Gene BBX15 for regulating heading stage of millet and application

Abstract

The invention provides a gene BBX15 for regulating and controlling the heading stage of millet and application, and belongs to the technical field of functional genes. The invention provides a gene BBX15 capable of being used for regulating and controlling the heading stage of millet. The gene BBX15 is subjected to frame-shift mutation by using a gene editing technology, so that the coded protein is terminated in advance, and a mutant material is constructed. Compared with a wild type, the mutant material has the advantage that the heading period is obviously advanced. Therefore, the gene BBX15 provides a novel regulation and control way for regulating and controlling the heading period characters of the plants, and has important value for plant character improvement and breeding practice.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional genes, and in particular relates to a gene BBX15 for regulating the heading period of millet and its application. Background Art

[0002] Heading date is a key agronomic trait that determines the promotion area and planting season of crops, and is one of the important factors affecting crop yield. Plants sense changes in external factors such as light and temperature, and activate different signal regulation mechanisms to induce heading and flowering to adapt to changes in the growing environment. Millet has outstanding advantages such as drought resistance, resistance to poor soil conditions, and efficient photosynthesis. However, millet is very sensitive to the light cycle, and the existing bred varieties have obvious regional ecological dependence. Spring millet is mainly planted in high-latitude areas, while summer millet is mainly planted in low and medium latitudes. The heading date of millet varieties varies greatly in different planting areas. Many excellent varieties are difficult to promote and apply on a large scale due to regional dependence, and the utilization rate of improved varieties is not high. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a gene BBX15 that regulates the heading period of millet, providing an effective gene target for millet genetic breeding and providing a genetic basis for the large-scale promotion of millet planting.

[0004] The present invention provides a millet heading period regulatory protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0005] The present invention provides a gene BBX15 for encoding the protein, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0006] The present invention provides the use of the millet heading period regulatory protein or the gene BBX15 as a target in at least one of the following: regulating the millet heading period, improving the agronomic traits of the millet heading period, screening millet varieties with early heading period, and carrying out millet genetic breeding.

[0007] Preferably, the gene BBX15 advances the heading period of millet through negative regulation.

[0008] The present invention provides the use of inhibiting the function of the millet heading period regulatory protein or inhibiting the expression of the gene BBX15 in promoting the early heading period of millet and / or constructing a millet variety with an early heading period.

[0009] The present invention provides an sgRNA targeting the gene BBX15, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4.

[0010] The present invention provides a gene editing system comprising the sgRNA.

[0011] The present invention provides a recombinant strain comprising the sgRNA.

[0012] The present invention provides the use of the sgRNA, the gene editing system or the recombinant strain in promoting the early heading period of millet and / or constructing a millet variety with an early heading period.

[0013] The present invention provides a method for promoting the early heading period of millet, which inhibits the expression of the gene BBX15 in millet or inhibits the function of the millet heading period regulatory protein according to claim 1 in millet.

[0014] The present invention provides a gene BBX15 for encoding a protein regulating the heading period of millet, the nucleotide sequence of which is shown in SEQ ID NO: 2. The gene BBX15 has the biological effect of regulating the heading period of millet, specifically promoting the early heading period of millet through negative regulation. The present invention uses Agrobacterium-mediated to create millet BBX15 gene-edited mutant materials. The results show that compared with wild-type millet materials, the heading period of millet BBX15 gene-edited mutant materials is significantly advanced. Using the gene BBX15 as a target, millet varieties with early heading period or improving the agronomic traits of millet heading period are constructed through gene editing, thereby expanding the planting range of millet. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The phenotypes of the millet mutant sibbx15 and the wild type material at the heading stage after 30 days of growth, where the scale bar = 10 cm;

[0016] Figure 2 Comparison results of the heading days between the foxtail millet mutant sibbx15 and the wild-type material; Note: Genotype: genotype, WT: wild type (ci846), sibbx15: BBX15 gene-edited mutant material, Heading days: heading days; ***: p<0.001. DETAILED DESCRIPTION

[0017] The present invention provides a millet heading period regulatory protein, the amino acid sequence of which is shown in SEQ ID NO: 1 (MKVQCDVCAAEAASVFCCADEAALCDACDRRVHRANKLAGKHRRFSLL HPSSSSSAAQTKPPLCDICQERRGFLFCKEDRAILCRECDVPVHTANELTRRHSRFLLTGVRLSSAPVDSPAPSEGEDQEEEELENSGSPCNADSCSGGAGATTAASASDGSSISEYLTKTLPGWHVEDFLVDDASAGACSDDALYQQGEQGQIGGLLQEAYTPWTGREQMLADVVVTTDERASRERWVPQMHAEFAACKRARASPPCSYW*, wherein * represents a terminator).

[0018]

[0019] The present invention provides the use of the millet heading period regulatory protein or the gene BBX15 as a target in at least one of the following: regulating the millet heading period, improving the agronomic traits of the millet heading period, screening millet varieties with early heading period, and carrying out millet genetic breeding.

[0020] In the present invention, considering that the gene BBX15 preferably advances the heading period of millet through negative regulation, the method for regulating the heading period of millet or improving the agronomic traits of the heading period of millet is to inhibit the expression of gene BBX15, such as mutating gene BBX15 in millet, or performing gene knockout or gene editing.

[0021] In the present invention, the method for screening millet varieties with an early heading period comprises detecting the relative expression level of the gene BBX15 or its encoded protein in the millet to be tested, and selecting the millet variety based on the relative expression level of the gene BBX15 or its encoded protein: when the relative expression level of the gene BBX15 or its encoded protein is downregulated, the millet to be tested is a variety with an early heading period. The method for detecting the relative expression level of the gene BBX15 in the millet to be tested comprises RT-qPCR technology. The method for detecting the relative expression level of the protein encoded by the gene BBX15 comprises immunoblotting technology. The reference sample in the method preferably comprises millet ci846.

[0022] In the present invention, the method for carrying out millet genetic breeding preferably uses millet materials with relatively low expression levels of gene BBX15 as breeding parents. The reference sample in the method preferably includes millet ci846.

[0023] The present invention provides the use of inhibiting the expression of the gene BBX15 or inhibiting the function of the millet heading period regulatory protein in promoting the early heading period of millet and / or constructing a millet variety with an early heading period.

[0024] In the present invention, the method for inhibiting the expression of the gene BBX15 preferably includes a method of gene editing using sgRNA, a method of transiently silencing the expression of the gene BBX15 using small interfering RNA (siRNA), or a method of stably silencing the expression of the gene BBX15 in foxtail millet using short hairpin RNA (shRNA). The method for inhibiting the function of the foxtail millet heading period regulatory protein preferably includes targeting the foxtail millet heading period regulatory protein with a monoclonal antibody against the protein, or using an inhibitor to act on the protein, causing the protein to lose its original biological function.

[0025] The present invention provides an sgRNA targeting the gene BBX15, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4.

[0026] The present invention provides a gene editing system comprising the sgRNA.

[0027] In the present invention, the gene editing system comprising the sgRNA preferably comprises a CRISPR / Cas9 recombinant vector comprising the sgRNA. In an embodiment of the present invention, the CRISPR / Cas9 recombinant vector comprising the sgRNA is preferably a CRISPR / Cas9-BBX15-sgRNA vector.

[0028] The present invention provides a recombinant strain comprising the sgRNA.

[0029] In the present invention, the host bacteria of the recombinant strain preferably include Agrobacterium. In the present invention, the Agrobacterium strain is preferably AGL1. The present invention has no special restrictions on the construction method of the recombinant strain, and the construction method of the recombinant strain well known in the art can be adopted. For example, the CRISPR / Cas9-BBX15-sgRNA vector is transformed into Agrobacterium, and after culture, screening and verification, a recombinant strain containing the sgRNA is obtained. The present invention has no special restrictions on the transformation method, and the transformation method well known in the art can be adopted, such as freeze-thaw method or electroporation. The screening preferably includes antibiotic culture screening. The verification method preferably includes PCR detection.

[0030] The present invention provides the use of the sgRNA, the gene editing system or the recombinant strain in promoting the early heading period of millet and / or constructing a millet variety with an early heading period.

[0031] In the present invention, the sgRNA has the function of efficiently targeting the gene BBX15. The gene editing system containing the sgRNA constructs a stable genetic transformation system under the mediation of Agrobacterium, causing the gene BBX15 to undergo permanent mutation, thereby obtaining a millet material with a mutation in the gene BBX15.

[0032] The present invention provides a method for promoting the early heading period of millet, which inhibits the expression of the gene BBX15 in millet or inhibits the function of the millet heading period regulatory protein in millet.

[0033] In the present invention, the method of inhibiting the expression of the gene BBX15 in millet or inhibiting the function of the millet heading period regulatory protein in millet is the same as the above technical solution, which will not be described in detail here.

[0034] The present invention does not impose any special restrictions on the variety of millet, and millet varieties known in the art can be used. In the embodiment of the present invention, the millet is preferably CI846. The light intensity of the millet is preferably 350 to 700 μmol·m -2 ·s -1The light / dark cycle of the millet is 12h / 12h. The day and night culture temperature of the millet is 28°C / 22°C.

[0035] In the present embodiment, the number of days to heading was used as a preparation to evaluate the early heading period of millet. The statistical method for the number of days to heading was from 25 days to 41 days. The average heading period of millet mutants edited with the sgRNA for the gene BBX15 was 7 days earlier than that of the wild-type (ci846) material, and statistically significant differences were found between the millet mutants and the wild-type in terms of the number of days to heading.

[0036] The following describes in detail the gene BBX15 for regulating the heading period of millet and its application provided by the present invention in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Analysis and Sequence Determination of Gene BBX15 in Foxtail Millet

[0039] Through the association analysis of millet whole genome resequencing information and heading period, it was found that gene BBX15 was associated with heading period.

[0040] Example 2

[0041] A method for creating millet mutant materials targeting gene BBX15

[0042] 1. Target design

[0043] Referring to the method disclosed in prior art 1 (Xie X, Ma X, Zhu Q, et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing [J]. Molecular plant, 2017, 10 (9): 1246-1249.), sgRNA (GACUUGCGCGCAGGAGCGGA, SEQ ID NO: 4) was designed for foxtail millet BBX15 gene (SEQ ID NO: 1) using the targetDesign-scau.edu.cn website (http: / / skl.scau.edu.cn / targetdesign / ).

[0044] 2. Method for constructing Crispr / Cas9-BBX15-sgRNA vector

[0045] The designed sgRNA targeting BBX15 was connected to the Crispr / Cas9 vector, and the Crispr / Cas9-BBX2-sgRNA vector was created with reference to the existing technology (Xingliang M.A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants[J].Molecular Plant,2015,8(8):1274-1284.).

[0046] 3. Genetic transformation of millet

[0047] Based on the construction of the Agrobacterium-mediated millet genetic transformation system, the Crispr / Cas9-BBX15-sgRNA vector was transformed into the wild-type millet material ci846. With specific reference to the prior art 2 (Yang Z, Zhang H, Li X, et al. A minifoxtail millet with an Arabidopsis-like life cycle as a C4 model system [J]. Nature plants, 2020, 6(9): 1167-1178.), the gene-edited mutant material sibbx15 of millet BBX15 was created.

[0048] The gene-edited mutant material sibbx15 of foxtail millet BBX15 was amplified by PCR and the amplified product was sequenced. The sequencing results showed that the BBX15 gene was mutated. The nucleotide sequence of the mutated BBX15 gene is shown in SEQ ID NO:4, and the expression of the corresponding encoded protein was terminated prematurely.

[0049] Example 3

[0050] Heading period regulation experiment

[0051] The gene-edited mutant material sibbx15 of millet BBX15 created in Example 2 and the wild-type millet material ci846 were planted in mixed soil (nutrient soil and vermiculite mixed in a volume ratio of 1:1), with 20 plants in each group. The two groups of plants were grown in an automatically controlled culture chamber equipped with a full-spectrum (420-730 nm) LED light source, under a day-night cycle of 28°C / 22°C, a light / dark cycle of 12h / 12h, and a light intensity of 350-700μmol·m -2 ·s -1 The time when the two groups of plants reached the heading stage was counted.

[0052] Depend on Figure 1 It can be seen that under the same growth time, the mutant material sibbx15 entered the heading stage earlier than the wild-type plants. The time taken by the two groups of plants to enter the heading stage was statistically analyzed, and the results are shown in Tables 1 and Figure 2 The results showed that the average heading date of the mutant material sibbx15 was 7 days earlier than that of the wild type, and the BBX15 mutation significantly shortened the time when millet plants entered the heading period compared to the wild type. This suggests that the negative regulation of the BBX15 gene promotes the early entry of millet into the heading period.

[0053] Table 1 Time (days) when the mutant material sibbx15 and the wild-type millet material ci846 entered the heading stage

[0054] plant average value Standard deviation Minimum Maximum Sibbx15 31.20 1.85 29 35 WT 38.55 1.43 36 41

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A millet heading period regulatory protein, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene BBX15 encoding the protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

2.

3. Use of the millet heading period regulatory protein according to claim 1 or the gene BBX15 according to claim 2 as a target in at least one of the following: regulating the heading period of millet, improving the agronomic traits of the heading period of millet, screening millet varieties with early heading period, and conducting millet genetic breeding.

4. The application according to claim 3, characterized in that The gene BBX15 advances the heading period of millet through negative regulation.

5. Use of inhibiting the function of the millet heading period regulatory protein according to claim 1 or inhibiting the expression of the gene BBX15 according to claim 2 in promoting the early heading period of millet and / or constructing a millet variety with an early heading period.

6. An sgRNA targeting the gene BBX15 according to claim 2, characterized in that The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

4.

7. A gene editing system comprising the sgRNA according to claim 6.

8. A recombinant strain comprising the sgRNA according to claim 6.

9. Use of the sgRNA according to claim 6, the gene editing system according to claim 7, or the recombinant strain according to claim 8 in promoting an early heading period of millet and / or constructing a millet variety with an early heading period.

10. A method for promoting the early heading period of millet, characterized in that: Inhibiting the expression of the gene BBX15 according to claim 2 in millet or inhibiting the function of the millet heading period regulatory protein according to claim 1 in millet.

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