Rice three-allele dominant genic male sterility gene SDGMS and application thereof

Abstract

This invention provides a rice tristiginal male sterility gene SDGMS and its applications, belonging to the field of functional gene technology. This invention determines the genomic location of the rice tristiginal male sterility gene SDGMS through fine mapping. This invention transforms the dominant male sterility gene SDGMS into wild-type rice varieties, creating dominant male sterile lines; simultaneously, increasing the expression level of SDGMS can increase the degree of sterility. The SDGMS gene provided by this invention has the biological function of dominant male sterility, can regulate the tristiginal male sterility trait in rice, and has applications in breeding and / or constructing sterile rice lines, as well as in detecting or identifying tristiginal male sterile rice lines.

Description

Technical Field

[0001] This invention belongs to the field of functional gene technology, specifically relating to a rice tristiginal nuclear male sterility gene SDGMS and its application. Background Technology

[0002] Male sterility is a widespread phenomenon in nature. Based on the location of the controlling genes, it can be divided into cytoplasmic male sterility and nuclear male sterility. Nuclear male sterility can be further divided into dominant nuclear male sterility and recessive nuclear male sterility. Cytoplasmic male sterility and photoperiod-sensitive male sterility have been widely applied in production, making significant contributions to the utilization of heterosis in crops and hybridization breeding.

[0003] Dominant nuclear male sterility is a relatively scarce material, with only more than 20 cases identified in 10 crops. As an ideal breeding method, the recurrent selection system based on dominant nuclear male sterility materials can quickly break genetic linkage and aggregate superior loci, and has been applied in a variety of crops.

[0004] Currently, three genes controlling dominant nuclear male sterility have been cloned in crops. In 1972, Gao Zhongli identified a male-sterile plant in Taigu County, Shanxi Province. After a series of identifications, it was determined to be a dominant pollenless male sterility controlled by a single gene, and the material was named wheat Taigu male sterility. Liu Binghua et al. (1986) located the gene controlling Taigu nuclear male sterility on the short arm of chromosome 4D through a study of backcross progeny of tetraploid durum wheat and Taigu male sterility. Liu Binghua and Yang Li (1991) found that the wheat dwarf gene (Rht10) and the Taigu male sterility gene (MS2) are closely linked, thus breeding dwarf abortive wheat, making it easier to distinguish between sterile and fertile single plants, and improving cross-pollination. Xia et al. (2017) found that the insertion of a terminal repeat small retrotransposon (TRIM) element into the MS2 promoter region specifically activated the expression of the MS2 protein in the anther, resulting in a dominant male sterility phenotype. Further research by Liu et al. (2022) revealed that the activated MS2 protein interacts with the reactive oxygen species regulator Taromo1 and promotes its polymerization, thereby reducing the ROS level in anthers and inhibiting anther development in wheat.

[0005] In 1972, the Yibin Agricultural Research Institute in Sichuan Province discovered dominant nuclear male-sterile material in Brassica napus and further bred the dominant nuclear male-sterile line Yi3A. Xin et al. (2016) showed that male sterility in the Yi3A type of Brassica napus is controlled by three multiple alleles at the same locus: MS5a (restor gene), MS5b (sterility gene), and MS5c (preservation gene). MS5 encodes a Brassica genus-specific protein, which plays an important role in chromosome synapsis and the localization of the meiotic condensing subunit SYN1. MS5b, due to the insertion of the MULE transposon in its second intron, cannot form the correct synaptic complex and recombination element during meiosis, leading to male sterility. In a subsequent study, Xin et al. (2022) found that the proteins encoded by MS5a and MS5b are essential for normal male development in Brassica napus. The chimeric protein encoded by MS5b can competitively bind to both MS5a and MS5b, forming a non-functional dimer. The expression levels of MS5b and MS5c are comparable, and the resulting dimer produces a dominant effect, leading to a male sterility phenotype. However, the expression level of MS5a is much higher than that of MS5b, thus maintaining sufficient MS5a homodimers, leading to the restoration of fertility.

[0006] MS44 is a dominant male sterility mutant of maize identified from EMS mutations. Tim et al. (2017) showed that maize Ms44 encodes a lipid transfer protein that is specifically expressed in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein eliminates protein processing and prevents the protein from being secreted from tapetum cells into the chamber, resulting in dominant male sterility.

[0007] Six dominant male-sterile rice materials have been identified so far: Pingxiang dominant male-sterile, tri-dominant male-sterile, 8987 low-temperature sensitive dominant male-sterile, Zhe9248 mutant dominant male-sterile, 1783 and 1789 dominant male-sterile mutant materials, and OsDMS-2 dominant male-sterile. There are currently no reports of gene cloning.

[0008] The dominant nuclear male sterility gene in Pingxiang was isolated by Yan Long'an et al. in the F2 generation of Ping'ai 58 / Huaye in 1978 (Yan Long'an et al., 1984). Chen Xuewei et al. (2000) used SSLP markers to locate the Pingxiang dominant nuclear male sterility gene Ms-p on chromosome 10, between markers RM228 and RM258, with a genetic distance of 14.9 cM from RM228. Further analysis of the parents and population using RFLP markers adjacent to RM228 and RM258 revealed that RFLP marker G2155 is linked to the gene Ms-p, with a genetic distance of 2.6 cM. He Haohua et al. (2004) located it near the SSR marker RM239, with a genetic distance of 5.65 cM between them. Based on this, Huang et al. (2007) located Ms-P between SSR markers RM171 and RM6745, with genetic distances of 0.3 cM and 3.0 cM from the two markers, respectively.

[0009] Low-temperature-sensitive dominant male sterility (8987) was discovered by Deng Xiaojian and Zhou Kaida (1994) in 1989 in the 3304 / Minghui 63F5 generation. It is characterized by complete sterility at low temperatures and normal fruit setting at higher temperatures, and is controlled by a single gene pair. Li Shigui et al. (1999) used RFLP and microsatellite markers to locate its controlling gene, TMS, between two markers, C235 and RM50, on chromosome 6. The genetic distances of TMS from these two markers were 6.4 cM and 12.9 cM, respectively.

[0010] The tristigious nuclear male sterility trait in rice was discovered in 2001 by the Sanming Agricultural Science Research Institute in Fujian Province in the F2 generation of the hybrid SE21S / Basmati370. Compared with other rice sterile materials, its male sterility is stable and complete, with good stigma exposure, and female fertility is unaffected. Analysis showed that tristigious nuclear male sterility is regulated by a single site (Huang et al., 2008). Yang et al. (2012) located the tristigious nuclear male sterility gene between two In-Del markers ZM30 and ZM9 on chromosome 8, with an interval of approximately 99 kb referenced from the Nipponbare genome. However, there are currently no reports on the tristigious nuclear male sterility gene in rice.

[0011] References

[0012] [1] Yu Fengqun, Fu Tingdong. Cell morphology study on anther development of several male sterile lines of Brassica napus [J]. Wuhan Botanical Research, 1990, (03): 209-216+301-302.

[0013] [2] Yang Zemao, Xie Xiaofang, Huang Xianbo, et al. Localization of dominant nuclear male sterility gene in rice “Sanming”[J]. Heredity, 2012, 34(05):615-620.

[0014] [3] Yang Guangsheng, Qu Bo, Fu Tingdong. Anatomical study on anther development of dominant nucleus male sterile line Yi 3A of Brassica napus [J]. Journal of Huazhong Agricultural University, 1999, (05): 405-408+501-503.

[0015] [4] Yan Longan, Zhang Juncai, Zhu Cheng, et al. Preliminary report on the identification of dominant male sterility gene in rice [J]. Acta Agronomica Sinica, 1989, (02): 174-181.

[0016] [5] Liu Binghua, Yang Li. Breeding and utilization prospects of “dwarf” wheat [J]. Chinese Science Bulletin, 1991, (04): 306-308.

[0017] [6] Liu Binghua, Deng Jingyang, Yang Li. Chromosomal localization of Ta1, a single gene for dominant male sterility in wheat [J]. Heredity 1986(02):13-14+50.

[0018] [7] Liu Binghua, Deng Jingyang. Chromosome location of single gene for dominant male sterility in wheat [J]. Heredity, 1984, (06): 10-12+49.

[0019] [8] Li Shigui, Zhou Kaida, Zhu Lihuang. Genetic analysis and molecular marker localization of thermosensitive dominant nuclear male sterility gene in rice [J]. Chinese Science Bulletin, 1999, (09): 955-958.

[0020] [9] Huang Xianbo, Tian Zhihong, Deng Zeqin, et al. Preliminary identification of tristigious nuclear male sterility gene in rice [J]. Acta Agronomica Sinica, 2008, (10): 1865-1868.

[0021]

[10] Huang Tingyou, Wang Yuping, Ma Bingtian, et al. Location and genetic analysis of fertility-related genes in dominant male-sterile rice in Pingxiang (English) [J]. Acta Genetica Sinica, 2007, (07): 616-622.

[0022]

[11] He Haohua, Liu Xiaoqiang, Zhu Changlan, et al. Localization of male sterility gene in dominant male sterile rice in Pingxiang[J]. Journal of Agricultural Biotechnology, 2004, (06): 727-728.

[0023]

[12] Deng Xiaojian, Zhou Kaida. Fertility conversion and genetic study of low-temperature sensitive dominant male sterile rice “8987” [J]. Journal of Sichuan Agricultural University, 1994, (03): 376-382.

[0024]

[13] Chen Xuewei, Li Shigui, Li Hanyun, et al. Localization of dominant nuclear male sterility gene in rice in Pingxiang[J]. Chinese Science Bulletin, 2000, (15): 1644-1648.

[0025]

[14] XIN Q,WANG

[0026]

[15]

[0027]

[16] XIA C,ZHANG L,ZOU C,et al.A TRIM insertion in the promoter of Ms2causes male sterility in wheat[J].Nat Commun,2017,8:15407.

[0028]

[17] LIU J,

[0029]

[18] FOX T,DEBRUIN J,HAUG COLLET K,et al.A single point mutation in Ms44resultsindominantmalesterilityandimprovesnitrogenuseefficiencyinmaize[J].PlantBiotechnolJ,2017,15(8):942-952. Summary of the Invention

[0030] In view of this, the purpose of this invention is to provide a dominant nuclear male sterility gene SDGMS for rice, providing a new genetic resource for breeding male-sterile rice.

[0031] This invention provides a rice tristiginal nuclear male sterility gene SDGMS, comprising the following DNA fragments:

[0032] 1) A DNA fragment with a nucleotide sequence as shown in SEQ ID NO:1;

[0033] 2) A DNA fragment that is based on item 1) with several base substitutions, deletions or insertions, and whose expression product has the same function as the expression product of the DNA fragment shown in SEQ ID NO:1.

[0034] 3) A DNA fragment encoding a protein with the amino acid sequence shown in SEQ ID NO:2;

[0035] 4) DNA fragments with nucleotide sequences as shown in SEQ ID NO:3.

[0036] This invention provides a protein encoded by the rice tristiginal nuclear male sterility gene SDGMS, the amino acid sequence of which is shown in SEQ ID NO:2.

[0037] This invention provides a recombinant vector containing the rice tristiginal nuclear male sterility gene SDGMS.

[0038] Preferably, the scaffold carrier of the recombinant vector includes pCAMBIA1301.

[0039] Preferably, the rice tristiginal nuclear male sterility gene SDGMS is inserted into the backbone vector at the KpnI and BamHI multiple cloning sites.

[0040] This invention provides the application of the rice tridistal nuclear male sterility gene SDGMS, the protein, or the recombinant vector in regulating the tridistal nuclear male sterility trait in rice.

[0041] This invention provides the application of the rice tristiginal male sterility gene SDGMS or the recombinant vector in the breeding and / or construction of male sterile rice lines.

[0042] Preferably, the application of the rice tristiginal male sterility gene SDGMS combined with elements that enhance SDGMS expression in the breeding of male sterile rice lines.

[0043] Preferably, the element comprises a maize Ubiquitin promoter; the nucleotide sequence of the maize Ubiquitin promoter is shown in SEQ ID NO:4.

[0044] This invention provides the application of the rice tristiginal male sterility gene SDGMS and the protein as a biomarker in the detection or identification of tristiginal male sterility rice lines.

[0045] This invention provides a rice tristiginal male sterility gene, SDGMS. Building upon previous research that the rice tristiginal male sterility gene is located between two In-Del markers ZM30 and ZM9 on rice chromosome 8, this invention conducts polymorphism analysis on multiple rice parents and pedigree-related parents from which the tristiginal male sterility mutation originates during population construction. Molecular markers were developed, and through fine mapping, the target gene was located between markers xch7 and SM1. Then, based on a genome-wide BAC library of the near-isogenic line (BC7F1) with a tristiginal male sterility background (938), the gene SDGMS was obtained through screening using molecular markers xch7 and xch95. This invention utilizes genetic transformation technology to transfer the SDGMS gene into wild-type rice varieties, creating dominant male sterile lines; simultaneously, increasing SDGMS expression levels increases the degree of sterility. Therefore, this invention demonstrates that the SDGMS gene possesses the biological function of dominant male sterility. Attached Figure Description

[0046] Figure 1 This is a map-based cloning of the SDGMS gene of the present invention. In the figure, (A) shows the position of SDGMS on the genetic linkage map of rice chromosome 8 and the fine localization of SDGMS; the numbers between the markers represent the physical distance between each marker, and the numbers below the markers represent the number of recombinant plants.

[0047] Figure 2 This is a diagram of the vector used for SDGMS transgenic complementation verification in this invention, specifically a diagram of the functional vector pCAMBIA1301.

[0048] Figure 3 These are phenotypic images of pollen iodine staining and seed setting rate of T1 generation transgenic single plants of the present invention. In the figures: AC represents the phenotype of Nipponbare transformed by SEQ ID NO: 1 ligated to pCAMBIA1301; A represents the whole plant phenotype of transgenic negative (left) and transgenic positive (right); B represents the mature ear phenotype of transgenic negative (left) and transgenic positive (right); C represents the anther and pollen iodine staining phenotype of transgenic negative (left) and transgenic positive (right); DF represents the phenotype of Nipponbare transformed by replacing the bases 1-1723 of SEQ ID NO: 1 with the maize Ubiquitin promoter and ligating to pCAMBIA1301; D represents the whole plant phenotype of transgenic negative (left) and transgenic positive (right); E represents the mature ear phenotype of transgenic negative (left) and transgenic positive (right); F represents the anther and pollen iodine staining phenotype of transgenic negative (left) and transgenic positive (right). Figure 4 The spectrum of the recombinant vector pCAMBIA1301u. Detailed Implementation

[0049] This invention provides a rice tristiginal nuclear male sterility gene SDGMS, comprising the following DNA fragments:

[0050]

[0051] 2) A DNA fragment that is based on item 1) with several base substitutions, deletions or insertions, and whose expression product has the same function as the expression product of the DNA fragment shown in SEQ ID NO:1.

[0052] 3) A DNA fragment encoding a protein with the amino acid sequence shown in SEQ ID NO:2 (MAPKKEQPVKEPPASDEFDLVLN DLPESYKKLMEDAIPTRLEQIIIQEAPSNSKGKKLFSKVAERLYTPKGGTFLVKLKPTKSSPDEEIVTLLFRWKDLYFEAFHAKGKWYRMSDAEESLPPRSQLHYSKKEKEGVFNMNNISTSYNDVGGHNIEVGRRAFKNCHQSLLMAEELVRQKRLKEELGSGPLSLPVVTISESIRFPLLQRWVLGTFSAPPTAKSEKKVPKKFSCEFNEWGKYSRALFTQELPVGCELTFAQIAEKLRVLKYRAAWVPQPAQKHVKDV).

[0053]

[0054] AAATCGCCCTCCCAGAGGGCGGTAGGCGACTATTTCCGTCAATTTTCAAATTGG

[0055] AAAATTATTTTTGTAAAACTTTTAATAAAAAAATTATAAATAAAAAAATTCATC

[0056] ACCTGACCACCCAGAAATGCCTCTCTTCTCCCAAGTCTCCATTGCTCAAGAGCT

[0057] CCGCCCCCGCCGCGCGCCTCCGCCTACTCCTCCTCTCCAGTGATACATACGTCC

[0058] GCCATGACTGCGAGGAGGAGGAGGGAGATGTCCAAGTAGGAGAAGCCTTGGC

[0059] CACCGGCAAGATACATTGTCCTTCTTGTTGGCTCAGCCCAGGAACACTCTCACA

[0060] CTCACGAACACAAACGCCGGTGAGGGAGACAATGAACAATGGATTTTACAGCG

[0061] ACGAACGCCGCGGAGCCGATCGCTCCTGTATCTTGGCTTTATTACTCACTGAAC

[0062] CTTGCTAAGAAACAACGAAGGAGTACAGGGGCCTCTTATAGACCGGTGACACA

[0063] CTGGGAGCTAGCCTCCATGCCGGCCATTGCGCCACGTTCCGCAGAACCCGCTCG

[0064] ACGTCGCCGTGAGCCACACGCTCGCCCGGACGACGCGGCTTCGTCCGCAAGAA

[0065] CCGACGCCACGCACACCATGCACACCCACGACTCGATCAACACTGCAACGGCC

[0066] ATGCAAACAACCATGCAGAAGCCATGCACTCGCGATCACGACTCAATCAAACT

[0067] AACAGAACACACACGACGCACACATGCAGCAGCACGACACACACACACTGACT

[0068] CAACCACGCCAGGAATACACCATGACGTGTTTATTTCCAACAATCTCCCCCTAA

[0069] ACACGACATGGCACACTTCGTTATCACAGCAGCGTCGGCTCCTCATCGACGTTA

[0070] CAAAGCAACGCCTGTTCCTTCTTCTTCCGCGGACAGTTCCTCGCCATGTGACCG

[0071] CGTTCTCCGCAGTCGAAGCACTTTCCTCGGTAGCGGCTCCTACCGTGCCCCGAG

[0072] CTTGTGCTGCTGCCATCGTCGTCGTCGTCGTCGTGGCCGCCGCCCTTCTCGCTGC

[0073] CGCCGCCGTGCACATGCTCCTTGCCACGGCGTTGACGCCGCCTCGCCTCCCATT

[0074] GCTCCTCCGTGAGCAGGAGTTGCCCAGCGTGCTCCACGCTAGACGCCGCTTGAT

[0075] CCTCAGCGTCCGCGTCCTCCGCCACCTGCAAGCGGCCGACGAGCTCCTCGATTT

[0076] CCATGGTGTCGATGTCGGCGAGCATCTCAATCGCCACCGCGACTTGCTTCAGCC

[0077] TCTTGGGGACCACGCGCAGAACTTTCTTCACGACGCGACTGTCCTCCATCTCCT

[0078] CACCAAGCTCGCGCAGGCTGGCGACGAGCCCGTTGATGCGCATTGCGAAGTCG

[0079] CCGACGTTCTCTCCATCACGGAACGCCATGTTCTCGAACTGCTTCATGAGACGT

[0080] TGGACACTTGCAGCCTTCACGCGGTCGTCGCCAGCGTGCATCTTCTTCACTGCG

[0081] TCCCACGCCTCCTTTGCCGACTTCTTCACGGCAAGCCCAGCCTTCATCTCCCTTG

[0082] GAACGGCGCGGATGATCGTGGCCAGCGCGCGTCGATCCTTGCCCCGGTCCTTGC

[0083] TCACCGCCTCCACCGCGTCCCACTGCTCCAACGCCTCCATGCTCACCTGCATCA

[0084] CCAAAGACCACTCATGGTAGTTGGTCCTTGTCAATAGCGGGAGCTCCACGACG

[0085] CTGGAGTTGGCCATGACGCGCTCGGCGGCACCCGAGCTCGAGCCTTCTCCGATC

[0086] TTCGGTGGCGAGCTATCTCCGATCTTCGTCATCCGGCTCTGATACCAATTGTTG

[0087] GCTCAGCCCAGGAACACTCTCACACTCACGAACACAAACGCCGGTGAGGGAGA

[0088] CAATGAACAATGGATTTTACAGCGACGAACGCCGCGGAGCCGATCGCTCCTGT

[0089] ATCTTGGCTTTATTACTCACTGAACCTTGCTAAGAAACAACGAAGGAGTACAGG

[0090] GGCCTCTTATAGACCGGTGACACACTGGGAGCTAGCCTCCATGCCGGCCATTGC

[0091] GCCACGTTCCGCAGAACCCGCTCGACGTCGCCGTGAGCCACACGCTCGCCCGG

[0092] ACGACGCGGCTTCGTCCGCAAGAACCGACGCCACGCACACCATGCACACCCAC

[0093] GACTCGATCAACACTGCAACGGCCATGCAAACAACCATGCAGAAGCCATGCAC

[0094] TCGCGATCACGACTCAATCAAACTAACAGAACACACACGACGCACACATGCAG

[0095] CAGCACGACACACACACACTGACTCAACCACGCCAGGAATACACCATGACGTG

[0096] TTTATTTCCAACACTTCTGCTCCGATCCTCTGCTTCAAAAACCCTTCAACCCCCA

[0097] CCTGCTCGCTTCAAAACCCCCTTCCAAGAACAAGAAAAGCATCTTCTATGAATC

[0098] GCCATGGCCCCAAAGAAAGAACAGCCAGTGAAGGAGGTACGGAATCTCAACA

[0099] CTCCTTATTCCTGTTTGTGTGTCGTGCGCCATGACTTTGGTTCTTCAAAGATCAT

[0100] GGGGATTTCAAATGAAAAAAATGATCTTTTTTTTTCCTCTCCTTGCTTGTTGTTT

[0101] CTTGTCAAGCCCCCTGCATCAGATGAATTCGATCTCGTGCTGAATGATTTGCCT

[0102] GAAAGCTACAAGAAGCTCATGGAGGACGCCATTCCAACAAGATTAGAACAGAT

[0103] AATCATACAGGAAGCTCCGTCCAATAGCAAAGGCAAAAAGCTCTTCTCCAAGG

[0104] TTGCTGAGAGACTCTACACGCCCAAAGGTGGGACTTTTCTGGTGAAGTTGAAGC

[0105] CGACCAAGAGCAGTCCAGATGAGGAAATCGTAACACTCCTGTTCCGATGGAAA

[0106] GACCTCTACTTTGAGGCCTTTCATGCTAAAGGTAAGATTCTTTGCTAAATGCAA

[0107] ATGCATTTCCATTCTTGATAATTAAGGGAGGATCAGAGATTGATGCTAGCTGCT

[0108] TACAGAGGAGGAGAAGGTTGTGTTTGTGGCCTGTGGGACGCCGGAAGGGAATG

[0109] ATAGGATAGAGAGGGAGAAAATATGAACATTGGGGTTTGCAGCACCTTATGTG

[0110] CCAACTGTTCACAACACGAAAAACTCTTGCTGTGGCAACTGAATCTGTGATATC

[0111] TGCAATATTTGACATGATAAGTTTTATCAAGAGTAATTTTTTAGATAAGGAAGA

[0112] TATTTAAAATTATCATTTGTAATCCAAAGCTAGACTTAGATCATGCCTAGCTAG

[0113] ATCATTAACATGCGCATGTTCACACGCATTTTTTTCCTTGTCCTAGTATGCCTTA

[0114] TGATGTATTCTGATAAAGAAAAATCACCATGTTAACACTTATGTAGCACAATTTC

[0115] CTGTGGGTTAAGAATCAATACACCATCGTATCGTGCATGTTGTTATTATGAAT

[0116] ACAATCTGTAATTTCACATCATACACTAAATCTATTTTGTAACTTCACATCAAA

[0117] ATTTTAATCTATTATGTGTTATAACTAGCTAAAATTTTGTGAGGGGGATGAGAA

[0118] CTCTAAAGAGATGAACTTTGCCTTGCTTCTATGGTTTTGAAAGTGTGAGAACAT

[0119] GTTTACAAATGTAAAATACAGAATGCCATGCAATGCAACTACTCAAATAGACC

[0120] AACAGTTTGGGTTTTTCCATCCATGGAATTTTGTTGCCCTTCATGCACATTAACT

[0121] GCACTACAAATTAGCATGTATTCTGATAGTTCTACATATTGCTACAGTTTTTTTC

[0122] CCCTTATAGTATATATCGTTCTGTACTTCTTTTGTATACATTAGCATGTTTCAAA

[0123] ATCACTAGCTACTTGCTTCAGTTGGTTTTTCGCACAATATTACCGATGATTATATT

[0124] ATCAACAACTGCTTAACTAGACTACAAAACATGCACTTCTAGTAGTTGCCTTTT

[0125] TTCTGCTGTTGTTTTATTGCATTAGGATAAAACAGTCTGACATGCATGTATTTTC

[0126] TTTTCCAAAATCCAGACTTGCTTTTAATTGGTTGGTGGTAGCATATTAGCATCA

[0127] GTTAGAAGGGTGCACATACCTGCCCAATGAGGGTGCACATACCTGCCCAATGT

[0128] GTACTGTGAATATTCCTCTGCATCCTGTACCACCAGTTTAACTAGCTCACAAGT

[0129] CGGTGGTTATTATCTTAGCTTCTACTAGTAGATACCAGCATGCATGTGTTCCCC

[0130] AATTATAATGTACTATGTTGTCTTCTTACTGTGACAGGAAAATGGTATAGGATG

[0131] AGCGACGCTGAAGAGAGTCTGCCTCCGCGTTCTCAACTGCACTACTCCAAGAA

[0132] GGAGAAGGAAGGGGTATTTAATATGAACAATATTAGCACAAGTTATAATGATG

[0133] TAGGTGGCCATAACATTGAAGTAGGCAGAAGGGCATTCAAGAATTGTCACCAA

[0134] TCCCTACTCATGGCAGAAGAGTTGGTCAGACAGAAGCGCCTGAAGGAAGAACT

[0135] TGGGTCAGGCCCATTGAGTCTGCCGGTCGTGACCATCTCAGAATCCATCCGCTT

[0136] CCCGCTCTTGCAGCGATGGGTTTTAGGCACTTTCTCTGCACCTCCTACAGCTAA

[0137] GTCTGAGAAGAAGGTTCCTAAGAAATTTTCCTGTGAGTTCAACGAATGGGGCA

[0138] AGTACTCTCGTGCTTTGTTCACACAGGAGCTTCCAGTTGGCTGTGAACTAACTT

[0139] TTGCTCAGATTGCTGAAAAACTGCGGGTACTCAAGTACAGAGCGGCATGGGTG

[0140] CCTCAACCAGCACAGAAGCATGTCAAGGACGTGTAACTGGAGTAGCTGGAGAC

[0141] CAGCTGATCTTTTCGCGTGGAGAAACCCTACATGCCATTTTGGACATTCTGTTT

[0142] GTGTAGGAGTCCTGGAGTAGCTAGTTGGTCTTAATTAATATGTTTCAGTTAGCC

[0143] ATATGCTGGTATCTCTGTTCTTTTGACAAGACATGTACATTGATATCAGTCATTT

[0144] TTGTAAGGATCTTTCCCCCCTCTCCCTCTATATATATCTCCTGATGATCATTGCA

[0145] AACCAACATCATCAGCAATATACATATTCTGGGAATTGAGATTCTACTATATTC

[0146] TTCCATGGGGCTTTTAGCCAACTCGTGGTAGTTAATCTTTATTACCTCCGTGGAG

[0147] AATGTGCTTGTTCAAAAAAGAATCTTCACTTATGTCGACTGAGCAAAAGTTTGC

[0148] AATGCATGAACGAATACGTAACCTGGGTCTTATGCATGATGATTATTTAATGTA

[0149] CAGTAGTTAGACTAACGTCATTAGTTTAAACAAGGGGAGTAATAATAAATAAG

[0150] CACACTACTGCTAGTACGTGACGTAGGCTGTCTACTTGTCTAGTCTAATTCAAC

[0151] CCGGGCACTTCGCTTCAGTCTTCACCACGTATCTAGCTAAACCATTCCCAAAAT

[0152] TTTATCATATCGCAGTATAACTTGCGGCGAAAAATGCCGGAAAAACGCTCAAA

[0153] ACATTTATTTACTCTTTTTTTCTTCAAAACGAAATGGGTGTGGTAGAGTCAGAG

[0154] ACCATAACGATATCAATACTATCAAAACATCTTAAAATACCAGAAACGGAATT

[0155] ATCGGAATAGAGCCTCGGAAACAACGATATTTGAATACATCTGTCGGCGACAT

[0156] GGGTCCGGGAGTATCATGACTAGAGGCTTGAGGTAGACACAATCGCCCACGTG

[0157] GCCTGGCACCCTCGGGGGACGTCGGGCCCGAGGGTGACGTGTTCGCCCTCCTCT

[0158] TAGTCTCCCCGAGGGGGTCGGACTGCTCCCGCCTCGGCCCCGAGGGCCGAGGC

[0159] GCCCCGACCCCTCGTGGGTTCTGCTCCGCGTGTGCGGGTTAGGTGAGCACAGCG

[0160] GCGCTCACCTAACCGCATTTATGGCGGTTTGGCTGAGCGTGTCATGCCGCATGT

[0161] AGCGTAGTGCAATGCACTCTTTTATCCGGTCTGTGACCAGTCACAGACCGGTCA

[0162] GATCGCGGGTTAGGTGGCGACAGGCGATCTGACACACGCCTCGCCCCATCCCG

[0163] TCAGGACGAGGGCTTCTAAGCGCTCGTTCCCAGCTGGAGCTAGCGTGTTATCTC

[0164] CCAGAGATGGCACGTTAGTTCTGGTTAGATATATGCCAGGCTTCATCCTAACCA

[0165] TTACAGGCAAGATGTTTTGTGAAGAAGGGCAAACATGCATGTTGCTAAGCTGA

[0166] CGCGTGGTGGACAAGAATGACCGATTTGTGACCAGTCTGACACTGATCATGTC

[0167] GTCAGCAGACAGCCACGTTCCCACGTCGCGCCTGCCTCCGGCGGAAGTGGAGG

[0168] TAGGTATGTGACGTCCCATCAGAAGGTCATTCGGACGGCAACCATACAAATCT

[0169] CCGTCCATTTATGAAGAAAAGGCAAGTCCAGTTTAGAGAAAGGGAGCATGTGG

[0170] TATCCCCTTGAGATATAAAAGGAGGACCTTGCCCAGTTAGAGAGGGGGACTGG

[0171] ACCTTTTCCAGATTCGGAACCCAGAACAAGGGAGAGGCTGGTTCATACTTTGTA

[0172] GCTCCTTCATACACAGATCCACCAAAACACAGGAGTAGGGTATTACGCTTCTCA

[0173] GCGGCCCGAACCTGTATACATCGCCCGTGTCTTGTGCTCTTTTCGCGGTCGCGA

[0174] ATCTTCCCACATACAGAGAGAGCTTAGAATTTCATCCTGAGCCCCCGGCCGAAC

[0175] CGGCAAAGGGGGGCCTGCGCGGTCTCCCGGTGAGGAGCCCCACGCTCCGTCAT

[0176] CTGGCGCGCCAGGTAGGGGGCTTAGTGTGTGTTTTCCGAGCTCTCGTACTATTC

[0177] CGTGTGCGCCAAGCTTTTTCCTGTTCAGCCTGATGGCCGAGCACGCAAAGGCGCA

[0178] CGACCTCTCTCGAGGTGTGAGTGGCGACGACGGGGAGCCAAACCCCCGCCGTC

[0179] GAGCTCGTTCTCCGCCGCCCCCTCCACGCCAAAGTCCTAAGCGGGGGGAGGCGCTCGAGAGGGTCGAAAGATCCGCGACTTCA). In the complementary transformed SDGMS genome sequence, bases 2559-4536 are a retrotransposon insertion, bases 4631-6924 are an open reading frame (ORF) containing 3 exons (bases 4631-4663, 4799-5087, 6392-6924) and 2 introns (bases 4364-4798, 5088-6391), and bases 6925-9064 are the 3' untranslated region.

[0180] This invention provides a protein encoded by the rice tristiginal male sterility gene SDGMS, the amino acid sequence of which is shown in SEQ ID NO:2. The encoded protein is a ribosome-inactivating protein with rRNA N-glycosidase activity, capable of negatively regulating protein translation, and its biological function is to cause dominant male sterility in plants.

[0181] This invention provides a recombinant vector containing the rice tristiginal nuclear male sterility gene SDGMS.

[0182] In this invention, the backbone vector of the recombinant vector preferably includes pCAMBIA1301. The rice tristiginal nuclear male sterility gene SDGMS is preferably inserted into the backbone vector at the KpnI and BamHI multiple cloning sites.

[0183] In this invention, the method for constructing the recombinant vector preferably includes the following steps:

[0184] PCR amplification of the rice triple-dominant nuclear male sterility gene SDGMS;

[0185] The amplified rice tristiginal nuclear male sterility gene SDGMS was inserted into a backbone vector via homologous recombination, and the recombinant vector was verified.

[0186] In this invention, the primers used for amplifying the rice tristiginal nuclear male sterility gene SDGMS preferably include nucleotide sequences such as SEQ ID NO:5(ATGATTACGAATTCGAGCTC). GGTACC The forward primer and nucleotide sequence shown in SEQ ID NO:6 (GCCTGCAGGTCGACTCTAGAG, where the underlined part is the restriction endonuclease KpnI cleavage site) are as follows: GATCC The reverse primer is shown in GAAGACTGAAGCGAAGTGCC (the underlined area indicates the BamHI restriction enzyme site). The amplification reaction system is preferably 50 μl, containing the following components: 1× reaction buffer, 200 μM dNTPs, 200 ng BAC-DNA, 0.3 μM each of forward and reverse primers, and 1.0 U KODFX polymerase. The preferred amplification reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 10 s, 68℃ for 10 min, 35 cycles; 68℃ for 15 min; 25℃ for 1 min.

[0187] In this invention, the exogenous fragment of the rice tristiginal male sterility gene SDGMS is inserted into the pCMABIA1301 vector at positions 11046-11050 bp, specifically at the KpnI / BamHI restriction site. This invention does not impose any particular limitation on the homologous recombination method; any homologous recombination method well-known in the art can be used. For example, the amplified rice tristiginal male sterility gene SDGMS fragment and the backbone vector can be double-digested with restriction endonucleases, and the digested gene fragment is ligated with a linear vector to obtain a recombinant vector. The verification method involves transforming the ligation product into competent *E. coli* cells, selecting for positive transformants through antibiotic resistance, and then sequencing the positive transformants. If the sequenced fragment matches the expected sequence, the recombinant vector has been successfully constructed.

[0188] Given that the SDGMS gene is a dominant nuclear male sterility gene that can alter the fertility of wild-type rice, this invention provides the application of the rice trispecific nuclear male sterility gene SDGMS, the protein, or the recombinant vector in regulating the trispecific nuclear male sterility trait in rice.

[0189] Given that the SDGMS gene is a dominant nuclear male sterility gene, its introduction into wild-type rice varieties can create dominant nuclear male sterility lines. This invention provides the application of the rice tristigious nuclear male sterility gene SDGMS or the recombinant vector in the breeding and / or construction of male sterile rice lines.

[0190] In this invention, the method for cultivating sterile rice lines involves hybridizing a rice line carrying the rice tristiginal male sterility gene SDGMS with a rice line exhibiting the target trait, and obtaining sterile rice lines from the hybrid offspring. Preferably, the method for constructing sterile rice lines involves cloning the rice tristiginal male sterility gene SDGMS into a vector, transferring the resulting recombinant vector into rice callus tissue under Agrobacterium-mediated transformation, and then cultivating it through a genetic transformation system to obtain sterile rice lines.

[0191]

[0192] This invention provides the application of the rice tristiginal male sterility gene SDGMS and the protein as a biomarker in the detection or identification of tristiginal male sterility rice lines.

[0193] In this invention, the preferred method for detecting the rice tristiginal male sterility gene SDGMS is to detect the presence of the SDGMS gene in the rice variety under test using PCR amplification. The PCR amplification method described above is the same as in this invention and will not be repeated here.

[0194] The following detailed description, in conjunction with embodiments, illustrates the rice tristiginal nuclear male sterility gene SDGMS and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0195] Example 1

[0196] Fine mapping of the rice tristiginal male sterility gene SDGMS

[0197] Yang et al. (2012) located the gene controlling the tristigious nuclear male sterility in rice between two In-Del markers ZM30 and ZM9 on chromosome 8, with the interval approximately 99 kb from the Nipponbare genome. The inventors used the 938 background near-isogenic line (BC7F1) as material (https: / / www.ricedata.cn / variety).

[0198] The gene controlling the tristigious nuclear male sterility in rice was finely mapped using the database / varis / 614634.htm. Building upon previous research, the candidate region was expanded to approximately 500 kb of the NIP reference genome. To clone this gene, the inventors backcrossed a background sterile 938 variety with wild-type 938, planted the backcross progeny in Lingshui, Hainan, and examined the fertility of 393 individual plants. The number of fertile and sterile plants were 189 and 204, respectively, meeting the 1:1 segregation (xi / varis / 614634.htm). 2=0.57), indicating that tristiginal male sterility is a single-point dominant inheritance. Since the parental materials used for mapping lacked a reference genome, polymorphism analysis was performed on the rice parents 938 and Basmati370 used in population construction, as well as the pedigree-related parents ZS97 and Nongken 58 (parents from which the tristiginal male sterility mutation originated, SE21S), based on information from the RiceVarMap2 website (http: / / ricevarmap.ncpgr.cn / ). Molecular markers were developed for possible In-Del variations within candidate intervals. Recombinant single plants were screened using molecular markers xch43 and xch95, resulting in 188 recombinant single plants. SDGMS was mapped between markers xch7 and SM1, corresponding to a region of approximately 53.3 kb in the Nipponbare reference genome (rice TIGR / MSU annotation version 6.1). http: / / rice.plant biology.msu.edu / ).

[0199] The corresponding primers for the molecular markers are:

[0200] xch43-F:5'-GAGTTGAGAAGGCTAAAACGACT-3' (SEQ ID NO:20);

[0201] xch43-R:5'-TGTTTCCCCTTCTTGTTTGCC-3' (SEQ ID NO:7);

[0202] xch95-F:5'-ATTCCCTAGTGGTTGCAGCT-3' (SEQ ID NO:8);

[0203] xch95-R:5'-AGGCTTCAGCTCCGACTTAG-3' (SEQ ID NO:9);

[0204] xch7-F:5'-GAGATAGTGGTGGAGGTGGATGC-3' (SEQ ID NO: 10);

[0205] xch7-R:5'-GCACTTGTACTCCCATTTCTCAACC-3' (SEQ ID NO:11);

[0206] SM1-F:5'-GACTTTCTAGCATTGCCCATAT-3' (SEQ ID NO:12);

[0207] SM1-R: 5'-GGCGAAGGGGAATTGGAATAAC-3' (SEQ ID NO: 13).

[0208] Example 2: Construction of a tristigious nuclear sterility genomic BAC library and sequencing of the target BAC.

[0209] During the fine mapping process, the inventors hypothesized that significant structural variations might exist within the mapped region. Therefore, they constructed a whole-genome BAC library of the near-isogenic line 938 (BC7F1) with tristigious nuclear male sterility. This library contained 36,480 single clones, stored in 95 384-well plates, with an average insert size of approximately 110 kb, covering about 10 times the rice genome. Using the markers xch7 and xch95 for the mapped region, two BAC fragments containing the target region, 62-H-5 and 9-B-10, were screened from the BAC library. Sequencing of these two markers confirmed that 62-H-5 was the fertile genotype, while 9-B-10 was the male-sterile genotype. Sequencing of these two BAC fragments, followed by sequence alignment with the reference genomes of Nipponbare and Zhenshan 97, identified SDGMS (SEQ ID NO:1) as the unique candidate gene within the mapped region.

[0210] xch7-F:5'-GAGATAGTGGTGGAGGTGGATGC-3' (SEQ ID NO: 14);

[0211] xch7-R:5'-GCACTTGTACTCCCATTTCTCAACC-3'(SEQ ID NO:15)

[0212] xch95-F:5'-ATTCCCTAGTGGTTGCAGCT-3'(SEQ ID NO:16)

[0213] xch95-R: 5'-AGGCTTCAGCTCCGACTTAG-3' (SEQ ID NO: 17).

[0214] Example 3: Transgenic Complementation Experiment of SDGMS

[0215] Based on the predicted full-length candidate gene sequence, a pair of oligonucleotide primers with KpnI and BamHI restriction endonuclease linkers were designed for PCR amplification. The primer sequence is shown in SEQ ID NO:5(5'-ATGATTACGAATTCGAGCTC). GGTAC C GACTCGACGGGGACAGCTAG-3' (underlined is the restriction endonuclease KpnI cleavage site) and SEQ ID NO:6 (5'-GCCTGCAGGTCG ACTCTAGA GGATCC The underlined line GAAGACTGAAGCGAAGTGCC-3' indicates the restriction endonuclease BamHI cleavage site.

[0216] A 9064 bp fragment containing a promoter, coding region, and downstream termination sequence was amplified from the BAC fragment 9-B-10 using PCR. The PCR reaction system was as follows: 50 μl of reaction mixture contained 1× reaction buffer, 200 μM dNTPs, 200 ng BAC-DNA, 0.3 μM primers, and 1.0 U KODFX polymerase. The reaction program was as follows: Step 1: 98℃ for 2 min. Step 2: 98℃ for 10 s, 58℃ for 10 s, 68℃ for 10 min (35 cycles). Step 3: 68℃ for 15 min. Step 4: 25℃ for 1 min. After the reaction, the PCR product was purified. The vector pCAMBIA1301 (CAMBIA, Canberra, Australia) was digested with restriction endonucleases KpnI and BamHI, and the PCR product was ligated into the vector via homologous recombination. Sequencing was performed to select the correct, mutation-free clone vector, which was then introduced into Agrobacterium EHA105. Callus tissue was induced from mature seeds of Nipponbare on an induction medium.

[0217] EHA105, containing the target gene transformation vector, was used to infect callus tissue of Nipponbare rice. After co-culture, screening for callus resistant to hygromycin, differentiation, rooting, hardening, and transplanting, transgenic rice plantlets were obtained.

[0218] The main steps of genetic transformation, the culture medium, and its preparation method are described below:

[0219] (1) Abbreviations for reagents and solutions

[0220] The abbreviations for plant hormones used in the culture medium in this invention are as follows: 6-BA (6-Benzylaminopurine); CN (Carbenicillin); KT (Kinetin); NAA (Napthaleneacetic acid); IAA (Indole-3-aceticacid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); CH (Casein Enzymatic Hydrolysate); HN (Hygromycin B); DMSO (Dimethyl Sulfoxide); N6max (N6 macronutrient solution); N6mix (N6 micronutrient solution); MSmax (MS macronutrient solution); MSmix (MS micronutrient solution)

[0221] (2) Main solution formulation

[0222] 1) N6 max Culture medium stock solution of macroelements (prepared according to 10x concentrate (10×)):

[0223]

[0224] Dissolve each of the above reagents one by one, then dilute to 1000 mL with distilled water at room temperature and store at room temperature.

[0225] 2) N6 min Culture medium trace element stock solution (prepared according to 100x concentrate):

[0226]

[0227] Dissolve the above reagents at room temperature and bring the volume to 1000 mL with distilled water. Store at room temperature.

[0228] 3) Iron salts (Fe 2+ EDTA stock solution (prepared according to 100× concentrate):

[0229] Dissolve 3.73g of disodium ethylenediaminetetraacetate (Na2EDTA·2H2O) and 2.78g of FeSO4·7H2O separately, mix them, and bring the volume to 1000mL with distilled water. Incubate at 70℃ for 2 hours, and store at 4℃ for later use.

[0230] 4) Vitamin stock solution (prepared according to 100× concentrate):

[0231]

[0232] Add distilled water to a final volume of 1000 mL and store at 4°C for later use.

[0233] 5) MS medium for macro-elements (MS) max Mother liquor (prepared according to 10× concentrate):

[0234]

[0235] Dissolve the above reagents at room temperature and bring the volume to 1000 mL with distilled water. Store at room temperature.

[0236] 6) MS medium trace element stock solution (MS) min Mother liquor (prepared according to 100× concentrate):

[0237]

[0238] Dissolve the above reagents at room temperature and bring the volume to 1000 mL with distilled water. Store at room temperature.

[0239] 7) Preparation of 2,4-D stock solution (1 mg / mL):

[0240] Weigh 100 mg of 2,4-D, dissolve it in 1 mL of 1N potassium hydroxide for 5 min, then add 10 mL of distilled water to dissolve completely and bring the volume to 100 mL. Store at room temperature.

[0241] 8) Preparation of 6-BA stock solution (1 mg / mL):

[0242] Weigh 100 mg of 6-BA, dissolve it in 1 mL of 1N potassium hydroxide for 5 min, then add 10 mL of distilled water to dissolve completely and bring the volume to 100 mL. Store at room temperature.

[0243] 9) Preparation of naphthaleneacetic acid (NAA) stock solution (1 mg / mL):

[0244] Weigh 100 mg of NAA, dissolve it in 1 mL of 1N potassium hydroxide for 5 min, then add 10 mL of distilled water to dissolve completely and bring the volume to 100 mL. Store at 4°C protected from light.

[0245] 10) Preparation of indoleacetic acid (IAA) stock solution (1 mg / mL):

[0246] Weigh 100 mg of IAA, dissolve it in 1 mL of 1N potassium hydroxide for 5 min, then add 10 mL of distilled water to dissolve completely and bring the volume to 100 mL. Store at 4°C protected from light.

[0247] 11) Preparation of glucose stock solution (0.5 g / mL):

[0248] Weigh 125g of glucose, then dissolve it in distilled water and bring the volume to 250mL. After sterilization, store at 4℃ for later use.

[0249] 12) Preparation of AS stock solution:

[0250] Weigh 0.392g of AS, dissolve it in 10mL of DMSO, dispense it into 1.5mL centrifuge tubes, and store at -20℃ for later use.

[0251] 13) Preparation of 1N potassium hydroxide stock solution:

[0252] Weigh 5.6g of potassium hydroxide, dissolve it in distilled water and bring the volume to 100mL. Store at room temperature for later use.

[0253] (3) Culture medium formulation for rice genetic transformation:

[0254] 1) Induction medium

[0255]

[0256] Add distilled water to 900 mL, adjust the pH to 5.9 with 1N potassium hydroxide, boil and bring the volume to 1000 mL, dispense into 50 mL Erlenmeyer flasks (30 mL / flask), seal and sterilize using conventional methods (e.g., sterilize at 121°C for 15 min; the sterilization method for the culture medium described below is the same as the sterilization method for this culture medium).

[0257] 2) Subculture medium:

[0258]

[0259] Add distilled water to 900 mL, adjust the pH to 5.9 with 1N potassium hydroxide, boil and bring the volume to 1000 mL, dispense into 50 mL Erlenmeyer flasks (30 mL / flask), seal, and sterilize as described above.

[0260] 3) Pre-culture medium (this step can be omitted for japonica rice):

[0261]

[0262] Add distilled water to 250 mL, adjust the pH to 5.6 with 1 N potassium hydroxide, seal, and sterilize as described above.

[0263] Before use, heat and dissolve the culture medium, then add 5 mL of glucose stock solution and 250 μL of LAS stock solution, and dispense into petri dishes (25 mL / dish).

[0264] 4) Suspension culture medium:

[0265]

[0266] Add distilled water to 100 mL, adjust the pH to 5.4, dispense into two 100 mL Erlenmeyer flasks, seal, and sterilize as described above.

[0267] Add 1 mL of sterile glucose stock solution and 100 μL of LAS stock solution before use.

[0268] 5) Co-culture medium:

[0269]

[0270] Add distilled water to 250 mL, adjust the pH to 5.6 with 1 N potassium hydroxide, seal, and sterilize as described above.

[0271] Before use, heat and dissolve the culture medium, then add 5 mL of glucose stock solution and 250 μL of LAS stock solution, and dispense into petri dishes (25 mL / dish).

[0272] 6) Screening culture medium:

[0273]

[0274] Add distilled water to 250 mL, adjust the pH to 6.0, seal, and sterilize using the method described above.

[0275] Dissolve the culture medium before use, add 250 μL HN (50 mg / mL) and 400 μL L CN (10 g CN / 36 mL water), and dispense into petri dishes (25 mL / dish). (Note: The carbenicillin concentration in the first selective medium is 400 mg / L, and the carbenicillin concentration in the second and subsequent selective mediums is 250 mg / L).

[0276] 7) Predifferentiation medium (this step can be omitted for japonica rice):

[0277]

[0278] Add distilled water to 250 mL, adjust the pH to 5.9 with 1 N potassium hydroxide, seal, and sterilize as described above.

[0279] Dissolve the culture medium before use: 250 μL HN (50 mg / mL) and 250 μL LCN (250 mg / mL), and dispense into petri dishes (25 mL / dish).

[0280] 8) Differentiation medium:

[0281]

[0282]

[0283] Add distilled water to 900 mL, and adjust the pH to 6.0 with 1 N potassium hydroxide.

[0284] Boil and dilute to 1000mL with distilled water, dispense into 100mL Erlenmeyer flasks (50mL / flask), seal, and sterilize as described above.

[0285] 9) Rooting medium

[0286]

[0287] Add distilled water to 900 mL, and adjust the pH to 5.8 with 1N potassium hydroxide.

[0288] Boil the contents and dilute to 1000 mL with distilled water. Dispense the contents into rooting tubes (25 mL / tube), seal the tubes, and sterilize them as described above.

[0289] (4) Steps of Agrobacterium-mediated genetic transformation:

[0290] 3.1 Callus Induction

[0291] 1) Remove the husks from mature Zhonghua 11 rice seeds, then treat them sequentially with 70% ethanol for 1 min and 0.15% mercuric chloride (HgCl2) for 15 min.

[0292] 2) Wash the seeds 4-5 times with sterile water;

[0293] 3) Place 8-10 seeds on the induction medium;

[0294] 4) Place the inoculated culture medium in the dark for 4 to 5 weeks at a temperature of 26±1℃.

[0295] 3.2 Callus succession:

[0296] Select bright yellow, firm and relatively dry seed embryogenic callus from Nipponbare seeds and culture them in the dark for 2 weeks on a subculture medium at a temperature of 25±1℃.

[0297] 3.3 Pre-culture:

[0298] Select firm and relatively dry embryogenic callus and culture them in the dark for 2 weeks on pre-medium at a temperature of 26±1℃.

[0299] 3.4 Agrobacterium culture:

[0300] 1) Streaking and pre-culturing Agrobacterium EHA105 (this strain is from the publicly available Agrobacterium strain from CAMBIA) on LA medium with corresponding resistance selection (LA medium preparation refers to J. Sambrug et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, translated by Jin Dongyan et al., Science Press, 2002, Beijing) for two days at 28°C.

[0301] 2) Transfer Agrobacterium to suspension culture medium and incubate on a shaker at 28°C for 2-3 hours.

[0302] 35 Agrobacterium infection:

[0303] 1) Transfer the pre-cultured callus to a sterilized bottle;

[0304] 2) Adjust the Agrobacterium suspension to OD. 600 To 0.8–1.0;

[0305] 3) Soak the callus in Agrobacterium suspension for 30 minutes;

[0306] 4) Transfer the callus to sterilized filter paper and blot dry; then place it on a co-culture medium and incubate for 3 days at a temperature of 19-20℃.

[0307] 3.6 Callus washing and selective culture:

[0308] 1) Wash the callus with sterile water until Agrobacterium is no longer visible;

[0309] 2) Soak in sterile water containing 400 mg / L carbenicillin (CN) and shake for 30 minutes;

[0310] 3) Transfer the callus to sterilized filter paper and blot dry;

[0311] 4) Transfer the callus to a selective medium and culture it 2-3 times, 2 weeks each time, until good resistant callus grows.

[0312] 3.7 Differentiation:

[0313] 1) Transfer the resistant callus to predifferentiation medium and culture in the dark for 5-7 days;

[0314] 2) Transfer the predifferentiated callus to differentiation medium, with three independent callus distributed evenly in each bottle. Culture under light for 5-6 weeks until large seedlings grow, at a temperature of 26℃.

[0315] 3.8 Rooting and hardening off:

[0316] 1) Cut off the old roots that develop during differentiation;

[0317] Then transfer it to a rooting medium and culture it under light for 2-3 weeks until large seedlings grow. After that, remove the sealing film, add some tap water to harden the seedlings for a week, and then transplant them at a temperature of 26℃.

[0318] 3.9 Transplanting

[0319] Wash away any residual culture medium from the roots, transfer seedlings with good root systems to a greenhouse, keep them moist for the first few days, and then transplant them to the field when they are growing vigorously.

[0320] 4.0 Identification of transgenic positive plants

[0321] Leaves of transgenic plants were collected and total DNA was extracted. Transgenic positive plants were identified using primers for amplifying the hygR gene (which is located on the transformation vector and serves as a selection resistance gene, and will be inserted into the rice genome along with the target gene).

[0322] hygR-F:5'-ACACTACATGGCGTGATTTCAT-3' (SEQ ID NO: 18);

[0323] hygR-R: 5'-TCCACTATCGGCGAGTACTTCT-3' (SEQ ID NO: 19).

[0324] This invention yielded 25 independent transgenic complementary T0 generation rice plants, including 18 positive plants and 7 negative plants. The positive plants were planted in the field, and pollen was collected during heading and flowering. The pollen was stained with potassium iodide and examined under a microscope to assess the spikelet fertility of the T0 generation. The spikelet fertility was assessed based on the seed setting rate, as shown in Formula I.

[0325] Seed setting rate (%) = (Number of grains per spike / Number of spikelets per spike) × 100% Formula I

[0326] The spikelet fertility of transgenic negative single plants was not significantly different from that of wild-type Nipponbare (NIP). Five transgenic positive single plants had a spikelet fertility of 0, which is completely sterile. The spikelet fertility of the remaining single plants was also less than 35%, which is significantly lower than that of transgenic negative and wild-type NIP.

[0327] After harvesting seeds from the T0 generation plants, they were planted in the field to continue observing the T1 generation phenotype. Phenotypic examination was conducted at maturity, and positive tests were performed on individual T1 generation plants (using the same method as above). At the same time, spikelet fertility was examined, and pollen iodine staining was performed.

[0328] The results showed that the spikelet fertility of positive plants carrying the transgenic fragment was significantly reduced, all below 30%, while the spikelet fertility of negative plants was approximately 70%. Simultaneously, we backcrossed two completely sterile plants from the T0 generation with wild-type NIP, and conducted positive tests and spikelet fertility assessments on the backcross families. The results showed that plants carrying the transgenic fragment exhibited a sterile phenotype, with a large number of plants showing 0% spikelet fertility, and only a few plants producing a small amount of seeds. Pollen iodine staining results showed that sterile plants in the families had no pollen grains or only a small number of unstained, wrinkled pollen grains. The seed production of negative transgenic plants was similar to that of wild-type NIP, showing normal fertility.

[0329] Replacing the promoter with the corn Ubiquitin promoter increased SDGMS expression levels. The specific steps are as follows:

[0330] Based on the predicted full-length candidate gene sequence, a pair of oligonucleotide primers with KpnI and BamHI linkers for PCR amplification were designed. The primer sequence is shown in SEQ ID NO:5(5'-ACCATTTACGAACGATAGCC). GGTAC C TGGCAGCAAACCAAATATGA-3' (underlined is the restriction endonuclease KpnI cleavage site) and SEQ ID NO:6 (5'-TGATTTTTGCGGACTCTAGA) GGATCC CCCATCTCGCCATTCTTCT-3' (the underlined area is the restriction endonuclease BamHI cleavage site) is shown.

[0331] A 7377 bp fragment containing a promoter, coding region, and downstream termination sequence was amplified from BAC fragment 9-B-10 using PCR. The PCR reaction system was as follows: 50 μl of reaction mixture contained 1× reaction buffer, 200 μM dNTPs, 200 ng BAC-DNA, 0.3 μM primers each, and 1.0 U KODFX polymerase. The reaction program was as follows: Step 1: 98℃ for 2 min. Step 2: 98℃ for 10 s, 58℃ for 10 s, 68℃ for 10 min (35 cycles). Step 3: 68℃ for 15 min. Step 4: 25℃ for 1 min. After the reaction, the PCR product was purified. The vector pCAMBIA1301u (CAMBIA, Canberra, Australia, Characterization of Os bZIP23 as a Key Player of the Basic Leucine Zipper Transcription Factor Family. Yong Xiang, Ning Tang, Hao Du, Haiyan Ye, Lizhong Xiong Plant Physiology, 2008, 148(4):1938-1952 DOI:10.1104 / pp.108.128199) was digested with restriction endonucleases KpnI and BamHI, and the PCR product was ligated to the vector by homologous recombination (see Figure 4 The correct cloning vector without mutations was selected through sequencing and introduced into Agrobacterium EHA105. Mature seeds of Nipponbare were then used to induce callus formation on induction medium.

[0332] The successfully constructed recombinant vector carrying the maize Ubiquitin promoter and SDGMS gene was transformed into wild-type Nipponbare using the genetic transformation system construction method described above. The transgenic positive plants were identified using the above method, and the spikelet fertility of the transgenic positive plants was also tested.

[0333] The results showed that the transgenic positive single plantlets exhibited a completely sterile phenotype.

[0334] The results of this experiment demonstrate that transforming the dominant nuclear male sterility gene SDGMS into wild-type rice varieties can create dominant nuclear male sterility lines; simultaneously, increasing the expression level of SDGMS can increase the degree of sterility. This experiment also proves that this gene has the biological function of dominant nuclear male sterility. This invention identifies the gene cloned from the SDGMS locus of tristigious nuclear male sterility material as the target gene, namely the tristigious nuclear male sterility gene SDGMS.

[0335] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rice tristiginal male sterility gene SDGMS Its characteristics are, The nucleotide sequence of the gene SDGMS is shown as SEQ ID NO:

3.

2. A rice tristiginal male sterility gene comprising the one described in claim 1 SDGMS Recombinant carriers.

3. The recombinant vector according to claim 2, characterized in that, The scaffold vector of the recombinant vector includes pCAMBIA1301.

4. The recombinant vector according to claim 3, characterized in that, The rice triadic nuclear male sterility gene SDGMS The insertion position in the skeleton carrier is Kpn I and Bam HI multiple cloning site.

5. Overexpression of the rice tristiginal male sterility gene as described in claim 1 SDGMS Or the application of the recombinant vector according to any one of claims 2 to 4 in enhancing the degree of tristiginal male sterility in rice.

6. Overexpression of the rice tristiginal male sterility gene as described in claim 1 SDGMS Or the application of the recombinant vector described in any one of claims 2 to 4 in the cultivation and / or construction of tristigious male-sterile rice lines.

7. The application according to claim 6, characterized in that, The rice triadic nuclear male sterility gene SDGMS Jointly improve the SDGMS The application of expressed elements in breeding and / or constructing tristigrinogenic male sterile rice lines.

8. The application according to claim 7, characterized in that, The element includes corn. Ubiquitin Promoter; the corn Ubiquitin The nucleotide sequence of the promoter is shown in SEQ ID NO:

4.

9. The rice tristiginal male sterility gene as described in claim 1 SDGMS Application as a biomarker in the detection or identification of tristiginal male sterile rice lines.

Images