Application of brachypodium distachyon BdSTAR1 protein and coding gene thereof in regulating and controlling grain length of plant seeds

Abstract

The invention discloses an application of brachypodium distachyon BdSTAR1 protein and a coding gene thereof in regulating and controlling the grain length of plant seeds, and belongs to the technical field of plant genetic engineering. The invention finds that the brachypodium distachyon BdSTAR1 gene is related to the grain length of plant seeds for the first time, and overexpression of the brachypodium distachyon BdSTAR1 gene can increase the grain length of the plant seeds; the Brachypodium distachyon BdSTAR1 gene is knocked out or silenced, so that the grain length of plant seeds can be shortened. Therefore, by regulating the brachypodium distachyon BdSTAR1 gene, a corresponding plant variety can be cultivated according to the requirement on the grain shape of the plant seed, and the brachypodium distachyon BdSTAR1 gene has important application value in agricultural production.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to the application of the BdSTAR1 protein of Brachypodium distachyon and its encoding gene in regulating the grain length of plant seeds. Background Art

[0002] Brachypodium distachyon ( Brachypodium distachyon ) has the smallest genome among the known gramineous plants so far, only 272 Mbp. Brachypodium distachyon is very closely related to wheat, and the similarity between their genomes is more than 95%. In addition, as a weed, Brachypodium distachyon has the advantages of small plant type, easy cultivation, short life cycle, self-pollination, easy genetic transformation, etc. Therefore, Brachypodium distachyon is a new ideal model material for studying wheat and rice crops.

[0003] ABC transporters, also known as ATP-binding cassette transporters (ABC), are named because they contain an adenosine triphosphate (ATP) binding cassette. They can rely on the energy generated by ATP hydrolysis to achieve the transmembrane transport of substrates inside and outside the cell, and are currently the class of proteins with the largest known number and functions. So far, 130 and 128 gene family members have been successfully identified in Arabidopsis thaliana and rice respectively. However, there are few reports on its related family members. In addition, there are also reports on ABC family members in species such as wheat, corn and tomato, but the transport substrates and functions of most members are still unclear.

[0004] According to the international nomenclature system, plant ABC proteins are divided into eight subfamilies: ABCA-ABCG and ABCI. At present, no protein of the ABCH subfamily has been found in plants, and the ABCI family is a special type of fungus. Among the ABC families, more research has been done on the four subfamilies: ABCA, ABCB, ABCC, and ABCG.

[0005] The ABCA subfamily includes the full molecule protein AOH and the half molecule protein ATH. So far, only 12 subfamily members of ABCA have been identified in Arabidopsis thaliana, and it is speculated that they play a certain role in the lipid transport of plants. In addition, some ABCA family proteins have also been identified in species such as cucumber and tomato, and they may be involved in the secretion activity of roots and have specific functions in flower organs respectively.

[0006] ABCB is the second largest subfamily of ABC family proteins. Currently, 29 ABCB members have been found in Arabidopsis thaliana. Most of the identified ABCB members are mainly related to the transport and regulation of hormonal substances. In addition, studies in tomatoes have found that ABCB is also involved in ion and heavy metal transport. For example, SlABCB is highly expressed in roots, suggesting that it may be involved in ion and heavy metal transport in roots. In addition, ABC also plays a role in transporting alkaloids and maintaining iron homeostasis.

[0007] The functions of ABCC subfamily proteins include transporting metabolites (such as anthocyanins, phytic acid, folic acid, etc.). For example, ZmMRP3 in maize, VvABCCl in grapes, and AtABCC2 in Arabidopsis thaliana have all been proven to have the function of transporting anthocyanins in vacuoles. Another major function of the C subfamily is detoxification. In Arabidopsis thaliana, AtABCCl and AtABCC2 have been proven to be tolerant to arsenic, cadmium, and mercury.

[0008] ABCG subfamily proteins are the largest subfamily of ABC family proteins and are also the type of subproteins with the most functions and numbers. It includes two types: full-molecule PDR proteins and half-molecule WBC proteins. Currently, ABCG subfamily proteins have been identified in many plants. For the functions they perform, they mainly include: transporting metabolites in plants, including lipids, alkenes, alkaloids, etc. Secondly, they mediate the transport of hormones such as ABA substances. For example, AtABCG25 and AtABCG31 both have the function of transporting abscisic acid (ABA). In addition, ABCG is involved in heavy metal stress / adverse stress responses and can improve the stress resistance of plants to abiotic stresses. Except for the relatively more reports on the four subfamilies of ABCA, ABCB, ABCC, and ABCG in the ABC family proteins, most of the other subfamily members have not been identified or their functions have not been discovered.

[0009] In rice, Os STAR1 genes have been identified to be involved in regulating the aluminum toxicity tolerance pathway. Os STAR1 genes belong to the ABC transporter family and encode an NBD domain, which is closely related to At NAP3 in Arabidopsis thaliana. This gene is located in the vesicular granules of root cells. Its function of aluminum toxicity tolerance is achieved by interacting with Os STAR2 to form a complex, which is located on the vesicle membrane of root cells. Os STAR2 genes also belong to the ABC family transporter family and encode a TMD domain. The complex formed by the two functions as an ABC transporter. The ABC transporter transports UDP-glucose, and UDP-glucose can be activated to form UDP-glc, which is used as a substrate for glycosyltransferases to synthesize various glycosides. Os STAR1 and Os STAR2The formed complex has an efflux transport activity for UDP-glc. For Os STAR1 and Os STAR2 The connection between the complex and aluminum tolerance needs to be further studied. There are currently two possibilities. One is that UDP-Glc may act as a chelating agent like organic acid anions, so it relieves aluminum toxicity by forming non-toxic complexes with aluminum; the other is OsSTAR1 / OsSTAR2 the complex transports UDP-Glc from the cytoplasm to vesicles. Then, UDP-Glc or its derived glycosides are released from the vesicles to the apoplast by exocytosis and are used to modify the cell wall to mask aluminum binding sites, thereby improving the aluminum tolerance of rice.

[0010] Currently, for STAR1 genes and their homologous genes in other species, including Arabidopsis thaliana, rice, and buckwheat, etc., it has only been studied that these genes and their homologous genes can improve the aluminum tolerance of plants, and there is no relevant report on their effects on other agronomic traits. SUMMARY OF THE INVENTION

[0011] In view of the above prior art, the object of the present invention is to provide the application of Brachypodium distachyon BdSTAR1 protein and its encoding gene in regulating the grain length of plant seeds.

[0012] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, there is provided the application of Brachypodium distachyon BdSTAR1 protein in regulating the grain length of plant seeds; the Brachypodium distachyon BdSTAR1 protein is a protein shown in the following (A1) or (A2): (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0013] In the above application, the plant is Brachypodium distachyon, wheat, rice, and / or Arabidopsis thaliana.

[0014] In the second aspect of the present invention, there is provided the application of Brachypodium distachyon BdSTAR1 gene in the following (1) or (2): (1) Regulating the grain length of plant seeds; (2) Plant breeding; The Brachypodium distachyon BdSTAR1 gene is a DNA molecule shown in the following i), ii), or iii): i) A DNA molecule with a nucleotide sequence shown in SEQ ID NO.2; ii) A DNA molecule with a nucleotide sequence shown in SEQ ID NO.3; iii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than i) or ii).

[0015] In the above application, the plant is Brachypodium distachyon, wheat, rice and / or Arabidopsis thaliana.

[0016] The present invention discovers through research that Brachypodium distachyon BdSTAR1 gene and its encoded protein are related to seed grain length. Overexpressing the Brachypodium distachyon BdSTAR1 gene can make the seed grain length longer; while knocking out or silencing the Brachypodium distachyon BdSTAR1 gene will make the seed grain length shorter. Therefore, using the Brachypodium distachyon BdSTAR1 gene and its encoded protein as a target, the seed grain length trait can be regulated accordingly by regulating its expression; and corresponding varieties can be cultivated according to the requirements of plant seed grain shape.

[0017] In the third aspect of the present invention, there is provided the use of a recombinant expression vector or a genetically engineered bacterium containing the Brachypodium distachyon BdSTAR1 gene in the following (1) or (2): (1) Regulating the seed grain length of plants; (2) Plant breeding.

[0018] In the above application, the recombinant expression vector is an overexpression vector of the Brachypodium distachyon BdSTAR1 gene, or a knockout vector targeting the Brachypodium distachyon BdSTAR1 gene.

[0019] In the fourth aspect of the present invention, there is provided a method for regulating the seed grain length of plants, comprising the following steps: Exogenously transferring the Brachypodium distachyon BdSTAR1 gene into a wild-type plant to overexpress the Brachypodium distachyon BdSTAR1 gene, obtaining a transgenic plant, and the seed grain length of the transgenic plant is greater than that of the wild-type plant; Or, knocking out or silencing the Brachypodium distachyon BdSTAR1 gene in the wild-type plant to obtain a mutant plant, and the seed grain length of the mutant plant is less than that of the wild-type plant.

[0020] In the above method, the method for exogenously transferring the Brachypodium distachyon BdSTAR1 gene into a wild-type plant includes: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

[0021] In the above method, the Brachypodium distachyon BdSTAR1 gene can be knocked out or silenced by genetic engineering means to achieve the purpose of inhibiting the expression of the Brachypodium distachyon BdSTAR1 gene or reducing the activity of the BdSTAR1 protein.

[0022] Advantages of the present invention: The present invention for the first time discovers that the Brachypodium distachyon BdSTAR1 gene is related to the grain length of plant seeds, and overexpression of the Brachypodium distachyon BdSTAR1 gene can increase the grain length of plant seeds; knocking out or silencing the Brachypodium distachyon BdSTAR1 gene can shorten the grain length of plant seeds. Therefore, by regulating the Brachypodium distachyon BdSTAR1 gene, corresponding plant varieties can be cultivated according to the requirements for the grain shape of plant seeds, which has important application value in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : bdstar1 Insertion site (A) of the mutant Ds and bdstar1 transcription level (B) of the BdSTAR1 gene in the mutant.

[0024] Figure 2 : Seed grain length phenotypes (A) and statistical data of grain length (B) of wild-type Brachypodium distachyon (WT) and bdstar1 the mutant.

[0025] Figure 3 : PCR amplification BdSTAR1 Electrophoresis bands for gene detection.

[0026] Figure 4 : BdSTAR1 Results of tissue-specific expression analysis of the gene.

[0027] Figure 5 : A: BdSTAR1 PAM site of the gene B: BdSTAR1 Analysis of the expression level of the T0 generation positive seedlings with gene knockout, C: BdSTAR1 Statistical analysis of the grain length of the T1 generation positive seedlings with gene knockout.

[0028] Figure 6 : A: BdSTAR1 Construction of the overexpression vector of the gene, B: BdSTAR1 Analysis of the expression level of the T0 generation positive seedlings with gene overexpression, C: BdSTAR1 Statistical analysis of the grain length of the T1 generation positive seedlings with gene overexpression.

[0029] Figure 7 : A: BdSTAR1 Construction of the complementary vector of the positive seedlings of the gene, B: BdSTAR1 Analysis of the expression level of the T0 generation positive seedlings with gene complementation, C: BdSTAR1 Statistical analysis of the grain length of the T0 generation positive seedlings of the gene. DETAILED DESCRIPTION OF THE INVENTION

[0030] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0031] The grain shape of plant seeds includes grain length, grain width, grain thickness, length / width ratio, etc. The grain shape of seeds is not only an important factor affecting yield traits but also an important indicator for measuring the appearance quality of products. For plants such as rice and wheat, the grain length of seeds particularly reflects their appearance quality. Therefore, it is necessary to study the genes that regulate the grain length of plant seeds.

[0032] The present invention utilized a previously constructed Brachypodium distachyon mutant library to discover a Ds mutant with an element inserted into the intron of the Brachypodium distachyon BdSTAR1 gene. bdstar1 Upon observing the seed phenotype of the bdstar1 mutant, it was found that the grain length of its seeds was significantly shorter than that of the wild type. From this, it can be inferred that the BdSTAR1 gene may be related to the grain length of plant seeds.

[0033] Then, using the genomic database website EnsemblPlants the candidate gene BRADI_1g33777v3 was found, and this gene was named BdSTAR1 , BdSTAR1 The full length of the gene is 3482 bp, and its nucleotide sequence is as shown in SEQ ID NO.1. BdSTAR1 The CDS sequence of the gene is as shown in SEQ ID NO.2; the amino acid sequence of the BdSTAR1 protein is as shown in SEQ ID NO.3.

[0034] To further study the function of the BdSTAR1 gene, the present invention respectively constructed knockout plants, overexpression plants, and complementary BdSTAR1 gene plants of the BdSTAR1 gene. The results showed that: after knocking out the BdSTAR1 gene, the grain length of seeds was reduced by 10%-12% compared with the wild type under the same growth conditions; after overexpressing the BdSTAR1 gene, the grain length of seeds was increased by 5%-8% compared with the wild type under the same growth conditions; the grain length of the complementary BdSTAR1 gene plants changed from a 18%-20% reduction to an 8%-12% increase compared with the wild type plants, and could rescue the phenotype of the shortened grain length of the mutant to a certain extent.

[0035] In summary, BdSTAR1 the gene is involved in regulating the grain length of plant seeds and can be applied in agricultural production.

[0036] To enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0037] In the embodiments of the present invention, the test materials not specifically described are all conventional test materials in the art and can be obtained through commercial channels. For those not indicating specific experimental conditions and methods in the embodiments of the present invention, they are usually carried out under conventional conditions, such as "Molecular Cloning: A Laboratory Manual" (Third Edition), edited by J. Sambrook et al., Science Press, 2002; "Cell: A Laboratory Manual", edited by D. L. Spector et al., Science Press, 2001; or according to the conditions recommended by the manufacturer. Among them: Callus induction medium: 4.74 g of MS medium (without agar and sucrose), 2.5 mL of 2,4-D (1 mg / mL), 30 g of sucrose, 0.5 g of 4-morpholineethanesulfonic acid, 0.5 g of acid hydrolysate casein, add ddH 2 O to 1 L.

[0038] First screening medium: 4.74 g of MS medium (without agar and sucrose), 2.5 mL of 2,4-D (1 mg / mL), 100 μL of 6-BA (1 mg / mL), 30 g of sucrose, 0.5 g of 4-morpholineethanesulfonic acid, 100 mg of inositol, CuSO 4 (8mg / L) 75 μL, add ddH 2 O to 1 L.

[0039] Second screening medium: 4.74 g of MS medium (without agar and sucrose), 2.5 mL of 2,4-D (1 mg / mL), 100 μL of 6-BA (1 mg / mL), 18 g of sucrose, 0.5 g of 4-morpholineethanesulfonic acid, 100 mg of inositol, CuSO 4 (8mg / L) 75 μL, add ddH 2 O to 1 L.

[0040] Differentiation medium: 4.74 g of MS medium (without agar and sucrose), 30 g of sucrose, 0.5 g of 4-morpholineethanesulfonic acid, 400 μL of KT (1 mg / mL), 500 mg of hydrolyzed casein, 100 mg of inositol, 400 μL of 6-BA (1 mg / mL), add ddH 2 O to 1L.

[0041] Rooting medium: 4.74 g of MS medium (without agar and sucrose), 10 g of sucrose, 0.5 g of 4-morpholineethanesulfonic acid, 400 μL of NAA (1 mg / mL), add ddH 2 O to 1 L.

[0042] Example 1: bdstar1 Obtaining mutants and phenotypic detection I. bdstar1 Obtaining mutants and BdSTAR1 Gene expression level detection: 1. Obtaining mutants: Previously, the laboratory constructed a Brachypodium distachyon mutant library by AC / DS transposon tagging method (DOI: https: / / doi.org / 10.1104 / pp.18.00875). A mutant containing only DS element was screened from the constructed mutant library. Ds The insertion was in the intron of the BdSTAR1 gene in Brachypodium distachyon, so this mutant was named bdstar1 .

[0043] 2. BdSTAR1 Gene expression level detection: Use TransZol of TransGen Biotech to extract bdstar1 RNA of the mutant and reverse transcribe to synthesize the first strand of cDNA. For the region before the Ds insertion site (region 1), the region across the Ds region (region 2), and the region after the insertion site (region 3) of the bdstar1 mutant, as shown in Figure 1 A below, design relative fluorescence quantitative PCR primers for transcriptional level analysis. According to the requirements of qRT-PCR primer design, use Primer Premier 3.0 software to design specific primers. The primer pair sequences are as follows: qRT-Bd-STAR1-F1: 5′-AGACAATGTACGATACGGGC-3′; (SEQ ID NO.4) qRT-Bd-STAR1-R1: 5′-GAGCACTTCTGGGTCGTTG-3′; (SEQ ID NO.5) qRT-Bd-STAR1-F2: 5′-CAGACAATGTACGATACGGGCCG-3′; (SEQ ID NO.6) qRT-Bd-STAR1-R2: 5′-CAGGAGCACTTCTGGGTCGTTG-3′; (SEQ ID NO.7) qRT-Bd-STAR1-F3: 5′-ACCCTCGCCAACGACCCAGAA-3′; (SEQ ID NO.8) qRT-Bd-STAR1-R3: 5'-TTCACGCTGTGCGACACCATCA-3'. (SEQ ID NO.9) Using a 96-well plate dedicated for qRT-PCR and a high light transmittance sealing film, qRT-PCR analysis was performed with the Icycler real-time PCR system. Each sample was repeated 3 times. Using the cDNA obtained above as a template, a reaction system was established. The reaction system was referred to the instruction manual of SYBR Green Realtime PCR Master Mix (QPK-201). The reaction conditions were as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds, annealing at 58 ± 5.0°C for 30 seconds, extension at 72°C for 15 seconds, and a total of 40 cycles were repeated; incubation at 65°C for 30 seconds, melting curve from 65 - 95°C, reading once every 0.5°C, and maintaining for 1 second.

[0044] Mix multiple samples and perform the first amplification to detect whether the primers are available. Verify the specificity of primer amplification according to the melting curve. A single peak is considered specific amplification. If there are two peaks, appropriately adjust the annealing temperature and primer dosage. Use the mixed template to serially dilute by 10-fold concentration, a total of 4 times, and construct a relative standard curve with 5 concentrations of samples to verify the amplification efficiency of all primers and whether there is a linear amplification relationship for the target sequence within this concentration range. Using BdUBC18 as an internal reference, adjust the concentration of each template so that the difference in Ct values of the internal reference is less than 2. Each gene amplification has the internal reference amplified simultaneously, and the Ct value is read under default conditions. Each sample is repeated three times.

[0045] The results are as Figure 1 shown in B below, BdSTAR1 the gene transcription level was significantly reduced, indicating that bdstar1 the mutant caused BdSTAR1 gene silencing expression. And due to Ds the expression level of the insertion segment (region 2) was severely down-regulated, which also indicated that Ds the transposon existed at this position, which was the mutation position.

[0046] II. bdstar1 Phenotype detection of the mutant: Using wild-type Brachypodium distachyon as a control. Observe bdstar1 the phenotype of the mutant Brachypodium distachyon plants and count the length of ten seeds of the plants.

[0047] The results Figure 2 are shown bdstar1 as follows. The seeds of the mutant Brachypodium distachyon plants were significantly shorter than those of the wild type in terms of seed length, indicating that BdSTAR1 after the loss-of-function mutation of the gene, it could significantly shorten the seed length of Brachypodium distachyon plants.

[0048] Example 2: BdSTAR1 Cloning of the gene Extract using TransZol from TransGen Biotech bdstar1 RNA of the mutant, reverse transcribe to synthesize the first strand of cDNA, and use it as the cDNA template.

[0049] According to Brachypodium distachyon BdSTAR1 Design the gene primer pair based on the CDS sequence of the gene BdSTAR1 The primer pair sequence is (5'-3'): Forward primer: 5′-ATGGGCTCAGCATCAGATGACA-3′; (SEQ ID NO.10) Reverse primer: 5′-TCAGCTGAGCTCCAGGAAGC-3′. (SEQ ID NO.11) Use 2×Phanta Max MasterMix for PCR amplification. The reaction system is: Mix 25 μL, forward primer 2 μL, reverse primer 2 μL, cDNA template 1 μL, and make up to 50 μL with water.

[0050] PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 2 minutes, repeat 35 cycles in total; post-extension at 72°C for 5 minutes; hold at 15°C.

[0051] After the reaction, perform agarose gel electrophoresis. After detecting the target band ( Figure 3 ), cut the gel and perform gel extraction. The gel extraction method is carried out according to the CW Biotech Agarose Gel DNA Extraction Kit. Sequence the amplified target band, and its nucleotide sequence is shown in SEQ ID NO.2. It shows that the gene of the present invention has been successfully cloned BdSTAR1 gene.

[0052] Example 3: BdSTAR1 Analysis of tissue-specific expression of the gene For the aerial part (1-Ov) and roots (1-R) of wild-type Brachypodium distachyon at the one-leaf stage; the first leaf (2-1L), the leaf sheath of the first leaf (2-1Sh), the first internode (2-1St), the second leaf (2-2L), the second internode (2-2St), the leaf sheath of the second leaf (2-2Sh), the emerging part (2-3Ou) and the enclosed part (2-3I) of the third leaf at the three-leaf stage; the fourth leaf (3-4L), the leaf sheath of the fourth leaf (3-4Sh), the emerging part (3-5Ou) and the enclosed part (3-5I) of the fifth leaf at the five-leaf stage; the young spike part (S), the fifth leaf (S-5L), the sixth leaf (S-6L), and the seventh leaf (S-7L) at the young spike stage of the plant. Samples were taken from different parts, RNA was extracted, and cDNA was synthesized by reverse transcription.

[0053] Use BdSTAR1 gene primer pairs for qRT-PCR analysis BdSTAR1 of tissue expression patterns: Forward primer: 5′-ACCCTCGCCAACGACCCAGAA-3′; (SEQ ID NO.12) Reverse primer: 5′-TTCACGCTGTGCGACACCATCA-3′. (SEQ ID NO.13) Using a 96-well plate dedicated to qRT-PCR and a high light transmittance sealing film, qRT-PCR analysis was performed using an Icycler real-time PCR system. Each sample was repeated 3 times. Total RNA was extracted from the one-leaf stage of the plant and reverse transcribed to obtain cDNA, which was used as a template to establish a reaction system. The reaction system was referred to the SYBR Green Realtime PCR Master Mix (QPK-201) instruction manual. The reaction conditions were as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds, annealing at 58 ± 5.0°C for 30 seconds, extension at 72°C for 15 seconds, and a total of 50 - 60 cycles were repeated; incubation at 65°C for 20 seconds, melting curve at 65 - 95°C, reading once every 0.5°C, and maintaining for 1 second. Using BdUBC18 as an internal reference, each sample was repeated three times.

[0054] The results are as Figure 4 shown. The BdSTAR1 gene of Brachypodium distachyon was expressed in all parts at all stages of Brachypodium distachyon, and the highest expression levels were observed in the young and un-emerged parts at the five-leaf stage.

[0055] Example 4: Construction of the genetic transformation vector of Brachypodium distachyon 1. BdSTAR1 Construction of the knockout vector of the gene (1) BdSTAR1The nucleotide sequence was input into the online website CRISPR-P v2.0 for knockout target design to obtain the target target, as shown in Figure 5 shown in A. Primers were designed according to the target sequence to amplify three fragments of U6 (nucleotide sequence shown in SEQ ID NO.14), SG1 (nucleotide sequence shown in SEQ ID NO.15), and SG2 (nucleotide sequence shown in SEQ ID NO.16) respectively. The primer sequences are as follows: U6-upstream primer: 5′-CGATGGTACCGTGATGCTTGTAACTTTGTA-3′; (SEQ ID NO.17) U6-downstream primer: 5′-ACACGCCCATGACCACCCCGCGTGCACCAGCCGGGAATCGAA-3′. (SEQ IDNO.18) SG1-upstream primer: 5′-CACGCGGGGTGGTCATGGGCGTGTTTTAGAGCTAGAAATAGC-3′; (SEQ IDNO.19) SG1-downstream primer: 5′-ACCGACCTCGCCGTCGACCACGTGCACCAGCCGGGAATCGAA-3′. (SEQ IDNO.20) SG2-upstream primer: 5′-CACGTGGTCGACGGCGAGGTCGGTTTTAGAGCTAGAAATAGC-3′; (SEQ IDNO.21) SG2-downstream primer: 5′-GCTAACTAGTCTCGAGCGGCCGCCAGTGTG-3′; (SEQ ID NO.22) (2) Use 2×phanta Max Master Mix high-fidelity enzyme for amplification. The reaction system is: 25 μL of 2×phantaMax Master Mix high-fidelity enzyme, 2 μL of deoxyribonucleic acid (dNTP), 1 μL of upstream primer, 1 μL of downstream primer, and make up to 50 μL with water.

[0056] The PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 2 minutes, for a total of 40 cycles; post-extension at 72°C for 5 minutes; keep warm at 15°C.

[0057] After the reaction, agarose gel electrophoresis was carried out. A small amount of the amplified product was taken for identification. After detecting the target band, the amplified fragment was digested with Spe1 and Kpn1 restriction endonucleases on the vector, and the reaction was carried out at 37 °C for two hours. After the reaction, agarose gel electrophoresis was carried out, the gel was cut and gel extraction was performed. The gel extraction method was carried out according to the BioTeke agarose gel DNA extraction kit in Taiwan, China. (3)Digestion of the knockout vector: The plasmid DNA of the CRISPR knockout vector was extracted using the BioTeke high-purity plasmid miniprep kit in Taiwan, China, and the vector was digested with Spe1 and Kpn1 restriction endonucleases. The reaction was carried out at 37 °C for two hours. After the reaction, agarose gel electrophoresis was carried out, the gel was cut and gel extraction was performed. The gel extraction method was carried out according to the BioTeke agarose gel DNA extraction kit in Taiwan, China.

[0058] The gel-extracted product of gene amplification and the gel-extracted product of vector digestion were subjected to T4 ligation. The reaction system was as follows: 1 μL of 10× reaction buffer, 1 μL of T4 ligase, and the gel-extracted product was made up to 10 μL. The reaction was carried out overnight at 16 °C.

[0059] (4)Take 5 μL of the above ligation product and ligate it with the cloning vector. The ligation product was transformed into Escherichia coli DH5α strain by heat shock method and grown overnight on an LB plate containing SPE resistance. Single white colonies were picked and streaked on an LB plate for colony PCR, and positive colonies were selected and grown overnight at 37 °C in an LB liquid medium.

[0060] (5)Extraction of plasmid DNA: The plasmid DNA was extracted using the BioTeke high-purity plasmid miniprep kit in Taiwan, China, and was verified by sequencing by BioTeke Corporation (Shanghai, China), and the gene knockout vector was successfully constructed. BdSTAR1 Gene knockout vector.

[0061] 2、 BdSTAR1 Construction of the gene overexpression vector (1) BdSTAR1 Gene cloning: The wild-type BdSTAR1 RNA was extracted using TransZol from TransGen Biotech, and the first strand of cDNA was synthesized by reverse transcription and used as the cDNA template.

[0062] According to the CDS sequence of the Brachypodium distachyon BdSTAR1 gene, the following primer pair was designed, and its sequence was (5'-3'): Forward primer: 5′-gtgttacttctgcag gagctc ATGGGCTCAGCATCAGATGACA-3′; (SEQ ID NO.23) Reverse primer: 5′-tctagaggatccccg ggtaccTCAGCTGAGCTCCAGGAAGC-3′; (SEQ ID NO.24) The underlined parts are restriction sites. The restriction site of the upstream primer is Sac1, and the restriction site of the downstream primer is Kpn1.

[0063] PCR amplification was carried out using 2×Phanta Max MasterMix. The reaction system was as follows: Mix 25 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA template 1 μL, and water was added to make up to 50 μL.

[0064] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 2 minutes, and this cycle was repeated 40 times; post-extension at 72°C for 5 minutes; incubation at 15°C.

[0065] After the reaction, agarose gel electrophoresis was performed. After detecting the target band, the gel was cut and gel extraction was carried out. The gel extraction method was carried out according to the BioSune Agarose Gel DNA Extraction Kit from Taiwan, China. Sequencing of the amplified target band showed that the gene containing the correct restriction sites had been successfully cloned. BdSTAR1 gene.

[0066] (2) Digestion of pPZP211 vector: The plasmid DNA of the pPZP211 vector containing the UBI promoter was extracted using the BioSune High Purity Plasmid Mini Extraction Kit from Taiwan, China, and the vector was digested with Sac1 and Kpn1 restriction endonucleases. The reaction was carried out at 37°C for two hours. After the reaction, agarose gel electrophoresis was performed, the gel was cut and gel extraction was carried out. The gel extraction method was carried out according to the BioSune Agarose Gel DNA Extraction Kit from Taiwan, China.

[0067] (3) Homologous recombination: The gel extraction product of the digested vector and the gene amplification product were subjected to homologous recombination. The reaction system was as follows: homologous recombination enzyme 5 μL, gel extraction product of the digested vector 1 μL, gene amplification product 1 μL, and water was added to make up the total volume to 10 μL. The reaction was carried out at 50°C for 10 minutes.

[0068] (4) Take 5 μL of the above ligation product and ligate it with the pPZP211 cloning vector. The operation steps were carried out according to the pPZP211 vector instruction manual. Then the ligation product was transformed into Escherichia coli DH5α strain using the heat shock method and grown overnight on an LB plate containing SPE resistance. Single white colonies were picked and streaked on an LB plate, and colony PCR was performed. Positive colonies were selected and cultured overnight at 37°C in LB liquid medium.

[0069] (5) Extraction of plasmid DNA: The plasmid DNA of the positive colonies was extracted using the BioSune High Purity Plasmid Mini Extraction Kit from Taiwan, China, and verified by sequencing by Shanghai Bioengineering Co., Ltd., asFigure 6 As shown in A, the overexpression vector of the gene was successfully constructed. BdSTAR1 Overexpression vector of the gene.

[0070] 3. BdSTAR1 Construction of the complementary vector of the gene (1) BdSTAR1 Cloning of the gene and its 3000 bp upstream and downstream sequences: The DNA of wild-type Brachypodium distachyon was extracted using the CTAB method and used as a template. The following primer pairs 1 - primer pair 3 were designed to amplify BdSTAR1 the gene and 3000 bp upstream and downstream of this gene as BdSATR1 the promoter and terminator of the gene; BdSTAR1 The nucleotide sequence of 3000 bp upstream of the gene is shown in SEQ ID NO.25, BdSTAR1 and the nucleotide sequence of 3000 bp downstream of the gene is shown in SEQ ID NO.26.

[0071] Primer pair 1 - upstream primer: 5′-catgattacgaattc gagctc TTACTGTTTGCTTGATTATGTGGCG-3′; (SEQ ID NO.27) Primer pair 1 - downstream primer: 5′-ggcatcttcAGCCTGTTACTGGAATCGGAATC-3′. (SEQ ID NO.28) Primer pair 2 - upstream primer: 5′-agtaacaggctGAAGATGCCCTTGTAACCATGC-3′; (SEQ ID NO.29) Primer pair 2 - downstream primer: 5′-ggaatagcgagagaaatAGCAAATACGTTCATGAGCCTTT-3′. (SEQ ID NO.30) Primer pair 3 - upstream primer: 5′-gctATTTCTCTCGCTATTCCCTTGTGG-3′; (SEQ ID NO.31) Primer pair 3 - downstream primer: tctagaggatccccg ggtacc AATAAGGTACGAAGCTTCTCTCCTCC-3′. (SEQ ID NO.32) The underlined parts are restriction enzyme sites. The restriction enzyme site of the upstream primer is Sac1, and the restriction enzyme site of the downstream primer is Kpn1.

[0072] PCR amplification was carried out using 2×Phanta Max MasterMix. The reaction system was as follows: Mix 25 μL, forward primer 2 μL, reverse primer 2 μL, DNA template 1 μL, and water was added to make up to 50 μL.

[0073] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 2 minutes, and this cycle was repeated 40 times; final extension at 72°C for 5 minutes; incubation at 15°C.

[0074] After the reaction, agarose gel electrophoresis was performed, the gel was cut and gel extraction was carried out. The gel extraction method was carried out according to the BioSune Agarose Gel DNA Extraction Kit from Taiwan, China.

[0075] (2)Digestion of pPZP211 vector: The empty pPZP211 vector plasmid DNA was extracted using the BioSune High Purity Plasmid Mini Extraction Kit from Taiwan, China, and the vector was double-digested with Sac1 and Kpn1 restriction endonucleases. The reaction was carried out at 37°C for two hours. After the reaction, agarose gel electrophoresis was performed, the gel was cut and gel extraction was carried out. The gel extraction method was carried out according to the BioSune Agarose Gel DNA Extraction Kit from Taiwan, China.

[0076] (3)Homologous recombination: The gel extraction product of the digested vector and the gene amplification product were subjected to homologous recombination. The reaction system was as follows: homologous recombination enzyme 5 μL, gel extraction product of the digested vector 1 μL, each gene amplification product 1 μL, and water was added to make the total volume 10 μL. The reaction was carried out at 50°C for 10 minutes.

[0077] (4)Take 5 μL of the above ligation product and ligate it with the pPZP211 cloning vector. The operation steps were carried out according to the pPZP211 vector instruction manual. Then the ligation product was transformed into Escherichia coli DH5α strain using the heat shock method and grown overnight on an LB plate containing SPE. Single white colonies were picked and streaked on an LB plate, and colony PCR was performed. Positive colonies were selected and grown overnight in LB liquid medium.

[0078] (5)Extraction of plasmid DNA: Plasmid DNA was extracted using the BioSune High Purity Plasmid Mini Extraction Kit from Taiwan, China, and verified by sequencing by BioSune Biotechnology (Shanghai) Co., Ltd. As shown in A below, the complementary vector of the gene was successfully constructed. Figure 7 as shown in BdSTAR1 A, the complementary vector of the gene was successfully constructed.

[0079] Example 5: Genetic transformation of Brachypodium distachyon 1. Induction of callus (1)Wild-type Brachypodium distachyon (WT) and bdstar1 mutants that grew normally for about 1 month were selected respectively. The lemma and palea of the seeds were removed, and young and fresh seeds were selected. (2) Put the collected seeds in a laminar flow hood, disinfect them with 25% NaClO disinfectant diluted with Tween for 5 min, and finally wash them 3 times with sterile deionized water; (3) Adjust the stereomicroscope to be clear, use clean and sterile forceps to tear the epidermis, take out the white, tender and pointed young embryo (preferably 0.3 - 0.7 mm), and gently place it on the surface of the callus induction medium; (4) Incubate in an incubator at 28 °C in the dark for about 25 days, observe at any time, and remove the buds; (5) In order to make the utilization efficiency of callus higher, select yellowish - bright and relatively hard callus, divide it into small pieces of 1 - 2 mm in size and then culture for about 15 days, and observe in time; 2. Infection of Brachypodium distachyon (1) Respectively transform the knockout vector, over - expression vector and complementary vector of the BdSTAR1 gene into the Agrobacterium tumefaciens strain EHA105; Pour the transformed Agrobacterium tumefaciens strain into a shaking flask containing 10 ml of YEP, then add 10 μL of antibiotics and rifampicin, and culture at 200 rpm and 28 °C for 24 h; After the culture is completed, centrifuge at 4000 rmp at 4 °C for 20 min and discard the supernatant; (2) Add the Agrobacterium suspension to the shaking flask from which the supernatant has been discarded, and dilute the OD 600 value of the bacterial solution to 0.7 - 1.3; (3) Add the Agrobacterium bacterial solution transformed with the BdSTAR1 gene knockout vector and over - expression vector to a beaker containing the callus of wild - type Brachypodium distachyon (WT), and add the Agrobacterium bacterial solution transformed with the BdSTAR1 gene complementary vector to a beaker containing the callus of the bdstar1 mutant; And make the bacterial solution completely cover the callus. Manually shake the beaker, shake for 1 min each time, let it stand for 3 min, and repeat this step three times. After completion, remove the waste liquid and suck off the residual bacterial solution; (4) Transfer the callus to a sterile glass dish with filter paper and blow it to an appropriate dryness; (5) Continue to infect in a 28 °C dark incubator for 3 days; 3. Screening of Brachypodium distachyon (1) Transfer the callus that has been dark - cultured for 3 days to the first screening medium. Select the yellow - bright and healthy callus with good infection effect. So try to select the callus with sufficient yellow color and hardness, and screen in a 28 °C dark incubator for 10 - 12 days; (2) After the first screening, select the healthy callus and transfer it to the second screening medium, and screen in a 28 °C dark incubator for 14 days.

[0080] 4. Differentiation of Brachypodium distachyon (1) After the second screening, select new healthy calli and transfer them to the differentiation medium, and culture them continuously under extremely long-day conditions (18 h light / 6 h dark) at 28 °C; (2) Observe irregularly whether there are tender green buds growing; (3) Let the grown seedlings continue to grow for about 1 week, then try to remove the callus blocks at the bottom in the workbench, and then press the tender buds into the rooting medium, and culture them continuously under extremely long-day conditions (18 h light / 6 h dark) at 28 °C.

[0081] 5. Transplanting of Brachypodium distachyon after rooting (1) Take out the tissue culture seedlings with roots about 3 - 5 cm long, remove the miscellaneous buds, and rinse the remaining parts with deionized water; (2) Mix nutrient soil and vermiculite in a volume ratio of 1:1, fill them into the same flowerpots, and soak them thoroughly with water. Then carefully transplant the roots of the seedlings into the flowerpots with forceps; (3) After transplanting the seedlings, cover them with plastic wrap and plant them in weak light or no light for 3 days. Then remove the plastic wrap and transfer them to the greenhouse for normal cultivation. Observe the growth status of the seedlings regularly to ensure the normal growth of the plants until inoculation and seed harvesting.

[0082] Through the above genetic transformation operations, using BdSTAR1 the knockout vector of the gene to knockout the BdSATR1 gene of wild-type Brachypodium distachyon (WT) to obtain Brachypodium distachyon BdSATR1 knockout positive seedlings; using BdSTAR1 the overexpression vector of the gene to transform wild-type Brachypodium distachyon (WT) to obtain Brachypodium distachyon BdSATR1 overexpression positive seedlings; and the Brachypodium distachyon bdstar1 obtained after transferring the complementary vector of the BdSTAR1 gene into the BdSATR1 mutant.

[0083] Example 6: Brachypodium distachyon BdSTAR1 Identification of transgenic positive seedlings of Brachypodium distachyon and statistics of grain length 1. Identification of the T0 generation of Brachypodium distachyon BdSATR1 knockout positive seedlings and statistics of the grain length of the T1 generation Take samples of the transgenic seedlings obtained by tissue culture after transferring the BdSTAR1 knockout vector of the gene in Example 5, extract the RNA of the transgenic seedlings using TransZol of TransGen Biotech, and reverse transcribe it into cDNA. Use BdSTAR1 gene primer pairs for qRT-PCR analysis of the BdSTAR1 expression level in the transgenic seedlings. The results are shown in Figure 5 B. The BdSTAR1 expression level in the transgenic seedlings decreased, indicating that BdSATR1Knockout positive seedlings. Harvest the seeds of T0 generation positive seedlings, continue to sow the T1 generation, randomly select T1 generation lines for grain length statistics, and the results are as Figure 5 shown in C, indicating that after knocking out BdSTAR1 the gene, the grain length of the seeds becomes shorter.

[0084] 2. Brachypodium distachyon BdSATR1 Identification of T0 generation of overexpression positive seedlings and grain length statistics of T1 generation Take samples of the transgenic seedlings obtained by tissue culture after transferring the BdSTAR1 overexpression vector of the gene in Example 5, use TransZol of TransGen Biotech to extract the RNA of the transgenic seedlings, reverse transcribe it into cDNA, and use BdSTAR1 gene primer pairs to perform qRT-PCR analysis on the BdSTAR1 expression level in the transgenic seedlings. The results are as Figure 6 shown in B. The BdSTAR1 expression level in the transgenic seedlings increases, indicating that BdSATR1 overexpression positive seedlings are obtained. Harvest the seeds of T0 generation positive seedlings, continue to sow the T1 generation, randomly select T1 generation lines for grain length statistics, and the results are as Figure 6 shown in C, indicating that after overexpressing BdSTAR1 the gene, the grain length of the seeds becomes longer.

[0085] 3. Brachypodium distachyon BdSATR1 Identification of T0 generation of complementary positive seedlings and grain length statistics of T0 generation Take samples of the transgenic seedlings obtained by tissue culture after transferring the BdSTAR1 complementary vector of the gene in Example 5, use TransZol of TransGen Biotech to extract the RNA of the transgenic seedlings, reverse transcribe it into cDNA, and use BdSTAR1 gene primer pairs to perform qRT-PCR analysis on the BdSTAR1 expression level in the transgenic seedlings. The results are as Figure 7 shown in B. The BdSTAR1 expression level has no obvious difference from that of the wild type, indicating that BdSATR1 complementary positive seedlings are obtained. Harvest the seeds of T0 generation positive seedlings, randomly select T0 generation lines for grain length statistics, and the results are as Figure 7 shown in C, indicating that in the mutant background, complementing BdSTAR1 the gene can rescue the phenomenon of shorter grain length of seeds to a certain extent.

[0086] In summary, using transgenic technology, plants with knocked-out, overexpressed, and complementary Bd STAR1 genes are obtained. By observing the seed grain length phenotypes, the results show that the Bd STAR1 gene is involved in regulating the process of plant seed grain length development. When this gene is overexpressed, the grain length of plant seeds becomes longer, and the grain length of seeds is increased by about 5%-8% compared with that of the wild type (the average increase is 6.5%±1.5%, n = 30). After knocking outBdSTAR1 After the gene was knocked out, the grain length was reduced by about 10%-12% compared with that of wild-type seeds under the same growth conditions (the average reduction was 11%±1%, n = 30). Complementary expression of this gene in the mutant background could rescue the short grain length phenotype, and the grain length of the obtained positive plants changed from a reduction of about 18%-20% to 8%-12% compared with that of wild-type plants (the average reduction was 10%±2%, n = 30).

[0087] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Brachypodium distachyon BdSTAR1 protein in regulating plant seed length; the Brachypodium distachyon BdSTAR1 protein is a protein as shown in (A1) or (A2): (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; (A2) Fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The use according to claim 1, characterized in that: The plant is Brachypodium distachyon, wheat, rice and / or Arabidopsis thaliana.

3. Brachypodium distichum BdSTAR1 The use of genes in the following (1) or (2): (1) Regulate plant seed length; (2) Plant breeding; Brachypodium distachyon BdSTAR1 A gene is a DNA molecule as shown in i) or ii) or iii) below: i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; ii) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.3; iii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than i) or ii).

4. The use according to claim 3, characterized in that: The plant is Brachypodium distachyon, wheat, rice and / or Arabidopsis thaliana.

5. Contains Brachypodium distachyon BdSTAR1 Application of gene recombinant expression vectors or genetically engineered bacteria in the following (1) or (2): (1) Regulate plant seed length; (2) Plant breeding.

6. The use according to claim 5, characterized in that: Brachypodium distachyon BdSTAR1 Gene overexpression vectors or targeting Brachypodium distachyon BdSTAR1 Gene knockout vector.

7. A method for regulating plant seed length, characterized in that: The following steps are involved: Brachypodium distachyon BdSTAR1 Brachypodium distachyon BdSTAR1 Gene overexpression to obtain transgenic plants, wherein the seeds of the transgenic plants are larger than those of wild-type plants; Alternatively, wild-type Brachypodium distachyon BdSTAR1 Gene knockout or silencing can obtain mutant plants, wherein the seed length of the mutant plants is shorter than that of wild-type plants.

8. The method according to claim 7, characterized in that Brachypodium distachyon BdSTAR1 Methods for transferring exogenous genes into wild-type plants include: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

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