wet weight after fertilization of WT and tcbg1 mutants, respectively, where n is 20; and

FIG. 10 is a TCBG1 chloride ion efflux property obtained from a typical whole-cell current recording system using hyperpolarized pulses; in panels A and B, water containing no RNase was injected into Xenopus oocytes (as a pair)Lighting), the perfusion fluid is ND96 solution; panels C and D record currents of TCBG 1-expressing oocytes with ND96 (Panel C) and altered ND96 (Panel D), respectively, as perfusates, where the altered ND96 is NaCl substituted with NaGlu₂(ii) a Panels E and F record the current of oocytes expressing the TCBG1 mutant TCBG1 (deletion 251T) as perfusion using ND96 (Panel E) and altered ND96 (Panel F) as described above, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. Based on the examples of the present invention, all other examples obtained by one of ordinary skill in the art without making the inventive result of the function of TCBG1 protein are within the scope of the present invention.

In the following detailed description, reference is made to the accompanying drawings that form a part hereof and in which is shown by way of illustration specific embodiments of the application. In the drawings, like numerals describe substantially similar components throughout the different views. Various specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the present application. It is to be understood that other embodiments may be utilized or changes in the structure, logic, or properties of the TCBG1 protein may be made to the embodiments of the present application.

Unless otherwise indicated herein, nucleic acids are written from left to right in a 5 'to 3' direction.

A number of terms are used in the present invention. For a clear and consistent understanding of the present invention, the following definitions are provided:

gene: refers to nucleic acid fragments that express a functional molecule such as, but not limited to, a particular protein, including regulatory sequences located before (5 'non-coding sequences) and after (3' non-coding sequences) the coding sequence. "native gene" refers to a gene that has its own regulatory sequences as it exists in nature.

Genome: the total DNA or the entire set of genes that make up the genome of an organism is carried by a chromosome or set of chromosomes. When applied to plant cells, it encompasses not only chromosomal DNA present in the nucleus, but organelle DNA present in subcellular components of the cell (e.g., mitochondria or plasmids).

ORF: the Open Reading Frame (Open Reading Frame), starting from the start codon, is a base sequence with the potential of encoding protein in the DNA sequence, without interruption by a stop codon.

Mutant genes: is a gene that has been altered by human intervention. The sequence of such a mutant gene differs from the sequence of the corresponding non-mutant gene by comprising at least one nucleotide addition, deletion or substitution. In certain embodiments of the invention, the mutant gene comprises an alteration caused by a CRISPR/Cas endonuclease system as disclosed herein. A mutant plant is a plant that comprises a mutant gene.

Targeted mutation: is a mutation in a native gene that is made by: the target sequence within the native gene is altered using methods involving DNA capable of inducing double-stranded changes in the target sequence disclosed herein or known in the art.

Recombinant DNA: refers to a combination of nucleic acid fragments that do not normally occur together in nature. Thus, a recombinant DNA may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.

CRISPR (clustered regulated Short Palindromic repeats)/Cas (CRISPR-associated): is an engineered nuclease system, is a bacterial system useful for genome engineering, and is part of the adaptive immune response of many bacteria and archaea.

Cas 9: relates to a CAS protein-encoded endonuclease CAS9 gene capable of introducing double-strand breaks into DNA target sequences. It is typically coupled to, associated with, or near or in the vicinity of a flanking CRISPR locus.

Restriction enzymes: an endonuclease which hydrolyzes double-stranded DNA at a specific nucleotide sequence. NcoI is a restriction enzyme that specifically recognizes and cleaves the sequence CCATGG.

Target site, target sequence, sequence of interest: used interchangeably herein, refers to a sequence specifically recognized by an endonuclease gene in a polynucleotide sequence of a genome (including chloroplast and mitochondrial DNA). For example, CCATGG is the target site for the restriction endonuclease NcoI.

And (3) transformation: the transfer of foreign genes into the plant genome is called transformation. All techniques are contemplated by which nucleic acid molecules can be introduced into such cells. Examples include, but are not limited to: transfection with viral vectors; transforming with a plasmid vector; electroporation; carrying out liposome transfection; microinjection (Mueller et al, (1978) Cell 15: 579-85); agrobacterium-mediated transfer; direct DNA uptake; WHISKERSTM-mediated transformation; and particle bombardment. These techniques can be used for both stable and transient transformation of plant cells.

TALEN: namely Transcription Activator-like (TAL) Effector nucleotides, Transcription Activator Effector protein Nucleases. TALENs are enzymes that can target the modification of specific DNA sequences by means of TAL effectors, a natural protein secreted by plant bacteria, to recognize specific DNA base pairs. TAL effectors can be designed to recognize and bind all DNA sequences of interest. Adding a nuclease to the TAL effector generates a TALEN. TAL effector nucleases bind to DNA and cleave DNA strands at specific sites, thereby introducing new genetic material.

RNAi: the blocking effect of double-stranded RNA on gene expression is called RNA interference (RNAi), and double-stranded RNA is cleaved by enzyme to form many small fragments called siRNA, which, once complementarily bound to a homologous sequence in messenger RNA (mRNA), cause the mRNA to become nonfunctional, reducing the expression level of the protein, i.e., "silencing" the gene.

Additions, enhancements or enhancements herein are interchangeable. For example, yield or grain size, etc. is increased by at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% or more compared to wild type.

Phenotype, phenotypic trait: may refer to observable expression of a gene or series of genes. The phenotype may be observable to the naked eye or any other means of assessment known in the art, such as weighing, counting, measuring (length, width, angle, etc.), microscopy, biochemical analysis, and the like. In some cases, the phenotype is directly controlled by a single gene or locus, i.e., a "monogenic trait" or a "simple genetic trait". In the absence of a large level of environmental change, the individual genetic traits may be isolated in a population to give a "qualitative" or "discrete" distribution, i.e. the phenotype belongs to a discrete class. In other cases, a phenotype is the result of several genes and can be considered a "polygenic trait" or a "complex trait. The multigenic trait is segregated within a population to give a "quantitative" or "continuous" distribution, i.e., phenotypes are not classifiable into discrete classes. Both single gene and polygenic traits can be affected by the environment in which they are expressed, but polygenic traits are more closely related to the environment.

And (3) plant: can be the whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term "plant" can refer to any of the following: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny thereof. A plant cell is a cell of a plant obtained from the plant or a culture derived from a cell obtained from the plant.

The fact that the deletion or silencing of a single gene TCBG1 in rice affects plant filling and further affects yield directly or indirectly is described in detail herein.

Description of the genes of interest

The target gene targeted by the invention is TCBG1 gene. FIG. 1 shows the CDS sequence of TCBG1 gene shown in SEQ ID NO. 1. The ORF of the gene has overall 1440 bases, and the GC content of 70.49% and no intron. The amino acid sequence of the TCBG1 gene is shown in figure 2 or SEQ ID NO.2, the gene encodes 480 amino acids, has nine transmembrane domains and has typical MATE family secondary structure characteristics.

TCBG1 belongs to the DTX family, which is widely found in bacteria, fungi, animals and plants. The family can be divided into four subfamilies according to evolutionary relations, and the TCBG1 belongs to a third subfamily and has typical MATE family secondary structure characteristics. More than ten members have been reported in Arabidopsis thaliana, and are functionally diverse and distinct. Recent researches prove that some members of the family mediate the transport of organic acid and anion to regulate the cellular turgor of arabidopsis thaliana and have drought tolerance. Only OsFRDL1 and OsFRDL2 reported in rice have the function of transporting citric acid and are related to iron nutrient absorption, and the functions of most other members are not analyzed.

II, analysis of expression pattern of TCBG1

The expression pattern of a gene determines the function of the gene. To explore the expression pattern of TCBG1, we introduced the 3204bp promoter (3.2kb upstream of the start code) PRO of this gene_TCBG1Fused to the β -Glucuronidase (GUS) receptor, and the recombinant plasmid was transferred to wild-type rice (WT). For PRO_TCBG1The activity of the TCBG1 promoter in GUS transgenic plants was analyzed and the results showed that: the expression level of TCBG1 increased gradually as the glume developed.

As shown in FIG. 3, GUS activity was detected at different developmental stages of the reproductive organs (FIGS. 3a-g) of rice WT plants. In the figure, blue color indicates a tissue having GUS activity, and darker blue color indicates a higher expression level of TCBG1 gene in the tissue. FIGS. a, b and d show that TCBG1 is highly expressed in rice stamens. Further enlargement of the stamen, as shown in panel e, clearly shows that TCBG1 is expressed primarily in anthers of the stamen. As shown in fig. f, further observation of the expression level of TCBG1 in the anthers at different times revealed that TCBG1 expression level in the anthers increased with the development of the anthers. In fig. f, the degree of anther maturation is gradually increased from right to left. The graphs b, c and g show the expression pattern analysis of TCBG1 in pistil, and it can be seen that the expression level of TCBG1 gradually decreases with the development of stamen. Graph h shows that TCBG1 is expressed in higher amounts in seeds. The result proves that the TCBG1 is highly expressed in the rice reproductive development process, and the TCBG1 is predicted to be involved in the grain development process.

In some embodiments of the invention, in the process of screening rice mutants, the grain filling speed and time of rice grains are increased by changing the cell turgor pressure or osmotic potential aiming at the TCBG1 gene and the protein coded by the TCBG1 gene, so that the grain size of rice is increased. This finding is different from previously reported grain size control genes, and this family neither regulates the cell division of grain nor the synthesis and transport of grain assimilates. Therefore, the invention has pioneering property in increasing rice yield.

In the following specific examples, the rice tcbg1 mutant is used to further illustrate the present invention.

The first embodiment is as follows:

identification of tcbg1 and mutants using CRISPR/Cas9 knockdown:

the TCBG1 gene is edited by using CRISPR/Cas9 technology. Several suitable sites of the ORF of this gene were selected as target sequences, and 6 suitable sites as shown in Table 1 were obtained by sequence analysis. The identification of the tcbg1 gene knockout and mutants by using the CRISPR/Cas9 technology will be briefly described below by taking the target sequence No.3 as an example.

Table 1. target sequences of suitable CRISPR/Cas 9. Wherein, the single-line part base is a proper enzyme cutting site, and the double-line part base is PAM.

The 242bp-265bp is used as a CRISPR target sequence (figure 4A), and comprises an NcoI (CCATGG) restriction enzyme cutting site (single-lined partial base), so that the identification of subsequent positive plants is facilitated. The constructed fusion plasmid is transferred into wild rice by utilizing an agrobacterium-mediated genetic transformation system, and the harvested seeds are reseeded. The obtained positive plants are numbered (e.g., 1-12) and genomic DNA is extracted. And (2) selecting primers designed at two ends of the CRISPR target sequence from the base sequence, so that the distance between the CRISPR target sequence and primers at two ends is different by at least dozens of bases, and only one restriction endonuclease, namely NcoI, can be contained in two primer intervals. The CRISPR target region was PCR amplified using primers designed above, and the PCR product was then cleaved with NcoI overnight at 37 ℃. Electrophoresis detection shows that the plants with the

numbers

1,2 and 7 can not be cut at all, which indicates that the enzyme cutting site is damaged, namely the homozygous mutant; although some of the base fragments of the plants numbered 3,4,5,8,9,10,11, and 12 were not cut, some of the base fragments were cut with NcoI, indicating that the plants were heterozygous mutants; the last column is the control group, i.e.the Agrobacterium containing the fusion plasmid was not transferred to the plant and the corresponding alkaline fragment could be completely cut into two pieces by the restriction endonuclease (see FIG. 4B). Sequencing analysis is carried out on the plants with the

numbers

1,2 and 7, and the three plants are all subjected to one base deletion (Figure 4C and D), the CDS sequence after mutation is shown in SEQ ID NO.3, and the 251 th thymine (T) of the CDS base sequence is deleted.

Example two:

knock-out of tcbg1 using TALEN technology and identification of mutants:

the specific modification of the rice TCBG1 gene is realized by using a transcription activator-like effector nuclease (TALEN) technology to obtain a mutant with a function deletion.

The appropriate target sequence is selected as the recognition site for the TALEN. In the invention, several suitable sites of the ORF of the gene are selected as recognition region sequences, and several sets of TALEN recognition region sequences are designed through sequence analysis to obtain 3 suitable sites shown in Table 2.

Table 1. recognition sequences for suitable TALENs. Wherein the bases of the double underlined parts in the same sequence are the left target sequence and the right target sequence respectively.

The identification of knock-out tcbg1 gene and its mutants by TALEN technology will be briefly described below using the sequence of recognition region No.2 as a TALEN recognition site as an example. As shown in FIG. 5a, 219-271bp was selected as the recognition sequence of TALEN, wherein the left double underlined bases constitute the left target sequence and the right double underlined bases constitute the right target sequence. The left and right target sequences can be specifically identified and combined with the appointed position of the target gene TCBG1, and partial bases between the target sequences are edited.

And selecting and constructing a related TALEN knockout vector, and transferring the TALEN knockout vector into wild rice by using the method in the embodiment I. And collecting seeds, and reseeding the harvested seeds. The screened second generation transgenic rice can identify the positive plants with the damaged target gene from 140 screened transgenic plants, and different identification methods can be provided according to the designed identification region sequence and the used carrier, for example, the enzyme digestion method and the antibiotic screening method described in the embodiment are utilized, and the details are not repeated herein.

Extracting genome DNA of the transgenic plants, amplifying DNA fragments of a target gene TCBG1, performing sequencing analysis on the DNA fragments, and finally identifying eight (eleven) positive plants with genomes edited to different degrees. The ten mutant types, with different degrees or different positions of base deletions, are shown in figure 5 b.

Example three:

obtaining and analysis of RNAi strains

The function of a target gene is researched by inhibiting the expression of TCBG1 by using an RNA interference technology, and a 241 th base to a 540 th base sequence, namely a fragment with the length of 300bp, of the ORF of the gene close to the 5' end position is selected as a target sequence (as shown in FIG. 6A), and the base sequence is detailed in SEQ ID NO. 5. The target sequence is recombined into a rice expression vector pTCK309 (shown in figure 6B) and then transferred into wild rice.

The obtained positive plants are firstly subjected to DNA level identification, and whether a sequence which is specific to the expression vector pTCK309 is included in the transgenic plants is detected to determine whether the transgenosis is successful. Then, the expression level of TCBG1 in the transgenic positive plants is analyzed, and the expression quantity of the TCBG1 gene after the RNAi technology editing is quantitatively detected. Through the expression detection of TCBG1 by RT-PCR, plants in which the expression of TCBG1 is inhibited by more than sixty percent are finally obtained by screening and named as Ri-1 and Ri-2 (as shown in FIG. 6C). As shown in FIG. 6C, the vertical axis shows the relative expression of TCBG1, the black bars represent wild-type plants, and the gray bars represent the plants of TCBG1 mutant obtained by RNAi technique having the same growth period as the wild-type plants, wherein the middle gray bars are Ri-1 and the right side is Ri-2.

Phenotypic traits and analysis of examples one, two and three

The phenotypic characters of TCBG1 are analyzed by taking a base deletion mutant obtained by a CRISPR/Cas9 technology and a gene silencing mutant obtained by an RNAi technology as examples. Wherein the mutant obtained by the CRISPR/Cas9 technology is named as tcbg1-1, and the mutant obtained by the RNAi technology is named as tcbg 1-2.

A. Comparative analysis of wild type and mutant grain types: thousand grain weight of tcbg1 mutant grain is obviously improved compared with wild type

Grain type directly determines the weight of the kernel, including grain length, grain width and grain thickness. the tcbg1 mutant grain increased in length, width, and thickness to varying degrees compared to the wild type, with an increase in grain length of about 10.2% (see fig. 8A and C), an increase in grain width of about 7.1% (see fig. 8B and D), and an increase in grain thickness of about 6.5% (see fig. 8E), while statistics were also made on the kernels of brown rice, which were found to increase in length, width, and thickness by 8%, 9%, and 3%, respectively (see fig. 8F-J), thereby increasing thousand kernel weight by about 13% compared to the wild type (see fig. 7C).

The phenotype of the RNA interference strain of TCBG1 is similar to that of the TCBG1 mutant, the plant height is shorter than that of a wild type, the grain is bigger than that of the wild type, the expression level of TCBG1 is reduced by about sixty percent compared with that of the wild type, and the grain size is correspondingly bigger.

B. the tcbg1 mutant showed significantly faster filling rate than the wild type.

To investigate that the stage in which TCBG1 affected the filling process resulted in the occurrence of individual size differences between the mutant and wild type, the filling process was followed for both wild type and mutant and samples were taken every third day to measure the wet and dry weights of rice and brown rice grains. As shown in FIGS. 9A and B, rice and brown rice are similar, the filling speed of the mutant is obviously higher than that of the wild type twelve days after pollination, both the dry weight and the wet weight of the mutant are obviously higher than those of the wild type, the filling reaches the highest value 21 days after pollination, the wet weight and the dry weight of the mutant rice are respectively improved by 18.1 percent and 21.1 percent compared with the wild type, the wet weight and the dry weight of the brown rice are respectively improved by 16.0 percent and 18.2 percent, and in order to search the reason for accelerating the filling of the mutant, the expression levels of five genes, namely AP2/EREBP, AGPL2, GBSSI, GLUCAN and BEI, related to starch synthesis in the filling process are detected, and the expression levels of the five genes in the mutant are obviously more than two times higher than that of the wild type. It can thus be concluded that the rate of mutant filling is significantly increased in the middle and late stages of filling so that the final mutant accumulates significantly more organic than the wild type.

C. Molecular function of TCBG 1: TCBG1 having chloride ion outward transport activity

To investigate the electrophysiological activity of TCBG1, in vitro transcribed TCBG1 was injected into xenopus laevis oocytes (hereinafter frog eggs) by microinjection. Control frog egg cells were injected with an equal volume of water without rnase using the same method. The frog eggs injected with the cRNA and water are cultured in an environment of 18 ℃ for about 36 hours and then subjected to electrophysiological activity experiments. Analysis of experimental results shows that TCBG1 has similar function to DTX33/35, and has chloride ion outward transport activity. As shown in FIG. 10, no significant current activity was detected in the control frog eggs (injected with water containing no RNase) (see FIGS. 10A and B), while significant current activity was detected in the frog eggs expressing TCBG1 (injected with TCBG1 in vitro transcribed cRNA) when 96mM NaCl was added to the perfusate (see FIG. 10C).

We used the above technology to express the TCBG1 mutant cRNA with the 251 th T deletion in frog eggs, and since the protein is terminated early in the translation process, the amino acid sequence is shown in SEQ ID NO.4, and no obvious current activity is detected (as shown in FIGS. 10E and F), which indicates that the mutation of the site results in the loss of the function of TCBG 1.

Further, based on the transport activity and substrate specificity of TCBG1, to confirm that the current activity was caused by chloride ions, 96mM sodium glutamate was added to the perfusate in place of chloride ions, and the current activity of cRNA-injected frog egg cells was recorded in the same manner. No significant current generation was detected at this time (see fig. 10D), and it was preliminarily confirmed that the generation of TCBG1 current was due to outward transport of chloride ions. Thus, deletion of this channel in the mutant prevents chloride from being eliminated intracellularly, increasing the osmotic potential of the cell and thereby altering the turgor pressure of the cell.

Thus, the TCBG1 gene mediates chloride ion transport. The size of rice grains can be increased by knocking out or silencing the TCBG1 gene to regulate the turgor pressure or osmotic potential of cells. In the first and second embodiments, the mutant of TCBG1 gene lacks chloride efflux channel, so that chloride can not be excreted from the cell, and the osmotic potential of the cell is increased to change the turgor pressure of the cell, and further change the physiological process of rice growth, so that the cell of the seed is enlarged, more organic matters are accumulated, and the weight of the seed is increased. The invention represents a brand new mechanism for controlling the size of rice grains, and has creativity in the aspect of increasing the rice yield.

The above embodiments are provided for illustrative purposes only and are not intended to limit the present invention, and various changes and modifications may be made by those skilled in the art without departing from the scope of the present invention, and therefore, all equivalent technical solutions should fall within the scope of the present disclosure.

SEQUENCE LISTING

<110> university of capital education

<120> channel protein with anion transport activity and genome modification thereof for regulating and controlling crop grain size

<130> 90

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1440

<212> DNA

<213> Rice (Oryza sativa)

<400> 1

atgacgcctc caccgccgtc gccgccgcac gagaggaaaa cgtgggcgga gtcggtggcc 60

agcgagtttc gggcgcagcg cggcatcgcg ttccctctca tcgccatgaa cctcacctgg 120

ttcgccaagc tggccgtcac caccgccttc ctcggccgcc tcggcgacct ccagctcgcc 180

gccggcaccc tcggcttcag cttcgccaat gtcaccggct tcgccgtcct caccggcctc 240

tgcgccgcca tggaccccat ctgcgggcag gcgcacggcg ccagcaacgg gaagctcctc 300

cgcaagacgc tggtgatggc caccatcctg ctgctgggcg cgtccatccc catcgccttc 360

ctgtggctgc acgtggacgc cgtcctcctc cggttcggac agcaggcgga catgagcagc 420

aacgcacgca gctacgtggt gtgcctcctc ccggacctcg ccgtcacctc cttcgtcaac 480

ccgctcaagt cgtacctgag cgcgcagggg gtgacgctcc ccacgctgtt cgcctccgcc 540

ctggccctgg cgctccacgt ccccctcacc atgtggatgg ccaggaccag gggcatccag 600

ggcgtcgcca ccgccgtgtg ggtcagcgac ctggccgtgg ccgtcatgct cgccggctac 660

gtgctcgtct cggagcgacg acggaaggcg ggagggggcg gcggatgggt ggagcagacg 720

aggggtgagt gggtccggct cctccggctg gccgttccca gctgcctcaa cacctgcctg 780

gagtggtggt gctacgagat actggtgctc ctgacgggac gcctcccgga cgcccggcgc 840

acggtggcgg tgatggccgt gacgctcaac ttcgactacc tgctgttcgc ggggatgctg 900

tccctgtcgg tgagcgcgtc ggtgcgcgtg tcgaacgagc tgggcgcggg ggaggcgtgg 960

gcggcgaggc gcgcgggcat ggtgtcgatc gtgggcggcg cggtgggcgg ggtgggcggc 1020

ggggtggcga tggtggcggc gcggcgggcg tgggggagca tatacagctc agacgccggg 1080

gtgcgggagg gggtggggag ggcgatggag gtgatggcgg tgctggaggt ggtgaacttc 1140

ccgctgaacg tgtgcggggg gatagtgcga gggacggcga ggccggcggt ggggatgtac 1200

gccgtggtgg ccggcttcta cgtgctggcg ctgccgctcg gggtcgcgct cgccttcaag 1260

gccagacttg ggatccaggg cctcctcctg ggcttcctgg tgggcgccgc ggccagcttg 1320

gcggtgctcc tcaccttcat cgcgcgcatg gattggcccg ccgaggccca aaaggcgcgg 1380

actagaacca cagcaaccgt ggcccaattc caccaacacg acgaggtcgt ccagccttga 1440

<210> 2

<211> 479

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Thr Pro Pro Pro Pro Ser Pro Pro His Glu Arg Lys Thr Trp Ala

1 5 10 15

Glu Ser Val Ala Ser Glu Phe Arg Ala Gln Arg Gly Ile Ala Phe Pro

20 25 30

Leu Ile Ala Met Asn Leu Thr Trp Phe Ala Lys Leu Ala Val Thr Thr

35 40 45

Ala Phe Leu Gly Arg Leu Gly Asp Leu Gln Leu Ala Ala Gly Thr Leu

50 55 60

Gly Phe Ser Phe Ala Asn Val Thr Gly Phe Ala Val Leu Thr Gly Leu

65 70 75 80

Cys Ala Ala Met Asp Pro Ile Cys Gly Gln Ala His Gly Ala Ser Asn

85 90 95

Gly Lys Leu Leu Arg Lys Thr Leu Val Met Ala Thr Ile Leu Leu Leu

100 105 110

Gly Ala Ser Ile Pro Ile Ala Phe Leu Trp Leu His Val Asp Ala Val

115 120 125

Leu Leu Arg Phe Gly Gln Gln Ala Asp Met Ser Ser Asn Ala Arg Ser

130 135 140

Tyr Val Val Cys Leu Leu Pro Asp Leu Ala Val Thr Ser Phe Val Asn

145 150 155 160

Pro Leu Lys Ser Tyr Leu Ser Ala Gln Gly Val Thr Leu Pro Thr Leu

165 170 175

Phe Ala Ser Ala Leu Ala Leu Ala Leu His Val Pro Leu Thr Met Trp

180 185 190

Met Ala Arg Thr Arg Gly Ile Gln Gly Val Ala Thr Ala Val Trp Val

195 200 205

Ser Asp Leu Ala Val Ala Val Met Leu Ala Gly Tyr Val Leu Val Ser

210 215 220

Glu Arg Arg Arg Lys Ala Gly Gly Gly Gly Gly Trp Val Glu Gln Thr

225 230 235 240

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

245 250 255

Asn Thr Cys Leu Glu Trp Trp Cys Tyr Glu Ile Leu Val Leu Leu Thr

260 265 270

Gly Arg Leu Pro Asp Ala Arg Arg Thr Val Ala Val Met Ala Val Thr

275 280 285

Leu Asn Phe Asp Tyr Leu Leu Phe Ala Gly Met Leu Ser Leu Ser Val

290 295 300

Ser Ala Ser Val Arg Val Ser Asn Glu Leu Gly Ala Gly Glu Ala Trp

305 310 315 320

Ala Ala Arg Arg Ala Gly Met Val Ser Ile Val Gly Gly Ala Val Gly

325 330 335

Gly Val Gly Gly Gly Val Ala Met Val Ala Ala Arg Arg Ala Trp Gly

340 345 350

Ser Ile Tyr Ser Ser Asp Ala Gly Val Arg Glu Gly Val Gly Arg Ala

355 360 365

Met Glu Val Met Ala Val Leu Glu Val Val Asn Phe Pro Leu Asn Val

370 375 380

Cys Gly Gly Ile Val Arg Gly Thr Ala Arg Pro Ala Val Gly Met Tyr

385 390 395 400

Ala Val Val Ala Gly Phe Tyr Val Leu Ala Leu Pro Leu Gly Val Ala

405 410 415

Leu Ala Phe Lys Ala Arg Leu Gly Ile Gln Gly Leu Leu Leu Gly Phe

420 425 430

Leu Val Gly Ala Ala Ala Ser Leu Ala Val Leu Leu Thr Phe Ile Ala

435 440 445

Arg Met Asp Trp Pro Ala Glu Ala Gln Lys Ala Arg Thr Arg Thr Thr

450 455 460

Ala Thr Val Ala Gln Phe His Gln His Asp Glu Val Val Gln Pro

465 470 475

<210> 3

<211> 1439

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

atgacgcctc caccgccgtc gccgccgcac gagaggaaaa cgtgggcgga gtcggtggcc 60

agcgagtttc gggcgcagcg cggcatcgcg ttccctctca tcgccatgaa cctcacctgg 120

ttcgccaagc tggccgtcac caccgccttc ctcggccgcc tcggcgacct ccagctcgcc 180

gccggcaccc tcggcttcag cttcgccaat gtcaccggct tcgccgtcct caccggcctc 240

tgcgccgcca ggaccccatc tgcgggcagg cgcacggcgc cagcaacggg aagctcctcc 300

gcaagacgct ggtgatggcc accatcctgc tgctgggcgc gtccatcccc atcgccttcc 360

tgtggctgca cgtggacgcc gtcctcctcc ggttcggaca gcaggcggac atgagcagca 420

acgcacgcag ctacgtggtg tgcctcctcc cggacctcgc cgtcacctcc ttcgtcaacc 480

cgctcaagtc gtacctgagc gcgcaggggg tgacgctccc cacgctgttc gcctccgccc 540

tggccctggc gctccacgtc cccctcacca tgtggatggc caggaccagg ggcatccagg 600

gcgtcgccac cgccgtgtgg gtcagcgacc tggccgtggc cgtcatgctc gccggctacg 660

tgctcgtctc ggagcgacga cggaaggcgg gagggggcgg cggatgggtg gagcagacga 720

ggggtgagtg ggtccggctc ctccggctgg ccgttcccag ctgcctcaac acctgcctgg 780

agtggtggtg ctacgagata ctggtgctcc tgacgggacg cctcccggac gcccggcgca 840

cggtggcggt gatggccgtg acgctcaact tcgactacct gctgttcgcg gggatgctgt 900

ccctgtcggt gagcgcgtcg gtgcgcgtgt cgaacgagct gggcgcgggg gaggcgtggg 960

cggcgaggcg cgcgggcatg gtgtcgatcg tgggcggcgc ggtgggcggg gtgggcggcg 1020

gggtggcgat ggtggcggcg cggcgggcgt gggggagcat atacagctca gacgccgggg 1080

tgcgggaggg ggtggggagg gcgatggagg tgatggcggt gctggaggtg gtgaacttcc 1140

cgctgaacgt gtgcgggggg atagtgcgag ggacggcgag gccggcggtg gggatgtacg 1200

ccgtggtggc cggcttctac gtgctggcgc tgccgctcgg ggtcgcgctc gccttcaagg 1260

ccagacttgg gatccagggc ctcctcctgg gcttcctggt gggcgccgcg gccagcttgg 1320

cggtgctcct caccttcatc gcgcgcatgg attggcccgc cgaggcccaa aaggcgcgga 1380

ctagaaccac agcaaccgtg gcccaattcc accaacacga cgaggtcgtc cagccttga 1439

<210> 4

<211> 104

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

Met Thr Pro Pro Pro Pro Ser Pro Pro His Glu Arg Lys Thr Trp Ala

1 5 10 15

Glu Ser Val Ala Ser Glu Phe Arg Ala Gln Arg Gly Ile Ala Phe Pro

20 25 30

Leu Ile Ala Met Asn Leu Thr Trp Phe Ala Lys Leu Ala Val Thr Thr

35 40 45

Ala Phe Leu Gly Arg Leu Gly Asp Leu Gln Leu Ala Ala Gly Thr Leu

50 55 60

Gly Phe Ser Phe Ala Asn Val Thr Gly Phe Ala Val Leu Thr Gly Leu

65 70 75 80

Cys Ala Ala Arg Thr Pro Ser Ala Gly Arg Arg Thr Ala Pro Ala Thr

85 90 95

Gly Ser Ser Ser Ala Arg Arg Trp

100

<210> 5

<211> 300

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

tgcgccgcca tggaccccat ctgcgggcag gcgcacggcg ccagcaacgg gaagctcctc 60

cgcaagacgc tggtgatggc caccatcctg ctgctgggcg cgtccatccc catcgccttc 120

ctgtggctgc acgtggacgc cgtcctcctc cggttcggac agcaggcgga catgagcagc 180

aacgcacgca gctacgtggt gtgcctcctc ccggacctcg ccgtcacctc cttcgtcaac 240

ccgctcaagt cgtacctgag cgcgcagggg gtgacgctcc ccacgctgtt cgcctccgcc 300

Claims

1. A method for adjusting grain size of rice, comprising:

The DNA fragment of rice is modified by genetic engineering technology, so that the TCBG1 gene in rice is knocked out, knocked down or gene silenced; the CDS sequence of the TCBG1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2 .

2. The method according to claim 1, wherein the genetic engineering technical means is CRISPR/Cas9.

3. The method of claim 2, wherein the CRISPR/Cas9 target sequence is one or more of the following:

CGCCGTCGCCGCCGCACGAGAGG;

CCTCGGCCGCCTCGGCGACCTCC;

CCGCCATGGACCCCATCTGCGGGG;

CCCAGCTGCCTCAACACCTGCC;

GCGGGCATGGTGTCGATCGTGGGG;

CAAGGCCAGACTTGGGATCCAGG.

4. The method according to claim 3, wherein the CDS sequence of the recombinant DNA fragment obtained by CRISPR/Cas9 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

5. The method of claim 1, wherein the genetic engineering means is TALEN technology.

6. The method of claim 5, wherein the TALEN recognition region sequence is one or more of the following:

GGCCGTCACCACCGCCTTCCTCGGCCGCCTCGGCGACCTCCAGCTCGCCGCCGGC;

CGCCGTCCTCACCGGCCTCTGCGCCGCCATGGACCCCATCTGCGGGCAGGCGC;

CGCCTCCGCCCTGGCCCTGGCGCTCCACGTCCCCCTCACCATGTGGATGGCCAGG.

7. The method according to claim 1, wherein the genetic engineering technical means is RNAi.

8. The method of claim 7, wherein the RNAi target sequence is shown in SEQ ID NO.5.

9. A DNA fragment of rice modified by genetic engineering technology is used in adjusting rice grain size; wherein, the DNA fragment of rice modified by genetic engineering technology makes TCBG1 gene knockout, knockdown or gene silencing in rice , the CDS sequence of the TCBG1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

10. The application according to claim 9, wherein the genetic engineering technology means is CRISPR/Cas9.

11. The application of claim 10, wherein the CRISPR/Cas9 target sequence is one or more of the following:

CGCCGTCGCCGCCGCACGAGAGG;

CCTCGGCCGCCTCGGCGACCTCC;

CCGCCATGGACCCCATCTGCGGGG;

CCCAGCTGCCTCAACACCTGCC;

GCGGGCATGGTGTCGATCGTGGGG;

CAAGGCCAGACTTGGGATCCAGG.

The application according to claim 10, wherein the CDS sequence of the recombinant DNA fragment obtained by CRISPR/Cas9 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

13. The use according to claim 9, wherein the genetic engineering means is TALEN technology.

14. The application of claim 13, wherein the TALEN recognition region sequence is one or more of the following:

GGCCGTCACCACCGCCTTCCTCGGCCGCCTCGGCGACCTCCAGCTCGCCGCCGGC;

CGCCGTCCTCACCGGCCTCTGCGCCGCCATGGACCCCATCTGCGGGCAGGCGC;

CGCCTCCGCCCTGGCCCTGGCGCTCCACGTCCCCCTCACCATGTGGATGGCCAGG.

15. The application according to claim 9, wherein the genetic engineering technical means is RNAi.

16. The use according to claim 15, wherein the RNAi target sequence is shown in SEQ ID NO.5.

17. A method of adjusting grain size of rice, comprising:

The DNA fragment of rice is processed by genetic engineering technology, so that the DNA fragment of rice cannot be transcribed after editing, resulting in silencing of the TCBG1 gene; the CDS sequence of the TCBG1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQID NO.2 shown.

18. An application in adjusting turgor pressure or osmotic potential by transforming a DNA fragment of rice by means of genetic engineering technology wherein, the DNA fragment of transforming rice by means of genetic engineering technology makes TCBG1 gene knockout, knockdown or For gene silencing, the CDS sequence of the TCBG1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

19. The application according to claim 18, wherein the genetic engineering technology means is CRISPR/Cas9.

20. The application of claim 19, wherein the CRISPR/Cas9 target sequence is one or more of the following:

CGCCGTCGCCGCCGCACGAGAGG;

CCTCGGCCGCCTCGGCGACCTCC;

CCGCCATGGACCCCATCTGCGGGG;

CCCAGCTGCCTCAACACCTGCC;

GCGGGCATGGTGTCGATCGTGGGG;

CAAGGCCAGACTTGGGATCCAGG.

21. The application according to claim 20, wherein the CDS sequence of the recombinant DNA fragment obtained by CRISPR/Cas9 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

22. The use according to claim 18, wherein the genetic engineering means is TALEN technology.

23. The application of claim 22, wherein the TALEN recognition region sequence is one or more of the following:

GGCCGTCACCACCGCCTTCCTCGGCCGCCTCGGCGACCTCCAGCTCGCCGCCGGC;

CGCCGTCCTCACCGGCCTCTGCGCCGCCATGGACCCCATCTGCGGGCAGGCGC;

CGCCTCCGCCCTGGCCCTGGCGCTCCACGTCCCCCTCACCATGTGGATGGCCAGG.

24. The application according to claim 18, wherein the genetic engineering technical means is RNAi.

25. The use according to claim 24, wherein the RNAi target sequence is shown in SEQ ID NO.5.

26. A method for adjusting the turgor pressure or osmotic potential of rice cells, comprising:

The DNA fragment of rice is treated by genetic engineering technology to silence the TCBG1 gene after translation of the DNA fragment of rice; the CDS sequence of the TCBG1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.