Use of LTPL120 protein and gene encoding same in regulation of plant yield trait

Abstract

Provided is the use of an LTPL120 protein and a gene encoding same in the regulation of a plant yield trait. Further provided is the use of an LTPL120 protein or an LTPL120 gene in the regulation of a plant trait. The trait is a yield trait and / or a growth trait. The regulation means that a decrease in the LTPL120 protein results in an increase in the plant yield. The use of an LTPL120 protein or an LTPL120 gene as an inhibition target in plant breeding, wherein an objective of the plant breeding is to cultivate a plant with an altered trait. The trait is a yield trait and / or a growth trait, and the plant with the altered trait is a plant with an increased yield. The present invention can be used for plant breeding, especially rice breeding, and has application and popularization value.

Description

Application of LTPL120 protein and its encoding gene in regulating plant yield traits TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to application of LTPL120 protein and its encoding gene in regulating plant yield traits. BACKGROUND

[0002] Rice is one of the important food crops for human beings, and has a very long history of cultivation and consumption. Half of the world's population consumes rice, mainly in Asia, southern Europe and parts of the Americas and Africa. The total yield of rice accounts for the third place in the world's grain crop yield, lower than corn and wheat, but can maintain a larger population, so the United Nations designated 2004 as the "International Rice Year".

[0003] China is one of the original habitats of rice. The farmland in southern China is mainly paddy field, and the main grain crop is rice. 6000 years ago, Hemudu people domesticated rice, adding human factors to the simple relationship between plants and land. 2000 years ago, rice was introduced to Japan. Later, rice was introduced to Iran, Greece and Africa, and then to Spain, Italy, France, Germany and the United Kingdom. After the discovery of the New World, rice also entered the Americas.

[0004] In recent years, with the increase of world population, the decrease of arable land and the increasing stress of biotic and abiotic factors, rice production is facing unprecedented challenges. Traditional breeding techniques have made important contributions to improving yield and quality of rice, but due to the lack of germplasm resources of Oryza, changes in ecological environment and limitations of traditional breeding methods, it brings certain difficulties to further genetic improvement of rice. With the in-depth study of molecular biology, the isolation, cloning and recombination technology of gene are becoming mature, and transgenic technology has become an effective means of genetic improvement of rice. SUMMARY

[0005] Rice is one of the important food crops for human beings, and it is of great significance to improve the genetics of rice through transgenic technology to obtain high-yield rice. The present application provides application of LTPL120 protein in regulating plant traits, and the traits are yield traits and / or growth traits. The reduction of LTPL120 protein increases the yield of plants. The reduction of LTPL120 protein increases the root length and / or root weight and / or the volume of cortical cells in root tips of plants. The present application can be used for plant breeding, especially rice breeding, and has great application and promotion value. TECHNICAL SOLUTION

[0006] The purpose of the present application is to provide application of LTPL120 protein and its encoding gene in regulating plant yield traits.

[0007] The present application provides application of LTPL120 protein in regulating plant traits.

[0008] The traits are yield traits and / or growth traits.

[0009] The meaning of the regulation is that the decrease of LTPL120 protein increases the yield of the plant.

[0010] The meaning of the regulation is that the decrease of LTPL120 protein increases the root length and / or root weight and / or the volume of cortical cells in root tips of the plant.

[0011] The present application also provides application of LTPL120 gene in regulating plant traits.

[0012] The traits are yield traits and / or growth traits.

[0013] The meaning of the regulation is that the knockout of LTPL120 gene or the inhibition of expression of LTPL120 gene or the editing of LTPL120 gene increases the yield of the plant.

[0014] The meaning of the regulation is that the knockout of LTPL120 gene or the inhibition of expression of LTPL120 gene or the editing of LTPL120 gene increases the root length and / or root weight and / or the volume of cortical cells in root tips of the plant.

[0015] The present application also protects application of LTPL120 protein or LTPL120 gene as a target for inhibition in plant breeding; the goal of the plant breeding is to breed plants with changed traits.

[0016] The traits are yield traits and / or growth traits.

[0017] The plants with changed traits are plants with increased yield.

[0018] The plants with changed traits are plants with increased root length and / or root weight and / or the volume of cortical cells in root tips.

[0019] The present application also protects application of substances for inhibiting LTPL120 gene and / or substances for inhibiting LTPL120 protein in plant breeding; the goal of the plant breeding is to breed plants with changed traits.

[0020] The traits are yield traits and / or growth traits.

[0021] The plants with changed traits are plants with increased yield.

[0022] The plants with changed traits are plants with increased root length and / or root weight and / or the volume of cortical cells in root tips.

[0023] The application also provides a plant breeding method for changing the traits of plants, comprising the following steps: changing the traits of plants by inhibiting the expression of the LTPL120 gene in the plants.

[0024] The application also provides a plant breeding method for changing the traits of plants, comprising the following steps: performing gene editing on the LTPL120 gene in a recipient plant to obtain a gene edited plant, and screening a plant with changed traits from the gene edited plant relative to the recipient plant. The method further comprises the following steps: selfing the screened plant and obtaining a progeny plant.

[0025] The LTPL120 gene in a recipient plant refers to the LTPL120 gene in the genome of the recipient plant.

[0026] The traits are yield traits and / or growth traits.

[0027] The plant (or plant) with changed traits is a plant (or plant) with increased yield.

[0028] The plant (or plant) with changed traits is a plant (or plant) with increased root length and / or increased root weight and / or increased volume of cortical cells in root tips.

[0029] The application also provides a method for preparing a plant with changed traits, comprising the following steps: replacing the DNA segment shown in SEQ ID NO: 7 in the plant genomic DNA with the DNA segment shown in SEQ ID NO: 8 to obtain a plant with changed traits.

[0030] The application also provides a method for preparing a plant with changed traits, comprising the following steps: replacing the DNA segment shown in SEQ ID NO: 7 in the plant genomic DNA with the DNA segment shown in SEQ ID NO: 9 to obtain a plant with changed traits.

[0031] Specifically, the replacement is a homozygous replacement, i.e., the same replacement occurs in homologous chromosomes.

[0032] The DNA segment shown in SEQ ID NO: 7 is located in the LTPL120 gene in the plant genomic DNA.

[0033] Any of the above LTPL120 proteins is as follows (a1) or (a2) or (a3) or (a4):

[0034] (a1) the protein shown in SEQ ID NO: 1;

[0035] (a2) a fusion protein obtained by connecting a tag to the N terminus or / and C terminus of the protein in (a1);

[0036] (a3) a protein derived from rice and having 98% or more identity to (a1) and being associated with a plant trait;

[0037] (a4) a protein derived from rice and having 98% or more identity to (a1) and being associated with a plant trait.

[0038] Any of the above LTPL120 genes is a gene encoding a LTPL120 protein.

[0039] Any of the above LTPL120 genes is as follows (b1) or (b2) or (b3) or (b4):

[0040] (b1) a DNA molecule whose coding region is as set forth in SEQ ID NO: 2;

[0041] (b2) a DNA molecule as set forth in SEQ ID NO: 3;

[0042] (b3) a DNA molecule derived from rice and having 95% or more identity to (b1) or (b2) and encoding the protein;

[0043] (b4) a DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined in (b1) or (b2) and encoding the protein.

[0044] Any of the above LTPL120 genes is a gene encoding a LTPL120 protein.

[0045] Any of the above LTPL120 genes is a gene encoding a LTPL120 protein.

[0046] The gene editing material for editing the LTPL120 gene can be specifically sgRNA and Cas9 protein. The gene editing material for editing the LTPL120 gene can be specifically a DNA molecule encoding sgRNA and a DNA molecule encoding Cas9 protein. The gene editing material for editing the LTPL120 gene can be specifically an expression vector with a DNA molecule encoding sgRNA and an expression vector with a DNA molecule encoding Cas9 protein. The gene editing material for editing the LTPL120 gene can be specifically an expression vector with a DNA molecule encoding sgRNA and a DNA molecule encoding Cas9 protein. The target sequence binding region in the sgRNA is shown as SEQ ID NO: 6. The sgRNA is shown as SEQ ID NO: 5. The gene editing material for editing the LTPL120 gene is specifically a recombinant plasmid shown as SEQ ID NO: 4.

[0047] The Cas9 protein can be specifically a protein encoded by nucleotides 2697-6968 in SEQ ID NO: 4.

[0048] The plant can be specifically a monocotyledon or a dicotyledon.

[0049] The plant can be specifically a plant of the family Poaceae.

[0050] The plant can be specifically a plant of the genus Oryza.

[0051] The plant can be specifically rice, for example, Nipponbare. Beneficial effects

[0052] The present application can be used for plant breeding, in particular, rice breeding, and has great application and promotion value for improving rice yield. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a photograph showing the expression site of OsLTPL120 gene in roots observed by in situ hybridization.

[0054] Figure 2 is a photograph showing the phenotype of seedlings after 2 weeks of seed germination or plants at the jointing stage or the statistical results of root traits.

[0055] Figure 3 is an exemplary photograph showing the tillering of plants at the mature stage and the statistical results of the number of tillers.

[0056] Figure 4 is a photograph showing the number of grains per plant of plants at the mature stage and the statistical results of the yield per plant (reflected by the weight of grains). Embodiments of the present application

[0057] The application will be described in further detail below with specific reference to the embodiments. The examples given are merely for the purpose of illustrating the application and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0058] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged, unless otherwise specified.

[0059] The OsLTPL120 protein is shown in SEQ ID NO: 1. In the cDNA of rice Nipponbare, the CDS encoding the OsLTPL120 protein is shown in SEQ ID NO: 2. In the genomic DNA of rice Nipponbare, the gene encoding the OsLTPL120 protein is shown in SEQ ID NO: 3 (the gene has no intron). In the prior art, the function of the OsLTPL120 protein is unknown.

[0060] Example 1, preparation of gene-edited plants

[0061] I. Construction of recombinant plasmid

[0062] A recombinant plasmid (circular plasmid) was prepared, as shown in SEQ ID NO: 4. The recombinant plasmid expresses a Cas9 protein and a specific sgRNA. In SEQ ID NO: 4, nucleotides 2697-6968 encode the Cas9 protein. The coding region of the specific sgRNA is shown in nucleotides 520-614 of SEQ ID NO: 4. The specific sgRNA is shown in SEQ ID NO: 5, and the target sequence binding region in the sgRNA is shown in SEQ ID NO: 6.

[0063] II. Genetic transformation and obtaining regenerated plants

[0064] The recombinant plasmid prepared in step I was introduced into Agrobacterium EHA105 to obtain a recombinant Agrobacterium. The recombinant Agrobacterium was used to perform genetic transformation on embryogenic callus of rice Nipponbare by Agrobacterium dipping method, and then resistant callus was screened (resistance screening used 50 mg / L hygromycin), then differentiation and regeneration culture was performed, and then rooting culture was performed to obtain regenerated plants.

[0065] III. Obtaining gene-edited plants and their progeny plants

[0066] The regenerated plant obtained in step two is identified as follows: leaf blades are taken, genomic DNA is extracted, a primer pair composed of primer F1 and primer R1 is used for PCR amplification, and then the PCR amplification product is sequenced.

[0067] F1 (SEQ ID NO: 10): 5'-ACCACACAGCAATCGAGCTA-3';

[0068] R1 (SEQ ID NO: 11): 5'-GAGGTTGAGGTTGATGCCGA-3'.

[0069] Through the above identification, two homozygous mutant plants (homozygous mutant, i.e., the mutations on the two chromosomes are consistent) are screened from the regenerated plants obtained in step two, which are named osltpl120-1 plant and osltpl120-2 plant, respectively. Sequencing identification shows that, compared with the genomic DNA of rice Nipponbare, the difference of the osltpl120-1 plant is only that 3 consecutive nucleotides are deleted in the gene encoding the LTPL120 protein (i.e., the DNA mutation in SEQ ID NO: 7 in the genomic DNA is mutated to the DNA shown in SEQ ID NO: 8). Sequencing identification shows that, compared with the genomic DNA of rice Nipponbare, the difference of the osltpl120-2 plant is only that 2 consecutive nucleotides are deleted in the gene encoding the LTPL120 protein (i.e., the DNA mutation in SEQ ID NO: 7 in the genomic DNA is mutated to the DNA shown in SEQ ID NO: 9; causing a frame shift and premature termination).

[0070] The osltpl120-1 plant is selfed and seeds are harvested, which are cultivated into plants, i.e., T1 generation plants. The T1 generation plants are selfed and seeds are harvested, i.e., T2 generation seeds. The osltpl120-1 plant and its offspring are called osltpl120-1 line.

[0071] The osltpl120-2 plant is selfed and seeds are harvested, which are cultivated into plants, i.e., T1 generation plants. The T1 generation plants are selfed and seeds are harvested, i.e., T2 generation seeds. The osltpl120-2 plant and its offspring are called osltpl120-2 line.

[0072] Example 2, Expression characteristics of OsLTPL120 gene

[0073] After rice Nipponbare seeds are germinated for 5 days, root tips are taken, and the expression site of OsLTPL120 gene in roots is observed by in situ hybridization experiment. The results are shown in Figure 1, and it can be observed that the OsLTPL120 gene is specifically expressed in the cortical cells in the root tip.

[0074] Example 3, growth performance and production performance of plants

[0075] The test seeds were: rice Nipponbare seeds, T2 generation seeds of osltpl120-1 strain, T2 generation seeds of osltpl120-2 strain. Rice Nipponbare was denoted as WT.

[0076] The test plants were cultured under parallel conditions, specifically: seeds were taken, germinated and seedlings were obtained in a greenhouse (counting from the beginning of germination, a total of 3 weeks of culture), and 3-week-old seedlings were obtained; the 3-week-old seedlings were transplanted to a field in Langfang, Hebei, and normally cultivated and managed.

[0077] An exemplary photo of the seedlings 2 weeks after seed germination is shown in FIG. 2A. Two weeks after seed germination, the root tip part (the sampling site is shown in the rectangular frame in FIG. 2A) was taken, cross-sectioned to make a section, and subjected to aniline blue staining, and an exemplary photo is shown in FIG. 2C. Two weeks after seed germination, the root length was measured, and the results are shown in FIG. 2D (average value of 20 plants). After aniline blue staining, the surface area of the mesocortex cells was measured using PS software, and the results are shown in FIG. 2F (average value of 20 plants). After aniline blue staining, the number of mesocortex cells was measured using PS software, and the results are shown in FIG. 2G (average value of 20 plants).

[0078] An exemplary photo of the root phenotype of the plants at the jointing stage is shown in FIG. 2B. At the jointing stage, the root part (i.e., the entire underground part) was taken, dried at 80°C to a constant weight, and the root dry weight was obtained, and the results are shown in FIG. 2E (average value of 20 plants).

[0079] An exemplary photo of the tillering of the plants at the maturation stage is shown in FIG. 3A, and the statistical results of the tiller number are shown in FIG. 3B (average value of 20 plants).

[0080] A photo showing the grain number per plant of the plants at the maturation stage is shown in FIG. 4A, and the statistical results of the yield per plant (reflected as grain weight) are shown in FIG. 4B (average value of 20 plants).

[0081] Compared with the rice Nipponbare plants at the same stage, the roots of the osltpl120-1 strain plants and the osltpl120-2 strain plants were more developed (reflected as longer root length and heavier root dry weight). Compared with the rice Nipponbare plants at the same stage, the number of mesocortex cells at the root tip of the osltpl120-1 strain plants and the osltpl120-2 strain plants did not differ significantly, but the volume of the mesocortex cells was significantly increased (reflected as increased cell surface area). Compared with the rice Nipponbare plants at the same stage, the osltpl120-1 strain plants and the osltpl120-2 strain plants had more tillers, more grains, and higher yield.

[0082] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives specific examples, it should be understood that the application can be further improved. In summary, according to the principle of the application, the present application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application. Some basic features can be applied within the scope of the following attached claims. Industrial applicability

[0083] The application discloses application of LTPL120 protein in regulating plant traits, the traits are yield traits and / or growth traits. LTPL120 protein reduction increases plant yield. LTPL120 protein reduction increases root length and / or root weight and / or cortex cell volume in root tip. The application can be used in plant breeding, especially rice breeding, and has great application and popularization value.

Claims

1. Use of LTPL120 protein in regulating plant traits; The LTPL120 protein is as follows (a1) or (a2) or (a3) or (a4): (a1) the protein shown in SEQ ID NO: 1; (a2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the protein in (a1); (a3) a protein obtained by substitution and / or deletion and / or addition of one or several amino acid residues of (a1) and related to plant traits; (a4) a protein derived from rice and having more than 98% identity with (a1) and related to plant traits; The traits are yield traits and / or growth traits.

2. The use according to claim 1, wherein: The meaning of the regulation is that the LTPL120 protein reduces the yield of the plant.

3. Use of LTPL120 gene in regulating plant traits; The LTPL120 gene is a gene encoding the LTPL120 protein; The LTPL120 protein is the LTPL120 protein described in claim 1.

4. The use according to claim 3, wherein: The meaning of the regulation is that the LTPL120 gene is knocked out or the expression of the LTPL120 gene is inhibited or the LTPL120 gene is edited to increase the yield of the plant.

5. Use of LTPL120 protein or LTPL120 gene as a target for inhibition in plant breeding; the goal of the plant breeding is to breed plants with changed traits; the LTPL120 protein is the LTPL120 protein described in claim 1; the LTPL120 gene is a gene encoding the LTPL120 protein.

6. Use of a substance that inhibits the LTPL120 gene and / or a substance that inhibits the LTPL120 protein in plant breeding; the goal of the plant breeding is to breed plants with changed traits; the LTPL120 protein is the LTPL120 protein described in claim 1; the LTPL120 gene is a gene encoding the LTPL120 protein.

7. A plant breeding method for changing the traits of plants, comprising the following steps: changing the traits of the plants by inhibiting the expression of the LTPL120 gene in the plants; the LTPL120 gene is a gene encoding the LTPL120 protein, and the LTPL120 protein is the LTPL120 protein described in claim 1.

8. A plant breeding method for changing the traits of plants, comprising the following steps: performing gene editing on the LTPL120 gene in the recipient plants to obtain gene edited plants, and screening plants with changed traits from the gene edited plants relative to the recipient plants; the LTPL120 gene is a gene encoding the LTPL120 protein, and the LTPL120 protein is the LTPL120 protein described in claim 1.

9. A method for preparing a plant with altered traits, comprising the step of replacing the DNA segment as set forth in SEQ ID NO: 7 in the plant genomic DNA with the DNA segment as set forth in SEQ ID NO: 8 to obtain the plant with altered traits.

10. A method for preparing a plant with altered traits, comprising the step of replacing the DNA segment as set forth in SEQ ID NO: 7 in the plant genomic DNA with the DNA segment as set forth in SEQ ID NO: 9 to obtain the plant with altered traits.

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