Gene ltd1 for controlling rice tillering and application thereof

Abstract

The application discloses a gene LTD1 for controlling rice tillering and application, and belongs to the technical field of genetic engineering breeding. The indica rice restoration line 676R is subjected to chemical mutagenesis, and a few tiller mutant ltd1 is obtained. Through map-based cloning, high-throughput sequencing and MutMap analysis, a candidate gene LTD1 is obtained. It is verified that the LTD1 gene has an important influence on the growth and development and yield of rice. In the analysis of the variation of the LTD1 in a rice germplasm resource group, it is found that the haplotype Hap7 (indica rice specific) of the LTD1 gene has high tillering capacity and low plant height characteristics, and has great application potential in rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering breeding technology, specifically involving the application of the LTD1 gene and / or its encoded protein in regulating the number of rice tillers. Background Technology

[0002] Increasing rice yield per unit area is a primary measure to address challenges such as global population growth, decreasing arable land, and frequent extreme weather events. The three key factors affecting rice yield are the number of effective panicles, the number of grains per panicle, and grain weight. The number of effective tillers directly determines the number of effective panicles per plant, and an appropriate number of tillers is a crucial foundation for increasing rice yield. Too few tillers reduce the number of effective panicles, thus affecting yield per plant; while too many tillers affect ventilation and sunlight exposure within the rice plant population, making it more susceptible to disease and ultimately reducing yield. Therefore, in-depth research into the mechanism of rice tillering and the rational and effective control of tiller number are of great significance for ensuring high rice yields.

[0003] Based on the tillering process, genes involved in regulating the number of rice tillers can be divided into two categories: genes that regulate tiller bud formation and genes that regulate the outward growth of tiller buds. Over the past 20 years, numerous studies have been conducted on the discovery of genes involved in rice tillering and development, and their molecular mechanisms. Examples include MOC1, the first key gene controlling tillering to be cloned in rice, and other important genes such as MOC3 and FON1. However, discovering and cloning new tillering mutants or genes is of great significance for further elucidating the genetic regulatory network of rice tillering. Summary of the Invention

[0004] In this invention, the inventors chemically mutagenesis of the indica rice restorer line 676R obtained the low-tillering mutant ltd1, which exhibits reduced tillering and shorter plant height. Observation of tiller formation and growth revealed that ltd1 can form tillers, but tiller elongation is slow. Therefore, its low-tillering phenotype is mainly due to the inhibition of tiller elongation. Genetic analysis revealed that this mutant phenotype is controlled by a pair of recessive nuclear genes. Through map-based cloning combined with high-throughput sequencing and MutMap analysis, the candidate gene LTD1 (LOC_Os01g19760) was obtained.

[0005] Based on the above findings, one objective of this invention is to provide the application of the LTD1 gene and / or its encoded protein, an expression vector containing the LTD1 gene, and engineered bacteria containing the expression vector in regulating the number of rice tillers; a second objective of this invention is to provide a method for regulating the number of rice tillers; and another objective of this invention is to provide the application of the LTD1 gene haplotype Hap7 in screening rice varieties with a high number of tillers and short plant height.

[0006] This invention includes the following technical solutions:

[0007] In a first aspect, the present invention provides the use of the LTD1 gene and / or its encoded protein, or a substance for detecting the LTD1 gene and / or its encoded protein, in any of the following:

[0008] a1) Its application in regulating the number of rice tillers;

[0009] a2) Application in screening or assisting in screening rice plants, lines, or varieties with a large number of tillers;

[0010] a3) Application in rice hybrid breeding;

[0011] a4) Application in rice genetic engineering breeding.

[0012] The DNA sequence of the LTD1 gene is shown in SEQ ID NO:1, the cDNA sequence of the LTD1 gene is shown in SEQ ID NO:2, and the amino acid sequence of the protein encoded by the LTD1 gene is shown in SEQ ID NO:3.

[0013] The substance used to detect the LTD1 gene is any one of the following:

[0014] b1) A combination of the PCR primers shown in SEQ ID NO:4 and SEQ ID NO:5;

[0015] b2) PCR reagents containing the PCR primer combination described in b1);

[0016] b3) A kit containing the PCR primer composition described in b1) and / or the PCR reagents described in b2).

[0017] The substances used to detect the LTD1 gene-encoded protein include reagents for detecting the LTD1 gene-encoded protein by immunohistochemistry, or reagents that can detect the expression level of nucleic acids related to the LTD1 gene-encoded protein or the content of the LTD1 gene-encoded protein. The reagents include, but are not limited to, antibodies, peptides, proteins or nucleic acid molecules that bind to the LTD1 gene-encoded protein.

[0018] In a second aspect, the present invention provides an application of an expression vector in any of the following:

[0019] c1) Application in regulating the number of rice tillers;

[0020] c2) Application in screening or assisting in screening rice plants, lines, or varieties with a large number of tillers;

[0021] c3) Application in rice hybrid breeding;

[0022] c4) Application in rice genetic engineering breeding;

[0023] The expression vector contains the LTD1 gene described in the first aspect of this invention.

[0024] In a third aspect, the present invention provides the use of an engineered bacterium in any of the following:

[0025] d1) Application in regulating the number of rice tillers;

[0026] d2) Application in screening or assisting in screening rice plants, lines, or varieties with a high number of tillers;

[0027] d3) Application in rice hybrid breeding;

[0028] d4) Application in rice genetic engineering breeding;

[0029] The engineered bacteria contain the expression vector described in the second aspect of this invention.

[0030] Specifically, the engineered bacteria are prepared by transforming the expression vector described in Part II of this invention into a host bacterium, which includes, but is not limited to, Escherichia coli or Agrobacterium.

[0031] The regulation of rice tiller number described in this invention includes positively regulating the LTD1 gene and / or its encoded protein to promote the emergence and growth of rice tiller shoots.

[0032] In a fourth aspect, the present invention provides a method for regulating the number of tillers in rice, the method comprising transforming rice cells using any of the following methods, and cultivating the transformed rice cells into plants:

[0033] e1) The LTD1 gene as described in the first aspect of this invention;

[0034] e2) An expression vector containing the LTD1 gene described in e1);

[0035] e3) Engineered bacteria containing the expression vector described in e2).

[0036] In a fifth aspect, the present invention provides a method for increasing the number of tillers in rice, the method comprising the following steps:

[0037] f1) Culture of rice embryogenic callus;

[0038] f2) Using Agrobacterium-mediated transformation, engineered bacteria containing an LTD1 gene overexpression vector were used to infect the rice embryogenic callus obtained in step f1). Transgenic embryogenic callus was obtained through resistance screening. This was followed by screening, differentiation, rooting, and hardening-off processes to obtain transgenic embryogenic callus.

[0039] f3) Inducing the differentiation of the transgenic embryogenic callus, rooting, hardening off, and obtaining rice plants with high tiller numbers.

[0040] In a sixth aspect, the present invention provides a rice plant with a high tillering number, which is prepared by the method described in the fifth aspect of the present invention.

[0041] In a seventh aspect, the present invention provides the use of LTD1 gene haplotype Hap7 or a substance for detecting LTD1 gene haplotype Hap7 in any of the following:

[0042] g1) Application in identifying or assisting in the identification of rice tillering and plant height;

[0043] g2) Application in screening or assisting in screening rice individual plants, lines or varieties with high tiller number and low plant height;

[0044] Application of g3 in rice hybrid breeding;

[0045] Application of g4 in rice genetic engineering breeding.

[0046] The LTD1 gene haplotype Hap7 has the following bases: 394th base is G, 675th base is A, 994th base is A, 1290th base is C, 1451st base is A, 1791st base is G, 2164th base is G, 2394th base is C, 2428th base is T, 2621st base is G, and 2773rd base is G.

[0047] The substance used to detect the LTD1 gene haplotype Hap7 is any one of the following:

[0048] b1) A combination of the PCR primers shown in SEQ ID NO:4 and SEQ ID NO:5;

[0049] b2) PCR reagents containing the PCR primer combination described in b1);

[0050] b3) A kit containing the PCR primer composition described in b1) and / or the PCR reagents described in b2).

[0051] The technical solution provided by this invention has the following technical contributions:

[0052] First, the inventors obtained the mutant ltd1 through chemical mutagenesis. In addition to the obvious phenotype of fewer tillers, the mutant also showed a phenotype of shorter plant height. Through map-based cloning combined with high-throughput sequencing and MutMap analysis, the candidate gene LTD1 (LOC_Os01g19760) was obtained. Furthermore, LTD1 overexpressing plants and RNAi transgenic plants were constructed using an LTD1 overexpression vector. The results showed that the LTD1 gene has an important influence on the number of tillers in rice.

[0053] Furthermore, the inventors' analysis of the variation of LTD1 in rice germplasm resource populations showed that the distribution frequency of the eight haplotypes of LTD1 differed significantly between indica and japonica rice. In particular, haplotype Hap7 (unique to indica rice) not only has high tillering ability but also low seedling height, and can be used for rice breeding. Attached Figure Description

[0054] Figure 1 Phenotypic and agronomic traits of rice ltd1 mutant and wild-type parent plants: (A) Seedling plants of wild-type 676R (WT) and ltd1; (B) Mature plants of WT and ltd1, scale bar = 10 cm; (CG) Tiller number (C), plant height (D), number of grains per panicle (E), thousand-grain weight (F), and seed setting rate (G) of WT and ltd1 at maturity. The values ​​in the figure are the mean and standard error of three biological replicates. One-way ANOVA was used. * indicates P < 0.05; ** indicates P < 0.01; (H) Tillering buds of WT and ltd1 at the four-leaf stage; (I) Enlarged view of the boxed area at the base of the ltd1 stem in Figure H; yellow arrows indicate tillering buds.

[0055] Figure 2 Linkage analysis and MutMap analysis of the rice ltd1 mutant gene: (A) The ltd1 mutant gene was initially located between molecular markers RM259 and RM24 on chromosome 1; (B) The ltd1 mutant gene was located between molecular marker RM23 and InDel marker S2, with a distance of 1026 kb; (C) SNP index scatter plot of the ltd1 mutant gene on chromosome 1.

[0056] Figure 3 LTD1 gene functional complementation analysis: (A) Positive detection of T0 generation of LTD1 functionally complementary transgenic plants, M represents DNA marker, P represents positive control (transgenic expression vector plasmid pCAMBIA2300-LTD1), N represents negative control (ltd1 mutant plant); (BC) Phenotypes of WT, ltd1 and functionally complementary plants at tillering and maturity stages, scale bar, 10 cm; (DF) Comparison of tiller number (D), plant height (E) and grain number per ear (F) at maturity of WT, ltd1 and functionally complementary plants. The values ​​in (DF) are the mean and standard error of three biological replicates. One-way ANOVA was used. ** indicates P < 0.01.

[0057] Figure 4LTD1 gene RNAi interference analysis: (A) Maturity phenotype of japonica rice variety Nip and LTD1-RNAi interference lines; (B) Expression level of LTD1-RNAi interference lines under the Nip background, with LOC_Os07g02340 as an internal reference gene; (CE) Tiller number (C), plant height (D), and number of grains per panicle (E) at maturity of Nip and LTD1-RNAi interference lines. The values ​​in (CE) are the mean and standard error of three biological replicates. One-way ANOVA was used, and ** indicates P < 0.01.

[0058] Figure 5 Violin plots comparing tiller number and seedling height phenotypes of different haplotypes in the indica and japonica subgroups: (A) Violin plot comparing tiller number of haplotype 1-8 (Hap1-8) in the indica and japonica subgroups; (B) Violin plot comparing seedling height phenotype of haplotype 1-8 (Hap1-8) in the indica and japonica subgroups. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] Cloning and validation of rice tillering gene LTD1

[0061] Example 1: Discovery of Genes

[0062] Phenotypic characteristics of mutant ltd1

[0063] The present invention describes a method for chemically mutagenesis of the rice restorer line 676R to obtain a low-tillering mutant, ltd1, which exhibits reduced tillering and shorter plant height. Figure 1 AD). Agronomic traits surveyed at maturity showed that ltd1 had significantly lower grains per ear and 1000-grain weight compared to the wild type. Figure 1 EF). Observations on the formation and growth of tillers revealed that ltd1 could form tillers, but the tiller elongation was slow (EF). Figure 1 Therefore, its low tillering phenotype is mainly due to the inhibition of tiller elongation.

[0064] Identification of rice tillering gene LTD1

[0065] Genetic analysis revealed that the mutant phenotype is controlled by a pair of recessive nuclear genes. Through map-based cloning combined with high-throughput sequencing and MutMap analysis, the candidate gene LTD1 (LOC_Os01g19760) was identified. LTD1 is located on the short arm of chromosome 1. Figure 2The ltd1 mutant encodes a protein of unknown function. Its full-length DNA sequence is 8238 bp, and its full-length cDNA sequence is 2904 bp, encoding 967 amino acids with a molecular weight of approximately 107 kDa. In the ltd1 mutant, the 46th base is changed from G to A, resulting in the 16th amino acid position of the protein being changed from glutamic acid (Glu) to lysine (Lys).

[0066] Example 2 Cloning of the rice LTD1 gene

[0067] Synthesize the following primer pairs:

[0068]

[0069] DNA extracted from the leaves of the wild-type parent 676R was used as a template. PCR amplification was performed using primers P1 (underlined Xba I restriction site) and P2 (underlined Sal I restriction site), yielding an 8238 bp fragment. This fragment was ligated into the pMD-20-T vector to construct pMD-LTD1, which was then transformed into *E. coli* strain JM109. Positive clones were screened and sequenced. Sequencing results showed that the amplified LTD1 fragment possessed the sequence shown in SEQ ID NO:1 in the sequence listing, with its coding region from position 1 to position 8238. The sequenced fragment was named LTD1.

[0070] Example 3: Obtaining the cDNA sequence of the LTD1 gene

[0071] Subsequently, leaves were taken from the wild-type parent 676R during the seedling and tillering stages. Total RNA was extracted from the leaf samples using an RNA extraction kit, and then cDNA was obtained by reverse transcription using a reverse transcription kit.

[0072] Reverse transcription reaction system based on R223-01 kit:

[0073] ① Genomic DNA removal:

[0074]

[0075] After mixing with an enzyme-free pipette tip, incubate at 42°C for 2 min in a PCR instrument, then immediately remove and place on ice.

[0076] ②Preparation of the reverse transcription experimental system:

[0077]

[0078] After mixing with an enzyme-free pipette tip, incubate at 50°C for 15 min in a PCR instrument, then incubate at 85°C for 5 sec to obtain the first-strand cDNA.

[0079] The cDNA of this gene was amplified by RT-PCR using primers P1 and P2, yielding a fragment of approximately 2900 bp. This fragment was ligated into the pMD-20-T vector to construct pMD-LTD1-1, which was then transformed into *E. coli* strain JM109. Positive clones were screened and sequenced. Sequencing results showed that the amplified LTD1-1 fragment has the sequence shown in SEQ ID NO:2 in the sequence listing. Its open reading frame (ORF) from position 1 to position 2904 encodes 967 amino acids with a molecular weight of approximately 107 kDa.

[0080] Example 4 Construction of LTD1 gene overexpression vector

[0081] Synthesize the following primer pairs:

[0082]

[0083] Using the verified pMD-LTD1-1 from step one as a template, the cDNA of this gene was amplified with primers P3 (underlined is the XbaI restriction site) and P4 (underlined is the PstI restriction site), resulting in a fragment of approximately 2900 bp. This fragment was then ligated into the expression vector pCAMBIA2300, which contained the act 1 promoter from rice and had been double-digested with XbaI and PstI, to construct the transgenic expression vector pCAMBIA2300-LTD1.

[0084] Example 5: Obtaining LTD1 gene overexpressing plants

[0085] The constructed transgenic expression vector pCAMBIA2300-LTD1 was used to infect the callus tissue of the mutant ltd1 seeds via Agrobacterium-mediated transformation. After screening, differentiation, rooting, and hardening, transgenic T0 generation seedlings were obtained, resulting in 8 positive transgenic plants. The phenotype of the progeny positive transgenic plants was restored to the wild-type level. Figure 3 If the number of tillers, plant height, and number of grains per ear are not significantly different from those of the wild type, then... Figure 3 The LTD1 gene overexpression line prepared in this embodiment (actually also an LTD1 overexpression line in the ltd1 mutant background) was obtained from... Figure 3 It can be observed that LTD1 overexpression lines under the ltd1 mutant background have significantly increased tiller number, plant height and grains per ear compared with ltd1 mutant plants.

[0086] Example 6 Construction of RNAi interference vector

[0087] Synthesize the following primer pairs:

[0088]

[0089] Using the verified pMD-LTD1-1 from step one as a template, the cDNA of this gene was amplified with primers P5 (underlined is the XhoI restriction site) and P6 (underlined is the BamHI restriction site), yielding a fragment of approximately 230 bp. This fragment was ligated to the pUCCRNAi vector, which had undergone the same double digestion. Sequencing yielded the correct recombinant plasmid. The plasmid was then digested with the corresponding isoskeletal enzymes BamHI and SalI of XhoI and BglII. The fragment was then reverse-ligated into the pUCCRNAi vector. The correctly ligated forward and reverse fragment vectors were digested with the restriction enzyme PstI and then ligated into the PstI-digested expression vector pCAMBIA2300 to construct the transgenic expression vector pCAMBIA2300-LTD1 RNAi.

[0090] Example 7: Obtaining RNAi transgenic plants

[0091] The constructed pCAMBIA2300-LTD1 RNAi expression vector was used to infect callus tissue of the japonica rice variety Nipponbare (Nip) via Agrobacterium tumefaciens-mediated transformation. After screening, differentiation, rooting, and hardening, T0 generation interfering transgenic seedlings were obtained. Quantitative analysis of the obtained positive transgenic lines showed that the LTD1 gene expression level was significantly reduced. Figure 4 (AB), and the plants exhibited a phenotype similar to ltd1, characterized by reduced tillering and shorter plant height. Agronomic trait surveys showed that the number of tillers, plant height, and number of grains per ear were significantly reduced in both interference lines. Figure 4 This further demonstrates the function of the LTD1 gene and indicates that LTD1 is involved in the regulation of rice tillering.

[0092] LTD1 Haplotype Analysis

[0093] To investigate the variation of LTD1 in rice germplasm resources, this invention used the genome sequencing results of 2792 rice germplasm resources from the 3K Rice Genome Project (3KRG) to perform haplotype analysis on LTD1. Using 11 non-synonymous SNPs in the LTD1 coding region, LTD1 was divided into 8 major haplotypes (Hap1-8), as shown in Table 1. Among them, the indica rice subgroup (Xian) mainly contains 6 haplotypes (Hap1, Hap2, Hap4, Hap5, Hap6, Hap7), the japonica rice subgroup (Geng) mainly contains 5 haplotypes (Hap1, Hap2, Hap3, Hap5, Hap8), the Aus mainly contains 3 haplotypes (Hap1, Hap2, Hap5), the Bas mainly contains 3 haplotypes (Hap1, Hap2, Hap3), and the Adm mainly contains 5 haplotypes (Hap1, Hap2, Hap3, Hap5, Hap6).

[0094] Table 1. LTD1 genotype analysis

[0095]

[0096] Comparison of the tillering phenotypes of different haplotypes revealed that haplotypes Hap3, Hap5, and Hap7 exhibited significantly higher tillering than other haplotypes, while haplotypes Hap2 and Hap8 showed significantly lower tillering than other haplotypes. Figure 5 A); Comparison of seedling height phenotypes revealed that the seedling height of haplotypes Hap3 and Hap7 was significantly lower than that of other haplotypes ( Figure 5 B). From the perspectives of tillering and seedling height, there are significant differences between haplotype Hap7 (specific to indica rice) with its high tillering ability and low seedling height, and Hap8 (specific to japonica rice) with its low tillering ability and high seedling height. These differences warrant further research and utilization. Hap7 and Hap8 exhibit four base differences in their coding sequences (Table 1), which is speculated to be one of the key factors contributing to the differences in tillering and seedling height phenotypes between indica and japonica rice.

[0097] This invention's analysis of LTD1 variation in rice germplasm populations shows that the distribution frequencies of the eight LTD1 haplotypes differ significantly between indica and japonica rice. Notably, haplotype Hap7 (unique to indica rice) exhibits both high tillering ability and low seedling height. Subsequently, representative varieties or germplasm resources from superior LTD1 haplotypes (such as Hap7) can be selected to explore the relationship between LTD1 expression levels and yield traits. Furthermore, marker-assisted backcrossing can be used to introduce LTD1 into main cultivated varieties to create new breeding materials, thereby exploring the application potential of the LTD1 gene in rice breeding.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. LTD1 The application of genes in reducing rice tiller number is characterized by, By constructing LTD1-RNAi interference lines to reduce rice tiller number, the following... LTD1 The DNA sequence of the gene is shown in SEQ ID NO:

1. LTD1 The cDNA sequence of the gene is shown in SEQ ID NO:

2. LTD1 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:3.

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