Application of rice OsBBX3 gene

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to application of a rice OsBBX3 gene. Bioinformatics and detection analysis tests show that the protein OsBBX3 containing the B-box structural domain of the rice has the function of improving the drought tolerance of the rice, the drought tolerance of crops can be improved by improving the expression level of the OsBBX3 in the crops, the OsBBX3 gene can also be introduced into plants to construct drought-tolerant transgenic varieties, and the gene can also be used for identifying the drought-tolerant varieties.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and specifically, the present invention relates to an application of rice OsBBX3 gene. Background Art

[0002] Rice is one of the most important food crops, but in the context of changing climate, rice production is often affected by drought weather. Severe drought will affect rice yield and quality, endangering food security. Existing studies have shown that rice drought resistance is controlled by genes. Therefore, discovering drought-resistant genes and using them to improve rice drought resistance is an effective way to cope with drought and ensure rice production and food security. CN202410920410.1 discloses methods such as ozone fumigation screening, cloning a gene OsOSR1 that controls rice drought resistance and high temperature resistance, and biological function verification shows that it can improve rice drought and high temperature resistance. Co-segregation detection experiments show that osr1 mutants are closely related to drought and drought and high temperature sensitive phenotypes. CN202410421108.1 discloses HGW genes and their applications in improving rice stress tolerance. HGW genes can improve rice drought tolerance and salt tolerance. However, more research is needed on the related genes and mechanisms for improving rice drought tolerance.

[0003] B-box (BBX) proteins are a class of zinc finger proteins containing one or two B-box domains. They play an important role in plant growth and development, including responding to the diurnal cycle, photoperiodic regulation of flowering, and responding to external stress. Recently, some people have analyzed the BBX genes of rice using bioinformatics methods and found that the BBX genes of rice can be divided into five subtypes, and the expression patterns and expression conditions of these genes are also different. The expression level of most rice BBX members at the heading stage is higher than that at the seedling stage and the booting stage. Some rice OsBBX genes can be induced by external stimuli such as cold, some can respond to the induction of plant hormones, and some are greatly affected by heavy metals. At present, the nature, regulatory relationship, and function of rice OsBBX genes have not been thoroughly studied and need further exploration. Summary of the invention

[0004] In view of the insufficiency of the development of rice drought-resistant genes in the prior art, the present invention has found through extensive research and a large number of tests that rice OsBBX3 has the effect of improving rice drought resistance, thereby completing the present invention.

[0005] The technical solution of the present invention is as follows:

[0006] In one aspect of the present invention, the present invention discloses an application of a rice OsBBX3 gene in improving drought resistance of rice. The nucleotide sequence of the rice OsBBX3 gene is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0007] In one aspect of the present invention, a method for identifying drought-tolerant rice is disclosed, the method comprising detecting the expression level of the OsBBX3 gene in rice, and if the OsBBX3 gene is highly expressed, the rice is likely to be a drought-tolerant variety. The high expression means that the expression level of the OsBBX3 gene in rice is significantly increased relative to the normal level in wild rice.

[0008] In one aspect of the present invention, a method for preparing drought-tolerant rice is disclosed. The method comprises transducing a vector containing the rice OsBBX3 gene into rice, and constructing and screening transgenic plants.

[0009] In one embodiment, a method for preparing drought-tolerant rice comprises:

[0010] (4) PCR amplification of the full-length CDS sequence of OsBBX3 was performed using rice cDNA as a template;

[0011] (5) constructing the sequence obtained in step (1) into a gene expression vector;

[0012] (6) Using Agrobacterium transgenic method, the vector constructed in step (2) is transformed into rice, and drought-tolerant rice is screened.

[0013] Preferably, the vector used in the present invention is pCAMBIAubi1300 vector; the Agrobacterium strain used is EHA105 strain; and the rice variety is Nipponbare rice. The sequence of OsBBX3 is shown in SEQ ID NO.1.

[0014] Beneficial Effects

[0015] (1) The present invention utilizes the overexpression technology of rice drought-resistant gene OsBBX3 to significantly improve the drought-resistant performance of rice; after being treated with 20% PEG600 for 15 days, the survival rate and water loss rate after 5 days of recovery are significantly better than those of wild-type rice, and the root, stem and leaf growth are better than those of the control.

[0016] (2) The present invention provides a rice drought tolerance positive regulatory gene OsBBX3. ​​Rice overexpressing the OsBBX3 gene has a significant drought tolerance effect. In rice-growing areas prone to drought weather, this gene can significantly improve the drought tolerance of rice, cope with high temperature and drought encountered during the rice growing period, and ensure rice production.

[0017] (3) The present invention provides a rice drought tolerance positive regulatory gene OsBBX3, which is derived from rice. Therefore, rice modified with the OsBBX3 gene reduces concerns about food safety. In-depth research on this gene can also reveal its molecular mechanism for regulating rice drought tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a trend chart of the expression level of OsBBX3 in Example 1 of the present invention as the drought treatment time increases.

[0019] Figure 2 This is a graph showing the expression levels of OsBBX3 in roots, stems and leaves in Example 1 of the present invention.

[0020] Figure 3 This is a diagram showing the results of drought treatment of drought-tolerant rice in Example 2 of the present invention.

[0021] Figure 4 This is a diagram showing the location and information of the gene target sequence in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Embodiment 1:

[0024] The rice drought tolerance positive regulatory gene OsBBX3 of this embodiment is located at the rice drought tolerance QTL site on chromosome 2 located by whole genome association analysis through bioinformatics analysis. OsBBX3 has two BBX domains and one CCT domain, and belongs to the group I member of the B-box transcription factor family. The promoter cis-acting element analysis found that the OsBBX3 promoter contains cis-elements that respond to light, hormones, stress, growth and development, etc. The nucleotide sequence of the rice OsBBX3 gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0025] Experiment 1: Analysis of the expression trend of rice drought tolerance positive regulatory gene OsBBX3

[0026] (1) Three-week-old Nipponbare seedlings were placed in a light incubator for drought treatment, with 12 hours of light and 75% humidity for 3 days, and leaf samples were taken at 0, 6, 12, 24, 48, and 72 hours.

[0027] (2) Total RNA was extracted from leaf samples and reverse transcribed to synthesize the first-chain cDNA.

[0028] (3) Based on the cDNA sequence of OsBBX3 gene, primers were synthesized and real-time PCR was used to detect the expression of OsBBX3 at each treatment period.

[0029] Results Figure 1 The expression level of OsBBX3 was induced at 1 hour and reached the maximum at 3 hours, then decreased at 6 hours and then gradually increased. Based on this, it was speculated that overexpression of OsBBX3 in Nipponbare could enhance its drought tolerance.

[0030] Experiment 2: Analysis of the specific expression of rice drought tolerance gene OsBBX3 in root, stem and leaf tissues

[0031] (1) Take the roots, stems and leaves of 3-week-old Nipponbare rice.

[0032] (2) Total RNA was extracted from root, stem and leaf samples and reverse transcribed to synthesize the first-chain cDNA.

[0033] (3) We also detected the specificity of OsBBX3 expression in root, stem, and leaf tissues under normal growth conditions.

[0034] Results Figure 2 : This showed that the expression level of OsBBX3 in leaves was significantly higher than that in other tissues.

[0035] Example 2

[0036] Application of rice drought tolerance positive regulatory gene OsBBX3 in producing drought-tolerant rice

[0037] Based on Example 1, this example uses OsBBX3 and overexpression technology to prepare drought-tolerant rice. The method of this example includes the following steps:

[0038] (1) PCR amplification of OsBBX3 CDS sequence

[0039] Primers were designed using BioXM2.6 (primer sequences are shown in Table 1), and the enzyme used was a high-fidelity polymerase MaxSuper-Fidelity DNA Polymerase; PCR amplification of the full-length CDS sequence of OsBBX3 was performed using cDNA with NPB as the background as the template. The reaction system was as follows (50 μl):

[0040] The primer sequences are shown in Table 1:

[0041] Table 1 Primer sequences

[0042]

[0043]

[0044] Components Dosage cDNA template 1μl Upstream primer 2μl Downstream primer 2μl dntpMix(10mMeach) 1μl 2×PhantaMaxBuffer 25μl PhantaMaxSuper-FidelityDNAPolymerase 1μl <![CDATA[ddH2O]]> 18μl

[0045] The reaction procedure is as follows:

[0046] Cycle steps Reaction conditions Number of cycles Pre-denaturation 95℃3min 1 transsexual 95℃15s 35 annealing 58℃15s 35 extend 72℃1min 35 Secondary extension 72℃5min 1

[0047] (2) Construction of pCAMBIAubi1300-OSBBX3 vector

[0048] The vector pCAMBIAubi1300 was purchased from a commercial company; the strain used was competent Escherichia coli DH5α.

[0049] pCAMBIAubi1300 vector was double-digested with the following reaction system (50 μl):

[0050] Components Dosage pCAMBIAubi1300 vector 4μl(1μg) FDSacI 1μl FDHindIII 1μl FDGreenBuffer 5μl <![CDATA[ddH2O]]> 40μl

[0051] The PCR product and the pCAMBIAubi1300 vector after double restriction digestion at 37°C for 2 to 3 hours were detected by agarose gel electrophoresis, and the target fragment and linearized vector were recovered by cutting the gel (see the kit manual for specific methods).

[0052] The homologous recombination reaction system is as follows (10 μl):

[0053] Components Dosage pCAMBIAubi1300 linearized vector 2μl(100ng) Insert 3μl(200ng) 5×CEIIBuffer 2μl ExnaseII 1μl <![CDATA[ddH2O]]> 2μl

[0054] The linearized vector and the target fragment were homologously recombined using a recombinase, connected at 37°C for 30 min, and immediately placed on ice, followed by E. coli transformation.

[0055] The homologous recombination product was transformed into competent Escherichia coli DH5α by heat shock method. After inverted culture at 37°C overnight, single colonies were picked for bacterial liquid PCR identification (paired amplification with vector forward primer and target fragment rear primer). The obtained positive clones were used for sequencing and plasmid extraction. The correctly sequenced bacterial liquid and recombinant plasmid were named: pCAMBIAubi1300-OSBBX3.

[0056] (3) Obtaining OsBBX3 overexpressing rice plants

[0057] 3.1 The pCAMBIAubi1300-OSBBX3 vector was introduced into the Agrobacterium EHA105 strain to infect the mature embryo callus of Nipponbare rice. Resistant callus was obtained through co-culture, recovery culture and screening culture (the hygromycin concentration in the screening medium was 50 mg / L).

[0058] 3.2 The resistant callus is cultured through differentiation, rooting, etc. to obtain regenerated plants.

[0059] 3.3 The regenerated plants were transplanted into nutrient pots and planted in a greenhouse. The surviving overexpression strains were selected using 50 mg / L hygromycin B to screen positive plants, and the relative expression level of OsBBX3 in each positive plant was detected using qRT-PCR technology, and the strains with high expression levels were selected for cold-tolerant phenotype identification.

[0060] Experiment 3: Evaluation of drought tolerance in rice plants overexpressing OsBBX3

[0061] OsBBX3 overexpressing plants with high expression levels were planted in nutrient pots, 6 plants were planted in each pot, and repeated 3 times. Nipponbare rice was used as a control and grown normally in the greenhouse for 3 weeks. Then, the 3-week-old plants were treated with 20% PEG600 for 15 days, and the phenotype was observed after 5 days of recovery.

[0062] Results Figure 3 :It was found that the survival rate of the transgenic plants with high expression of OsBBX3 was significantly higher than that of the control Nipponbare. The results showed that overexpression of OsBBX3 can significantly improve the drought tolerance of rice. After three repeated experiments, the overexpression of OsBBX3 strains showed strong drought tolerance. Therefore, the experimental results prove that overexpression of the OsBBX3 gene can significantly improve the drought tolerance of rice and is a positive regulatory gene for rice drought tolerance.

[0063] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. An application of rice OsBBX3 gene in improving rice drought resistance, characterized in that: The nucleotide sequence of the rice OsBBX3 gene is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.

2.

2. A method for identifying drought-tolerant rice, characterized in that: The method comprises detecting the expression level of the OsBBX3 gene in rice. If the OsBBX3 gene is highly expressed, the rice is likely to be a drought-resistant variety.

3. A method for preparing drought-tolerant rice, characterized in that: The method comprises the steps of transducing a vector containing the rice OsBBX3 gene into rice, and constructing and screening transgenic rice plants.

4. A method for preparing drought-tolerant rice, characterized in that: The method comprises (1) PCR amplification of the full-length CDS sequence of OsBBX3 was performed using rice cDNA as a template; (2) constructing the sequence obtained in step (1) into a gene expression vector; (3) The vector constructed in step (2) is transformed into rice using the Agrobacterium transgenic method, and drought-tolerant rice is screened out.

5. The preparation method according to claim 4, characterized in that: The sequence of OsBBX3 is shown in SEQ ID NO.

1.

6. The preparation method according to claim 4, characterized in that: The vector is pCAMBIAubi1300.

7. The preparation method according to claim 4, characterized in that: The Agrobacterium strain used was EHA105.

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