Rice mutant capable of improving light adaptability and yield and application of rice mutant

Abstract

The invention discloses a rice mutant capable of improving light adaptability and yield and application of the rice mutant, the mutant is a mutant with OSBCM protein deletion, and the amino acid sequence of the OSBCM protein is shown as SEQ ID NO.1. The invention further discloses a preparation method of the rice mutant capable of improving light adaptability and yield. By obtaining the osbcm rice mutant, the mechanism of the mutant for improving the light adaptability and the yield of the rice is disclosed, a new thought is provided for plants to resist stress under high light and improve the yield, and the osbcm rice mutant has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a rice mutant with improved light adaptation ability and yield and application thereof. Background Art

[0002] Plants growing in the natural environment face a variety of stresses, which are mainly divided into biotic stress and abiotic stress. Plants can produce stress responses in the face of various stress signals, and the plant body integrates multiple responses to cope with them. Rice is a light-loving plant, and its light saturation point is generally 1000 μmol m -2 s -1 Rice growing areas are generally distributed in areas with strong light intensity. However, the light intensity in these areas can reach up to 2000 µmol / m around noon in summer. -2 ·s -1 (Kasajima, I., Ebana, K., Yamamoto, T., Takahara, K., Yano, M., Kawai-Yamada, M. and Uchimiya, H. Molecular distinction in genetic regulation of nonphotochemical quenching in rice. Proc Natl Acad Sci USA. 2011, 108: 13835-13840), far exceeding the maximum light intensity that rice can utilize. Therefore, the photoinhibition of rice in the field is very common, and the damage and repair of PSII (photosystem II) are extremely frequent, which seriously affects the photosynthetic efficiency of rice. It is estimated that the absorption of excessive light intensity may cause the photosynthetic efficiency to decrease by about 30%, the yield to decrease by 10-20%, and cause economic losses.

[0003] Under normal circumstances, the number of chlorophyll molecules in PSII and PSI (photosystem I) in plants is 157 and 204 respectively (Junfei, Gu, Zhenxiang, Zhou, Zhikang, Li, Ying, Chen, Zhiqin and Wang Rice (Oryza sativa L.) with reduced chlorophyll content exhibit higher photosynthetic rate and efficiency, improved canopy light distribution, and greater yields than normally pigmented plants. Field Crops Research. 2017). Some chlorophyll molecules may exist in the large chlorophyll antenna molecules in an inactive form as energy storage molecules (Li, Y., Ren, B., Gao, L., Ding, L., Jiang, D., Xu, X., Shen, Q. and Guo, S. LessChlorophyll Does not Necessarily Restrain Light Capture Ability andPhotosynthesis in a Chlorophyll-Deficient Rice Mutant. Journal of Agronomyand Crop Science, 2012, 199: 49-56), so it is feasible to reduce the chlorophyll content to improve photosynthetic efficiency. In 2012, Zoee used RNAi technology to regulate the expression of CAO (chlorophyllide aoxygenase) in Chlamydomonas reinhardtii, thereby regulating the level of chlorophyll b. The content of LHCII (light-harvesting complex II) changed, decreasing by 20%-30% compared with the wild type. Compared with the wild type, the mutant improved the light utilization efficiency, and the culture density of Chlamydomonas increased by 40% (Perrine, Z., Negi, S., andSayre, RT. Optimization of photosynthetic light energy utilization bymicroalgae. Algal Research, 2012, 1: 134-142.).In 2016, Jin discovered that HPE1 (high photosynthetic efficiency 1) encodes a chloroplast protein containing an RNA recognition motif in Arabidopsis. The loss of this gene may lead to a decrease in the expression of genes encoding chlorophyll synthesis. Compared with the wild type, the mutant plants have reduced Chl a and Chl b contents, increased Chla / b ratio, smaller light-harvesting antennae, and exhibit higher. Fv / FmThe maximum photochemical efficiency of the PSII and the actual photochemical efficiency of PSII, faster photochemical quenching and electron transfer rates, increased carbohydrate content and biomass production (Jin, H., Li, M., Duan, S., Fu, M., Dong, X., Liu, B., Feng, D., Wang, J., and Wang, HB. Optimization of Light-Harvesting Pigment ImprovesPhotosynthetic Efficiency. Plant Physiol, 2016,172: 1720-1731.). In 2017, Gu found that the chlorophyll content in the pgl (chlorophyll a oxygenase) mutant in rice was reduced, and it had a smaller light-harvesting antenna. Compared with Z802, the mutant had more biomass under high light, and the electron transfer efficiency, PSII efficiency, and photochemical quenching were increased, and NPQ (nonphotochemical quenching) was reduced. Therefore, the photosynthesis of the mutant was improved, and the photoinhibition was reduced (Gu, J., Zhou, Z., Li, Z., Chen, Y., Wang, Z., Zhang, H., and Yang, J. Photosynthetic Properties and Potentials for Improvement of Photosynthesis in Pale Green Leaf Rice under High Light Conditions. Front Plant Sci, 2017, 8: 1082.). The above studies show that the photosynthetic efficiency of plants can be effectively improved by regulating the chlorophyll content in plants to optimize the absorption, transfer and conversion of light energy in plants. In addition, chlorophyll synthesis is a finely regulated process involving multiple enzymes (Czarnecki, O., and Grimm, B. Post-translational control of tetrapyrrolebiosynthesis in plants, algae, and cyanobacteria. J Exp Bot, 2012, 63: 1675-1687). By regulating the key genes of chlorophyll synthesis, the precise regulation of chlorophyll content can be experimentally achieved. However, previous studies have shown that when the pigment content decreases too much, it often seriously affects the supplementary light, resulting in insufficient light reaction and affecting the photosynthetic rate.Therefore, finding and utilizing the key factors that regulate light absorption plays an important role in helping plants resist high-light stress. Summary of the invention

[0004] Technical problems solved: In response to the above technical problems, the present invention provides a rice mutant with improved light adaptability and yield and its application, which can effectively solve the shortcomings of existing rice that has a significant reduction in yield due to high light stress, thereby causing economic losses.

[0005] Technical solution: In the first aspect, the present invention provides a rice mutant with improved light adaptation ability and yield, wherein the mutant is a mutant lacking OSBCM protein, and the amino acid sequence of the OSBCM protein is shown in SEQ ID NO.1: SEQ ID NO.1: MELAPFAAASSSSSSSTVTNTCKLRLRLSYTHCSIGFAARDAELAMWERLGAVVRLSYGIGIYAAMALTGRFICQMAGIDCTGGFHPSLSALVEGLGYAAPPIMALLFILDDEVVKYSPHARAIRDVEDEELRSFFYGMSPWQFILIVTASSIGEELFYRVAV QGALADIFLRGTELMKDARGIASLSGMVPPFVPFAQTFAAAITAALTGSLYYVATAPKDPTYVVTPAMRSNTGRENLKKLFAAWYERRQMRKIYSPLLEGILAFYLGFEWIQTDNILAPMITHGIYSTVVLCHGLWKIHDHRRRLRQRIQEIRQEGTTNSDTL.

[0006] Preferably, the OSBCM protein is encoded by osbcm The nucleotide sequence of the gene is shown in SEQ ID NO.2: SEQ ID NO.2: ATGGAGCTGGCCCCTTTTGCGGCGGCCTCCTCCTCCTCCTCCTCTTCCACAGTCACAAACACGTGCAAGCTCAGGCTCAGGCTCAGCTACACCCACTGTTCCATCGGCTTCGCAGCCCGGGACGCGGAGCTGGCCATGTGGGAGAGGCTGGGAGCGGTTGTCAGGCTCAGCTATGGCATTGGGATTTATGCCGCCATGGCACTTACTGGGAGATTCATATGCCAGATGGCCGGAATAGACTGCACAGGGGGCTTCCATCCATCTCTCTCTGCGCTTGTTGAGGGACTCGGATATGCAGCTCCGCCGATTATGGCTTTGCTCTTTATCCTCGACGATGAAGTTGTCAAATATTCACCTCATGCTCGCGCCATCAGGGATGTCGAGGATGAGGAGCTTCGCAGCTTCTTCTATGGCATGTCACCATGGCAGTTCATTCTTATTGTAACTGCTAGCTCAATTGGAGAGGAGCTATTCTATCGTGTCGCTGTCCAGGGTGCGTTGGCTGATATTTTTCTGAGAGGCACAGAGCTCATGAAAGACGCTCGAGGGATTGCATCCCTGAGTGGCATGGTGCCTCCATTTGTTCCATTTGCTCAAACGTTTGCAGCAGCCATCACTGCGGCTCTCACAGGATCACTCTATTATGTGGCTACTGCCCCGAAAGATCCTACTTATGTGGTGACACCAGCAATGCGTTCAAATACTGGGCGAGAGAATCTCAAGAAACTATTTGCAGCATGGTATGAAAGGCGGCAAATGAGGAAGATATATTCCCCTCTTCTTGAAGGAATCTTGGCTTTCTACCTTGGCTTTGAGTGGATCCAGACAGATAATATTCTTGCACCTATGATCACCCACGGCATATACTCTACCGTTGTGCTTTGCCATGGGTTGTGGAAAATACACGACCACAGGAGAAGGCTACGGCAAAGAATTCAGGAAATCAGACAGGAAGGAACAACAAATTCAGACACGTTGTAA。

[0007] In a second aspect, the present invention provides a biological material containing the mutant described in the first aspect, wherein the biological material is an expression vector or a strain.

[0008] In a third aspect, the present invention provides use of the mutant described in the first aspect in improving the light adaptation ability and yield of rice.

[0009] In a fourth aspect, the present invention provides use of the mutant described in the first aspect in preparing a product for improving the light adaptability and yield of rice.

[0010] Beneficial effect: The present invention obtains osbcm A rice mutant, wherein the leaves of the mutant turn yellow, the chlorophyll content decreases, and the light absorption can be reduced. osbcm The rice mutant was subjected to physiological and biochemical analysis to explore the mechanism of the mutant's resistance to high light. osbcm The rice mutants were planted in the field and the agronomic traits were statistically analyzed. It was found that the yield was significantly improved compared with the wild type, which is an important method to increase rice yield. The present invention is a mechanism for improving the light adaptation ability and yield of rice, and provides a new idea for plants to resist high light stress and increase yield, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The OSBCM evolutionary tree and structure diagram of the present invention, wherein A is a comparison of BCM proteins in monocots and dicots to construct an evolutionary tree; B is a schematic diagram of the structure of the mutation site in the mutant; C is osbcm Sequencing peaks corresponding to the mutants, red boxes represent insertion and deletion sites; Figure 2 The present invention osbcm Phenotype and chlorophyll content determination, where A represents the wild type (WT) and osbcm Phenotypes of mutants at seedling, tillering and maturity stages. B is WT and osbcm The results of the determination of chlorophyll content of mutants at the seedling and tillering stages; Figure 3 This is a schematic diagram of the changes in Arabidopsis photosystem proteins after high light exposure in the present invention; Figure 4 The results of the in vitro light inhibition and light recovery experiment are shown in Figure 1, where A is the in vitro light inhibition and light recovery phenotype of rice, and B is the process of in vitro light inhibition and light recovery of rice. Fv / Fm Value statistics, C is the phenotype of Arabidopsis thaliana in vitro light inhibition and light recovery, D is the process of Arabidopsis thaliana in vitro light inhibition and light recovery Fv / Fm Value statistics; Figure 5These are the BN-PAGE and 2D-SDS-PAGE images of rice and Arabidopsis before and after high light, where A is the BN-PAGE image of the rice sample before and after high light, B is the 2D-SDS-PAGE image of the rice sample before and after high light, C is the BN-PAGE image of the Arabidopsis sample before and after high light, and D is the 2D-SDS-PAGE image of the Arabidopsis sample before and after high light; Figure 6 is a statistical graph of rice agronomic traits in one embodiment of the present invention, wherein A is the statistical result of rice agronomic traits, and B is the statistical result of WT and osbcm Thousand-grain weight analysis of mutants. DETAILED DESCRIPTION

[0012] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Embodiment 1: osbcm Obtaining Rice Mutants First, we used the Arabidopsis BCM1 protein to perform blast and found homologous genes in rice ( Figure 1 A), send the sequence to a biotechnology company for target design and genetic transformation ( Figure 1 B) to obtain osbcm Rice mutant seedlings. Then, specific primers were designed based on the sequence near the target (forward primer (SEQ ID NO.3): 5'-GCTCAGCTACACCCACTGTTC-3', reverse primer (SEQ ID NO.4): 5'-CCAATTGAGCTAGCAGTTAC-3'), and PCR amplification reaction was performed and sent for sequencing. The positive plants were compared with the OSBCM sequence (SEQ ID NO.2) to find positive plants and expand them to obtain osbcm The rice mutant seeds, the sequencing results are as follows Figure 1 As shown in C.

[0013] Embodiment 2: osbcm Phenotypes of rice mutants In Arabidopsis, BCM1 (Balance of Chlorophyll Metabolism 1) is a chloroplast protein encoded by a nuclear gene. The protein has six transmembrane domains and specifically acts on the magnesium branch of the TBS (Tetrapyrrole Biosynthesis) biosynthesis pathway. Its mutants show light green leaf color. BCM1 promotes Chl (chlorophyll) biosynthesis by stimulating MgCh (Magnesium Chelatase) activity through GUN4 (Genomes Uncoupled 4). In addition, it interacts with SGR1 (Stay-Green 1) and destroys its stability to prevent Chl degradation. At the beginning of leaf senescence, although BCM1 is greatly inhibited, BCM2 (Balance of Chlorophyll Metabolism 2) and SGR1 are both upregulated, and BCM2 can promote the inhibition of Chl decomposition. However, strong accumulation of SGR1 eventually enables plants to initiate Chl degradation.

[0014] In rice, whether in the seedling stage, tillering stage or maturity stage, osbcm The leaves also appear light green ( Figure 2 A); By measuring the chlorophyll content, it was found that the chlorophyll content decreased, and as the rice growth time increased, the chlorophyll content decreased ( Figure 2 B).

[0015] Embodiment 3: osbcm Rice mutants involved in high light stress The chlorophyll content is closely related to the LHCP content. bcm The chlorophyll content in the mutants (including Arabidopsis BCM1 protein-deficient mutants, BCM2 protein-deficient mutants and rice OSBCM protein-deficient mutants) was reduced. After detecting the content of photosystem proteins, it was found that the content of LHCII in the mutants was also reduced accordingly. LHCII is the light-harvesting antenna protein of PSII, which is responsible for the absorption and transmission of light energy. The results showed that the slight reduction in LHCII content did not affect the absorption and energy transmission of light energy. In Arabidopsis, the present invention uses high light stress to treat WT, bcm1 Mutants (BCM1 protein deficiency mutants) and bcm2 mutant (BCM2 protein deficiency mutant), and extracted thylakoid membrane proteins to detect the changes in the content of photosystem proteins after high light. The results showed that compared with the wild type, bcm1 The amount of D1 protein in the mutant was increased ( Figure 3), indicating that under high light conditions, bcm1 The PSII of the mutant was less damaged by light. bcm1 Mutants and bcm2 To determine whether the mutants responded differently to high light, rosette leaves grown in soil for four weeks were cut, placed in water, and then exposed to 900 μmol photons m −2 ·s −1 The leaves were treated with high light intensity and the Fv / Fm The results showed that under high light conditions, compared with wild type and bcm2 Mutants, bcm1 The mutant is less sensitive to bright light ( Figure 4 C and Figure 4 D).

[0016] Similarly, the wild type of rice detected by the present invention is different from osbcm To determine whether the mutants also differed in their response to high light, rice leaves were cut from seedlings, placed with the front side facing up in water, and then exposed to 1000 μmol photons m −2 ·s −1 The leaves were treated with high light intensity and the Fv / Fm The results showed that rice and Arabidopsis had similar results. Under high light conditions, compared with the wild type, osbcm The mutant is less sensitive to high light ( Figure 4 A and Figure 4 B).

[0017] In this study, thylakoid membranes were extracted from samples treated with high light and those not treated with high light, and BN-PAGE and 2D-SDS-PAGE were performed to explore bcm The mechanism of the mutant's insensitivity to high light. The results showed that the PSII supercomplex of the mutant was more stable under high light conditions, whether in rice or Arabidopsis, which can also be proved by the results of 2D-SDS-PAGE ( Figure 5 ).

[0018] Embodiment 4: osbcm Agronomic traits of rice mutants in the field The insensitivity to high light is beneficial to the growth of rice under stress conditions, especially in the natural environment of the field. osbcm Will the agronomic traits of the mutant be improved? Statistical analysis of rice thousand-grain weight, seed yield, number of effective tillers, and total number of grains revealed that ( Figure 6 ), compared with WT, osbcmThe mutant showed an increase in thousand-grain weight and effective tiller number. The number of effective ears, average number of grains per ear and thousand-grain weight are three key factors affecting seed yield. osbcm The mutants generally showed an increase in seed yield. From the perspective of thousand-grain weight, osbcm The mutant seeds were larger in length and width than those in WT, indicating that osbcm The mutation is beneficial to the improvement of the agronomic traits of plants under high light.

[0019] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A rice mutant with improved light adaptation ability and yield, characterized in that: The mutant is a mutant lacking OSBCM protein, and the amino acid sequence of the OSBCM protein is shown in SEQ ID NO.

1.

2. A rice mutant with improved light adaptation ability and yield according to claim 1, characterized in that: Encoding OSBCM protein osbcm The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A biological material containing the mutant according to claim 1 or 2, wherein the biological material is an expression vector or a strain.

4. Use of the mutant according to claim 1 or 2 in improving the light adaptation ability and yield of rice.

5. Use of the mutant according to claim 1 or 2 in preparing a product for improving the light adaptation ability and yield of rice.

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