Application of High Temperature-sensitive Rice Leaf Whitening Gene OsHCD1 in Regulating Rice Leaf Color Traits

Abstract

The application discloses application of a high-temperature sensitive rice leaf whitening gene OsHCD1 in regulation of rice leaf color traits, and a genomic sequence of the whitening gene OsHCD1 is shown as SEQ ID NO. 1. The high-temperature sensitive whitening gene OsHCD1 is cloned from a rice leaf whitening mutant oshcd1 by a map-based cloning technique; the OsHCD1 gene in a two-line sterile line of rice is knocked out by using a gene editing system, and a two-line sterile line carrying a leaf whitening trait is obtained; and the gene OsHCD1 can be applied to the field of rice breeding, and has important significance for false hybrid removal and identification of seed purity of a sterile line.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant genetic engineering and rice molecular breeding technology, and particularly relates to application of a high-temperature-sensitive rice leaf albino gene OsHCD1 in regulation of rice leaf color traits. BACKGROUND

[0002] Rice (Oryza Sativa L.) is one of the most important food crops in China, and its yield is closely related to the food security of China. Leaf is the most important source organ (Okita et al., Increasing rice productivity and yield by manipulation of starch synthesis. Novartis Found Symp, 2001, 236: 135-146; Julius et al., Sugar transporters in plants: new insights and discoveries. Plant Cell Physiol, 2017, 58: 1442-1460), and its most important function, photosynthesis, depends on the growth and development of chloroplasts. Chloroplast, as a unique organelle in plants, is believed to originate from endosymbiotic cyanobacteria (McFadden GI. Primary and secondary endosymbiosis and the origin of plastids. Journal of Phycology, 2001, 37(6): 951-959). The organelle belongs to a semi-autonomous organelle, has the ability to encode its own genetic material, but its assembly and development are not only regulated by nuclear genes, but also are extremely susceptible to external environment, especially light and temperature. As the only place in plants that can convert light energy into stable chemical energy and fix carbon dioxide, the development of chloroplasts directly affects the normal growth and development of plants.

[0003] Leaf color mutation is a common mutation trait, and its traits usually appear in the seedling stage, and a few of them show mutation traits until the late growth period. Among them, leaf albino is the most common leaf color mutation, which is mainly caused by the failure of chloroplast development in mesophyll cells under certain conditions, resulting in extremely low or even no chlorophyll content (Chent et al., Physiological character and gene mapping in a new green revertible albinomutant rice. J Genet Genomics, 2007, 34:331-338). In recent years, the utilization value of leaf color mutants has attracted more and more attention. Not only can they be used for basic research to broaden the understanding of a series of physiological metabolic processes such as photosynthesis, photomorphogenesis, hormone physiology, and disease resistance mechanisms, but also can be used for analyzing and identifying gene function and gene interaction. In breeding, leaf color variation can be used as a marker trait to simplify elite breeding and hybrid production, improve breeding efficiency, and eliminate false hybrids. Some leaf color mutants have specific traits in production practice and modern commodity economy, which can be studied in terms of physiological mechanisms and developed into new varieties. For example, through mutation or hybridization, sterile lines with white albino leaves have been bred, such as Yunfeng 66A (Li Xiaolin et al., Breeding of rice seedling white-to-green leaf color marker indica sterile line "Yunfeng 66A". Southwestern China Journal of Agricultural Sciences, 2011, 24:410-413), NHR111S (Wu Wei et al., Two-line hybrid rice sterile line NHR111S carrying white-to-green leaf color marker. Chinese Journal of Nuclear Agricultural Sciences, 2006, 20:103-105), Baifeng A (Shen Shengquan et al., Application research on practical green-type leaf color marker sterile line Baifeng A. Chinese Journal of Rice Science, 2004, 18:34-38), Yutu S (Zhao Haijun et al., Breeding of photoperiod-thermo-sensitive male sterile line Yutu S carrying white-to-green leaf color marker and its characteristics. Chinese Journal of Rice Science, 2004, 18:515-521), Yunfeng 88A (Li Xiaolin et al., Breeding of rice leaf margin white leaf color marker three-line sterile line Yunfeng 88A. Seed, 2011, 30:109-111), Nanfeng 8S (Lu Huajin et al., Breeding of rice photoperiod-thermo-sensitive male sterile line Nanfeng 8S with white-to-green leaf color marker at the seedling stage. Hybrid Rice, 2012, 27:16-18), and Anji White Tea, a mutant material with stage-specific whitening, has increased free amino acid content during the whitening period, resulting in unique flavor and high nutritional value, which has driven the economic value up. It has become a valuable tea tree germplasm resource (Cheng Hao et al. Physiological and biochemical nature of Anji White Tea's specific traits. Tea Science, 1999, 19(2):87-92). In addition, there is a type of mutation in leaf color mutation, which is evergreen phenotype, that is, the leaves delay senescence and the chlorophyll content and photosynthetic capacity remain unchanged during the late growth period.In the production practice of tobacco, the use of such mutations can increase the biomass and seed yield of tobacco by 40% and 52%, respectively (Gan and Amasino. Inhibition of leaf senescence by autoregulated production of cytokinin. Science, 1995, 270(5244): 1986-1988). Nowadays, crops can also be used as ornamental plants, and the use of suitable leaf color mutants can breed colorful plants. For example, the National Agriculture and Food Research Organization of Japan has developed two new varieties of colored rice, Oshu-Kan 378 and Oshu-Kan 379, which can be used for fresh cut flowers and dried flowers.

[0004] Pentatricopeptide repeat (PPR) proteins are a class of proteins containing pentatricopeptide repeat domains. This class of proteins is one of the largest families in plants, and is usually localized in mitochondria or chloroplasts, editing and modifying the RNA transcribed from the genomes of these organelles, participating in the regulation of many important histogenesis and organ formation processes, and also participating in the response to environmental factors. Rice contains 477 genes encoding PPR proteins. Based on the types and numbers of PPR tandem repeat motifs and their arrangement, these PPR proteins are divided into two subgroups, P and PLS (Chen et al. Genome-wide analysis of the rice PPR gene family and their expression profiles under different stress treatments. BMC Genomics, 2018, 19: 720). The P class is composed of a classic 35-amino acid motif arrangement, mainly involved in the transcriptional regulation of organelle genes; while the PLS subfamily is composed of short S, long L and classic P motif arrangements, mainly editing and modifying the RNA transcribed from the genomes of organelles. So far, several PPR genes regulating rice leaf albino traits have been cloned, such as YSA (Su et al. Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production. Plant Physiol, 2012, 159(1): 227-238), OspTAC2 (Wang et al. OspTAC2 encodes a pentatricopeptide repeat protein and regulates rice chloroplast development. J Genet Genomics. 2016, 43(10): 601-608), CDE4 (Liu et al. CDE4 encodes a pentatricopeptide repeat protein involved in chloroplast RNA splicing and affects chloroplast development under low-temperature conditions in rice.J Integr Plant Biol. 2021, 63(10): 1724-1739), OsPPR6 (Tang et al. OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. Plant Mol Biol. 2017, 95(4-5): 345-357), ALS3 (The rice ALS3 encoding a novel pentatricopeptide repeat protein is required for chloroplast development and seedling growth. Rice (NY). 2015, 8: 17), TCD10 (Wu et al. The rice pentatricopeptide repeat gene TCD10 is needed for chloroplast development under cold stress. Rice (NY). 2016, 9(1): 67), OsV4 (Gong et al. The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress. J Integr Plant Biol. 2014, 56(4): 400-410), and OsPPR1 (Gothandam et al. OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis. Plant Mol Biol. 2005, 58: 421-33), wherein the leaf white trait caused by mutation of YSA, CDE4, TCD10 and OsV4 genes is closely related to low temperature.

[0005] However, the temperature in the seeding season of single-crop rice and late rice of double-crop rice is relatively high, and therefore, in order to improve hybrid purity, a high-temperature sensitive albino gene marker sterile line and hybrid thereof are required. However, there are few reports on high-temperature sensitive rice albino mutants, and therefore, it is urgently needed to cultivate new high-temperature sensitive leaf albino materials by using modern molecular breeding methods. SUMMARY

[0006] The application not only provides the use of the high-temperature sensitive rice leaf albino gene OsHCD1 in regulating rice leaf color traits, but also provides a high-temperature sensitive rice leaf albino mutant gene oshcd1, which can be used for molecular breeding of rice leaf color traits.

[0007] The specific technical solutions are as follows:

[0008] The application provides the use of the high-temperature sensitive rice leaf albino gene OsHCD1 in regulating rice leaf color traits, and the genomic sequence of the gene is shown as SEQ ID NO. 1.

[0009] Further, the application provides the use of the high-temperature sensitive rice leaf albino gene OsHCD1 in participating in molecular breeding of rice leaf color traits, and the genomic sequence of the gene is shown as SEQ ID NO. 1.

[0010] Further, the application provides the use of the high-temperature sensitive rice leaf albino gene OsHCD1 in identifying hybrid purity of F1 generation of hybrid rice and / or identifying seed purity of sterile line self-pollination, and the genomic sequence of the gene is shown as SEQ ID NO. 1.

[0011] The application first screens a leaf albino mutant oshcd1 from offspring of EMS mutagenized indica rice restorer lines, and then finds the gene OsHCD1 for regulating rice leaf albino traits through fine mapping. Finally, it is determined that the leaves of mutant plants obtained by editing, interfering or knocking out the OsHCD1 gene appear albino traits under the condition of greater than 28 DEG C, by genetic complementation verification and construction of the OsHCD1 gene knockout vector. The editing refers to adding, substituting, inserting or deleting one or more nucleotides in the coding nucleotide sequence of the wild-type gene by using gene editing technology such as CRISRP / Cas9 technology to generate an allele.

[0012] The application further provides a high-temperature sensitive rice leaf albino mutant gene oshcd1, and the genomic sequence of the gene is shown as SEQ ID NO. 2.

[0013] The high-temperature sensitive rice leaf albino gene oshcd1 is a mutant gene of OsHCD1, that is, a mutant gene of a 2-oxoglutarate-dependent dioxygenase gene, and the genomic sequence of the gene is shown as SEQ ID NO. 2.

[0014] The application also provides application of the high-temperature sensitive rice leaf albino gene oshcd1 in molecular breeding of rice leaf color traits.

[0015] The application also provides application of the high-temperature sensitive rice leaf albino gene oshcd1 in hybrid rice F1 hybrid purity identification and / or sterile line self-pollination seed purity identification.

[0016] The application also provides a method for removing false hybrids in hybrid breeding, comprising the following steps:

[0017] (1) constructing a mutant plant in which the high-temperature sensitive rice leaf albino gene OsHCD1 is edited, interfered or knocked out; the genomic sequence of the high-temperature sensitive rice leaf albino gene OsHCD1 is shown as SEQ ID NO. 1.

[0018] (2) hybridizing the mutant sterile line with a normal-leaf-color rice restorer line to obtain F1 hybrids

[0019] If the F1 single plant appears a white phenotype under high-temperature conditions, it is a false hybrid of self-pollination of the sterile line, and the false hybrid is removed; the temperature is > 28 DEG C.

[0020] Further, the temperature is > 32 DEG C.

[0021] As preferred, in step (1), a mutant sterile line containing the high-temperature sensitive rice leaf albino gene OsHCD1 according to claim 4 is constructed.

[0022] The method for removing false hybrids in hybrid breeding of the application is suitable for three-line and two-line hybrid varieties.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application clones the high-temperature sensitive albino gene OsHCD1 from the rice leaf albino mutant oshcd1 by map-based cloning technology, and knocks out the OsHCD1 gene in a two-line sterile line of rice by using a gene editing system to obtain a two-line sterile line carrying a leaf albino trait; the gene OsHCD1 can be applied to the field of rice breeding, and has important significance for removing false hybrids and identifying the purity of sterile line seeds. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Phenotype and chlorophyll content of leaf bleaching mutant oshcd1 and its wild type control under different temperature conditions in Example 1;

[0026] Wherein, A, C, E and G are plant phenotype under 24℃, 28℃, 32℃ and 36℃ conditions respectively; B, D, F and H are chlorophyll content of leaf under 24℃, 28℃, 32℃ and 36℃ conditions respectively.

[0027] Figure 2 OsHCD1 gene map-based cloning result in Example 2;

[0028] Wherein, A is the linkage molecular marker of OsHCD1 gene; B is the fine mapping interval BAC clone; C is the structure of OsHCD1 gene; D is the mutation site sequencing analysis of OsHCD1 gene; E is the field phenotype of complementary transgenic offspring; F is the PCR detection of endogenous and exogenous OsHCD1 gene of transgenic offspring.

[0029] Figure 3 Creation of two-line bleaching sterile line oshcd1-2;

[0030] Wherein, A is the sequencing analysis of OsHCD1 gene mutation site; B is the field phenotype of two-line bleaching sterile line oshcd1-2; C is the chlorophyll content analysis of two-line bleaching sterile line oshcd1-2. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with specific examples, the following enumeration is only the specific examples of the present application, but the protection scope of the present application is not limited to this.

[0032] The molecular biology and biochemical methods used in the examples are all known techniques, which are described in detail in Current Protocols in Molecular Biology edited by Ausubel and published by John Wiley and Sons company, Molecular Cloning: A Laboratory Mannual, 3 rd ED. et al. The experimental materials used in the examples are all commercially available products unless otherwise specified.

[0033] Isolation and genetic analysis of mutant in Example 1

[0034] A leaf white mutant oshcd1 was screened from the progeny of a mutant library of conventional japonica rice Xiushui 134 by EMS mutagenesis. The mutant trait was stably inherited in the lines obtained by continuous backcrossing and selfing in Hangzhou and Hainan.

[0035] The mutant was planted under different temperature conditions (24℃, 28℃, 32℃ and 36℃), and the results showed that the mutant oshcd1 was sensitive to high temperature. At 28℃, the mutant showed visible white and green interlaced phenotype (C). At 32℃, the first leaf was white and other leaves were basically white (E). At 36℃, the whole plant was basically white (G). The total chlorophyll content of the mutant under 24℃, 28℃, 32℃ and 36℃ was only 91.6% (B), 72.8% (D), 9.2% (F) and 2.7% (H) of that of the wild type control, respectively. These results indicated that the white trait of the mutant was positively correlated with high temperature. Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1

[0036] The F1 plants showed normal phenotype. The F2 progeny obtained by selfing the F1 plants showed wild type and mutant phenotypes, and the segregation ratio of wild type to mutant was 3:1, indicating that the white trait was controlled by a recessive single gene.

[0037] Example 2 Fine mapping of the leaf white gene OsHCD1

[0038] 500 pairs of SSR markers and 50 pairs of InDel markers distributed on 12 chromosomes of rice were selected, and the polymorphism of each marker between the mutant oshcd1 and 93-11 was analyzed to obtain polymorphic molecular markers uniformly distributed on 12 chromosomes. The molecular markers linked to the target gene OsHCD1 were determined by BSA method, and the results showed that the SSR markers RM21095, RM21159, RM21178 and RM21243 on the short arm of chromosome 7 of rice were closely linked to the gene OsHCD1. Then, the genotypes of 50 recessive extreme single plants of oshcd1 / 93-11 F2 were analyzed by the three molecular markers, and the physical arrangement of the three markers and OsHCD1 gene on chromosome 7 was determined based on the position of each molecular marker in the chromosome, i.e. RM21095-RM21159-RM21178-OsHCD1-RM21243 (A). Figure 2

[0039] ​​​​​​​​​To further fine map the OsHCD1 gene, 15 pairs of SSR markers were designed between RM21178 and RM21243, among which 4 pairs of markers were polymorphic between the parents. Using these 4 pairs of markers, the genotype of the F2 albino leaf population was analyzed, and the OsHCD1 gene was finally mapped between SSR markers RM21183 and RM21214, with a physical distance of about 668 kb between them, spanning 8 BACs (AP003943, AP003837, AP003748, AP005098, AP005779, AP003931, AP004002 and AP003963) and 5 genes (LOC_Os07g11280, LOC_Os07g11290, LOC_Os07g11300, LOC_Os07g11310 and LOC_Os07g11320) (Figure 1). Figure 2 B).

[0040] Example 3 Prediction and sequencing alignment analysis of albino mutant gene

[0041] After whole genome sequencing of the mutant and its wild type control, it was found that the 251st base G in the 3rd exon of the candidate gene LOC_Os07g11280 in the mapping interval in Example 2 was mutated to A, resulting in the mutation of the encoded cysteine to tyrosine Figure 2 C-D).

[0042] Example 4 Genetic complementation verification of mutant oshcd1

[0043] Construction of gene complementation vector: a pair of primers was designed according to the sequence of the full-length cDNA of OsHCD1 gene in GenBank (TIGR Locus: LOC_Os07g11280):

[0044] Upstream primer: OsHCD1F: 5'-GCTCTCACACCGCCCCAACG-3';

[0045] Downstream primer: OsHCD1R: 5'-CAGT CCCGGG CTCACGTTTGCTTGTCTCTC-3'(containing a SmaI enzyme cutting site);

[0046] The cDNA of OsHCD1 gene was amplified from total RNA of Huaizan using RT-PCR technology, and the OsHCD1-cDNA fragment was obtained by electrophoresis purification;

[0047] Meanwhile, with the help of high-fidelity enzyme PrimerSTAR (Takara), the following primers were used:

[0048] OsHCD1PF: 5'-CAGT CCCGGG CTCGAGAAGTAAAGAAGCAAG-3'(containing a SmaI enzyme cutting site)

[0049] OsHCD1 PR: 5'-CGTTGGGGCGGTGTGAGAGC-3'

[0050] PCR reaction system:

[0051]

[0052] Reaction condition parameters: 98°C denaturation for 10 seconds, 56°C annealing for 15 seconds, 72°C extension for 1 minute and 30 seconds, a total of 40 cycles. 72°C extension for 5 minutes.

[0053] The promoter of the OsHCD1 gene was amplified using the Oryza sativa genome as a template, and the OsHCD1-Promoter fragment was obtained by purification. Then, the OsHCD1-cDNA fragment was fused with the OsHCD1-Promoter fragment using fusion PCR technology, and the primer OsHCD1PF and OsHCD1R were used for extension. Finally, the OsHCD1 fragment containing the promoter and cDNA was obtained, and the purified PCR product was digested with SmaI, and then connected to the Agrobacterium plasmid pCAMBIA1300-Nos to form pOsHCD1. The plasmid with correct insertion was selected for sequencing to determine the correct inserted fragment.

[0054] The Agrobacterium-mediated method was used to transform Agrobacterium EHA105, and was used to transform the mutant oshcd1.

[0055] The results show that the transgenic offspring transformed with pOsHCD1 have normal leaf color and no albino phenomenon Figure 2 E-F).

[0056] Example 5: Creation of a two-line albino sterile line

[0057] Step 1: Construction of OsHCD1 gene knockout vector

[0058] Based on the genomic (SEQ ID NO. 1) sequence, a specific target sequence was searched online (http: / / crispr.dbcls.jp / ), and the target sequence "5'-ACTATATGCGAGGCATAACC-3'" was selected. The complementary primer was synthesized according to the target sequence:

[0059] Upstream primer: 5'-GGCACTATATGCGAGGCATAACC-3';

[0060] Downstream primer: 5'-AAACGGTTATGCCTCGCATATAGT-3';

[0061] The above primers were fused in a PCR instrument to obtain a fusion fragment containing the target sequence.

[0062] The linearized vector was obtained by gel recovery after the vector pHun4c12 was digested with BsaI restriction enzyme. The fusion fragment was ligated into the linearized pHun4c12 (Xu et al., 2014, Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice 7:5), and the correct vector was identified by enzyme digestion and further confirmed by sequencing that the target sequence was introduced into the vector. The correct vector was named as pHCD1. Meanwhile, the pHCD1 was introduced into Agrobacterium strain EHA105 by electroporation for subsequent genetic transformation.

[0063] Step 2 Genetic transformation of two-line japonica sterile line

[0064] The genetic transformation of rice was performed according to the method of Pan et al. (2006) with slight modifications.

[0065] Briefly, the sterilized two-line japonica sterile line D239S seeds were inoculated into N6 medium containing 2.5 mg L -1 2,4-D, and induced callus for 7-10 days under continuous light at 32°C. About 40 μL of EHA105 Agrobacterium liquid containing pHCD1 plasmid was added to 20 mL of LB liquid medium containing 25 mg L -1 Rif and 50 mg L -1 Kan, and the bacteria were cultured overnight at 28°C with shaking. The bacteria were resuspended in 10 mM MgSO4and centrifuged, and the supernatant was removed and resuspended in AA liquid medium containing 200 μM AS to OD 600 = 0.1-0.2. After the callus was infected with the bacteria for 30 min, the callus was transferred to a sterile filter paper to absorb the excess bacteria. The callus was transferred to a culture dish with a single layer of sterile filter paper, and cultured in the dark at 28°C for 36-48 h, and then transferred to N6 medium containing 500 mg L -1 Cef and 50 mg L -1 Hyg for screening culture, and subcultured every two weeks until resistant callus was obtained. The resistant callus was regenerated to obtain T0 transgenic seedlings and planted in the field until T1 was harvested.

[0066] Step 3 Molecular identification and phenotype identification of transgenic rice

[0067] The T0 generation transgenic rice was grown to 3-4 leaf stage, and the young leaves were selected to extract genomic DNA by CTAB method. The endogenous OsHCD1 gene of the transgenic rice was amplified by using HCD1 specific primers (upstream primer: 5'-CAACTTCCCTCTCCTCATCC-3'; downstream primer: 5'-ATAGACAAGTTACCGACGAGA-3'), and the PCR product was verified by sequencing.

[0068] The results showed that the OsHCD1 gene had two base deletions, i.e. the transgenic pure line with loss of OsHCD1 gene function was obtained, and was named as oshcd1-2. Figure 3 A).

[0069] The results showed that the OsHCD1 gene had two base deletions, i.e. the transgenic pure line with loss of OsHCD1 gene function was obtained, and was named as oshcd1-2. Figure 3 B), and the total chlorophyll content was only 8.23% of the control ( Figure 3 C).

Claims

1. Application of high-temperature sensitive rice leaf whitening gene OsHCD1 in regulating rice leaf color traits, characterized in that, The gene sequence is shown as SEQ ID NO.

1.

2. The application of high-temperature sensitive rice leaf whitening gene OsHCD1 in molecular breeding of rice leaf color traits, characterized in that, The wild-type gene sequence is shown as SEQ ID NO. 1, and the mutant gene sequence is shown as SEQ ID NO.

2.

3. The application of high-temperature sensitive rice leaf albino gene OsHCD1 in hybrid rice F1 hybrid purity identification and / or sterile line self-seed purity identification, characterized in that, The wild-type gene sequence is shown as SEQ ID NO. 1, and the mutant gene sequence is shown as SEQ ID NO.

2.

4. A method of hybrid breeding to eliminate false hybrids, characterized by, The method comprises the following steps: (1) constructing a mutant plant of a high-temperature-sensitive rice leaf albino gene OsHCD1 interference or knockout; the gene sequence of the high-temperature-sensitive rice leaf albino gene OsHCD1 is shown as SEQ ID NO. 1; (2) crossing the mutant sterile line with a normal-leaf-color rice restorer line to obtain F1 hybrids by seed production; If an F1 single plant appears an albino phenotype under high-temperature conditions, it is a false hybrid of the sterile line self-crossing, and the false hybrid is removed; the temperature is greater than 28 DEG C.

5. The method of hybridization breeding to eliminate false hybrids of claim 4, wherein, In step (1), a sterile line containing a high-temperature-sensitive rice leaf albino mutant gene oshcd1 is constructed, and the gene sequence of the high-temperature-sensitive rice leaf albino mutant gene oshcd1 is shown as SEQ ID NO. 2.

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