Millet control tillering number and effective ear number gene Sisd1 and application thereof

Abstract

The present application relates to genes for regulating the number of tillers and the number of effective ears of millet and application thereof. Specifically, the present application provides application of gene Sisd1 or its alleles in regulating the number of tillers and the number of effective ears of millet, and thereby improving the yield of millet. The present application also provides application of gene SiSD1 or its alleles in regulating the number of tillers and the number of effective ears of millet, and thereby improving the yield of millet. The present application also discloses a method for cultivating plants with increased number of effective ears, increased number of effective tillers and / or improved yield. The genes SiSD1 and Sisd1 have wide application prospects in millet breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology. Specifically, it relates to the millet gene Sisd1, which controls the number of tillers and the number of effective ears, and its applications. Background Technology

[0002] Millet (Setaria italica (L.) P. Beauv.) is one of the main dryland crops in northern my country, with a cultivation history of nearly 8,000 years, nurturing the agricultural civilization of the Yellow River basin. Compared with the three major staple crops of rice, wheat, and corn, millet is characterized by its resistance to adverse conditions and poor soil conditions, and its wide adaptability. However, in the past 50 years, the yield increase of millet has been relatively slow, leading to a gradual decrease in its planting area. Therefore, how to increase the yield per unit area of ​​millet has become one of the core issues in the development of the millet industry (Liu Jiean, 2019; Li Shunguo, 2022). Crop yield is a complex quantitative trait, influenced by both its own genetic factors and the external environment. Research on staple crops such as rice shows that increasing the number of effective tillers per unit area is one of the effective ways to increase yield. For example, the number of effective tillers in rice determines the number of effective panicles per unit area, thus affecting rice yield. Applying nitrogen fertilizer can significantly promote the elongation of tiller buds, increase the number of tillers, and thus increase yield. For example, NGR5 is a key tillering regulator. Under high nitrogen induction, it inhibits the expression of tillering repressor genes D14 and OsSPL14 through epigenetic modification. Simultaneously, as a novel, non-SLR1-dependent target protein of the GA-GID1-SCFGID2 signaling pathway, it can be ubiquitinated by GID2 and subsequently degraded by the proteasome. SLR1 weakens the NGR5-GID1 interaction, enhances NGR5 stability, and promotes tillering, thereby increasing rice yield (Wu et al., 2020; Guo et al., 2013).

[0003] Gibberellins are a large class of diterpenoid carboxylic acids that regulate cell elongation, cell division, flowering, and fruit development in plants. After binding to the receptor GID1, gibberellins promote the degradation of DELLA proteins, relieving their inhibitory effect on growth and development (Liu et al., 2022). Mutants in gibberellin synthesis or signal transduction lead to significant crop dwarfing. For example, the SD1 gene in rice encodes OsGA20ox2; deletion mutations in this gene result in decreased levels of active gibberellins (such as GA1 and GA3), significant accumulation of DELLA proteins, reduced plant height, stronger fertilizer tolerance and lodging resistance, and an increased harvest index (Sasaki et al., 2002). Currently, several alleles of the SD1 gene have been identified, such as sd1-d from 'Low-Legged Black-Tail' and its derivatives, sd1-r from Reimei, sd1-c from Calrose76, and sd1-j from Jikkoku. Subsequent research found that the HTD1 gene from Pitay was involved in the improvement of modern indica rice. HZ With the SD1 gene from the low-legged black vine DGWG Simultaneously, it was selected and widely used by breeders, promoting the "Green Revolution" in rice breeding (Wang et al., 2020). The widespread application of wheat genes Rht-B1b and Rht-D1b, which encode the DELLA protein, has also brought about similar effects, increasing the number of tillers per unit area, increasing the number of effective panicles, and significantly improving the yield of the population, thus triggering the first "Green Revolution" (Jia Jizeng et al., 1992; Peng et al., 1999). Mutations in the Rht1 (Rht-B1b) and Rht2 (Rht-D1b) genes both form a truncated protein that does not contain the DELLA domain, resulting in dwarfing and insensitivity to gibberellins. The gene Rht8, derived from Japanese red wheat (Akagomugi), encodes a protein containing a Ribonuclease H-like domain. This protein is located in the cell nucleus and can regulate the expression of gibberellin synthesis-related genes (such as GA13ox and GA20ox-2), reduce GA3 and increase GA4 content, thereby reducing plant height (Van De Velde et al., 2021; Chai et al., 2022; Xiong et al., 2022).

[0004] The millet gene SiDWARF1 (D1) is a homolog of the rice gene SLR1 and the wheat gene Rht1. Retrotransposon insertion leading to the n-terminal deletion of DELLA (D1-tt) results in millet dwarfing (Zhao et al., 2019). The gene SiDWARF2 (D2) encodes cytochrome P450, negatively regulating ABA-mediated inhibition of internode cell elongation (Xue et al., 2016). Resequencing and constructing genetic maps of 333 recombinant inbred lines constructed from Ai 88 and Liaogu 1 as parents identified 13 reproducible QTLs, among which Seita.1G242300, encoding GA20ox8, is the most critical gene regulating plant height (He et al., 2021). All these studies indicate that gibberellin (GA) is involved in the regulation of millet plant height. To date, whether gibberellin is involved in the regulation of millet tiller number has not been reported.

[0005] Current research on the molecular regulatory mechanisms of millet tiller number and yield is limited. Studies have found that the gene Tb1 (Teosintebranched1) is a key domestication gene associated with the transition from multi-tillering teosinte to single-stem maize, while the gene OsTb1 is a key gene for inhibiting tillering in rice (Doebley et al., 1997; Takeda et al., 2003). Although millet and maize are closely related, the gene Tb1 does not play a significant role in millet domestication, mainly because it only regulates tiller bud elongation and does not participate in regulating inflorescence structure or sex determination. Therefore, it is speculated that certain key genes in hormone synthesis and signaling pathways may be involved in regulating millet tiller number. Recent studies have shown that the genes SiTCP7 and SiTCP22 may synergistically regulate millet tiller number. Overexpression of the gene Sipf40 leads to an increase in tiller angle and a significant increase in tiller number (Luane et al., 2010). Millet breeding practices have shown that increasing the number of effective panicles per unit area (tillering millet) can significantly increase yield (Qian et al., 2012; Fan Guangyu, 2019). Summary of the Invention

[0006] Against this research background, we obtained the dwarfed multi-tillering millet mutant material dmt1 ( D warf and M ore T (iller1). It was crossed with Changsheng 19 (CS19) to construct the BC1F2 population. Then, the gene controlling tillering was obtained by map-based cloning. It was found that the gene encodes GA20ox2, which is the ortholog of the rice SD1 gene in millet, and it was named SiSD1 gene.

[0007] The mutation of this gene reduces the content of active gibberellin, leading to an increase in the number of millet tillers. Overexpression of SiSD1 results in reduced tillering. Cloning of this gene will help promote a "green revolution" in millet breeding, providing important genetic resources and a theoretical basis for improving millet plant architecture and increasing yield.

[0008] The inventors of this invention used the mutagen ethyl methanesulfonate (EMS) to mutagenesis and genetic screening of the millet variety Jingu 10, and isolated and identified a mutant dmt1 (dwarf and more tiller1) with increased tiller number. Then, using map-based cloning, they cloned the key gene DMT1 (hereinafter referred to as the SiSD1 gene), which controls the number of millet tillers. Phenotypic analysis of the mutant and genetic complementation experiments demonstrated the function of this gene.

[0009] Therefore, the purpose of this invention is to provide a key factor for increasing the number of tillers and yield of millet, and to provide genetic resources and theoretical guidance for breeding high-yield millet varieties.

[0010] In a first aspect, the present invention provides a gene SiSD1 that controls the number of millet tillers, said gene SiSD1 encoding a SiSD1 protein, the amino acid sequence of which is shown in any of the following:

[0011] 1) The amino acid sequence shown in SEQ ID NO. 3;

[0012] 2) An amino acid sequence that has one or more substitutions, deletions, and / or insertions of amino acid residues compared to the sequence shown in SEQ ID NO. 3, and has the same function as the amino acid sequence shown in SEQ ID NO. 3;

[0013] 3) An amino acid sequence having at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO. 3, and having the same function as the amino acid sequence shown in SEQ ID NO. 3; or

[0014] 4) An active fragment comprising any one of the amino acid sequences described in 1)-3);

[0015] The protein shown in SEQ ID NO. 3 consists of 423 amino acids.

[0016] The nucleotide sequence of the gene SiSD1 is shown in any of the following:

[0017] a) The nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0018] b) A nucleotide sequence that has one or more substitutions, deletions, and / or insertions of nucleotide sequences compared to the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and has the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0019] c) A nucleotide sequence having at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and having the same function as the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0020] d) Nucleotide sequences that differ from SEQ ID NO. 1 or SEQ ID NO. 2 due to the degeneracy of the genetic code;

[0021] e) An active fragment comprising any one of the nucleotide sequences described in a)-d);

[0022] f) A nucleotide sequence that hybridizes with the complementary sequence of any of the nucleotide sequences described in a)-e) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or

[0023] g) A nucleotide sequence complementary to any of the nucleotide sequences described in a)-e);

[0024] The gene SiSD1, which controls the number of millet tillers and the number of effective panicles, is an isolated nucleotide sequence.

[0025] Secondly, the present invention provides a gene Sisd1 that controls the number of tillers in millet, said gene Sisd1 encoding a Sisd1 protein, the amino acid sequence of which is shown in any of the following:

[0026] 1) The amino acid sequence shown in SEQ ID NO. 5;

[0027] 2) An amino acid sequence that has one or more substitutions, deletions, and / or insertions of amino acid residues compared to the sequence shown in SEQ ID NO. 5, and has the same function as the amino acid sequence shown in SEQ ID NO. 5;

[0028] 3) An amino acid sequence having at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO. 5, and having the same function as the amino acid sequence shown in SEQ ID NO. 3; or

[0029] 4) An active fragment comprising any one of the amino acid sequences described in 1)-3);

[0030] The nucleotide sequence of the Sisd1 gene is shown in any of the following:

[0031] a) The nucleotide sequence shown in SEQ ID NO. 4;

[0032] b) A nucleotide sequence that has one or more substitutions, deletions, and / or insertions of nucleotide sequences compared to the sequence shown in SEQ ID NO. 4, and has the same function as the nucleotide sequence shown in SEQ ID NO. 4;

[0033] c) A nucleotide sequence having at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO. 4, and having the same function as the nucleotide sequence shown in SEQ ID NO. 4;

[0034] d) A nucleotide sequence that differs from SEQ ID NO. 4 due to the degeneracy of the genetic code;

[0035] e) An active fragment comprising any one of the nucleotide sequences described in a)-d);

[0036] f) A nucleotide sequence that hybridizes with the complementary sequence of any of the nucleotide sequences described in a)-e) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or

[0037] g) A nucleotide sequence complementary to any of the nucleotide sequences described in a)-e).

[0038] In a preferred embodiment, the gene SiSD1 that controls the number of millet tillers and the number of effective panicles has the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; and, those skilled in the art should understand that, in a broader sense, the gene SiSD1 that controls the number of millet tillers and the number of effective panicles is a nucleotide sequence that has more than 90%, preferably more than 99%, homology with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 and encodes a protein with the same function.

[0039] In a preferred embodiment, the protein encoded by the gene SiSD1, which controls the number of millet tillers and the number of effective panicles as described in the first aspect, is an isolated protein with the amino acid sequence shown in SEQ ID NO. 3.

[0040] In a preferred embodiment, the gene Sisd1 that controls the number of millet tillers and the number of effective ears has the nucleotide sequence shown in SEQ ID NO. 4; and, those skilled in the art should understand that, in a broader sense, the gene Sisd1 that controls the number of millet tillers and the number of effective ears is a nucleotide sequence that has more than 90%, preferably more than 99%, homology with the nucleotide sequence shown in SEQ ID NO. 4 and encodes a protein with the same function.

[0041] In a third aspect, the present invention provides a recombinant vector comprising the gene SiSD1, as described in the first aspect, which controls the number of millet tillers and the number of effective panicles. The vector includes a plant expression vector and / or a plant gene editing vector.

[0042] The present invention also provides a recombinant vector comprising the gene Sisd1, which controls the number of tillers and effective panicles of millet as described in the second aspect. The vector includes plant expression vectors and / or plant gene editing vectors.

[0043] The plant expression vector is preferably pCAMBIA1300, and the plant expression vector / or plant gene editing vector contains a SiSD1 gene promoter or an enhancing promoter. Preferably, the plant expression vector or plant gene editing vector also contains a gene sequence encoding a tag protein. The SiSD1 gene promoter is preferably a sequence 2kb upstream of the SiSD1 gene coding region, the enhancing promoter is preferably an Actin promoter, the Actin promoter sequence is shown in SEQ ID NO. 6, and the gene sequence encoding the tag protein is preferably a GFP tag sequence as shown in SEQ ID NO. 7.

[0044] Preferably, the recombinant vector contains a foreign nucleotide fragment encoding the amino acid sequence shown in SEQ ID NO. 3. More preferably, the recombinant vector contains a foreign nucleotide fragment as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0045] Preferably, the recombinant vector contains a foreign nucleotide fragment encoding the amino acid sequence shown in SEQ ID NO. 5. More preferably, the recombinant vector contains a foreign nucleotide fragment as shown in SEQ ID NO. 4.

[0046] In one embodiment, the plasmid used to construct the recombinant vector may be selected from, but is not limited to, pCAMBIA1300.

[0047] The present invention also provides a host cell comprising the SiSD1 gene or its allele or the recombinant vector.

[0048] The present invention also provides a host cell comprising the Sisd1 gene or its allele or the recombinant vector.

[0049] In one embodiment, host cells containing the recombinant vector can be obtained by transforming or transfecting the recombinant vector into cells, which can then be used for applications such as amplifying expression vectors, expressing the protein, or obtaining transgenic plants.

[0050] The host cell may be selected from, but is not limited to, bacterial cells (e.g., Escherichia coli cells or Agrobacterium cells), fungal cells (e.g., yeast cells), or plant cells (e.g., millet cells).

[0051] In a fourth aspect, the present invention provides the use of the gene SiSD1 or the SiSD1 protein encoded thereon in the cultivation or acquisition of plants with increased effective panicle number, increased effective tiller number and increased yield, wherein the plants are grasses, preferably millet.

[0052] The present invention also provides the use of the gene Sisd1 or the Sisd1 protein encoded thereon in the cultivation or acquisition of plants with increased effective panicle number, increased effective tiller number and increased yield, wherein the plants are grasses, preferably millet.

[0053] In a fifth aspect, the present invention provides a method for cultivating or obtaining a plant with increased effective panicle number, increased effective tiller number and / or increased yield, the method comprising increasing the expression of the Sisd1 gene or its allele or increasing the amount of Sisd1 protein in the plant, or decreasing the expression of the SiSD1 gene or its allele or decreasing the amount of SiSD1 protein.

[0054] Preferably, the method further includes knocking out or reducing the expression of the SiSD1 gene in plants or causing the SiSD1 protein to lose its function.

[0055] In one embodiment, the method can be implemented by: editing the nucleic acid sequence of SiSD1 in wild-type plants or plant cells using gene editing technology, introducing or deleting one or more bases, or causing nucleotide substitutions that result in missense mutations, thereby altering the amino acid sequence encoded by the gene; or by using gene silencing technology to reduce or knock out the expression of the SiSD1 gene in plants.

[0056] In one embodiment, the method can be implemented by transforming or transfecting a recombinant vector containing the Sisd1 gene or its allele, or a host cell containing the recombinant vector, into plant cells to obtain a transgenic plant.

[0057] In one embodiment, the method can be implemented by constructing a Sisd1 gene overexpression vector containing an enhancing promoter (preferably an Actin promoter) and transforming or transfecting it into millet to increase the amount of protein encoded by the Sisd1 gene.

[0058] In one embodiment, the transformation or transfection of the Sisd1 gene expression vector, the Sisd1 gene overexpression vector, and the Sisd1 gene editing vector can be performed by Agrobacterium-mediated transformation or gene gun transformation; preferably, the transformation or transfection is performed by Agrobacterium-mediated transformation or gene gun transformation.

[0059] In one embodiment, the method can be implemented by establishing a near-isogenic line to obtain the plant; preferably, the method for establishing a near-isogenic line is as follows: a target parent carrying the Sisd1 gene or its allele is crossed with another parent line, and then the offspring of the cross are backcrossed multiple times with another parent line. During the backcrossing process, individuals carrying the target parent trait are continuously selected, and backcrossing continues until the target trait does not segregate when the offspring are self-crossed. At this point, a near-isogenic line with a genetic background similar to the other parent line and stably carrying the trait controlled by the Sisd1 gene or its allele in the target parent line is obtained; the target parent trait is the target parent trait controlled by the Sisd1 gene or its allele, and the target trait is an increase in the number of effective spikes, an increase in the number of effective tillers, and / or an increase in yield. This invention provides theoretical guidance and material basis for breeding high-yield crop varieties.

[0060] When the Sisd1 gene of the present invention is used to increase millet yield, the following methods can be used: (1) constructing a Sisd1 gene overexpression vector containing an enhancing promoter (preferably an Actin promoter); (2) transforming the constructed overexpression vector into regenerable millet tissues or organs; (3) culturing the transformed tissues or organs into plants and screening plants with significantly increased Sisd1 gene expression.

[0061] The increase in effective panicle number, increase in effective tiller number and / or increase in yield described in this invention are compared with wild-type plants, preferably compared with millet, more preferably compared with wild-type millet, more preferably compared with Jin Gu 21 (JG21), more preferably compared with Chang Sheng 19 (CS19), more preferably compared with millet ci846, and more preferably compared with Jin Gu 10.

[0062] In this invention, the plant is a grass, preferably millet.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] This invention clones the key genes SiSD1 and / or Sisd1 that control the number of tillers and effective panicles, which can be used in millet genetic engineering and molecular breeding to improve millet traits, increase the number of effective panicles and effective tillers, and improve millet yield. Attached Figure Description

[0065] Figure 1 The diagram shows the phenotypic analysis of CS19 and dmt1, as well as their hybrid F2, and the map-based cloning of the SiSD1 gene. A shows the phenotype of mature plants of CS19 and dmt1, as well as their hybrid BC1F2, with a scale bar of 20 cm. B shows the phenotype of ears of grain of CS19 and dmt1, as well as their hybrid BC1F2, with a scale bar of 10 cm. CD shows the statistical analysis of plant height and tiller number of CS19 and dmt1, as well as their hybrid BC1F2; the statistical count n = 20, analyzed using Duncan's test (p < 0.05). E shows the map-based cloning of the target gene SiSD1; in the diagram, horizontal lines represent chromosomes, and vertical lines represent molecular markers; in the SiSD1 gene structure, blue-green squares, black squares, and black lines represent non-coding regions, exons, and introns, respectively. F shows the protein sequence alignment analysis of CS19 and dmt1.

[0066] Figure 2 The gene phylogenetic tree of SiSD1, the subcellular localization of the encoded protein, and the tissue expression pattern are shown; where A is the gene phylogenetic tree analysis of SiSD1; B is the subcellular localization of the SiSD1-GFP fusion protein; and C is the tissue expression pattern of SiSD1.

[0067] Figure 3 The SiSD1 gene functional complementation vector was shown. Figure 3 A) and overexpression vector construction structure ( Figure 3 B).

[0068] Figure 4 The function of the SiSD1 gene was verified by complementation, showing that SiSD1 can complement the phenotype of Sisd1. A represents the phenotype of mature plants of JG21, Sisd1, and the transgenic complementary line (pSiSD1::SiSD1), with a scale bar of 20 cm. B represents the statistical analysis of plant height in JG21, Sisd1, and pSiSD1::SiSD1. C represents the statistical analysis of the number of effective tillers per plant in JG21, Sisd1, and pSiSD1::SiSD1. The statistical count was n = 10, and significance was analyzed using Duncan's test (p < 0.05). D represents the expression analysis of the SiSD1 gene in JG21, Sisd1, and pSiSD1::SiSD1 plants.

[0069] Figure 5The phenotypic and gene expression analyses of JG21 and its near-isogenic line Sisd1 are shown. A represents the phenotype of mature plants of JG21 and its near-isogenic line Sisd1 (scale bar: 20 cm); B represents the phenotype of panicles of JG21 and Sisd1 (scale bar: 10 cm); C represents the phenotype of internodes of JG21 and Sisd1; DF represents the statistical analysis of plant height, tiller number, and internode length of JG21 and Sisd1; the statistical counts for plant height, tiller number, and internodes were n = 20; significance analysis was performed using Duncan's test (p < 0.05); G represents the expression analysis of key genes regulating plant height and tillering.

[0070] Figure 6 The images show phenotypic and gene expression analyses of SiSD1-overexpressing plants. A represents the phenotype of mature plants of ci846 and the SiSD1-overexpressing line SiSD1-OE (scale bar: 20 cm); B represents the phenotype of internodes in ci846 and SiSD1-OE (scale bar: 5 cm); C represents the phenotype of panicles in ci846 and SiSD1-OE (scale bar: 5 cm); DF represents the statistical analysis of effective tiller number, plant height, and internode length per plant in ci846 and SiSD1-OE. The statistical counts for effective tiller number and plant height were n = 10, and the statistical counts for internode length were n = 14. Significance analysis was performed using Duncan's test (p < 0.05). G represents the expression analysis of the SiSD1 gene in ci846 and SiSD1-OE; H represents the content analysis of the SiSD1 protein in ci846 and SiSD1-OE; I represents the expression analysis of key genes regulating tillering.

[0071] Figure 7 The results showed that, compared to JG21, the near-isogenic line Sisd1 significantly increased yield at different planting densities. A represents the phenotypes of JG21 and the near-isogenic line Sisd1 at three different planting densities in the field; B represents the statistical analysis of the number of effective panicles in JG21 and Sisd1 at the three different planting densities; C represents the statistical analysis of the total yield of JG21 and Sisd1 at the three different planting densities. Significance analysis was performed using Duncan's test (p < 0.05). Detailed Implementation

[0072] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0073] Unless otherwise specified, the experimental methods described in the following examples are standard experimental methods. All reagents, kits, and instruments used in the experiments can be purchased from biological instrument and reagent companies unless otherwise specified.

[0074] Changsheng 19 (CS19), Jingu 21 (JG21), millet ci846, and Jingu 10 are all commercially available millet varieties.

[0075] Example 1. Map-based cloning of key regulatory genes for millet tiller number.

[0076] Using Jingu 10 as a background, the inventors conducted mutagenesis and genetic screening with the mutagen ethyl methanesulfonate (EMS), isolating and identifying a multi-tillering millet mutant material, dmt1. This mutant was crossed with Changsheng 19 (CS19), and the F1 generation was self-crossed to obtain the F2 generation. Individual plants from the F2 generation with phenotypes similar to dmt1 were backcrossed with CS19, and the BC1F1 generation was self-crossed to obtain the BC1F2 genetic population. The plant height and number of tillers in BC1F1 were between those of the two parents. In the BC1F2 population, the ratio of CS19-like phenotypes, BC1F1-like phenotypes, and dmt1-like phenotypes was 1:2:1, indicating that the dmt1 phenotype is caused by a semi-dominant gene (preliminarily named DMT1). The mature plant height of the dmt1 mutant plants in both dmt1 and BC1F2 was significantly lower than that of CS19, while the number of effective tillers and effective panicles was significantly increased, showing a certain yield-increasing potential. Figure 1 Based on this, it was decided to clone the superior allele regulating this desirable trait. Candidate genes were identified using the BC1F2 genetic population constructed from the Changsheng 19 and dmt1 mutants. Coarse mapping showed the gene was located between molecular markers P950 and P1500 on the long arm of chromosome 5. Further fine mapping located it between molecular markers PL20 and PR20, a candidate region of approximately 24 kb containing 3 genes. DNA sequencing revealed a deletion of one G base at 1015 bp in the coding region of gene Si5g40430, causing a frameshift mutation and producing a truncated protein (…). Figure 1 E and F). We also examined the expression of these three genes within the candidate region and found that the expression of the other two genes remained unchanged. However, gene Si5g40430 was slightly upregulated using two pairs of primers (primer sequences are shown in Table 1), further confirming Si5g40430 as a candidate gene.

[0077] Phylogenetic analysis revealed that the gene Si5g40430 encodes GA20ox2, which is a homolog of rice SD1 in millet; therefore, we named it SiSD1. Figure 2 A), whose nucleotide sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and whose amino acid sequence of the protein encoded by the SiSD1 gene (SiSD1 protein) is shown in SEQ ID NO. 3. Subcellular localization analysis showed that the SiSD1-GFP fusion protein was localized in the cytoplasm and nucleus ( Figure 2B). Tissue expression pattern analysis revealed that the gene SiSD1 was highly expressed in the roots and leaf sheaths of millet. Figure 2 C). The mutant SiSD1 gene in the dmt1 mutant (which, compared to the SiSD1 gene, lacks a single base G at 1015 bp in the coding region) is named the Sisd1 gene, and its nucleotide sequence is shown in SEQ ID NO. 4. The amino acid sequence of the protein encoded by the Sisd1 gene (Sisd1 protein) is shown in SEQ ID NO. 5.

[0078] Table 1

[0079]

[0080] Example 2. Construction of SiSD1 genetic vector and genetic transformation of millet.

[0081] The complete SiSD1 gene from the wild-type Jin Gu 21 (JG21) genome, whose nucleotide sequence is shown in SEQ ID NO.1 (including its own promoter sequence, genomic sequence, and 3' untranslated region sequence), was amplified by DNA polymerase PCR and then ligated to the multiple cloning site of the pCAMBIA1300 plasmid (purchased from CAMBIA) using restriction enzyme digestion and ligation methods to obtain the complementary vector pSiSD1::SiSD1( Figure 3 A).

[0082] The constructed complementary vector was transformed into E. coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and positive clones were screened using kanamycin. The cells were shaken and plasmids were extracted. Sequencing confirmed the presence of a completely accurate SiSD1 gene sequence in the positive clones. The plasmids of these positive clones were then electroporated into EHA105 Agrobacterium competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.).

[0083] Next, the clones that were successfully transformed and contained the target plasmid (complementary vector pSiSD1::SiSD1) were genetically transformed using Agrobacterium infection with the mutant dmt1 callus tissue as the recipient to obtain positive plants pSiSD1::SiSD1.

[0084] Example 3. Obtaining and phenotypic analyzing the near-isogenic line Sisd1.

[0085] To study the function of the gene SiSD1, we obtained the near-isogenic line Sisd1 through multiple backcrosses. The method for obtaining the near-isogenic line Sisd1 was as follows: the parent dmt1 carrying the target trait was crossed with JG21, a famous millet variety from Shanxi Province (as the recipient parent). The offspring were then backcrossed with the parent JG21 multiple times until the target trait did not segregate when the offspring were self-crossed. This yielded the Sisd1 line (which carries the Sisd1 gene) with a genetic background similar to JG21. This line and the parent JG21 (which carries the SiSD1 gene) constitute a near-isogenic pair.

[0086] We analyzed the phenotypes of mature plant height, effective tiller number per plant, and internodes of the JG21 and its near-isogenic line Sisd1. The plant heights of the JG21 and Sisd1 lines were 182 and 148 cm, respectively, and the lengths of the main spikelets were 18 and 21 cm, respectively. The results showed that the near-isogenic line Sisd1 differed significantly from the JG21 line in plant height, effective tiller number per plant, and internode phenotype. The plant height of the near-isogenic line Sisd1 was significantly lower than that of JG21, while the number of effective tillers per plant was significantly increased. Figure 5 AB); The number of internodes in the near-isogenic line Sisd1 remained unchanged compared to JG21, while the internode length was shortened. This indicates that the dwarfing of the Sisd1 line originates from the shortened internodes, but does not affect the number of nodes. Figure 5 CF); We also examined the expression of tillering regulatory genes SiTB1, SiTAD1, SiDLT1, SiMOC1, SiMOC3, SiSLR1, and SiSD1 in near-isogenic lines Sisd1 and JG21 (reverse transcription primer sequences are shown in Tables 1 and 2). We found that the tillering-inhibiting genes SiTB1, SiTAD1, and SiDLT1 were all downregulated in Sisd1, while the tillering-promoting genes SiMOC1 and SiMOC3 were upregulated. The key GA signaling pathway gene SiSLR1 was downregulated, and SiSD1 itself was slightly upregulated. Figure 5 (G); This indicates that genes SiSD1 and Sisd1 are key genes controlling the number of tillers or the number of effective tillers per millet plant.

[0087] Table 2

[0088]

[0089] Example 4. Phenotypic analysis of SiSD1 transgenic complementary plants

[0090] To examine whether the gene SiSD1 can complement the phenotype of Sisd1, the phenotypes of the positive plant pSiSD1::SiSD1 obtained through transgenic experiments in Example 2, the wild-type plant JG21, and the near-isogenic line Sisd1 described in Example 3 were analyzed. Plant height and tiller number were statistically analyzed for JG21, Sisd1, and pSiSD1::SiSD1. The results showed that compared to plant Sisd1, plant pSiSD1::SiSD1 had a significantly reduced number of effective tillers, which could restore the phenotype of Sisd1. Figure 4 AC). We examined the expression of the SiSD1 gene in plants JG21, Sisd1, and pSiSD1::SiSD1, and found that the expression level of this gene was significantly increased in plant pSiSD1::SiSD1, and slightly increased in plant Sisd1. Figure 4 (D) This indicates that the SiSD1 gene can restore the phenotype of the effective tiller number of Sisd1, suggesting that the SiSD1 gene is a key gene controlling the number of tillers or effective tillers in millet.

[0091] Example 5. Construction and genetic transformation of SiSD1 overexpression vector

[0092] We ligated the Actin promoter sequence (SEQ ID NO. 6), the SiSD1 gene CDS coding sequence (SEQ ID NO. 2), and the GFP tag sequence (SEQ ID NO. 7) into the pCAMBIA1300 plasmid via enzyme digestion to obtain the overexpression vector pActin1::SiSD1-GFP. Figure 3 B). The constructed overexpression vector was transformed into E. coli DH5α competent cells, and positive clones were screened using kanamycin. Plasmids were extracted and sequenced to identify positive clones with completely correct Actin promoter sequences, SiSD1 gene CDS coding sequences, and GFP tag sequences. The plasmids of these positive clones were then electroporated into EHA105 Agrobacterium competent cells (prepared using conventional methods, referring to *Plant Genetic Engineering*, Wang Guanlin and Fang Hongyun, Science Press, 2nd edition, 2004). The successfully transformed clones were then genetically transformed using Agrobacterium infection with wild-type millet ci846 callus tissue as the recipient. The transgenic operation method is as described in Example 2. Finally, the SiSD1 overexpression line SiSD1-OE was obtained.

[0093] Example 6. The number of effective tillers per plant decreased in plants overexpressing SiSD1.

[0094] To determine whether increasing SiSD1 expression could increase millet plant height, the phenotypes of the SiSD1-overexpressing line SiSD1-OE at maturity, including plant height, internode length, and panicle length, were analyzed. The plant heights of SiSD1-OE under the ci846 and ci846 backgrounds were 105 and 182 cm, respectively, and the main panicle lengths were 16 and 14 cm, respectively. The results showed significant differences in plant height, internode length, and panicle phenotype in the overexpressing line SiSD1-OE. The plant height of SiSD1-OE was significantly higher than that of ci846, while the number of effective tillers per plant decreased, the number of internodes increased, and the internode length elongated. Figure 6 AF); Simultaneously, we examined changes in SiSD1 gene expression and protein levels in the overexpression line SiSD1-OE, and the results showed that SiSD1 gene expression was significantly upregulated and protein content was significantly increased (AF); Figure 6 GH); We also examined the expression of tillering regulatory genes SiTB1, SiTAD1, SiDLT1, SiMOC1, SiMOC3, SiSLR1, and SiSD1, and found that the expression of the negative tillering regulators SiTB1 and SiDLT1 was upregulated, SiMOC3 was downregulated, SiSLR1 was upregulated, and SiSD1 was significantly upregulated (GH). Figure 6 (I) This indicates that increasing the expression level of the SiSD1 gene can significantly reduce the number of effective tillers per plant. It is understandable that decreasing the expression level of the SiSD1 gene can significantly increase the number of effective tillers per plant.

[0095] Example 7. Sisd1 can increase yield under different planting densities.

[0096] JG21 and Sisd1 plants obtained in Example 3 were sown at the Dongyang base of Shanxi Agricultural University. The field was divided into 18 equal-sized plots of 6 m² each. 2 In each plot, the environmental conditions, such as soil fertility, light, and moisture, were basically uniform. Based on a randomized controlled trial design, three tests were repeated, using three densities and two materials, totaling 18 plots. With consistent row spacing, compared to JG21, Sisd1 showed a 15% increase in effective panicles per unit area and a 16% increase in yield at a plant spacing of 10 cm; and a 28% increase at a plant spacing of 20 cm. Figure 7 Therefore, compared with the wild type, Sisd1 has an increased number of effective spikes per unit area and a higher yield under different planting density conditions.

[0097] It should be understood that although the invention has been specifically shown and described with reference to its exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made therein, and various combinations of embodiments may be made, without departing from the spirit and scope of the invention as defined by the appended claims.

[0098] References:

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[0105] 7. Wang Y, Shang L, Yu H, Zeng L, Hu J, Ni S, Rao Y, Li S, Chu J, Meng X, Wang L, Hu P, Yan J, Kang S, Qu M, Lin H, Wang T, Wang Q, Hu X, Chen H, Wang B, Gao Z, Guo L, Zeng D, Zhu X, Xiong G, Li J, Qian Q. AStrigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice.Mol Plant. 2020 Jun 1;13(6):923-932.

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[0122] Sequence List:

[0123] SEQ ID NO. 1: Genomic nucleotide sequence of the millet SiSD1 gene, including the 1.5kb promoter (underlined), 5'UTR (bold), and 3'UTR (underlined and bolded) sequences.

[0124]

[0125]

[0126]

[0127] SEQ ID NO. 2: Nucleotide sequence of the CDS of the SiSD1 gene in millet.

[0128]

[0129] SEQ ID NO. 3: Amino acid sequence of the protein encoded by the millet SiSD1 gene.

[0130] MVSQAQQEPALPHSSSTAKRAAASLMDARPAQPLLLRAPTPSIDLPASKPDRAAAAAGKAAAASVFDLRREPKIPAPFVWPHDDARPASAAELDVPLVDVGVLRNGDRAGLRRAAAQVAAACATHGFFQVCGHGVGADLARAALDGASDFFRLPLAEKQRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDGAASPVVVDYFAGTLG QDFEAVGRVYQRYCEEMKALSLTIMELLELSLGVERGYYRDFFEDSRSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGDWRPVRPVPGAMVINIGDTFMALSNGRYKSCLHRAVVNQRQERRSLAFFLCPREDRVVRPPASGAVGEAPRRYPDFTWADLMRFTQRHYRADTRTLDAFTRWLSHGPAQDAPVAAAAST

[0131] SEQ ID NO. 4: CDS nucleotide sequence of the millet Sisd1 gene (one base is missing after the underline, indicating a frameshift mutation).

[0132]

[0133] SEQ ID NO. 5: Amino acid sequence of the protein encoded by the millet Sisd1 gene.

[0134] MVSQAQQEPALPHSSSTAKRAAASLMDARPAQPLLLRAPTPSIDLPASKPDRAAAAAGKAAAASVFDLRREPKIPAPFVWPHDDARPASAAELDVPLVDVGVLRNGDRAGLRRAAAQVAAACATHGFFQVCGHGVGADLARAALDGASDFFRLPLAEKQRARRVPGTVSG YTSAHADRFASKLPWKETLSFGFHDGAASPVVVDYFAGTLGQDFEAVGRVYQRYCEEMKALSLTIMELLELSLGVERGYYRDFFEDSRSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGDWRPVRPVPGAMVINIGDTFMALSNGRYKSCLHRRW

[0135] SEQ ID NO. 6: Nucleotide sequence of the Actin promoter

[0136]

[0137] SEQ ID NO. 7: Nucleotide sequence of GFP protein

[0138]

[0139] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for obtaining plants with increased effective panicle number, increased effective tiller number, and / or increased yield, the method comprising: The expression of the SiSD1 gene in a plant was knocked down, thereby reducing the expression of the SiSD1 gene or the amount of SiSD1 protein in the plant. The amino acid sequence of the SiSD1 protein is shown in SEQ ID NO. 3; the nucleotide sequence of the SiSD1 gene is shown in SEQ ID NO. 1 or SEQ ID NO.

2. The plant is millet, specifically Jin Gu 21, Chang Sheng 19, and Jin Gu 10.

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