Setaria italica L. Beauv. Heading period regulation gene BBX2 and application thereof

Abstract

The invention provides a millet heading period regulation gene BBX2 and application thereof, and belongs to the technical field of functional genes. The invention provides a Setaria italica L. Beauv. Heading period regulation gene BBX2. The nucleotide sequence of the gene BBX2 is shown in SEQ ID NO: 2. The gene BBX2 is related to the morning and evening of the heading period of millet. The foxtail millet BBX2 gene editing mutant material is created by agrobacterium mediation, and the result shows that compared with a wild foxtail millet material, the heading period of the foxtail millet BBX2 gene editing mutant material is obviously advanced. The gene BBX2 promotes heading of foxtail millet through negative regulation. The gene BBX2 provided by the invention provides an effective gene locus for screening or constructing a millet variety with an advanced heading stage and developing millet genetic breeding work, and also lays a foundation for large-area popularization and planting of millet.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of functional gene technology, and particularly relates to a foxtail millet heading stage regulating gene BBX2 and application thereof. BACKGROUND

[0002] Foxtail millet is a short-day crop, which is widely distributed in China and is planted in all provinces and regions. However, foxtail millet is sensitive to photoperiod and has strong ecological dependence on varieties, and has narrow adaptability. Many excellent varieties cannot be widely applied due to regional limitations, and the utilization rate of good varieties is not high. In particular, in recent years, with the improvement of the level of foxtail millet light simplification and mechanized planting, the high latitude areas of Inner Mongolia, Liaoning and northern Jilin have formed the dominant areas of foxtail millet, and many excellent varieties selected in Hebei and Shanxi are difficult or late to head in the above-mentioned areas, which cannot meet the needs of the growth period, and it is urgent to breed widely adaptable varieties. Since the basic research on the heading stage of foxtail millet related to photoperiod started late, many key genes are not clear. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a foxtail millet heading stage regulating gene BBX2, which can advance the heading stage of foxtail millet through negative regulation, so as to adapt the planting of foxtail millet in high latitude areas.

[0004] The present application provides a foxtail millet heading stage regulating protein, and the amino acid sequence is shown as SEQ ID NO: 1.

[0005] The present application provides a gene BBX2 for encoding the protein, and the nucleotide sequence is shown as SEQ ID NO: 2.

[0006] The present application provides the application of the foxtail millet heading stage regulating protein or the gene BBX2 as a target in at least one of the following: regulating the heading stage of foxtail millet, improving the agronomic traits of the heading stage of foxtail millet, screening foxtail millet varieties with advanced heading stage, and carrying out foxtail millet genetic breeding.

[0007] Preferably, the gene BBX2 advances the heading stage of foxtail millet through negative regulation.

[0008] The present application provides the application of inhibiting the expression of the gene BBX2 or inhibiting the function of the foxtail millet heading stage regulating protein in promoting the heading stage of foxtail millet and / or constructing foxtail millet varieties with advanced heading stage.

[0009] The present application provides a target sequence targeting the gene BBX2, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 4.

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

[0011] The application provides a recombinant strain containing the sgRNA.

[0012] The application provides application of the sgRNA, the gene editing system or the recombinant strain in promoting early heading date of millet and / or constructing a millet variety with early heading date.

[0013] The application provides a method for promoting early heading date of millet, inhibiting expression of the gene BBX2 in millet or inhibiting function of the millet heading date regulatory protein in millet.

[0014] The application provides a gene BBX2 for encoding the protein, and a nucleotide sequence is shown as SEQ ID NO: 2. The gene BBX2 is related to early or late heading date of millet. The application creates a millet BBX2 gene editing mutant material by using agrobacterium mediation, and results show that, compared with wild-type millet material, the heading date of the millet BBX2 gene editing mutant material is significantly advanced. Therefore, the gene BBX2 promotes early heading of millet through negative regulation. The gene BBX2 provided by the application provides an effective gene site for screening or constructing a millet variety with early heading date, and carrying out millet genetic breeding work, and lays a foundation for large-area planting of millet. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Fig. 1 is a phenotype diagram of heading date of mutant material sibbx2 and wild type at 30 days of growth, wherein the scale = 10 cm;

[0016] Figure 2 Fig. 2 is a columnar chart of data analysis of heading date of mutant material sibbx2 and wild type, wherein Genotype: genotype, WT: wild type (ci846), sibbx2: BBX2 gene editing mutant material, Heading days: heading days; ***: p < 0.001. DETAILED DESCRIPTION

[0017] The application provides a millet heading stage regulation protein, and an amino acid sequence is shown in SEQ ID NO:1 (MKIQCDACEGAAATVVCCADEAALCARCDVEIHAANKLASKHQRLPLEALSARLPRCDVCQEKAAFIFCVEDRALFCRDCDEPIHVPGTLSGNHQRYLATGIRVGAASTCSAGGCDAHDSDHHAPPKATVEPPPQPAVSAEAQQVPSPPQFLPQGWAVDELLQFSDYESGDKLQKESPLGFKELEWFADIDLFHDQAAPKAGRTLAEVPELFGSQAASDAAYYRPSKAAGAGAGVRQSKKARIEVTDDEEDYLIVPDLG*,* represents a terminator).

[0018]

[0019] The application provides application of the foxtail millet heading stage regulating protein or the gene BBX2 as a target point in at least one of the following: regulating the foxtail millet heading stage, improving the foxtail millet heading stage agronomic character, screening a foxtail millet variety with an advanced heading stage and carrying out foxtail millet genetic breeding.

[0020] In the application, the gene BBX2 preferably makes the foxtail millet heading stage advanced through negative regulation, the method for regulating the foxtail millet heading stage or improving the foxtail millet heading stage agronomic character is inhibiting the expression of the gene BBX2, for example, mutation of the gene BBX2 in the foxtail millet or gene knockout or gene editing and the like.

[0021] In the application, the method for screening the foxtail millet variety with an advanced heading stage detects the relative expression level of the gene BBX2 or the coded protein thereof in the foxtail millet to be tested, and selects according to the relative expression level of the gene BBX2 or the coded protein thereof: when the relative expression level of the gene BBX2 or the coded protein thereof is down-regulated, the foxtail millet to be tested is a variety with an advanced heading stage. The method for detecting the relative expression level of the gene BBX2 in the foxtail millet to be tested comprises an RT-qPCR technology. The method for detecting the relative expression level of the protein coded by the gene BBX2 comprises an immunoblotting technology. The reference sample in the method preferably comprises a foxtail millet ci846.

[0022] In the application, the method for carrying out foxtail millet genetic breeding preferably uses a foxtail millet material with a lower relative expression level of the gene BBX2 or a gene function loss as a breeding parent.

[0023] The application provides application of inhibition of the expression of the gene BBX2 or inhibition of the function of the foxtail millet heading stage regulating protein in promoting the foxtail millet heading stage to be advanced and / or constructing a foxtail millet variety with an advanced heading stage.

[0024] In the application, the method for inhibiting the expression of the gene BBX2 preferably comprises a method for gene editing by using sgRNA, a method for transiently silencing the expression of the gene BBX2 by using small interfering RNA (siRNA) or stably silencing the expression of the gene BBX2 in the foxtail millet by using short hairpin RNA (shRNA). The method for inhibiting the function of the foxtail millet heading stage regulating protein preferably comprises targeted binding of the foxtail millet heading stage regulating protein by using a monoclonal antibody against the protein or making the protein lose the original biological function by using an inhibitor.

[0025] The application provides an sgRNA targeting the gene BBX2, and a nucleotide sequence of the sgRNA is shown as SEQ ID NO: 4.

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

[0027] In the present application, the gene editing system comprising the sgRNA preferably comprises a sgRNA-containing CRISPR / Cas9 recombinant vector. In the embodiments of the present application, the sgRNA-containing CRISPR / Cas9 recombinant vector is preferably a CRISPR / Cas9-BBX2-sgRNA vector.

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

[0029] In the present application, the host bacteria of the recombinant strain preferably comprise Agrobacterium. In the present application, the strain of Agrobacterium is preferably AGL1. The present application does not have special restrictions on the construction method of the recombinant strain, and the construction method of the recombinant strain known in the art can be used, for example, the CRISPR / Cas9-BBX2-sgRNA vector is transformed into Agrobacterium, and after cultivation, screening and verification, a recombinant strain comprising the sgRNA is obtained. The present application does not have special restrictions on the transformation method, and the transformation method known in the art can be used, for example, freeze-thaw method or electroporation. The screening preferably comprises antibiotic culture screening. The verification method preferably comprises PCR detection and first-generation sequencing.

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

[0031] In the present application, the sgRNA has the function of efficiently targeting the gene BBX2, and the gene editing system comprising the sgRNA constructs a stable genetic transformation system under the mediation of Agrobacterium, so that the gene BBX2 is permanently mutated, and a millet material with a mutated gene BBX2 is obtained.

[0032] The present application provides a method for promoting the heading date of millet, which inhibits the expression of the gene BBX2 in millet or inhibits the function of the millet heading date regulatory protein in millet.

[0033] In the present application, the method for inhibiting the expression of the gene BBX2 in millet or inhibiting the function of the millet heading date regulatory protein in millet is the same as the above technical solution, which will not be described here.

[0034] The present application does not have special restrictions on the variety of the millet, and the millet variety known in the art can be used. In the embodiments of the present application, the millet is preferably ci846. The light intensity of the millet is preferably 350-700 μmol·m -2 ·s -1 The light / dark cycle of the millet is 12h / 12h. The day / night culture temperature of the millet is 28℃ / 22℃.

[0035] In the embodiments of the present application, the heading days are used to evaluate the heading stage of foxtail millet. The statistical method of the heading days is from 25 days to 41 days. The average heading stage of the foxtail millet mutant edited by the sgRNA on the gene BBX2 is 8 days earlier than that of the wild type (ci846) material, and the statistical results show that there is a significant difference between the foxtail millet mutant and the wild type in the heading advance days.

[0036] The gene BBX2 for regulating the heading stage of foxtail millet and the application thereof provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0037] Example 1

[0038] Analysis and sequence acquisition of the gene BBX2 in foxtail millet

[0039] Through the whole genome resequencing information of foxtail millet and the correlation analysis of the heading stage, it is found that the gene BBX2 is correlated with the heading stage.

[0040] Example 2

[0041] A method for creating a foxtail millet mutant material targeting the gene BBX2

[0042] 1. Designing a target

[0043] According 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.), the sgRNA (CUGCGACGUCUGCCAGGUAC, SEQ ID NO: 4) is designed for the foxtail millet BBX2 gene (SEQ ID NO: 2) by using the targetDesign-scau.edu.cn website (http: / / skl.scau.edu.cn / targetdesign / ).

[0044] 2. Method for constructing a Crispr / Cas9-BBX2-sgRNA vector

[0045] The designed sgRNA targeting BBX2 is connected to the Crispr / Cas9 vector, referring to the prior art (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.) to create the Crispr / Cas9-BBX2-sgRNA vector.

[0046] 3. Genetic transformation of foxtail millet

[0047] Based on the Agrobacterium-mediated genetic transformation system of foxtail millet, the Crispr / Cas9-BBX2-sgRNA vector is transformed into wild-type foxtail millet material ci846, referring to the prior art 2 (Yang Z, Zhang H, Li X, et al. A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system[J]. Nature plants, 2020, 6(9): 1167-1178.) to create the gene editing mutant material sibbx2 of foxtail millet BBX2.

[0048] The gene editing mutant material sibbx2 of foxtail millet BBX2 is subjected to PCR amplification and sequencing of the amplification product, and the sequencing result is that the gene of BBX2 is mutated, and the nucleotide sequence of the mutated BBX2 gene is shown as SEQ ID NO: 4, and the corresponding expression of the encoded protein is terminated in advance.

[0049] Example 3

[0050] Heading stage regulation experiment

[0051] The gene editing mutant material sibbx2 of foxtail millet BBX2 created in Example 2 and the wild-type foxtail millet material ci846 are respectively planted in mixed soil (nutrient soil and vermiculite mixed at a volume ratio of 1:1), 20 plants per group. Two groups of plants are grown in an automatic control culture room equipped with full-spectrum (420-730 nm) LED light source, under a day-night cycle of 28℃ / 22℃, light / dark cycle of 12h / 12h, light intensity of 350-700 μmol·m -2 ·s -1 The time for the two groups of plants to reach the heading stage is counted.

[0052] From Figure 1It can be seen that the mutant material sibbx2 enters the heading stage earlier than the wild type plant in the same growth time. Figure 2 The results show that the average heading stage of the mutant material sibbx2 is 8 days earlier than the wild type, and the mutation of BBX2 makes the time of the millet plant to reach the heading stage significantly shortened compared with the wild type. It is proved that the BBX2 gene negatively regulates the promotion of millet to enter the heading stage in advance.

[0053] Table 1 Time (days) of the mutant material sibbx2 and the wild type millet material ci846 to enter the heading stage

[0054] Plant Mean Standard deviation Minimum Maximum Sibbx2 30.25 1.86 27 34 WT 38.55 1.43 36 41

[0055] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

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 BBX2 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 BBX2 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 BBX2 advances the heading period of millet through negative regulation.

5. Use of inhibiting the expression of the gene BBX2 according to claim 2 or inhibiting the function of the millet heading period regulatory protein according to claim 1 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 BBX2 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 BBX2 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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