CN116200411A - Rice salt tolerance gene OsINH2 and OsINH3 and application thereof - Google Patents

Abstract

The invention provides rice salt-tolerant genes OsINH2 and OsINH3 and applications thereof. When excavating stress-resistant genes, it was found that the rice mutants knocked out by CRISPR-Cas were sensitive to salt, while the overexpressed lines not only survived high under salt stress, but also did not significantly inhibit their growth. Based on these phenotypic results, we believe that the study of OsINH2 and OsINH3 genes will play a positive role in improving rice salt and stress resistance and expanding the stress resistance gene pool. Further studies have found that OsINH2 and OsINH3 genes also have functions in regulating plant fertility, regulating plant pollen activity, and scavenging reactive oxygen species (ROS).

Description

Salt tolerance genes OsINH2 and OsINH3 in rice and their application

technical field

The invention belongs to the field of rice resistance gene mining, and specifically relates to rice salt-tolerant genes OsINH2 and OsINH3 and applications thereof.

Background technique

Excessive soil salinity seriously damages plant growth, development and crop yield, while about 20% of the world's arable land and nearly half of irrigated land suffer from excessive salinity, and industrial pollution and unreasonable irrigation in agriculture are exacerbating this situation ; With the growth of population and the improvement of the demand for living materials, we urgently need to find some genes related to adversity stress that are widely present and conserved in plants to improve the ecological environment and increase the yield of crops in saline soil. Therefore, discovering the key regulatory proteins in the salt response pathway is particularly important for elucidating the molecular mechanism of plant response to salt and improving the salt-resistant quality of crops. Rice is one of the most important cereal crops, and it is the most salt-sensitive among all cereal crops, especially in the seedling and reproductive stages. Therefore, digging in-depth stress-resistant and salt-tolerant genes in rice and analyzing their physiological mechanisms are current biotechnology The first task of breeding.

Plants face various biotic and abiotic stresses during their growth and development. In order to cope with the changing environment, plants must precisely adjust the balance between growth and development and stress response. In a suitable environment, plants germinate, grow, meet and bear fruit, and complete their life cycle normally; while in a stress environment, plants promote the expression of stress-responsive genes and enhance plant resistance to stress, but at the same time inhibit the expression of growth-related genes, resulting in delayed growth or even Stasis, this growth-defense trade-off widely exists in crops, such as wheat, corn and rice, coordinates the growth and development and stress resistance of crops. Regarding the excavation of stress-response or yield-enhancing genes in rice breeding, often due to growth-defense trade-offs, they cause damage to the other party. At present, only a few literatures have reported some genes involved in growth and stress-resistance regulatory nodes, including some involved in Growth and development and stress response plant hormones ABA and BR signaling pathway nodes. Therefore, solving the growth-defense trade-off is still a difficult point in mining stress and salt tolerance genes in rice. Problems in the prior art: There is no report in the prior art that OsINH2 and OsINH3 have salt resistance.

Contents of the invention

The key technical problem to be solved by the present invention is the application of OsINH2 and OsINH3 with salt resistance. In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

Example 1

1. rice gene OsINH2, said rice gene OsINH2 has the nucleotide sequence shown in SEQ ID NO.1, and the full length is respectively 4741bp; The transcript (mRNA) of above-mentioned rice gene OsINH2 has as shown in SEQ ID NO.2 The nucleotide sequence shown, wherein 239bp-736bp is the CDS region, a total of 498bp sequence.

2. Rice gene OsINH3, comprising: rice gene OsINH3 has the nucleotide sequence shown in SEQ ID NO.3, and the full length is 1243bp respectively; The nucleotide sequence shown, wherein 88bp-465bp is the CDS region, a total of 378bp sequence.

3. Cloning of rice genes OsINH2 and OsINH3 and verification of interacting proteins, specifically including: (1) cloning of rice genes OsINH2 and OsINH3, (2) interaction between OsINH2 and OsINH3 and OsTOPPs.

4. Gene function verification method of rice genes OsINH2 and OsINH3, specifically including: (1) analysis of subcellular localization of OsINH2 and OsINH3, (2) analysis of expression patterns of OsINH2 and OsINH3, (3) construction of vectors for overexpression of OsINH2 and OsINH3 in rice , (4) Construction of rice mutants osinh2 and osinh3, (5) Rice genetic transformation and positive strain screening, (6) ABA response analysis, (7) Osmoregulation analysis.

5. Application of OsINH2 and OsINH3 to regulate plant fertility.

6. Application of OsINH2 and OsINH3 to regulate plant pollen vigor.

7. The application of OsINH2 and OsINH3 to scavenge reactive oxygen species (ROS).

8. The application of the interaction between OsINH3 and OsSnRK.

9. Application of OsINH2 and OsINH3 for salt resistance.

10. A vector comprising rice OsINH2 or OsINH3, characterized in that said OsINH2 has the nucleotide sequence shown in SEQ ID NO.1; said OsINH3 has the nucleotide sequence shown in SEQ ID NO.3.

Beneficial effects: When excavating stress-resistant genes, it was found that the rice mutants knocked out by CRISPR-Cas of these two genes were salt-sensitive, while the overexpression lines not only survived high under salt stress, but also their growth was not significantly inhibited. In addition, the OsINH2 and OsINH3 knockout lines had lower fertility, decreased pollen vigor, and significant accumulation of reactive oxygen species, while the overexpression lines had similar fertility and pollen vigor to the wild type, and significantly decreased reactive oxygen species. Based on these phenotypic results, we believe that the study of OsINH2 and OsINH3 genes will play a positive role in improving the ability of rice to scavenge reactive oxygen species and salt resistance, and to expand the stress resistance gene pool. Further research on the mechanism of action of the gene and its conservation in other crops, so as to promote it in crops.

Description of drawings

Figure 1 is the agarose gel electrophoresis of OsINH2 and OsINH3 clones. In the figure (a) OsINH2 gene amplification size is 495bp, (b) OsINH3 gene amplification size is 375bp.

Figure 2 shows the interaction and intracellular colocalization of OsINH2 and OsINH3 with OsTOPPs. Panel (a) shows the interaction of OsINH2 and OsINH3 with OsTOPPs in the yeast two-hybrid system. OsINH2-AD and OsSAPK1-BD or OsINH3-AD and OsSAPK1-BD were used as positive controls. OsINH2 and OsINH3 fused to empty AD and OsTOPPs fused to empty BD were used as negative controls. DDO, SD/-Leu/-Trp; QDO/X, SD/-Leu/-Trp/-His/-Ade supplemented with X-α-Gal. Panel (b) represents the BiFC analysis of the interaction of OsINH2 and OsINH3 with OsTOPPs. OsINH2-YC and OsSAPK1-YN and OsINH3-YC and OsSAPK1-YN were used as positive controls. OsINH2-YC and OsSAPK4-YN or OsINH3-YC and OsSAPK4-YN were used as negative controls. Figure (c) is a transient expression analysis showing that OsINH2 co-localized with OsTOPPs in vivo, and OsSAPK1-RFP and YFP-OsINH2 were used as positive controls. (d) Transient expression analysis shows that OsINH3 co-localizes with OsTOPPs in vivo, and OsSAPK1-RFP and YFP-OsINH3 are used as positive controls.

Figure 3 shows the tissue-specific expression of OsINH2 and OsINH3 and their protein subcellular localization. In the figure (a) is the expression pattern of OsINH2, (i) seedling, (ii) primary root, (iii) crown root, (iv) leaf, (v) stem, (vi) floret, (vii) flower, and ( viii) Pollen grains. In the figure (b) is the expression pattern of OsINH3, (i) seedling, (ii) primary root, (iii) crown root, (iv) leaf, (v) stem, (vi) floret, (vii) flower, and ( viii) Pollen grains. (c) is the subcellular localization of OsINH2. Figure (d) shows the subcellular localization of OsINH3.

Figure 4 is quantitative PCR identification of the expression levels of OsINH2-OE and OsINH3-OE. In the figure (a) the gene expression level of OsINH2 in OsINH2-OE lines #1 and #3 was significantly up-regulated compared with wild-type Zhonghua 11. (b) The gene expression level of OsINH3 in OsINH3-OE lines #1 and #4 was significantly up-regulated compared with Zhonghua 11.

Figure 5 shows that osinh2 and osinh3 mutants are sensitive to ABA. (a) and (b) are the seed germination rates of OsINH2 mutants and overexpression lines under control and ABA treatment, respectively. Figures (c) and (d) are statistical root and shoot lengths of OsINH2 mutants and overexpression lines seedlings. (e) and (f) are the seed germination rates of OsINH3 mutants and overexpression lines under control and ABA treatment. (g) and (h) in the figure are the statistical root and shoot lengths of OsINH3 mutant and overexpression line seedlings. Seedlings in the upper panel were grown on 1/2MS medium with 0 μM and 5 μM ABA, and the germination rate (%) was recorded for up to 7 days.

Figure 6 shows that OsINH2 and OsINH3 are involved in ROS scavenging and osmoregulation in response to ABA. In the figure (a) is the ROS accumulation of OsINH2 and OsINH3 mutants and overexpression lines in normal and ABA treatment. The seeds were grown on 1/2MS medium, and the one-week-old seedlings were transferred to the nutrient solution. 100 μM ABA was sprayed during the period, and leaf samples were incubated overnight in NBT solution, and ROS were observed after decolorization with 75% ethanol. (b) and (c) in the figure are the proline content in OsINH2 and OsINH3 mutants and overexpression lines. (d) and (e) are the expression profiles of OsP5CS1 in OsINH2 and OsINH3 mutants and overexpression lines.

Figure 7 shows the mature seeds and anther phenotypes of mutants and overexpression lines of OsINH2 and OsINH3. In the figure (a) is the phenotype of spikelet fertility of wild type (WT) and OsINH2 mutant and overexpression lines. (b) in the figure is the statistics of the number of seeds per panicle in WT and OsINH2 mutants and overexpression lines. (c) is the morphological observation of pollen grains of WT and OsINH2 mutants and overexpression lines. Scale bar, 30 μm. Figure (d) represents the percentage of active and inactive pollen of WT and OsINH2 mutants and overexpression lines. Panel (e) is the phenotype of spikelet fertility in WT and OsINH3 mutants and overexpression lines. (f) in the figure is the statistics of the number of seeds per panicle in WT and OsINH3 mutants and overexpression lines. (g) is the morphological observation of pollen grains of WT and OsINH3 mutants and overexpression lines. (h) in the figure represents the percentage of active and inactive pollen in WT and OsINH3 mutant and overexpression lines.

Figure 8 shows that OsINH2 and OsINH3 are involved in ROS scavenging in response to ABA. (a) and (b) in the figure are the MDA content in the mutants and overexpression lines of OsINH2 and OsINH3. (c) and (d) in the figure are the POD activities in the mutants and overexpression lines of OsINH2 and OsINH3. The seeds were grown on 1/2MS medium, and the one-week-old seedlings were transferred to the nutrient solution. The MDA content and POD activity were detected from the leaves after spraying 100μMABA. (e) and (h) are the expression profiles of OsLEA3 and OsLIP19 in OsINH2 and OsINH3 mutants and overexpression lines.

Figure 9 shows the interaction between OsINH2 and OsINH3 and OsSAPKs. (a) Yeast two-hybrid demonstrates the interaction of OsINH2 and OsINH3 with OsSAPKs. OsINH2-AD and OsTOPP4-BD or OsINH3-AD and OsSAPK4-BD were used as positive controls. OsINH2 and OsINH3 fused to an empty AD and OsSAPKs fused to an empty BD were used as negative controls. DDO: SD/-Leu/-Trp; QDO/X: SD/-Leu/-Trp/-His/-Ade supplemented with X-α-Gal. (b) BiFC results show that OsINH2-YC and OsINH3-YC interact with OsSAPK1/2/3 and OsSAPK8/9. OsINH2-YC and OsTOPP4-YN, OsINH3-YC and OsTOPP4-YN were used as positive controls. OsINH2-YC and OsSAPK5-YN, OsINH3-YC and OsSAPK5-YN were used as negative controls. BF, bright field. Scale bar, 50 μm.

Figure 10 shows the phenotypes of OsINH2 and OsINH3 mutants and overexpression lines at germination stage under salt stress.

Figure 11 is the stem length statistics of wild-type Zhonghua 11, OsINH2 and OsINH3 mutants and overexpression lines under salt stress. In the figure (a) when treated with 0mM NaCl, there was no difference in stem length between wild type Zhonghua 11, osinh2 and OsINH2-OE; while under salt treatment, compared with Zhonghua 11, the stem length of osinh2 was significantly smaller, while OsINH2-OE There is no difference in stem length. (b) When treated with 0mM NaCl, there was no difference in stem length between wild-type Zhonghua 11, osinh3 and OsINH3-OE; however, under salt treatment, compared with Zhonghua 11, the stem length of osinh3 was significantly smaller, while the stem length of OsINH3-OE No difference.

Fig. 12 is the root length statistics of wild-type Zhonghua 11, OsINH2 and OsINH3 mutants and overexpression lines under salt stress. (a) When treated with 0mM NaCl, there is no difference in the root length of Zhonghua 11, osinh2, and OsINH2-OE; when treated with salt, the root length of osinh2 is significantly smaller than that of Zhonghua 11, but there is no difference in the root length of OsINH2-OE . (b) When treated with 0mM NaCl, the root lengths of osinh3 and OsINH3-OE were not different from those of Zhonghua 11; however, when treated with salt, the root length of osinh3 was significantly smaller than that of Zhonghua 11, while the root length of OsINH3-OE was not. difference.

Figure 13 shows the salt tolerance phenotype of OsINH2-OE. (a) Before salt stress treatment, there was no difference in the growth and development of wild-type Zhonghua 11 and osinh2, but after 7 days of 150mM NaCl treatment, osinh2 was significantly shorter than Zhonghua 11, and the degree of yellowing of leaves was more serious; under normal rice nutrition Compared with Zhonghua 11, the survival rate of osinh2 was lower and the plant height did not recover after 14 days in solution. (b) Before salt stress treatment, there was no difference in the growth and development of Zhonghua 11 and OsINH2-OE, but after 7 days of 150mM NaCl treatment, OsINH2-OE suffered less salt damage than Zhonghua 11, with higher plant height and yellow leaves To a lesser extent; after 14 days of normal rice nutrient solution recovery, the survival rate and plant height of OsINH2-OE were significantly higher than those of Zhonghua 11.

Figure 14 shows the salt tolerance phenotype of OsINH3-OE. (a) Before salt stress treatment, there was no difference in the growth and development of wild-type Zhonghua 11 and osinh3, but after 150mM NaCl treatment for 7 days, the plant height of osinh3 was significantly shorter than that of Zhonghua 11, and the degree of yellowing of leaves was more serious; in normal Compared with Zhonghua 11, the survival rate of osinh3 was lower and the plant height did not recover after 14 days of recovery in rice nutrient solution. (b) Before salt stress treatment, there was no difference in the growth and development of Zhonghua 11 and OsINH3-OE, but after 7 days of 150mM NaCl treatment, OsINH3-OE suffered less salt damage than Zhonghua 11, with higher plant height and yellow leaves To a lesser extent; after 14 days of normal rice nutrient solution recovery, the survival rate and plant height of OsINH3-OE were significantly higher than those of Zhonghua 11.

Figure 15 shows the survival statistics of wild-type Zhonghua 11, OsINH2-OE, osinh2 and OsINH3-OE, osinh3 after salt stress treatment. In (a) compared with the wild type Zhonghua 11, the survival rate of osinh2 and osinh3 was significantly reduced after salt treatment. (b) Compared with Zhonghua 11, the survival rate of OsINH2-OE and OsINH3-OE was significantly increased after salt treatment.

Specific implementation method

The methods and devices used in the following examples of the patent of the present invention are conventional methods and devices unless otherwise specified; the equipment and reagents used are all conventional equipment and reagents purchased by reagent companies. In order to make the purpose, technical solution and advantages of the patent of the present invention clearer, the specific implementation of the patent of the present invention will be described in detail below in conjunction with specific examples. Examples of these preferred embodiments are illustrated in the specific examples. Here, it should also be noted that, in order to avoid obscuring the technical solution of the patent of the present invention due to unnecessary details, only the technical solutions and/or processes closely related to the patent of the present invention are shown in the embodiments steps, while omitting other irrelevant details.

Example 1

This embodiment provides the rice gene OsINH2, including: the rice gene OsINH2 has a nucleotide sequence as shown in SEQ ID NO.1, and the full length is 4741bp; the mRNA of the above rice gene OsINH2 has a nucleotide sequence as shown in SEQ ID NO.2 Nucleotide sequence, of which 239bp-736bp is the CDS region, a total of 498bp sequence (U is replaced by T for convenience of analysis).

Example 2

This embodiment provides the rice gene OsINH3, including: the rice gene OsINH3 has a nucleotide sequence as shown in SEQ ID NO.3, and the full length is 1243bp; the mRNA of the above rice gene OsINH3 has a nucleotide sequence as shown in SEQ ID NO.4 Nucleotide sequence, of which 88bp-465bp is the CDS region, a total of 378bp sequence (U is replaced by T for convenience of analysis).

Example 3

This example provides the cloning and interaction protein verification of rice genes OsINH2 and OsINH3, specifically including:

1. Cloning of rice genes OsINH2 and OsINH3

The inhibitor2 and inhibitor3 genes were first reported in mammalian cells and yeast. As the regulatory subunits of protein phosphatase PP1, they regulate cell division and other aspects by regulating the function of PP1. Then we found its homologous genes AtI2 and Atinh3 in Arabidopsis thaliana. Through protein sequence alignment, we found two genes OsINH2 and OsINH3 with high homology to Arabidopsis thaliana.

The CDS sequence of the target gene was obtained from the phytozome website, and primers were designed to clone the rice OsINH2 and OsINH3 genes: OsINH2-F-1: GGGGTACCCCATGAGCTCTCGTCGTGTGAAG, OsINH2-R-1: GGACTAGTCCTGTTTGCGGTGGGGGTG. OsINH3-F-1: GGGGTACCCCATGGCAACGCGCGCGCCGGCGA, OsINH3-R-1: GGACTAGTCCATGGTCGTGGCCGTGGCC.

Using OsINH2-F-1/OsINH2-R-1 and OsINH3-F-1/OsINH3-R-1 as upstream and downstream primers, respectively, and rice Zhonghua 11 seedling cDNA as a template, PCR amplification was performed to obtain the target gene. The PCR amplification reaction was carried out in a BIO-RADS1000 Thermal Cycler PCR instrument, and the program was: 95°C pre-denaturation for 4 minutes; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, cycle 35 times; 72°C extension for 5 min; 15°C storage . The PCR products were verified by agarose gel electrophoresis, as shown in Figure 1.

2. OsINH2 and OsINH3 interact with OsTOPPs

To detect whether OsINH2 and OsINH3 directly interact with OsTOPP1-5 in the yeast two-hybrid system, refer to (Zhang et al., 2020). OsINH2 and OsINH3 are fused to the Gal4 activation domain (AD), and OsTOPP1-5 are linked to the Gal4 DNA-binding domain (BD). Spread the yeast containing AD and BD plasmids on a DDO (SD/-Leu/-Trp) medium plate and incubate at 28°C for 3-4 days. After incubation, each sample was inoculated on a QDO (SD/-Leu/-Trp/-His/-Ade/+X-a-Gal agar plate, and incubated for 3-7 days. Then observe the colony morphology. Yeast two-hybrid detection as attached As shown in Figure 2, OsINH2 and OsINH3 can directly interact with OsTOPP1-5.

At the same time, bimolecular fluorescence complementation (BiFc) experiments were carried out in tobacco leaves, method reference (Hu et.al., 2018). OsINH2-cYFP or OsINH3-cYFP was co-expressed with OsTOPPs- _N YFP plasmid in tobacco, and the fluorescent signal was observed by confocal laser microscopy. The results are shown in Figure 2, OsINH2 and OsINH3 directly interact with OsTOPP1-5 in plants.

Example 4

This embodiment provides a gene function verification method for rice genes OsINH2 and OsINH3, specifically including:

1. Analysis of subcellular localization of OsINH2 and OsINH3

Primers were designed as follows: OsINH2-F-2: GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGAGCTCTCGTCGTGTGAAG, OsINH2-R-2: GGGGACCACTTTGTACAAGAAAGCTGGGTTTCATGTTTGCGGTGGGGGTG, OsINH3-F-2: GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGGCAAC GCGCGCGCCGGCGA, OsINH3-R-2: GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTCGTGGCCGTGGCC.

OsINH2-F-2/OsINH2-R-2 and OsINH3-F-2/OsINH3-R-2 were used as primers respectively, and Zhonghua 11 seedling leaf cDNA was used as template to carry out PCR amplification to obtain the target gene. The PCR amplification reaction was carried out in a BIO-RAD S1000 ThermalCycler PCR instrument. The program was: 95°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and 35 cycles; 72°C extension for 5 min; 15°C save. After the PCR product was recovered and purified, the target gene was recombined into the intermediate vector pDONR-Zeo with BP enzyme, and the ligated product was transformed into Escherichia coli competent DH5α cells, coated with LB culture dishes containing Zeo antibiotics and cultured overnight at 37°C. The plaques were taken for colony PCR with general primers M13F and M13R, and sent for testing and verification. The plaques with correct sequencing were suspended and cultured in Zeo-containing LB, and the plasmids were extracted. Recombine the pDONR-Zeo vector containing the target gene into the pEarleyGate 104 (N-YFP) expression vector with LR enzyme, transform the ligated product into E. coli competent DH5α cells, and spread it on LB culture containing kanamycin antibiotic Based on overnight culture at 37°C, plaques were picked and cultured in LB containing kanamycin, and the extracted plasmid was transformed into Agrobacterium GV3101. Transiently expressed in tobacco leaves, the results are shown in Figure 3, and OsINH2 was detected to be distributed in the cytoplasm and nucleus, while OsINH3 was localized in the nucleus.

2. Analysis of OsINH2 and OsINH3 expression patterns

The promoter sequences of OsINH2 and OsINH3 were respectively inserted into pCAMBIA1301 GUS plasmid, and the sequence sizes were 3.3Kb and 3Kb, respectively. Different tissues of GUS transgenic plants transformed into Zhonghua 11 (ProOsINH2:GUS/WT and ProOsINH2:GUS/WT) were stained with GUS working solution for 1 night at 37°C, and then decolorized with 70% ethanol, method reference (Qinet al., 2014). Imaged using a LEICA (M205A) stereomicroscope. The results are shown in Figure 3, the promoters of OsINH2 and OsINH3 are highly activated during germination and late germination, and are highly expressed in seedlings, tap roots, crown roots, leaves and aerial parts. It is expressed in ears, anthers and pollen grains during the reproductive stage.

3. Construction of rice overexpression OsINH2 and OsINH3 vectors

The genes were amplified with OsINH2-F-1/OsINH2-R-1 and OsINH3-F-1/OsINH3-R-1 primers respectively and recovered by gel. Digest the POX vector with BamHI and KpnI double enzymes to obtain a linear vector, react with the purified product recovered from the above PCR with infusion enzyme in a water bath at 15°C for 10 minutes to recombine the target gene into POX, and transform the ligated product into E. coli competent DH5α cells. Pick plaques and use OsINH2-F-1/OsINH2-R-1 or OsINH3-F-1/OsINH3-R-1 as primers for PCR amplification, and send them for testing and verification. Mycin LB suspension culture, extract the plasmid.

4. Construction of rice mutants osinh2 and osinh3

Enter the OsINH2 and OsINH3 gene sequences at https://crispr.dbcls.jp/, obtain the CRISPR editing specific target sequences GTTAGGCAGAAGATAACTGA and CATCCACGACCGTGACGGTG, design adapter primers, and construct mutants with CRISPR-Cas9.

5. Genetic transformation of rice and screening of positive strains

(1) Callus induction: Peel off the glume of rice seeds, soak in 70% ethanol for 1 min, and wash with sterile water for 3 times. Then put it into a sodium hypochlorite solution with 5% available chlorine and soak for 15 minutes, and wash it with sterile water for 5 times. Place on N6D medium and culture at 32°C for about two weeks to induce callus differentiation.

(2) Co-transformation with Agrobacterium: Add Agrobacterium for transformation into 200 mL of LB medium, and culture at 28° C. for about 16 hours. The shaken bacterial solution was centrifuged at 5000 rpm for 15 minutes, the supernatant was discarded, and the bacterial cells were suspended in AAM medium added with acetosyringone (AS), and the bacteria were shaken at 28°C for 2 hours. Cut off the rice callus and put it into a sterilized Erlenmeyer flask, pour the shaken bacterial solution into it and soak for 10 minutes. Pour off the bacterial liquid, place the callus on sterile filter paper to blot the bacterial liquid on the surface, and culture it on N6 AS medium at 25°C for 48-72 hours.

(3) Removal of Agrobacterium: 3 bottles of 500ml cefotaxime sodium (cef) solution were prepared with sterile water, the concentrations were 500mg/L, 1000mg/L and 2000mg/L respectively. The callus co-cultured with Agrobacterium was put into the Erlenmeyer flask, and the callus was washed with cef solution according to the concentration gradient from low to high.

(4) Differentiation culture: use sterile filter paper to blot the liquid on the surface of the degermed callus, place the callus on N6DS medium for two weeks at 32°C, and then transfer the newly grown callus to Continue to culture on RE medium, at this time the callus will gradually differentiate into mature seedlings.

(5) Rooting culture: When the rice seedlings differentiated on the RE medium grow to about 3 cm, the seedlings are transferred to the HF medium for about two weeks at 28° C., and then transplanted to sterile perlite for hardening for one week. Can be transplanted to the greenhouse.

For rice overexpressing OsINH2 and OsINH3 transformed lines, we performed real-time fluorescent PCR detection, as shown in Figure 4. Two strains, OsINH2OE-1, OsINH2OE-3, OsINH3OE-1, and OsINH3OE-4, whose expression levels were significantly higher than those of the wild type were screened.

For the transformed strain constructed by CRISPR-Cas technology knockout, we designed primers on the upstream and downstream of the target site, and identified frameshift mutants osinh2-6, osinh2-11, and osinh3 caused by two base insertions or deletions by gene sequencing. -4, osinh3-9.

6. ABA response analysis

Under normal conditions, the seed germination rate of OsINH2 and OsINH3 knockout and overexpression lines was the same as that of wild type. After ABA treatment, the seed germination rate of the knockout lines osinh2-6, osinh2-11, osinh3-4, osinh3-9 was significantly reduced, and the growth of seedlings was also inhibited. Compared with the wild type, the roots and shoots of the knockout lines The growth was obviously slowed down, as shown in Figure 5. Although the seed germination and seedling growth of OsINH2 and OsINH3 overexpression lines were similar to wild type, they showed tolerance to ABA stress compared with their respective knockout lines, as shown in Fig. 5.

7. Osmoregulation Analysis

Proline is a compatible and permeable substance that functions by stabilizing subcellular structures and promoting cellular recovery after stress injury, and can serve as a stress indicator affecting adaptive responses. Free proline content calculation reference (Lou et al., 2017). Calculate the standard concentration of proline with l-proline, and measure the absorbance at a wavelength of 530nm with a spectrophotometer. As shown in Figure 6, under the action of ABA, the accumulated free proline content of the OsINH2 and OsINH3 knockout lines was significantly reduced, while the free proline content of the overexpression line was significantly increased compared with the wild type. In addition, the expression levels of the proline biosynthesis gene OsP5CS1 were significantly decreased in the OsINH2 and OsINH3 knockout lines after ABA treatment, but significantly increased in the overexpression lines.

Example 5

This embodiment provides the application of rice genes OsINH2 and OsINH3, specifically including:

1. Application of OsINH2 and OsINH3 to regulate plant fertility

As shown in Figure 7, during reproductive development, the OsINH2 and OsINH3 knockout lines had lower fertility and significantly reduced seed number, while the OsINH2 and OsINH3 overexpression lines had the same fertility as the wild type, with no significant seed number difference. Therefore, OsINH2 and OsINH3 may play an important role in the regulation of rice fertility.

2. Application of OsINH2 and OsINH3 to regulate plant pollen viability

Refer to pollen grain staining method (Xiang et al., 2019). Select a flower that is fully developed and the anther is wrapped in the floret, blast an anther on a glass slide, add 1-2 drops of potassium iodide solution, and observe under a light microscope. Viable pollen turns dark blue, and inactive or stunted pollen turns brown. Anthers became dry and brown in OsINH2 and OsINH3 knockout lines, and inactive pollen grains increased compared with wild type. However, the proportion of active pollen in the OsINH2 and OsINH3 overexpression lines was similar to that of the wild type, as shown in Figure 7. Therefore, OsINH2 and OsINH3 may be critical in rice pollen development.

3. Application of OsINH2 and OsINH3 to scavenge reactive oxygen species (ROS)

The method for detecting ROS refers to (He et al., 2012). Leaves were stained with nitro blue tetrazolium (NBT). The results are shown in Figure 6, ROS accumulated in the OsINH2 and OsINH3 knockout lines, but decreased significantly in the overexpression lines. MDA content and POD enzyme activity were evaluated according to commercial kit instructions (Solarbio, Cat No: BC0020, Solarbio Cat No: BC0200). The results are shown in Figure 8. After ABA treatment, the amount of MDA formed in the OsINH2 and OsINH3 knockout lines increased, while the MDA content of the overexpression lines decreased significantly, indicating that OsINH2 and OsINH3 can be protected by reducing the formation of lipid peroxidation. Membrane damage. Compared with the wild type, after ABA treatment, the POD activity in the OsINH2 and OsINH3 knockout lines was lower, while the POD activity in the overexpression line was increased. After ABA treatment, the gene expressions of OsLEA3 and OsLIP9 were significantly downregulated in OsINH2 and OsINH3 knockout lines. In contrast, the expression of these genes was induced to upregulate in the overexpression lines. These results suggest that OsINH2 and OsINH3 may regulate ROS by stimulating the activity of antioxidant enzymes and triggering stress response genes to promote rice growth under stress.

4. Application of interaction between OsINH3 and OsSnRK

The results are shown in Figure 9. Yeast two-hybrid assays confirmed that OsINH3 interacted with OsSAPK1, OsSAPK2, OsSAPK3, OsSAPK8 and OsSAPK9, which are important regulators of the ABA signaling pathway. Therefore, OsINH3 may participate in the ABA signal transduction process through OsSAPKs

5. Application of OsINH2 and OsINH3 for salt resistance

The method for analyzing the salt tolerance of OsINH2-OE, OsINH3-OE, osinh2, and osinh3 at the seedling stage is as follows: put rice seeds in an oven at 45°C for a week to break dormancy, then sterilize the seeds with 70% alcohol for 60 seconds, and then use 50% Soak in sodium hypochlorite solution for 10 minutes, wash with sterilized water for 5 times, plant in 1/2MS medium, culture conditions: 14 hours of light (28°C)/10 hours of darkness (24°C), light intensity 800μmol·m-2·s -1, 70% relative humidity. After the seeds germinated, they were transferred to 1/2MS medium containing 150 mM NaCl, grown for 5 days, and the plant height and root length were counted. The results are shown in Fig. 10, Fig. 11 and Fig. 12, the OsINH2 and OsINH3 knockout lines are more sensitive to salt treatment at the seed germination stage, while the overexpression lines are similar to the wild type in response.

Salt stress treatment was carried out when the seedlings grew to the four-leaf stage, and 150mM NaCl was added to the rice nutrient solution, which was replaced every 3 days. After 7 days of salt treatment, the seedlings were re-placed in the Yoshida nutrient solution without adding NaCl. After 7 days, the phenotype was observed and the survival rate of the seedlings was counted. The results are shown in Figures 13 and 14, knocking out OsINH2 and OsINH3 genes reduced the salt tolerance of rice, while overexpressing OsINH2 and OsINH3 genes enhanced the salt tolerance of rice.

The above description is only the specific implementation of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present application, some improvements and modifications can also be made. It should be regarded as the protection scope of this application.

references:

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Claims (10)

1. The rice gene OsINH2 is characterized in that the rice gene OsINH2 has a nucleotide sequence as shown in SEQ ID NO.1, and its full length is 4741bp; its transcript has a nucleotide sequence as shown in SEQ ID NO.2 Sequence, wherein 239bp-736bp is the CDS region, a total of 498bp sequence. 2. The rice gene OsINH3 is characterized in that the rice gene OsINH3 has a nucleotide sequence as shown in SEQ ID NO.3, and its full length is 1243bp; its transcript has a nucleotide sequence as shown in SEQ ID NO.4 Sequence, in which 88bp-465bp is the CDS region, a total of 378bp sequence. 3. A method for cloning and interacting protein verification of rice genes OsINH2 and OsINH3, characterized in that the method comprises the following steps: (1) cloning of rice genes OsINH2 and OsINH3, (2) OsINH2 and OsINH3 interact with OsTOPPs. 4. The gene function verification method of rice genes OsINH2 and OsINH3 is characterized in that the method comprises the following steps: (1) analysis of subcellular localization of OsINH2 and OsINH3, (2) analysis of expression patterns of OsINH2 and OsINH3, (3) rice ultra- Construction of vectors expressing OsINH2 and OsINH3, (4) construction of rice mutants osinh2 and osinh3, (5) rice genetic transformation and positive strain screening, (6) ABA response analysis, (7) osmoregulation analysis. 5. The application of rice OsINH2 and OsINH3 to regulate plant fertility, characterized in that said OsINH2 has a nucleotide sequence as shown in SEQ ID NO.1; said OsINH3 has a nucleotide sequence as shown in SEQ ID NO.3 . 6. The application of rice OsINH2 and OsINH3 to regulate plant pollen vigor, characterized in that said OsINH2 has a nucleotide sequence as shown in SEQ ID NO.1; said OsINH3 has a nucleotide sequence as shown in SEQ ID NO.3 . 7. The application of rice OsINH2 and OsINH3 to scavenge active oxygen, characterized in that the OsINH2 has the nucleotide sequence shown in SEQ ID NO.1; the OsINH3 has the nucleotide sequence shown in SEQ ID NO.3. 8. The application of rice OsINH3 interacting with OsSnRK, characterized in that the OsINH3 has the nucleotide sequence shown in SEQ ID NO.3. 9. The application of rice OsINH2 and OsINH3 salt resistance, characterized in that the OsINH2 has the nucleotide sequence shown in SEQ ID NO.1; the OsINH3 has the nucleotide sequence shown in SEQ ID NO.3 . 10. A vector comprising rice OsINH2 or OsINH3, characterized in that said OsINH2 has a nucleotide sequence as shown in SEQ ID NO.1; said OsINH3 has a nucleotide sequence as shown in SEQ ID NO.3 .

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