Gene Sisd1 for controlling tillering number and effective ear number of foxtail millet and application of gene Sisd1

Abstract

The invention relates to a gene for regulating and controlling the tillering number and the effective ear number of foxtail millet and application of the gene. Specifically, the invention provides the application of the gene Sisd1 or the allele thereof in the aspect of improving the yield of the millet by regulating the tiller number and the effective ear number of the millet. The invention also provides application of the gene SiSD1 or the allele thereof in the aspect of improving the yield of the millet by regulating the tiller number and the effective ear number of the millet. Also disclosed are methods of growing plants with increased number of available ears, increased number of available tillers, and / or increased yield. The genes SiSD1 and Sisd1 have a wide application prospect in millet breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and specifically to a gene Sisd1 for controlling the number of tillers and effective ears of millet and its application. Background Art

[0002] Millet (Setaria italica (L.) P. Beauv.) is one of the main dryland crops in northern my country. It has a cultivation history of nearly 8,000 years and has nurtured the Chinese agricultural civilization in the Yellow River Basin. Compared with the three major staple crops of rice, wheat and corn, millet is resistant to adversity and drought and has wide adaptability. However, the increase in millet yield has been relatively slow in the past 50 years, causing its planting area to gradually decrease. Therefore, how to increase millet yield has become one of the core issues in the development of the millet industry (Liu Jie'an, 2019; Li Shunguo, 2022). Crop yield is a complex quantitative trait, which is affected by both its own genetic factors and the external environment. The research results of the staple crop rice show 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 of rice determines the number of effective panicles per unit area, which in turn affects the rice yield. The application of 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. When induced by high nitrogen, it inhibits the expression of tillering inhibitory genes D14 and OsSPL14 through epigenetic modification. At the same time, as a new target protein of GA-GID1-SCFGID2 in the gibberellin signaling pathway that is not SLR1-dependent, it can be ubiquitinated by protein GID2 and then degraded by proteasome. SLR1 weakens the interaction between NGR5 and GID1, enhances the stability of NGR5 and promotes the occurrence of tillering, thereby increasing rice yield (Wu et al., 2020; Guo et al., 2013).

[0003] Gibberellins are a large class of diterpene carboxylic acids that regulate plant cell elongation, cell division, flowering, fruit development, etc. After binding to the receptor GID1, gibberellins promote the degradation of DELLA proteins and relieve the inhibitory effect of DELLA proteins on growth and development (Liu et al., 2022). Gibberellin synthesis or signal transduction mutants all lead to significant dwarfing of crops. For example, the rice SD1 gene encodes OsGA20ox2. The deletion mutation of this gene leads to a decrease in the content of active gibberellins (such as GA1, GA3) in rice, a significant accumulation of DELLA proteins, a decrease in plant height, stronger fertilizer tolerance and lodging resistance, and an increased harvest index (Sasaki et al., 2002). At present, multiple alleles of the SD1 gene have been found, such as the gene sd1-d from "Dijiao Wujian" and its derivatives, the gene sd1-r from Reimei, the gene sd1-c from Calrose76, and the gene sd1-j from Jikkoku. Subsequent studies have found that the gene HTD1 from Pitai was involved in the improvement of modern indica rice. HZ The gene SD1 from the low-legged black tip DGWG At the same time, it was selected and widely used by breeders, promoting the "green revolution" in rice breeding (Wang et al., 2020). The widespread use of wheat genes Rht-B1b and Rht-D1b encoding DELLA proteins also brought similar effects, increasing the number of tillers per unit area, the number of effective panicles, and the population yield significantly, thus triggering the first "green revolution" (Jia Ji Zeng et al., 1992; Peng et al., 1999). Mutations in the Rht1 (Rht-B1b) and Rht2 (Rht-D1b) genes both form a truncated protein without a DELLA domain, resulting in dwarfing and insensitivity to gibberellins. The gene Rht8 from Japanese red wheat (Akagomugi) encodes a protein containing a Ribonuclease H-like domain. This protein is localized 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 the millet homolog of the rice gene SLR1 and the wheat gene Rht1. Retrotransposon insertion results in the N-terminal deletion of DELLA (D1-tt), which leads to millet dwarfing (Zhao et al., 2019). The gene SiDWARF2 (D2) encodes cytochrome P450, which negatively regulates ABA-mediated inhibition of internode cell elongation (Xue et al., 2016). By resequencing and constructing a genetic map of 333 recombinant inbred line populations constructed with Ai 88 and Liaogu No. 1 as parents, 13 reproducible QTLs were identified, among which Seita.1G242300 encodes GA20ox8, which is the most critical plant height regulatory gene (He et al., 2021). The above studies all pointed out that gibberellins (GA) are involved in the regulation of millet plant height. So far, there has been no report on whether gibberellins are involved in the regulation of millet tiller number.

[0005] At present, there are few studies on the molecular regulatory mechanisms of millet tiller number and yield. Studies have found that gene Tb1 (Teosintebranched1) is a key domestication gene related to the transition from multi-tillering of teosinte to single-stem maize, and gene OsTb1 is a key gene for rice tiller inhibition (Doebley et al., 1997; Takeda et al., 2003). Although millet and maize are closely related, gene Tb1 does not have an important function in the domestication of millet, mainly because in millet gene Tb1 only regulates the elongation of tiller buds and does not participate in the regulation of inflorescence structure and sex determination. Therefore, it is speculated that some key genes in hormone synthesis and signaling pathways may be involved in the regulation of millet tiller number. Recent studies have shown that genes SiTCP7 and SiTCP22 may synergistically regulate millet tiller number. Overexpression of gene Sipf40 leads to an increase in millet tiller angle and a significant increase in tiller number (Luan et al., 2010). Millet breeding practice shows that the yield can be significantly increased by increasing the number of effective ears per unit area (tillering millet) (Qian et al., 2012; Fan Guangyu, 2019). Summary of the invention

[0006] In this research context, we obtained a dwarf multi-tillering millet mutant material dmt1 ( D warf and M ore T iller1). It was hybridized with Changsheng 19 (CS19) to construct a BC1F2 population, and then the gene controlling tillering was obtained by map-based cloning. It was found that the gene encodes GA20ox2, which is the orthologous gene of the rice SD1 gene in millet and was named SiSD1 gene.

[0007] After the gene mutates, the active gibberellin content decreases, resulting in an increase in the number of millet tillers. Overexpression of SiSD1 leads to a decrease in tillers. The cloning of this gene will help promote the "green revolution" in millet breeding and provide important genetic resources and theoretical basis for millet plant type improvement and yield increase.

[0008] The inventors of the present invention used the mutagen ethyl methanesulfonate (EMS) to induce mutagenesis and genetic screening on Jingu No. 10, and isolated and identified a mutant dmt1 (dwarf and more tiller1) with increased millet tiller number. Then, the key gene DMT1 (hereinafter referred to as SiSD1 gene) controlling the millet tiller number was cloned by the map-based cloning method. The function of the gene was proved by phenotypic analysis of the mutant and genetic complementation experiments.

[0009] Therefore, the purpose of the present invention is to provide a key factor for improving the tillering number 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 for controlling the tillering number of millet, wherein the gene SiSD1 encodes a SiSD1 protein, and the amino acid sequence thereof is shown in any one of the following:

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

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

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

[0014] 4) An active fragment comprising the amino acid sequence of any one of 1) to 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 one of the following:

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

[0018] b) a nucleotide sequence having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the 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;

[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) a nucleotide sequence that differs in sequence from SEQ ID NO. 1 or SEQ ID NO. 2 due to the degeneracy of the genetic code;

[0021] e) an active fragment comprising the nucleotide sequence described in any one of a) to d);

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

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

[0024] The gene SiSD1 controlling the tillering number and effective ear number of millet is an isolated nucleotide sequence.

[0025] In a second aspect, the present invention provides a gene Sisd1 for controlling the tillering number of millet, wherein the gene Sisd1 encodes a Sisd1 protein, and the amino acid sequence thereof is shown in any one 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 amino acid residues substituted, deleted and / or inserted 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 that has at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO. 5 and has the same function as the amino acid sequence shown in SEQ ID NO. 3; or

[0029] 4) An active fragment comprising the amino acid sequence of any one of 1) to 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 having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 4, and having 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 in sequence from SEQ ID NO. 4 due to the degeneracy of the genetic code;

[0035] e) an active fragment comprising the nucleotide sequence described in any one of a) to d);

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

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

[0038] In a preferred embodiment, the gene SiSD1 that controls the number of millet tillers and effective ears 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 effective ears is a nucleotide sequence that has more than 90% homology, 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 for controlling the number of millet tillers and effective ears described in the first aspect is an isolated protein, and its amino acid sequence is shown in SEQ ID NO. 3.

[0040] In a preferred embodiment, the gene Sisd1 that controls the number of millet tillers and 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 effective ears is a nucleotide sequence that has more than 90% homology, 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 for controlling the number of tillers and effective panicles of millet as described in the first aspect. The vector comprises a plant expression vector and / or a plant gene editing vector.

[0042] The present invention also provides a recombinant vector comprising the gene Sisd1 for controlling the number of tillers and effective ears of millet as described in the second aspect. The vector comprises a plant expression vector and / or a plant gene editing vector.

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

[0044] Preferably, the exogenous nucleotide fragment contained in the recombinant vector encodes the amino acid sequence shown in SEQ ID NO. 3. More preferably, the exogenous nucleotide fragment contained in the recombinant vector is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0045] Preferably, the exogenous nucleotide fragment contained in the recombinant vector encodes the amino acid sequence shown in SEQ ID NO. 5. More preferably, the exogenous nucleotide fragment contained in the recombinant vector is shown in SEQ ID NO. 4.

[0046] In one embodiment, the plasmid used to construct the recombinant vector can be selected from, but 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, the recombinant vector can be transformed or transfected into cells to obtain a host cell containing the recombinant vector, which can be further used for amplifying the expression vector, expressing the protein, or obtaining transgenic plants.

[0050] The host cell may be selected from, but not limited to, bacterial cells (such as Escherichia coli cells or Agrobacterium cells), fungal cells (such as yeast cells) or plant cells (such as millet cells) and the like.

[0051] In a fourth aspect, the present invention provides use of the gene SiSD1 or the SiSD1 protein encoded thereby in cultivating or obtaining plants with increased effective panicle number, increased effective tiller number and improved yield, wherein the plant is a Poaceae plant, preferably millet.

[0052] The present invention also provides the use of the gene Sisd1 or the Sisd1 protein encoded by it in cultivating or obtaining plants with increased effective panicle number, increased effective tiller number and improved yield, wherein the plant is a grass plant, preferably millet.

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

[0054] Preferably, the method further comprises knocking out or knocking down the expression of SiSD1 gene in the plant or causing the loss of SiSD1 protein function.

[0055] In one embodiment, the method can be achieved by the following methods: editing the nucleic acid sequence of SiSD1 in a wild-type plant or plant cell by gene editing technology, introducing or deleting one or more bases, or causing nucleotide substitutions that lead to missense mutations, resulting in changes in the amino acid sequence encoded by the gene; or using gene silencing technology to achieve reduction or knockout of the expression of the SiSD1 gene in the plant.

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

[0057] In one embodiment, the method can be achieved by constructing a Sisd1 gene overexpression vector containing an enhanced promoter (preferably Actin promoter), and transforming or transfecting the vector into foxtail 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, Sisd1 gene overexpression vector, and Sisd1 gene editing vector can be performed by Agrobacterium-mediated method or gene gun method; preferably, the transformation or transfection is performed by Agrobacterium-mediated method or gene gun method.

[0059] In one embodiment, the method can be achieved by the following method: establishing a near-isogenic line to obtain the plant; preferably, the method for establishing the near-isogenic line is: hybridizing a target parent carrying the Sisd1 gene or its allele with another line parent, and then backcrossing the hybrid offspring with another line parent for multiple times, and in the backcrossing process, continuously selecting individuals with the target parent trait, and continuing backcrossing until the target trait does not separate when the offspring is self-pollinated, at this time, a near-isogenic line with a genetic background similar to that of another line parent 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 ears, an increase in the number of effective tillers, and / or an increase in yield. The present invention provides theoretical guidance and material basis for cultivating high-yield crop varieties.

[0060] When the Sisd1 gene of the present invention is used to increase millet yield, the following method can be used: (1) constructing a Sisd1 gene overexpression vector containing an enhanced promoter (preferably 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 levels.

[0061] The increase in the number of effective ears, the increase in the number of effective tillers and / or the increase in yield described in the present invention is compared with wild-type plants, preferably compared with foxtail millet, preferably compared with wild-type foxtail millet, more preferably compared with Jingu 21 (JG21), more preferably compared with Changsheng 19 (CS19), more preferably compared with foxtail millet ci846, and more preferably compared with Jingu No. 10.

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

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

[0064] The present invention clones the key genes SiSD1 and / or Sisd1 for controlling the number of tillers and the number of effective ears, which can be used for millet genetic engineering and molecular breeding to improve millet traits, increase the number of effective ears and the number of effective tillers, and improve millet yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The phenotypic analysis of CS19 and dmt1 and their hybrid offspring F2 and the positional cloning of SiSD1 gene are shown; A is the phenotype of mature plants of CS19 and dmt1 and their hybrid offspring BC1F2, with a scale bar of 20 cm; B is the phenotype of ear of CS19 and dmt1 and their hybrid offspring BC1F2, with a scale bar of 10 cm; CD are the statistical analysis of plant height and tiller number of CS19 and dmt1 and their hybrid offspring BC1F2; the statistical number n = 20, analyzed by Duncan's test (p < 0.05); E shows the positional cloning of target gene SiSD1; in the figure, horizontal lines represent chromosomes and vertical lines represent molecular markers; in the SiSD1 gene structure, cyan squares, black squares and black lines represent non-coding regions, exons and introns, respectively; F is the comparative analysis of protein sequences of CS19 and dmt1.

[0066] Figure 2 The gene evolution tree of SiSD1, the subcellular localization and tissue expression pattern of the encoded protein are shown; A is the gene evolution tree analysis of SiSD1; B is the subcellular localization of SiSD1-GFP fusion protein; 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 ( Figure 3 B).

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

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

[0070] Figure 6 The phenotypic analysis and gene expression analysis of SiSD1 overexpression plants are shown; A is the phenotype of mature plants of ci846 and SiSD1 overexpression line SiSD1-OE, with a scale of 20 cm; B is the phenotype of internodes of ci846 and SiSD1-OE, with a scale of 5 cm; C is the phenotype of ear of ci846 and SiSD1-OE, with a scale of 5 cm; DF is the statistical analysis of the number of effective tillers per plant, plant height and internode length of ci846 and SiSD1-OE; the statistical number of the number of effective tillers per plant and plant height is n = 10, and the statistical number of internode length is n = 14; the significance analysis is analyzed using Duncan's test (p < 0.05). G is the expression analysis of the gene SiSD1 in ci846 and SiSD1-OE; H is the content analysis of the protein SiSD1 in ci846 and SiSD1-OE; I is the expression analysis of key genes regulating tillering.

[0071] Figure 7 The results show that compared with JG21, the near-isogenic line Sisd1 can significantly increase yield under different planting densities; A is the phenotype of JG21 and the near-isogenic line Sisd1 under three different planting densities in the field; B is the statistical analysis of the effective panicle number of JG21 and Sisd1 under three different planting densities; C is the statistical analysis of the total yield of JG21 and Sisd1 under three different planting densities; significance analysis was performed using Duncan's test (p < 0.05). DETAILED DESCRIPTION

[0072] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

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

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

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

[0076] The inventors used Jingu No. 10 as the background, used the mutagen ethyl methanesulfonate (EMS) for mutagenesis and genetic screening, and isolated and identified a millet mutant material dmt1 with multiple tillers. They hybridized it with Changsheng 19 (CS19), self-pollinated F1 to obtain F2, selected individual plants with phenotypes similar to dmt1 in F2 and backcrossed with CS19, and self-pollinated BC1F1 to obtain the BC1F2 genetic population. The plant height and tiller number of BC1F1 were between those of the parents. In the BC1F2 population, the ratio of the number of individual plants with CS19-like phenotypes, BC1F1-like phenotypes, and dmt1 phenotypes was 1:2:1, indicating that the phenotype of dmt1 was caused by a semi-dominant gene (preliminarily named DMT1 gene). The plant height of the dmt1 mutant phenotype plants in dmt1 and BC1F2 at maturity was significantly lower than that of CS19, while the number of effective tillers and the number of effective ears increased significantly, showing a certain yield increase potential ( Figure 1 AD). Based on this, it was decided to clone the superior allele regulating this excellent trait. The candidate gene was identified using the BC1F2 genetic population constructed using Changsheng 19 and the dmt1 mutant. The coarse positioning results showed that the gene was located between the molecular markers P950 and P1500 on the long arm of chromosome 5. Further fine positioning located it between the molecular markers PL20 and PR20. The candidate interval was about 24 kb and contained 3 genes. DNA sequencing found that a base G was missing at 1015 bp in the coding region of the gene Si5g40430, causing a frameshift mutation and producing a truncated protein ( Figure 1 E and F). At the same time, we detected the expression of the three genes in the candidate interval and found that the expression of the other two genes did not change. The gene Si5g40430 was analyzed with two pairs of primers (primer sequences are shown in Table 1), and both showed a slight upregulation in expression, further confirming Si5g40430 as a candidate gene.

[0077] Gene phylogenetic tree analysis revealed that gene Si5g40430 encodes GA20ox2, which is the homologous gene of rice SD1 in millet, so we named it SiSD1 ( Figure 2 A), whose nucleotide sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the 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). Analysis of tissue expression patterns 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 (compared with the SiSD1 gene, a single base G was deleted at 1015 bp in the coding region) was named Sisd1 gene, and its nucleotide sequence was shown in SEQ ID NO. 4. The amino acid sequence of the protein encoded by the Sisd1 gene (Sisd1 protein) was 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 gene of SiSD1 gene from the wild-type Jingu 21 (JG21) genome, which is the nucleotide sequence shown in SEQ ID NO.1 (including the promoter sequence, genomic sequence and 3' non-coding region sequence), was amplified by PCR using DNA polymerase, and then ligated to the multiple cloning site of pCAMBIA1300 plasmid (purchased from CAMBIA) by restriction digestion and ligation 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 bacteria were shaken and the plasmids were extracted. The positive clones with completely correct SiSD1 gene sequences were obtained by sequencing, and then the plasmids of the positive clones were electroporated and transformed into EHA105 Agrobacterium competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.).

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

[0084] Example 3. Acquisition and phenotypic analysis of near-isogenic line Sisd1

[0085] In order to study the function of gene SiSD1, we obtained the near-isogenic line plant Sisd1 through multiple backcrossing methods. The method for obtaining the near-isogenic line plant Sisd1 is as follows: the parent dmt1 carrying the target trait is hybridized with the famous millet variety JG21 in Shanxi Province (as the recipient parent), and the offspring are then backcrossed with the parent JG21 for multiple times. The backcrossing is continued until the target trait does not separate when the offspring are self-pollinated. The line Sisd1 (which carries the Sisd1 gene) with a genetic background similar to JG21 but carrying the target trait is obtained. This line and the parent JG21 (which carries the SiSD1 gene) constitute a pair of near-isogenic lines.

[0086] We analyzed the plant height, number of effective tillers per plant and internode phenotypes of JG21 and its near isogenic line Sisd1 at maturity. The plant heights of JG21 and its near isogenic line Sisd1 were 182 and 148 cm, respectively, and the lengths of the main spikes were 18 and 21 cm, respectively. The results showed that there were significant differences in plant height, number of effective tillers per plant and internode phenotypes between the near isogenic lines Sisd1 and JG21. 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 internode number of the near-isogenic line Sisd1 did not change compared with JG21, but the internode length was shortened, which indicated that the dwarfing of line Sisd1 came from the shortening of internodes, but did not affect the number of nodes ( Figure 5 CF); At the same time, we detected the expression of tillering regulatory genes SiTB1, SiTAD1, SiDLT1, SiMOC1, SiMOC3, SiSLR1 and SiSD1 in the near-isogenic lines Sisd1 and JG21 (the reverse transcription primer sequences are shown in Tables 1 and 2). We found that the tillering-inhibiting genes SiTB1, SiTAD1, and SiDLT1 were all down-regulated in Sisd1, while the tillering-promoting genes SiMOC1 and SiMOC3 were up-regulated. The expression of SiSLR1, a key gene in the GA signaling pathway, was down-regulated, and the expression of SiSD1 itself was slightly up-regulated ( Figure 5 G); This indicates that gene SiSD1 and gene Sisd1 are key genes controlling the number of millet tillers or the number of effective tillers per plant.

[0087] Table 2

[0088]

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

[0090] In order to test whether the gene SiSD1 can complement the phenotype of Sisd1, the phenotypes of the positive plant pSiSD1::SiSD1 obtained by the transgenic experiment in Example 2, the wild-type plant JG21, and the near-isogenic line plant Sisd1 described in Example 3 were analyzed. The plant height and tiller number of JG21, Sisd1, and pSiSD1::SiSD1 were counted, and the results showed that compared with the plant Sisd1, the effective tiller number of the plant pSiSD1::SiSD1 was significantly reduced, which could restore the phenotype of Sisd1 ( Figure 4 AC). We detected the expression of the SiSD1 gene in plants JG21, Sisd1, and pSiSD1::SiSD1 and found that the expression level of the gene was significantly increased in the plant pSiSD1::SiSD1 and slightly increased in the plant Sisd1 ( Figure 4 D); This indicates that the gene SiSD1 can restore the phenotype of effective tiller number of Sisd1, indicating that the gene SiSD1 is a key gene controlling the tiller number or effective tiller number of millet.

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

[0092] We ligated the Actin promoter sequence (SEQ ID NO. 6), SiSD1 gene CDS coding sequence (SEQ ID NO. 2), and GFP tag sequence (SEQ ID NO. 7) into the pCAMBIA1300 plasmid by restriction 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. The plasmid was extracted and sequenced to obtain positive clones with completely correct Actin promoter sequence, SiSD1 gene CDS coding sequence and GFP tag sequence, and then the plasmid of the positive clone was electroporated into EHA105 Agrobacterium competent cells (refer to "Plant Genetic Engineering", Wang Guanlin, Fang Hongjun, Science Press, 2004, 2nd edition, prepared by conventional methods). The successfully transformed clones were genetically transformed using the Agrobacterium infection method with wild-type millet ci846 callus as the recipient. The transgenic operation method is as described in Example 2. Finally, the SiSD1 overexpression strain SiSD1-OE was obtained.

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

[0094] In order to determine whether increasing the expression of SiSD1 can increase the plant height of millet, the phenotypes of plant height, internodes and ears of SiSD1-OE overexpression line at maturity were analyzed. The plant heights of SiSD1-OE under the ci846 and ci846 backgrounds were 105 and 182 cm, respectively, and the lengths of the main ears were 16 and 14 cm, respectively. The results showed that the overexpression line SiSD1-OE had significant differences in plant height, internodes and ear phenotypes. 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 length of internodes was elongated ( Figure 6 AF); At the same time, we detected the changes in SiSD1 gene expression and protein levels in the overexpression line SiSD1-OE. The results showed that SiSD1 gene expression was significantly upregulated and protein content was significantly increased ( Figure 6 GH); We also detected the expression of tillering regulatory genes SiTB1, SiTAD1, SiDLT1, SiMOC1, SiMOC3, SiSLR1 and SiSD1, and found that the negative regulatory genes SiTB1 and SiDLT1 that inhibit tillering were upregulated, SiMOC3 was downregulated, SiSLR1 was upregulated, and SiSD1 was significantly upregulated ( Figure 6 I); This indicates that increasing the expression of the SiSD1 gene can significantly reduce the number of effective tillers per plant. It can be understood that reducing the expression of the SiSD1 gene can significantly increase the number of effective tillers per plant.

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

[0096] JG21 and the plant Sisd1 obtained in Example 3 were sown at the Dongyang base of Shanxi Agricultural University, and the field was divided into 18 equal plots of 6 m 2 The soil fertility, light, water and other environmental conditions of each plot were basically uniform. The test was repeated three times based on the random area design, with three densities and two materials for a total of 18 plots. When the row spacing was consistent, under the condition of 10 cm plant spacing, the number of effective ears per unit area of ​​Sisd1 increased compared with JG21, and the yield increased by 15%; under the condition of 15 cm plant spacing, the number of effective ears per unit area of ​​Sisd1 increased compared with JG21, and the yield increased by 16%; under the condition of 20 cm plant spacing, the number of effective ears per unit area of ​​Sisd1 increased compared with JG21, and the yield increased by 28% ( Figure 7 ). Therefore, compared with the wild type, the number of effective ears per unit area of ​​Sisd1 increased under different planting density conditions, and the yield was improved.

[0097] It should be understood that although the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it should be understood by those skilled in the art that various changes in form and details may be made therein and any combination of various embodiments may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

[0098] References:

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[0100] 2. Li Shunguo, Liu Fei, Liu Meng, et al. Historical evolution and future development direction of millet cultivation in China[J]. Journal of Cereals, Oils and Food Science and Technology, 2022, 30(04): 60-67+10. DOI: 10.16210 / j.cnki.1007-7561.2022.04.009.

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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.

[0106] 8. Sweetness, snow, snow, snow. International Journal of Chemical Engineering and Chemical Engineering [J].English Chemistry, 1992,(01): 1-5.

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[0108] 10. Van De Velde K, Thomas SG, Heyse F, Kaspar R, Van Der Straeten D,Rohde A. N-terminal truncated RHT-1 proteins generated by translationalreinitiation cause semi-dwarfing of wheat Green Revolution alleles. MolPlant. 2021 Apr 5;14(4):679-687.

[0109] 11. Chai L, Xin M, Dong C, Chen Z, Zhai H, Zhuang J, Cheng X, Wang N,Geng J, Wang X, Bian R, Yao Y, Guo W, Hu Z, Peng H, Bai G, Sun Q, Su Z, LiuJ, Ni Z. A natural variation in Ribonuclease H-like gene underlies Rht8 toconfer "Green Revolution" trait in wheat. Mol Plant. 2022 Mar 7;15(3):377-380.

[0110] 12. Xiong H, Zhou C, Fu M, Guo H, Xie Y, Zhao L, Gu J, Zhao S, DingY, Li Y, Zhang J, Wang K, Li X, Liu L. Cloning and functionalcharacterization of Rht8, a "Green Revolution" replacement gene in wheat. MolPlant. 2022 Mar 7;15(3):373-376.

[0111] 13. Zhao M, Zhi H, Zhang X, Jia G, Diao X. Retrotransposon-mediatedDELLA transcriptional reprograming underlies semi-dominant dwarfism infoxtail millet[J]. The Crop Journal, 2019, 7(4): 458-468.

[0112] 14. Xue C, Zhi H, Fang X, Liu X, Tang S, Chai Y, Zhao B, Jia G, DiaoX. Characterization and fine mapping of SiDWARF2 (D2) in foxtail millet[J].Crop Science, 2016, 56(1): 95-103..

[0113] 15. He Q,ZHI H,TANG S. QTL mapping for foxtail millet plant height inmulti-environment using an ultra-high density Bin map [J]. Theoretical andApplied Genetics,2021,134(2):557-572.

[0114] 16. Doebley J, Stec A, Hubbard L. The evolution of apical dominancein maize[J]. Nature, 1997, 386(6624): 485-488.

[0115] 17. Takeda T, Suwa Y, Suzuki M, Kitano H, Ueguchi-Tanaka M, AshikariM, Matsuoka M, Ueguchi C. The OsTB1 gene negatively regulates lateralbranching in rice. Plant J. 2003 Feb;33(3):513-20.

[0116] 18. Luan Y, Wang B, Zhao Q, Ao G, Yu J. Ectopic expression of foxtailmillet zip-like gene, SiPf40, in transgenic rice plants causes a pleiotropicphenotype affecting tillering, vascular distribution and root development.Sci China Life Sci. 2010 Dec;53(12):1450-8.

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[0122] Sequence Listing:

[0123] SEQ ID NO. 1: The genomic nucleotide sequence of foxtail millet SiSD1 gene, which also includes the 1.5kb promoter (underlined), 5'UTR (bold) and 3'UTR (underlined and bold) sequences

[0124]

[0125]

[0126]

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

[0128]

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

[0130] MVSQAQQEPALPHSSSTAKRAAASLMDARPAQPLLLRAPTPSIDLPASKPDRAAAAAGKAAAASVFDLRREPKIPAPFVWPHDDARPASAAELDVPLVDVGVLRNGDRAGLRRAAAQVAAACATHGFFQVCGHGVGADLARAALDGASDFFRLPLAEKQRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDGAASPVVVDYFAGTLG QDFEAVGRVYQRYCEEMKALSLTIMELLELSLGVERGYYRDFFEDSRSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGDWRPVRPVPGAMVINIGDTFMALSNGRYKSCLHRAVVNQRQERRSLAFFLCPREDRVVRPPASGAVGEAPRRYPDFTWADLMRFTQRHYRADTRTLDAFTRWLSHGPAQDAPVAAAAST

[0131] SEQ ID NO. 4: CDS nucleotide sequence of foxtail 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 foxtail millet Sisd1 gene

[0134] MVSQAQQEPALPHSSSTAKRAAASLMDARPAQPLLLRAPTPSIDLPASKPDRAAAAAGKAAAASVFDLRREPKIPAPFVWPHDDARPASAAELDVPLVDVGVLRNGDRAGLRRAAAQVAAACATHGFFQVCGHGVGADLARAALDGASDFFRLPLAEKQRARRVPGTVSG YTSAHADRFASKLPWKETLSFGFHDGAASPVVVDYFAGTLGQDFEAVGRVYQRYCEEMKALSLTIMELLELSLGVERGYYRDFFEDSRSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGDWRPVRPVPGAMVINIGDTFMALSNGRYKSCLHRRW

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

[0136]

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

[0138]

[0139] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a SiSD1 gene or a SiSD1 protein encoded thereby in obtaining plants with increased effective panicle number, increased effective tiller number and / or improved yield.

2. Use of the Sisd1 gene or the Sisd1 protein encoded by it in obtaining plants with increased effective panicle number, increased effective tiller number and / or improved yield.

3. The use according to claim 1, characterized in that The amino acid sequence of the SiSD1 protein is shown in any one of the following: 1) the amino acid sequence shown in SEQ ID NO. 3; 2) An amino acid sequence having one or more amino acid residue substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 3 and having the same function as the amino acid sequence shown in SEQ ID NO. 3; 3) an amino acid sequence that has at least 90%, preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO. 3, and has the same function as the amino acid sequence shown in SEQ ID NO. 3; or 4) An active fragment comprising the amino acid sequence of any one of 1) to 3); The nucleotide sequence of the SiSD1 gene is shown in any one of the following: a) the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; b) a nucleotide sequence having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the 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; 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; d) a nucleotide sequence that differs in sequence from SEQ ID NO. 1 or SEQ ID NO. 2 due to the degeneracy of the genetic code; e) an active fragment comprising the nucleotide sequence described in any one of a) to d); f) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence described in any one of a) to e) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or g) a nucleotide sequence complementary to the nucleotide sequence described in any one of a) to e).

4. The use according to claim 2, characterized in that The amino acid sequence of the Sisd1 protein is shown in any of the following: 1) the amino acid sequence shown in SEQ ID NO. 5; 2) An amino acid sequence having one or more amino acid residue substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 5 and having the same function as the amino acid sequence shown in SEQ ID NO. 5; 3) an amino acid sequence that has at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO. 5 and has the same function as the amino acid sequence shown in SEQ ID NO. 3; or 4) An active fragment comprising the amino acid sequence of any one of 1) to 3); The nucleotide sequence of the Sisd1 gene is shown in any of the following: a) the nucleotide sequence shown in SEQ ID NO. 4; b) a nucleotide sequence having one or more nucleotide sequence substitutions, deletions and / or insertions compared to the sequence shown in SEQ ID NO. 4, and having the same function as the nucleotide sequence shown in SEQ ID NO. 4; 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; d) a nucleotide sequence that differs in sequence from SEQ ID NO. 4 due to the degeneracy of the genetic code; e) an active fragment comprising the nucleotide sequence described in any one of a) to d); f) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence described in any one of a) to e) under moderately stringent hybridization conditions, preferably under highly stringent hybridization conditions; or g) a nucleotide sequence complementary to the nucleotide sequence described in any one of a) to e).

5. The use according to any one of claims 1 to 4, wherein the plant is a grass plant, preferably millet.

6. A method for obtaining a plant having an increased number of effective ears, an increased number of effective tillers and / or an improved yield, the method comprising: In the plant, the expression of Sisd1 gene or its allele is increased or the amount of Sisd1 protein is increased, or the expression of SiSD1 gene or its allele is reduced or the amount of SiSD1 protein is reduced, and the Sisd1 gene, Sisd1 protein, SiSD1 gene or SiSD1 protein is the gene or protein defined in claim 3 or 4.

7. The method of claim 6, wherein the method further comprises: Transforming or transfecting a recombinant vector comprising the Sisd1 gene or its allele as defined in claim 4 or a host cell comprising the recombinant vector into a plant cell to obtain a transgenic plant; or knocking out or knocking down the expression of the SiSD1 gene in the plant or causing the SiSD1 protein function to be lost.

8. The method of claim 6, wherein the method further comprises: Establish a near-isogenic line to obtain the plant; preferably, the method for establishing the near-isogenic line is: hybridize a target parent carrying the Sisd1 gene or its allele with another parent, and then backcross the hybrid offspring with the other parent for multiple times. During the backcrossing process, continuously select individuals with the target parent's traits, and continue backcrossing until the target trait does not separate when the offspring is self-pollinated. At this time, a near-isogenic line with a genetic background similar to that of the other parent is obtained, and the line stably carries the trait controlled by the Sisd1 gene or its allele in the target parent line; 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 ears, an increase in the number of effective tillers and / or an increase in yield.

9. The method of claim 7, wherein the transformation or transfection is performed by Agrobacterium-mediated method or gene gun method.

10. The method of any one of claims 6-8, wherein the plant is a grass plant, preferably millet.

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