Application of rice OsCP31B protein and coding gene thereof in improving abiotic stress resistance and yield of plants

Abstract

The invention belongs to the technical field of agricultural biology, and discloses a rice OsCP31B protein and application of a coding gene of the rice OsCP31B protein to improvement of abiotic stress resistance of plants. The rice OsCP31B gene is subjected to knockout mutation in rice by adopting a CRISPR / Cas9 gene editing technology, and according to the phenotypic change of a gene knockout rice plant, the capability of resisting low-temperature stress of the rice can be remarkably improved by knocking out the OsCP31B gene in the rice, so that the rice OsCP31B protein and the coding gene thereof can be applied to improving the resistance of the plant to abiotic stress, and the application of the rice OsCP31B protein and the coding gene thereof to the improvement of the resistance of the plant to the abiotic stress. And the yield is obviously increased. The method has application prospects in the aspects of improving and enhancing rice stress resistance, accelerating the stress-resistant molecular breeding process and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, and particularly relates to the use of rice OsCP31B protein and its coding gene in improving plant resistance to abiotic stress and yield. BACKGROUND

[0002] Low temperature chilling (lower than 15℃) is one of the most serious and influential natural disasters, and rice (Oryza sativa L.) is a kind of warm crop, which is extremely sensitive to low temperature. In recent years, global low temperature disasters occur frequently, which seriously harm the growth and development of rice, limit the geographical distribution of rice, cause a large amount of reduction in rice yield, and seriously degrade rice quality, thereby causing a serious threat to global food security. Therefore, it is of great significance to mine and clone key genes of plant cold tolerance for preventing and controlling rice low temperature and cultivating new rice varieties with low temperature stress resistance.

[0003] At present, more than 800 million people in the world are facing the problem of hunger. By 2050, the population of the earth will exceed 10 billion, and in order to feed these people, the global food production needs to be increased by 70%. Therefore, it is of great significance to mine and clone key genes for regulating rice yield and cultivate new rice varieties with high yield and high quality.

[0004] Chloroplast RNA binding proteins (cpRBPs) are a class of proteins encoded by the nucleus and located in chloroplasts, which mainly function to recognize and bind the C base at a specific position on a specific messenger RNA precursor (pre-mRNA) and convert it into a U base to promote the maturation of pre-mRNA and play its translation function. Studies have shown that the Arabidopsis cpRBP family protein AtCP31A specifically binds and edits part of pre-mRNA in Arabidopsis chloroplasts, and AtCP31A loss-of-function mutants exhibit leaf white phenotype under low temperature.

[0005] So far, there has been no report about the use of rice OsCP31B protein or its coding gene in improving plant resistance to low temperature stress. SUMMARY

[0006] The main purpose of the present application is to provide the use of rice OsCP31B protein or its coding gene in improving plant resistance to abiotic stress and yield.

[0007] The present application provides the use of rice OsCP31B protein or its coding nucleic acid in improving plant resistance to abiotic stress.

[0008] Further, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof, or the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0009] Further, the CP31B protein comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0010] Further, specifically comprising: (1) constructing a recombinant plant expression vector of rice OsCP31B functional deficiency; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) cultivating and screening to obtain transgenic plants with improved resistance to abiotic stress.

[0011] Further, the abiotic stress includes low temperature stress.

[0012] Further, the low temperature stress is low temperature stress in the germination stage.

[0013] Further, the low temperature stress is lower than 12℃.

[0014] Further, characterized in that the plant is rice.

[0015] Further, the abiotic stress includes low temperature stress. Preferably, the low temperature is lower than 12℃, 11℃, 10℃, 9℃, 8℃, 7℃, 6℃, 5℃, 4℃, 3℃, 2℃, 1℃, 0℃.

[0016] Further, the plant includes but is not limited to monocotyledonous plants or dicotyledonous plants; more preferably, the plant includes crops, vegetables or ornamental plants, fruit trees, etc., for example, can be rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, sugarcane or Arabidopsis, etc., preferably rice.

[0017] The present application has the following technical effects relative to the prior art: The application determines the tissue expression pattern and low temperature stress response mode of the cloned chloroplast RNA binding protein OsCP31B responding to abiotic stress in rice. The results show that the gene has the highest expression in green tissues such as leaves, and the lowest expression in roots and seeds. After being subjected to 6 DEG C low temperature stress treatment, the expression of OsCP31B in rice plants is significantly increased. In oscp31b mutants with ZhongHual1 (ZH11) as the genetic background, it is observed that the mutant lines have higher low temperature stress resistance than the wild type. It is found that the mutant lines have lower plant height at the mature stage, and the gene function loss of OsCP31B does not affect the seed setting rate and yield of rice. The application provides a new idea and direction for elucidating the response mechanism of rice resistance to abiotic stress, and the provided rice gene OsCP31B has important theoretical and practical significance in improving the stress resistance of rice and cultivating stress-resistant high-yield rice varieties. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 PCR positive identification of the transgenic plant of the rice OsCP31B CRISPR knockout material by using the hygromycin primer.

[0019] Figure 2 Knockout of the transgenic plant of the rice OsCP31B CRISPR knockout material of the application; Figure 3 Plant tissue expression pattern analysis diagram of the rice OsCP31B gene of the application; Figure 4 Expression pattern analysis diagram of the rice OsCP31B gene under low temperature treatment of the application; Figure 5 Cold tolerance phenotype of the transgenic plant of the rice OsCP31B CRISPR knockout material and the wild type plant under 6 DEG C low temperature at the seedling stage of the application. A is the phenotype diagram before 6 DEG C treatment (upper) and after 6 DEG C treatment for 4 days and recovery for 7 days (lower), and B is the survival rate statistical result before and after 6 DEG C treatment; Figure 6 Mature plant height phenotype of the transgenic plant of the rice OsCP31B CRISPR knockout material and the wild type plant of the application. A is the plant height phenotype diagram, and B is the plant height statistical result; Figure 7 Single plant yield statistical result of the transgenic plant of the rice OsCP31B CRISPR knockout material and the wild type plant of the application. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0021] Example 1: Construction of the OsCP31B gene CRISPR knockout vector Target sites 1 and 2 for knockout were designed based on the OsCP31B gene cDNA sequence (SEQ ID NO: 3) (Table 1).

[0022] Table 1 Knockout target sites (5'-3')

[0023] Primers CP31B-target1-BsF, CP31B-target1-F0, CP31B-arget2-R0, and CP31B-target2-BsR were designed based on the target. Four-primer PCR amplification was performed using a 100-fold diluted pCBC-MT1T2 plasmid as a template. The PCR product was purified and recovered, and the final vector was constructed using an enzyme digestion-ligation system. The vector was named CRISPR-oscp31b.

[0024] Table 2 Gene knockout primers (5'-3')

[0025] Example 2 Agrobacterium transformation The CRISPR-CP31B knockout vector was transferred into Agrobacterium EHA105 competent cells using the freeze-thaw method (competent cells were purchased from Shanghai Sangon Biotech Co., Ltd.). The specific experimental method was as described in the Molecular Cloning Laboratory Manual.

[0026] Example 3 Genetic Transformation 1) Sterilization: Remove the shells from healthy, plump Zhonghua 11 seeds, soak them in 70% ethanol for 1-2 minutes, add 50% bleach and place on a shaker (200 rpm) for about 1-1.5 hours. Rinse with sterile water 4-6 times, place the seeds on sterile filter paper to absorb excess moisture, and then evenly place them in NBD medium and incubate in the dark at 28 ℃. All the above steps are performed in a laminar flow hood.

[0027] 2) Subculture: After dark culture for about 10-15 days, the rice seed buds are separated and transferred to subculture medium NBD for continued dark culture at 28 ℃; after 10 days, the seeds are separated from the callus and the callus is transferred to a new subculture medium NBD. After dark culture at 28 ℃ for about 4-5 days, Agrobacterium transformation can be carried out. 3) During this period, Agrobacterium containing plasmids is streaked on the corresponding antibiotic culture medium. After 2 days, single clones are picked, streaked again, and cultured for 1 day. 4) Collect the bacterial cells from the culture medium, vortex them in NBC1 medium containing acetylsuccinone (AS), and adjust the OD to about 0.1-0.2; 5) Transformation: Select healthy callus tissue into a sterile Erlenmeyer flask, add the above-prepared suspension, gently shake at room temperature for about 10 minutes, discard the bacterial solution, place the callus tissue on sterile filter paper, absorb the excess bacterial solution, and place it in a laminar flow hood to blow air until the callus tissue turns slightly white. Then transfer the callus tissue to NBC2 medium with a layer of sterile filter paper, and co-culture at 22°C in the dark for 2 days. 6) Screening: Transfer the co-cultured callus to NBS1 medium containing the corresponding antibiotic, and culture in the dark at 28 ℃ for 10-12 days. Then transfer it to NBS2 medium and continue to culture in the dark at 28 ℃ for 10-12 days. 7) Differentiation: After transferring the callus to NBR1 medium, culture it in the dark at 28 ℃ for 6 days, then transfer it to an artificial climate incubator with 15 h light / 9 h darkness and culture it at 28 ℃ for 15-20 days. During this period, callus tissue with green spots is transferred to NBR2 medium and cultured until it differentiates into seedlings. 8) Cut the roots and leaves of the transgenic seedlings that are about 5 cm tall, transfer them to the rooting medium, and culture them at 28°C in an artificial climate incubator with 12 h light / 12 h darkness. 9) Hardening off seedlings: Once the root system of the transgenic seedlings is sufficiently developed, open the culture bottle for about 2 days, wash off the culture medium, place the seedlings in water for 1 week, and then transfer them to the soil for planting.

[0028] The culture medium formulations used in the above genetic transformation process are shown in Table 3; the preparation of hormone and antibiotic storage solutions in the culture medium is shown in Table 4.

[0029] Table 3 Genetic transformation culture medium

[0030] Table 4. Preparation of Hormone and Antibiotic Storage Solutions

[0031] Example 4 Molecular Identification of Transgenic Plants The T0 generation transgenic seedlings were validated by PCR and propagated. T1 generation transgenic seeds were harvested, and homozygous mutant materials were obtained through positive validation and sequencing. These homozygous mutant materials were then propagated, and the offspring were screened with hygromycin (50 mg / L) to obtain homozygous Cas9-free mutant materials. These materials were then propagated again, and the offspring were screened with hygromycin (50 mg / L) to obtain homozygous Cas9-free mutant materials.

[0032] The primers used for molecular identification of transgenic plants, including those for overexpression and mutant line identification, are shown in Table 5. The results are as follows: Figure 1 As shown.

[0033] Table 5 Primers for molecular identification of transgenic plants (5'-3')

[0034] Example 5 Molecular Identification of Transgenic Plants (1) Select seeds of T3 generation CP31B knockout mutant and wild-type Zhonghua 11.

[0035] (2) Rice cultivation in soil: After the newly harvested seeds break dormancy, soak them in a 28℃ incubator for 3 days until they germinate, and then sow them. Select seeds with uniform germination and sow them evenly in a mixture of nutrient soil and vermiculite prepared in a ratio of 3:1. Cover the surface with a layer of vermiculite and then let them grow normally in a 28℃ incubator. Water them once every 2-3 days during this period.

[0036] (3) Rice hydroponics: Select seeds with consistent germination and sow them in a 96-well PCR plate with the bottom removed. Place the plate in a 28 ℃ incubator and grow. Change the water every 2-3 days. Add an appropriate amount of nutrient solution when the third leaf just emerges. Change the water to clean water after the third leaf has fully unfolded.

[0037] (4) After the disinfected rice seeds germinate at room temperature, they are sown. Each experimental group has at least 3 replicates. After two weeks of cultivation under light at 28℃, the seeds are treated at 12℃ for 2-4 days (depending on the actual situation), and then transferred to 28℃ to resume growth for one week.

[0038] (5) For the identification of mutants, the genome near the target site is amplified as shown in SEQ ID NO.3 and sent to Sangon Biotech Co., Ltd. for sequencing. The mutation type is determined by comparing the sequencing results.

[0039] The results are as follows Figure 2 The figure shows the knockout status of the OsCP31B CRISPR knockout transgenic rice plants of the present invention.

[0040] Example 6: Analysis of the expression pattern of the rice OsCP31B gene The expression level of RNA in transgenic rice was detected using quantitative real-time PCR (AceQ qPCR SYBR Green Master Mix (vazyme)) and sequencing. Figure 3 The expression levels of the OsCP31B gene in different rice tissues. From... Figure 3 The OsCP31B gene is highly expressed in the green tissues of rice. Figure 4 The expression level of the OsCP31B gene changed after low-temperature treatment in wild-type ZH11 rice. Figure 3 As can be seen, low temperature significantly induces enhanced expression of OsCP31B.

[0041] Example 7 Molecular identification and stress resistance identification of transgenic plants (1) Select seeds of T2 generation OsCP31B gene CRISPR and wild type ZH11.

[0042] (2) Rice cultivation in soil: After the newly harvested seeds break dormancy, soak them in a 28℃ incubator for 3 days until they germinate, and then sow them. Select seeds with uniform germination and sow them evenly in a mixture of nutrient soil and vermiculite in a ratio of 3:1. Cover the surface with a layer of vermiculite and then let them grow normally in a 28℃ incubator. Water them once every 2 to 3 days during this period.

[0043] (3) Rice hydroponics: Select seeds with consistent germination and sow them in a 96-well PCR plate with the bottom removed. Place the plate in a 28℃ incubator and grow. Change the water every 2-3 days. Add an appropriate amount of nutrient solution when the third leaf just emerges. Replace the water with clean water after the third leaf has fully unfolded.

[0044] (4) After the disinfected rice seeds germinate at room temperature, they are sown. Each experimental group has at least 3 replicates. After two weeks of cultivation under 28℃ light according to the above conditions, they are treated at 6℃ for 2-4 days (depending on the actual situation), and then transferred to 28℃ to recover growth for 1 week.

[0045] (5) Regarding survival rate, the criterion in this study was whether new leaves had grown. If new leaves were present, the plant was considered alive; otherwise, it was considered dead. According to... Figure 5 The experimental results shown in Figures A and B demonstrate that mutating or knocking out the OsCP31B gene in rice significantly improves the rice's ability to resist low-temperature stress. Therefore, the rice OsCP31B protein, its encoding gene, and recombinant vector can be used to enhance the crop's resistance to abiotic stress.

[0046] Example 8: Yield determination of transgenic plants The plant height and yield per plant at maturity of OsCP31B knockout mutant plants were statistically analyzed. The phenotypes are shown in the figure. Figure 6 A, B and Figure 7The results showed that knocking out the OsCP31B gene in rice significantly reduced plant height and significantly increased yield per plant. Therefore, the rice OsCP31B protein, its encoding gene, and recombinant vector can be applied to enhance crop yield.

[0047] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. Use of rice OsCP31B protein or a nucleic acid encoding the same in improving plant resistance to abiotic stress.

2. The use according to claim 1, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 1 or a degenerate sequence thereof, or the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 3 or a degenerate sequence thereof.

3. The use according to claim 1, wherein the OsCP31B protein comprises an amino acid sequence as set forth in SEQ ID NO:

2.

4. Use according to claim 1, characterized in that, Specifically comprising: (1) constructing a recombinant plant expression vector for rice OsCP31B functional deficiency; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) cultivating and screening to obtain transgenic plants with improved resistance to abiotic stress.

5. The use according to claim 1, characterized in that, The abiotic stress includes low temperature stress.

6. Use according to claim 1, characterized in that, The low temperature stress is low temperature stress in the germination stage.

7. Use according to claim 5, characterized in that, The low temperature stress is lower than 12℃.

8. Use according to any one of claims 1 to 7, characterized in that, The plant is rice.

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