Application of rice leaf color mutation gene OsLCD2 in regulating leaf color traits of rice

Abstract

The application belongs to the field of plant genetic engineering, and particularly relates to application of a rice leaf albino gene OsLCD2 in regulating rice leaf color traits, and further provides a low-temperature sensitive rice leaf albino gene oslcd2. The OsLCD2 gene and the oslcd2 gene provided by the application have application prospects in hybrid F1 generation hybrid purity identification and sterile line self-cross propagation seed purity identification in rice breeding, and are of great significance in improving seed purity and reducing hybrid rice production risks.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application of the rice leaf albino gene OsLCD2 in regulating rice leaf color traits. Background Technology

[0002] Ribosomes are fundamental components of the translation system in all living cells of organisms. Similar to fungi and bacteria, plant chloroplasts contain 70S ribosomes, which are composed of a 30S small subunit and a 50S large subunit (Schmid LM, et al. Chloroplastribosome biogenesis factors. Plant Cell Physiol. 2024, 65: 516-536). The 30S small subunit consists of 25 plastid ribosomal proteins (PRPs) and 16S rRNA, of which 13 PRP proteins are encoded by the nuclear genome and the remaining 12 by the plastid genome. The 50S large subunit contains 23S, 5S, and 4.5S rRNA and 33 PRP proteins, of which 9 are encoded by the nuclear genome and the remaining 24 by the plastid genome (Yamaguchi K, Subramanian AR. The plastid ribosomal proteins. Identification of all the proteins in the 50S subunit of an organelle ribosome (chloroplast). J Biol Chem. 2000, 275: 28466-28482; Yamaguchi K, et al. The plastid ribosomal proteins. Identification of all the proteins in the 30S subunit of an organelle ribosome (chloroplast). J Biol Chem. 2000, 275: 28455-28465; Stoppel R, Meurer J. The cutting crew-ribonucleases are key players in the control of plastid gene expression. J Exp Bot. 2012, 63: 1663-1673.).Based on techniques such as two-dimensional protein electrophoresis and yeast double hybridization, PRPS1-5 and PRPS10, which constitute the 30S RP protein, have been identified and cloned from plants (Yamaguchi K, Subramanian AR. Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit. Eur J Biochem. 2003, 270: 190-205; Xiong HB, et al. AtNusG, a chloroplast nucleoid protein of bacterial origin linking chloroplast transcriptional and translational machinery, is required for proper chloroplast gene expression in Arabidopsisthaliana. Nucleic Acids Res. 2022, 50: 6715-6734). Studies have shown that the single-base substitution mutant rps5 of the Arabidopsis PRPS5 gene exhibits pale yellow new leaves and significantly delayed and weak plant growth and development. These results suggest that this type of protein is closely related to chloroplast formation and development (Zhang J, et al. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis. J Exp Bot. 2016, 67:2731-2744). Using gene editing technology, two mutants of the Arabidopsis PRPS10 gene, us10c-1 (with a single-base T insertion) and us10c2 (with a single-base substitution leading to amino acid replacement), were created. Studies showed that both mutants exhibited homozygous albino lethality, while the heterozygous state showed albino spotted leaves (Sun X, et al. The uS10c-BPG2 module mediates ribosomal RNA processing in chloroplastnucleoids. Nucleic Acids Res. 2024, 52:7893-7909). However, the temperature sensitivity of the PRPS10 gene has not yet been reported.

[0003] In hybrid rice breeding, especially two-line hybridization, sterile lines, such as temperature-sensitive two-line sterile lines, are typically fertile at low temperatures but sterile at high temperatures. Therefore, the fertility conversion of these sterile lines is affected by the sterility threshold temperature (usually required to be below 23.5℃). However, during two-line rice seed production, the complex and variable nature of natural temperatures often leads to self-pollination of sterile lines during seed production, affecting seed purity and even causing production failure. Leaf color is an important morphological marker for crops and can be used to simplify the propagation of superior varieties and improve hybrid production efficiency. Leaf color marking in rice has advantages such as being intuitive, reliable, and easy to use in identifying true and false hybrids. It has been applied in two-line rice breeding, such as the light green leaf high-quality fragrant male-sterile line Huangxiangzhan S (Eshendao 20210099) bred by Huanggang Academy of Agricultural Sciences and other units (Cai Xingxing et al. Breeding of high-quality fragrant rice two-line male-sterile line Huangxiangzhan S. Hybrid Rice, 2024, 39: 70-72). Using this male-sterile line, Huangliangyou 913 (Eshendao 20210017) was bred. Hunan Taohuayuan Agricultural Technology Co., Ltd. and Hunan Zhihe Seed Industry Technology Co., Ltd. have also used this line to breed other varieties. K Liangyou 22 (National Approval Rice No. 20196004) was bred using the pale yellow leaf temperature-sensitive male-sterile line KT27S, achieving fully mechanized seed production through direct seeding and drone-assisted pollination (Zhang Qing et al. Fully Mechanized Seed Production Technology of Hybrid Rice Based on Leaf Color Marker Male-sterile Lines. Hybrid Rice, 2021, 36: 25-28); Yunnan Academy of Agricultural Sciences Institute of Grain Crops and Zhejiang University Institute of Nuclear Agriculture bred Yunfuyou 26 (Yunnan Special (Honghe) Approval Rice No. 2008010) by crossing the albino-to-green leaf color marker two-line male-sterile line NHR111S with the restorer line Zhefu 26. (Deng Anfeng et al. "Yunfuyou 26", the first high-yield hybrid rice combination in Yunnan Province bred using a marker-controlled male-sterile line. Seeds, 2008, 27: 94-96); Hefei Fenghai Marked Rice Research Institute used the albino marker-carrying male-sterile line Luzhou 86A to breed rice varieties such as Luyou 855 (Anhui Variety Approval No. 07010606), Luyou 827 (Anhui Variety Approval No. 07010602), and Luyou 136 (Anhui Variety Approval No. 05010474); Liuzhou Agricultural Science Research Institute used the pale green leaf two-line male-sterile line QianS and the restorer line Liuhui 1552 to breed Qianliangyou. 1552 (Qin Ruide et al. Breeding and cultivation techniques of the new hybrid rice combination Qianliangyou 1552. Agricultural Science and Technology Communications, 2019, 7: 303-304); The Rice Research Institute of Guangxi Academy of Agricultural Sciences used the purple-red leaf marker male-sterile line Xianhong A to breed Xianhongyou 981 (Guishendao 2020046) and Xianhongyou 826 (Guishendao 2020149) with restorer lines Gui 981 and Gui 826 respectively (Deng Guofu et al. Breeding and application of the purple-red leaf marker male-sterile line Xianhong A. Chinese Rice, 2008, 5: 26-28). Summary of the Invention

[0004] To address the existing need for rice breeding screening using leaf color morphology markers, this invention provides the application of the rice leaf albino gene OsLCD2 in regulating rice leaf color traits. The specific technical solution is as follows:

[0005] In a first aspect, the present invention provides the application of the rice leaf albino gene OsLCD2 in regulating the rice leaf color trait, and the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1.

[0006] Secondly, the present invention provides the application of the rice leaf albino gene OsLCD2 in molecular breeding of rice leaf color traits, the nucleotide sequence of the OsLCD2 gene being shown in SEQ ID NO.1.

[0007] Thirdly, this invention provides the application of the rice leaf albino gene OsLCD2 in the identification of hybrid purity in F1 generation hybrid rice and / or the identification of purity of self-pollinated seeds of sterile lines, the genome sequence of which is shown in SEQ ID NO.1.

[0008] Fourthly, the present invention provides a low-temperature sensitive rice leaf albino gene oslcd2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] Fifthly, this invention provides the application of the above-mentioned oslcd2 gene in molecular breeding of rice leaf color traits.

[0010] Sixthly, the present invention provides the application of the above-mentioned oslcd2 gene in the identification of hybrid purity of F1 generation hybrid rice and / or the identification of purity of self-pollinated seeds of sterile lines.

[0011] The rice low-temperature sensitive leaf albinism gene oslcd2 of this invention was obtained by screening offspring from the EMS mutant library of Xiushui 134. The phenotype of mutant oslcd2 is that the leaves exhibit albinism under low temperature conditions, and this is a recessive trait. Further artificial temperature control experiments showed that the leaves of this mutant exhibited obvious albinism characteristics at 22℃. This invention used map-based cloning to initially locate the oslcd2 gene in an approximately 198kb interval on the short arm of chromosome 3, between RM14525 and RM14531, containing 36 genes. Further PCR cloning and sequencing comparative analysis of these genes identified the plastosome ribosomal protein gene PRPS10 as a candidate gene for OsLCD2. Subsequently, a genetic complementation expression vector was constructed and introduced into the mutant oslcd2. The transgenic offspring exhibited normal leaf color, thus confirming that the low-temperature sensitive albinism leaf characteristic of mutant oslcd2 is caused by a single base substitution in the OsLCD2 gene.

[0012] In a seventh aspect, the present invention provides the application of OsLCD2 protein in regulating the leaf color trait of rice, and the amino acid sequence of OsLCD2 protein is shown in SEQ ID NO.3.

[0013] Eighthly, the present invention provides a method for removing false hybrids in hybridization breeding, comprising the following steps:

[0014] (1) Construct mutant plants in which the rice leaf albino gene OsLCD2 was interfered with, knocked out, or silenced; the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1;

[0015] (2) The mutant plants obtained in step (1) are hybridized and backcrossed with rice maintainer lines and sterile lines to cultivate three-line and / or two-line sterile lines with low-temperature sensitive leaf whitening traits, and F1 hybrids are obtained through seed production.

[0016] Seedling stage identification was performed on the F1 generation of hybrids obtained from seed production. Plants with albino leaves were identified as pseudo-hybrids of the sterile line through self-pollination and were removed.

[0017] Further, in step (1), mutant plants containing the above-mentioned low-temperature sensitive rice leaf albino trait gene oslcd2 are constructed.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The OsLCD2 and oslcd2 genes provided by this invention have promising applications in identifying the purity of hybrid F1 hybrids and preserving the purity of self-pollination of sterile lines in rice breeding. They are of great significance for improving seed purity and reducing the production risks of hybrid rice. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the phenotype and chlorophyll content of the mutant oslcd2 and its wild-type control at different temperatures; where A represents the seedling phenotype under different temperature conditions; and B represents the statistical graph of leaf chlorophyll content under different temperature conditions.

[0021] Figure 2 The results of map-based cloning of the OsLCD2 gene are shown below; where A is the linkage molecular marker of the OsLCD2 gene; B is the BAC cloning of the fine-mapping region; C is the structure of the OsLCD2 gene; D is the sequencing analysis of the mutation sites of the OsLCD2 gene; E is the field phenotype of the complementary transgenic progeny; and F is the determination of the total chlorophyll content of the transgenic progeny.

[0022] Figure 3The image shows the plant creation analysis of Xiushui 134A, a three-line sterile line carrying the leaf albino gene oslcd2. In the image, A is a photograph of Xiushui 134A, a three-line sterile line carrying the leaf albino gene oslcd2, and B is the chlorophyll content analysis result of Xiushui 134A, a three-line sterile line carrying the leaf albino gene oslcd2. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0026] In the following examples, the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1, the amino acid sequence of the OsLCD2 protein is shown in SEQ ID NO.3, the nucleotide sequence of the oslcd2 gene is shown in SEQ ID NO.2, and the amino acid sequence of the oslcd2 protein is shown in SEQ ID NO.4.

[0027] SEQ ID NO.1:

[0028] ATGGCCGTCTCCACCTCCGCCTCCTCCACCCTCCCGCTCCTCTCCCTCCATCGCGCCGCGGGGAACCCCAACCCGGCCGCACTCTCCTTCCCGCCTCCCCTCCGCGCGCCCCCGCTCCGCTCCCGCGCCGCCGCTGCCGCGGCCTCCGCCGCAGCACCGCCCGCCGAGACCATCCCCGACATGCCCGAGGAGACGGAGGGGACGGGGATTCCGATGCCGTCGTCGATTGGGGATGATGGGGAGCAGTTGGCACCCAAGCAGAAGATCAGAATTAAGCTGAGGTCTTAC TGGGTACCATTGATCGAGGACTCCTGTAAGAAGATCATTGAAGCTGCAAAAACAACAAATGCAAAGACGATGGGTCCAGTTCCTCTGCCAACCAAGCGAAGAGTATATTGTGTGCTCAATTCTCCTCACGTGCACAAAGATTCAAGGTTCCATTTTGAGATCAGAACGCACCAGCGATTGATTGATATCATGTACCCGACTGCACAAACAATAGATTCCTTGATGCAGCTCCAGCTCCCTGCCGGGGTAGATGTTGAGGTTAAGCTATGA。

[0029] SEQ ID NO.2:

[0030] ATGGCCGTCTCCACCTCCGCCTCCTCCACCCTCCCGCTCCTCTCCCTCCATCGCGCCGCGGGGAACCCCAACCCGGCCGCACTCTCCTTCCCGCCTCCCCTCCGCGCGCCCCCGCTCCGCTCCCGCGCCGCCGCTGCCGCGGCCTCCGCCGCAGCACCGCCCGCCGAGACCATCCCCGACATGCCCGAGGAGACGGAGGGGACGGGGATTCCGATGCCGTCGTCGATTGGGGATGATGGGGAGCAGTTGGCACCCAAGCAGAAGATCAGAATTAAGCTGAGGTTTTACTGGGTACCATTGATCGAGGACTCCTGTAAGAAGATCATTGAAGCTGCAAAAACAACAAATGCAAAGACGATGGGTCCAGTTCCTCTGCCAACCAAGCGAAGAGTATATTGTGTGCTCAATTCTCCTCACGTGCACAAAGATTCAAGGTTCCATTTTGAGATCAGAACGCACCAGCGATTGATTGATATCATGTACCCGACTGCACAAACAATAGATTCCTTGATGCAGCTCCAGCTCCCTGCCGGGGTAGATGTTGAGGTTAAGCTATGA。

[0031] SEQ ID NO.3:

[0032] MAVSTSASSTLPLLSLHRAAGNPNPAALSFPPPLRAPPLRSRAAAAAASAAAPPAETIPDMPEETEGTGIPMPSSIGDDGEQLAPKQKIRIKLRSYWVPLIEDSCKKIIEAAKTTNAKTMGPVPLPTKRRVYCVLNSPHVHKDSRFHFEIRTHQRLIDIMYPTAQTIDSLMQLQLPAGVDVEVKL。

[0033] SEQ ID NO.4:

[0034] MAVSTSASSTLPLLSLHRAAGNPNPAALSFPPPLRAPPLRSRAAAAAASAAAPPAETIPDMPEETEGTGIPMPSSIGDDGEQLAPKQKIRIKLRFYWVPLIEDSCKKIIEAAKTTNAKTMGPVPLPTKRRVYCVLNSPHVHKDSRFHFEIRTHQRLIDIMYPTAQTIDSLMQLQLPAGVDVEVKL.

[0035] Example 1: Isolation and genetic analysis of the mutant oslcd2

[0036] Using EMS to induced mutagenesis of conventional japonica rice Xiushui 134, the leaf albino mutant oslcd2 was screened from the offspring of the mutant library. After multiple generations of backcrossing and self-crossing in Hangzhou and Hainan, materials with genetically stable mutant traits were obtained.

[0037] Mutants and wild-type plants were grown under different artificially controlled temperature conditions (22℃ and 28℃), such as Figure 1 As shown in Figure A, the mutant oslcd2 plant was found to be sensitive to low temperatures; specifically, it exhibited obvious chlorotic leaf symptoms at 22℃, while remaining pale green at 28℃. Further chlorophyll content measurements yielded the following results: Figure 1 As shown in Figure B, at 28℃, the chlorophyll and carotenoid contents of the mutant oslcd2 plant were basically the same as those of the wild-type plant, while at 22℃, the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the mutant oslcd2 plant were only 20.67%, 19.85%, 33.58%, and 20.11% of those in the wild-type control, respectively. These results indicate that the albino leaf trait of the mutant is a low-temperature sensitive type.

[0038] Using oslcd2 plants as the female parent, hybridization was carried out with Xiushui 134 and Indica rice restorer line 93-11 to obtain F1 plants. The F1 plants showed normal behavior. The F2 offspring obtained by self-pollination of F1 showed wild-type and mutant phenotypes, with a segregation ratio of wild-type to mutant = 3:1, indicating that the albino leaf trait of the mutant is controlled by a single recessive nuclear gene.

[0039] Example 2: Map-based cloning of the OsLCD2 gene

[0040] 1. Fine mapping of the OsLCD2 gene

[0041] Five hundred pairs of SSR markers and fifty pairs of InDel markers distributed on the 12 chromosomes of rice were selected. The polymorphism of each marker between the mutant oslcd2 and 93-11 was analyzed, obtaining polymorphic molecular markers evenly distributed across the 12 chromosomes. BSA was used to identify molecular markers linked to the target gene OsLCD2. The results showed that the SSR markers RM14487, RM14545, and RM14572 on the short arm of rice chromosome 3 were tightly linked to the OsLCD2 gene. Subsequently, the genotypes of 169 recessive extreme individuals in the oslcd2 / 93-11F2 population were analyzed using these three molecular markers. Simultaneously, the physical arrangement of these three markers and the OsLCD2 gene on chromosome 3 was determined by combining the position of each molecular marker on the chromosome, i.e., RM14487-OsLCD2-RM14545-RM14572 (e.g., RM14487-OsLCD2-RM14545-RM14572). Figure 2 (As shown in A).

[0042] To further refine the localization of the OsLCD2 gene, 10 pairs of SSR markers were designed between RM14487 and RM14545. Two of these pairs of markers showed polymorphism between the parents. Genotyping analysis was performed on recessive individuals in the F2 population that had single crossovers with RM14487 and RM14545 using these two pairs of markers. Ultimately, the OsLCD2 gene was located between SSR markers RM14525 and RM14531, with a physical distance of approximately 198 kb, containing 36 genes (such as...). Figure 2 (as shown in B).

[0043] 2. Prediction and sequencing comparison analysis of leaf albino mutant genes

[0044] PCR cloning and sequencing analysis were performed on the 36 genes located in the mutant and wild-type control regions. The results showed that the 284th base of the open reading frame (ORF) of the candidate gene LOC_Os03g10060 was mutated from C to T, resulting in a mutation of threonine to phenylalanine (e.g., ...). Figure 2 C and Figure 2 (as shown in D).

[0045] 3. Verification of genetic complementation of the mutant oslcd2

[0046] Construction of the gene complementation vector: A pair of primers was designed based on the sequence of the full-length cDNA of the OsLCD2 gene (TIGR Locus:LOC_Os03g10060) in GenBank:

[0047] Upstream primer: OsLCD2F1: 5′-GGAGGTGGAGACGGCCAT-3′;

[0048] Downstream primer: OsLCD2R1: 5′-TCGAGCTCGGTACCCAGCTCCACGTGAATCATG-3′;

[0049] Using total RNA from Xiushui 134 leaves as a template, the cDNA fragment of the OsLCD2 gene was obtained by RT-PCR.

[0050] 1 st The cDNA reaction system is shown in the table below:

[0051] Table 1 Reaction System

[0052]

[0053] After mixing the samples, incubate at 37°C for 5 min, then add 4 μL of 5×Easy RT MasterMix, mix well, and incubate at 42°C for 20 min. The cDNA fragment of the OsLCD2 gene is then obtained by conventional PCR using the reaction product as a template. The PCR reaction system is as follows:

[0054]

[0055] Reaction conditions: 98℃ denaturation for 10 seconds, 56℃ annealing for 15 seconds, 72℃ extension for 45 seconds, for a total of 40 cycles. 72℃ extension for 5 minutes.

[0056] Using the genomic DNA of Xiushui 134 as a template, the OsLCD2 fragment (containing the gene ORF and its promoter sequence) was obtained by PCR amplification using the following specific primers.

[0057] Upstream primer: OsLCD2F2: 5'-CTCTAGAGGATCCCTCTACTGCTAGCAATCTTCC-3';

[0058] Downstream primer: OsLCD2R2: 5′-CATGATTCACGTGGAGCT-3′;

[0059] PCR reaction system:

[0060]

[0061] Agrobacterium plasmid pCAMBIA1300-Nos was digested with SmaI, and then the cDNA fragment of OsLCD2 and its promoter were introduced into linearized pCAMBIA1300-Nos using In-Fusion technology to form pOsLCD2. The plasmid was then digested and sequenced to confirm its correctness. Agrobacterium-mediated transformation was used to transform Agrobacterium EHA105, which was then used to transform the mutant oslcd2 to obtain transgenic progeny (oslcd2-T1, oslcd2-T2). Results showed that the leaf color and chlorophyll content of the pOsLCD2 transgenic progeny returned to normal (e.g., ...). Figure 2 E and Figure 2 (as shown in F).

[0062] Example 3: Transformation of the albino leaf three-line sterile line

[0063] Xiushui 134 is a maintainer line, and its mutant oslcd2 is also a maintainer line. It can be crossed with the three-line sterile line Xiushui 134A to obtain F1. F1 is a sterile line with normal leaf color carrying the oslcd2 gene. Then, F1 is backcrossed with the mutant oslcd2 to obtain the BC1F1 population. From the offspring population, Xiushui 134A plants carrying the albino leaf marker (i.e., single plants carrying the homozygous oslcd2 genotype) are obtained (e.g.,...). Figure 3 As shown in A). Figure 3 As shown in B, under low temperature conditions, the total chlorophyll content of Xiushui 134A carrying the albino leaf marker is only 23.13% of that of normal Xiushui 134A.

Claims

1. A low temperature sensitive rice leaf bleaching gene of the application, characterized in that, oslc d2 The oslc d2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; the application is molecular breeding of rice leaf albino traits under low temperature conditions. ​ 2. A low temperature sensitive rice leaf bleaching gene oslc d2 Use in the identification of hybrid purity of F1 hybrids of hybrid rice and / or the identification of seed purity of selfed lines of sterile lines, characterized in that, The oslc d2 The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A method of hybrid breeding to eliminate false hybrids, characterized by, comprising the following steps: (1) Constructing a mutant plant containing a low-temperature sensitive rice leaf whitening gene shown in SEQ ID NO. 2 oslc d2 ; (2) crossing the mutant plant obtained from step (1) with a rice maintainer line and a sterile line material, backcrossing, cultivating a three-line and / or two-line sterile line with a low-temperature sensitive leaf whitening trait, and obtaining F1 hybrids by seed production; identifying the seed production hybrid F1 generation at the seedling stage, and removing the plants with white leaves under low-temperature conditions as false hybrids from self-pollination of the sterile line.

Images