Drought-tolerant related transcription factor osmdr in rice, and coding gene and application thereof

Abstract

The application belongs to the field of agricultural biotechnology, and particularly relates to a drought-tolerant related transcription factor OsMDR of rice, a coding gene thereof and application. The amino acid sequence of the drought-tolerant related transcription factor OsMDR of rice is shown as SEQ ID No:1. Overexpression of OsMDR reduces drought-tolerant correlation of transgenic rice at different development stages, simultaneously changes physiological indexes related to stress tolerance, makes the overall physiological level of cells not conducive to drought tolerance, and reduces the expression amount of drought-tolerant related marker genes. Therefore, the OsMDR gene can be used for cultivating drought-tolerant related varieties of crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the rice drought-resistant transcription factor OsMDR, its encoding gene, and its applications. Background Technology

[0002] Transcription factors play crucial roles in crop cell differentiation, growth and development, and tolerance to abiotic stresses. MYB family transcription factors regulate gene expression at the transcriptional level by specifically binding to the MBS (MYB binding site) element in the promoter sequence of target genes, participating in the regulation of various biological processes through both negative and positive regulation. The R2R3-MYB subfamily is a large proportion of MYB family transcription factors, possessing both R2 and R3 MYB domains, and is an important MYB transcription factor in response to abiotic stresses.

[0003] Existing research has demonstrated that multiple MYB family transcription factors are involved in plant stress tolerance processes across different species. For example, overexpression of the wheat R2R3-type MYB protein TaMpc1-D4 reduces drought tolerance in transgenic Arabidopsis thaliana. FtMYB 10, an R2R3-MYB gene from tartary buckwheat, is significantly induced by ABA and drought. Overexpression of FtMYB 10 reduces drought and salt tolerance in transgenic Arabidopsis thaliana.

[0004] Silencing the GhMYB3 gene reduces the rate of water loss in cotton, increases the rate of stomatal closure, and results in higher levels of anthocyanins and proanthocyanidins than in wild-type plants, thus enhancing the drought resistance of cotton seedlings and mature plants.

[0005] TaSIM gene expression is induced by drought, high salinity, low temperature, and ABA. Compared with the wild type, TaSIM gene overexpression improves plant tolerance to salt stress, and TaSIM-overexpressing plants have higher transcription levels of RD22, a gene involved in ABA-dependent signal transduction, and RD29A, a gene involved in ABA-independent signal transduction.

[0006] The expression of the maize ZmMYB48 gene was induced by drought and ABA treatment. Under drought stress, overexpression of ZmMYB48 in Arabidopsis thaliana significantly improved the drought tolerance of transgenic plants and also increased the expression of genes related to stress response or the ABA pathway, such as P5CS1, RD22, RD29B, and ABI1. It is speculated that ZmMYB48 may be a positive regulator of drought stress response through ABA signaling.

[0007] Expression of the soybean R2R3-MYB gene GmMYB84 was induced by drought, salt, H2O2, and ABA stress. Compared with wild-type soybean, soybeans overexpressing GmMYB84 under stress conditions exhibited higher survival rates, longer taproots, higher proline content, lower water loss and malondialdehyde (MDA) content, and higher antioxidant enzyme activity. Under abiotic stress, the expression of ROS-related genes was upregulated in GmMYB84 transgenic plants.

[0008] Overexpression of OsMYB55 in maize reduced the negative impact of high temperature and drought on the growth of transgenic plants. A large number of genes involved in abiotic stress responses such as high temperature, dehydration and oxidative stress were upregulated in transgenic maize with OsMYB55 overexpression, thus improving the heat resistance of transgenic maize.

[0009] Rice is a major food crop, with about half of the world's population relying on it for sustenance. Its growth and yield are severely affected by environmental problems such as drought and soil salinization. 192 MYB genes have been discovered and identified in rice, including 126 R2R3-MYB genes, the functions of most of which have not yet been determined. Summary of the Invention

[0010] The purpose of this invention is to provide a rice drought resistance-related transcription factor, OsMDR.

[0011] Another object of the present invention is to provide the encoding gene of the above-mentioned rice drought-resistant transcription factor OsMDR.

[0012] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned coding gene.

[0013] Another object of the present invention is to provide a recombinant strain containing the above-mentioned encoding gene.

[0014] Another object of the present invention is to provide the application of the above-mentioned rice drought resistance-related transcription factor OsMDR and its encoding gene.

[0015] The amino acid sequence of the rice transcription factor OsMDR of the present invention is shown in SEQ ID No:1:

[0016] MAAAEVQSAAGWGRQLQQDGGGWRKGPWTSQEDALLVEHVRQHGEGRWNSVSKLTGLKRSGKSCRLRWVNYLRPDLKRGKITPQEESIIVQLHALWGNRWSTIARSLPGRTDNEIKNYWRTHFKKGKPSKNIERARARFLK QRREMQQSQQLQQPTLMPTPTPQSKDIIVAETGDARTDDDAGGAAAAVAPSSSSSSLSMGGGEAEDLIMHQDAMDDLMMCPAMSYHLILHGAAVAGHQLSDGGGSCCASTSEDYGSSEDDGATWGSLWNLDGAAGACTLW.

[0017] The nucleotide sequence of the gene encoding the rice transcription factor OsMDR of the present invention is shown in SEQ ID No:2:

[0018] .

[0019] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0020] The present invention also provides a recombinant strain containing the above-mentioned encoding gene.

[0021] The beneficial effects of this invention are:

[0022] The experimental results showed that after 20% PEG treatment, the expression level of the OsMDR gene in the roots of wild-type rice could be upregulated by up to 9.95-fold. The OsMDR protein exhibited transcriptional activation activity, indicating that OsMDR is a transcription factor induced by PEG. Drought resistance identification of OsMDR transgenic rice revealed that upregulation of the OsMDR gene reduced drought resistance during the seedling and grain-filling stages, decreased chlorophyll content, proline content, and the ratio of completely closed stomata under stress conditions under PEG treatment, and increased malondialdehyde content and water loss rate. OsMDR gene overexpression downregulated the expression levels of stress resistance marker genes OsLEA3 and OsPOD1. Therefore, phenotypic, physiological, and molecular data of OsMDR transgenic rice under PEG treatment all indicate that upregulation of the OsMDR gene reduces the drought tolerance of transgenic rice.

[0023] OsMDR overexpression reduced drought resistance in transgenic rice at different developmental stages, while also altering stress-related physiological indicators, making the overall physiological level of cells unfavorable to drought resistance and reducing the expression levels of drought-related marker genes. Attached Figure Description

[0024] Figure 1 The expression level of the rice OsMDR gene was shown to change under 20% PEG6000 treatment.

[0025] Figure 2 Identification of the transcriptional activation activity of OsMDR protein: growth of OsMDR transgenic yeast on different culture media;

[0026] Figure 3 Identification of transcriptional activation activity of OsMDR protein: X-gal staining results of OsMDR transgenic yeast;

[0027] Figure 4 This shows the identification of drought resistance in OsMDR overexpressing transgenic rice during the seedling stage;

[0028] Figure 5 This shows the identification of drought resistance in OsMDR overexpressing transgenic rice during the grain-filling stage;

[0029] Figure 6 The results of measuring different physiological indicators of OsMDR-overexpressing transgenic rice before and after salt stress are shown.

[0030] Figure 7 This shows the expression levels of stress-related marker genes in OsMDR-overexpressing transgenic rice. Detailed Implementation

[0031] Experimental materials

[0032] The tested plant materials were wild-type rice (Oryza sativa), Nipponbare, and OsMDR overexpressing transgenic rice.

[0033] Test strains: Escherichia coli DH5α strain, Agrobacterium tumefaciens EHA105 strain, and yeast AH109.

[0034] Example 1: Analysis of OsMDR gene expression patterns under different treatment conditions

[0035] Wild-type rice was treated with 20% PEG6000. Roots were collected at 0h, 1h, 6h, 12h, 24h, and 72h after treatment. Total RNA was extracted from different materials using the Trizol method, and after reverse transcription, quantitative PCR analysis was performed to detect the expression level of OsMDR. The primer pairs used to detect the OsMDR gene are as follows:

[0036] The forward primer is: 5′-TCCAGCTCCATGCCTTGTG-3′;

[0037] The reverse primer is: 5′-GACGGCTTGCCCTTCTT-3′.

[0038] The primer pair used to detect the expression level of the internal reference gene (actin) is:

[0039] The forward primer is: 5′-TGGCATCTCTCAGCACATTCC-3′;

[0040] The reverse primer is: 5′-TGCACAATGGATGGGTCAGA-3′.

[0041] like Figure 1 As shown, the expression level of the OsMDR gene in rice roots was upregulated to about 8.3-fold after 1 h of 20% PEG treatment; after 6 h of stress, the upregulation reached 9.95-fold; the expression level was downregulated thereafter, but it was still more than 5-fold higher than before treatment at 72 h.

[0042] Therefore, OsMDR expression is induced by PEG-simulated drought stress and is a stress-resistance-related gene in response to drought stress.

[0043] Example 2: Cloning of the OsMDR gene and analysis of its encoded protein's transcription factor characteristics.

[0044] 1. Cloning the OsMDR gene

[0045] Using cDNA from leaf material subjected to 20% PEG stress for 6 hours in Example 1 as a template, PCR primer sequences were designed. The forward primer was 5′-TGCTGGTGAAAGAAGAACTG-3′, and the reverse primer was 5′-CCACGGATGATAAGTTTGCTG-3′. RT-PCR amplification was performed, and the amplified products were electrophoresed, recovered, and alpha-added. After recovery, the products were ligated into a T-vector and transformed into E. coli DH-5α. Positive clones were screened and sequenced. The clone with correct sequencing was named pT-OsMDR, thus completing the cloning of the OsMDR gene.

[0046] 2. Identification of the transcriptional activation activity of OsMDR protein

[0047] The pGBKT7-OsMDR recombinant plasmid was constructed and transformed into yeast AH109. After obtaining successfully transformed yeast cells, 5-6 clones were selected, suspended in ultrapure water, and serially diluted 10-fold and 100-fold. 2 μL of each suspension was spotted onto SD / -Trp / -His / -Ade medium and incubated at 28℃ for 3 days. The results were then observed. Yeast cells transformed with the empty pGBKT7 vector served as a control.

[0048] like Figure 2 As shown, yeast transformed with both pGBKT7 and pGBKT7-OsMDR could grow normally on SD / -Trp medium; however, the pGBKT7 transformant could not grow on SD / -Trp / -His / -Ade medium, while the pGBKT7-OsMDR transformant could grow normally. These results indicate that the OsMDR protein can initiate the expression of the His and Ade reporter genes.

[0049] Each Figure 2 Yeast culture containing plasmid pGBKT7 or pGBKT7-OsMDR, grown on a medium with a nutrient deficiency for 2 days, was spotted onto clean filter paper, lysed by liquid nitrogen freeze-thaw reaction, and then subjected to X-gal colorimetric reaction.

[0050] like Figure 3 As shown, yeast containing pGBKT7 was not stained blue, while yeast containing pGBKT7-OsMDR was stained blue, indicating that the OsMDR protein successfully activated the expression of the LacZ reporter gene.

[0051] The above results indicate that the OsMDR protein has transcriptional activation activity in yeast.

[0052] Therefore, the OsMDR protein of the present invention is a transcription factor with transcriptional activation activity.

[0053] Example 3: Identification of drought-related functions of OsMDR

[0054] The pTCK303-OsMDR overexpression vector was constructed and transformed into Agrobacterium EHA105. After obtaining correctly identified transformants, these transformants were transformed into callus tissue of Nipponbare rice. Transgenic callus tissue was screened and then regenerated to obtain OsMDR overexpressing transgenic rice lines. After DNA and RNA level identification of the different transgenic rice lines, they were cultured for 3-4 generations to obtain homozygous transgenic lines, which were then used for the following experiments.

[0055] 1. Detection of drought tolerance in OsMDR overexpression transgenic rice seedlings

[0056] Seedlings of OsMDR overexpressing transgenic rice lines and wild-type Nipponbare rice that had grown normally for 14 days after germination were transferred to 1 / 2 MS liquid medium containing 20% ​​PEG6000 for treatment. The normally growing material was used as a control. After 7 days of treatment, the growth of the seedlings was observed, and stress tolerance-related indicators of different materials were detected and statistically analyzed.

[0057] like Figure 4 As shown in Figures A and B, in the control group on 1 / 2 MS medium, the two OsMDR overexpressing transgenic lines showed no significant difference in growth compared to the wild-type Nipponbare. However, after 7 days of treatment with 20% PEG, the two OsMDR overexpressing transgenic lines exhibited significantly stronger drought resistance than the wild-type, and their survival rates were also lower than those of the wild-type Nipponbare. This indicates that the upregulation of the OsMDR gene reduces the drought tolerance of transgenic rice seedlings.

[0058] 2. Drought resistance of OsMDR overexpressing transgenic rice during the grain-filling stage

[0059] After 7 days of germination, rice seedlings of different strains were selected and transferred to flowerpots. The seedlings and flowerpots were then transplanted to the field for cultivation until the grain-filling stage. The rice plants and flowerpots were then removed and cultivated in a greenhouse without watering. The plants were subjected to drought treatment and their phenotypes were observed after 7 days.

[0060] like Figure 5 As shown, the OsMDR-overexpressing transgenic rice cultivated in the field exhibited similar growth patterns to Nipponbare rice. However, after drought treatment, both wild-type and transgenic rice lines turned yellow and wilted, but the OsMDR-overexpressing transgenic rice showed more pronounced drying, with both stems and leaves withering and turning yellow. Therefore, the upregulation of the OsMDR gene reduced the drought resistance of transgenic rice during the grain-filling stage.

[0061] In summary, upregulation of the OsMDR gene reduces drought resistance in transgenic rice and is a negative regulator related to drought resistance in rice.

[0062] Example 4: Stress-Related Physiological Indicators of OsMDR Transgenic Rice

[0063] 1. OsMDR overexpression altered stress-related physiological indicators in transgenic rice seedlings.

[0064] Seedlings of different rice lines subjected to drought stress for 7 days were used as materials to detect stress-related physiological indicators such as chlorophyll content, proline content, malondialdehyde content, and DAB staining analysis.

[0065] like Figure 6 As shown, under normal growth conditions, there were no significant differences in different physiological indicators between OsMDR overexpression transgenic lines and wild-type rice. However, under drought stress, the chlorophyll and proline content of the overexpression transgenic rice lines were lower than those of the wild type, while the MDA content was higher. DAB staining analysis showed that after 20% PEG treatment, the leaves of the OsMDR overexpression transgenic lines stained more deeply, indicating that their antioxidant capacity was reduced.

[0066] 2. OsMDR overexpression altered stomatal aperture in rice plants during the grain-filling stage.

[0067] like Figure 6 As shown, the stomata on the dorsal side of rice leaves after drought stress treatment were observed and counted. The results showed that under normal growth conditions, there was no significant difference in the distribution ratio of different types of stomata between OsMDR overexpression transgenic lines and wild-type rice leaves during the grain-filling stage. However, under drought treatment conditions, the proportion of completely closed stomata in OsMDR overexpression lines was lower than that in wild-type rice. This indicates that OsMDR overexpression makes transgenic rice more prone to water loss, which is consistent with the result that the water loss rate of transgenic rice is increased during the seedling stage.

[0068] Therefore, the data from the physiological indicators measured after drought stress were consistent with the phenotypic results. The upregulation of OsMDR expression led to comprehensive changes in leaf cells at the physiological level, reducing the drought-related abilities of rice.

[0069] 2. OsMDR overexpression reduces the expression of stress-resistance-related marker genes in transgenic lines.

[0070] Using the sequences in Table 1 as primers, real-time quantitative PCR was used to detect the expression levels of marker genes related to abiotic stress in OsMDR overexpression lines.

[0071] Table 1 Primers for real-time quantitative PCR of stress-resistance related genes

[0072]

[0073] like Figure 7As shown, OsMDR overexpression reduced the expression levels of OsLEA3 and OsPOD1. Both OsLEA3 and OsPOD1 are drought-resistance marker genes, associated with osmotic stress and reactive oxygen species scavenging in cells. Therefore, a decrease in their expression levels predicts a decline in the plant's drought resistance, consistent with the drought-resistance phenotype of rice. Thus, the molecular-level experimental results also indicate that OsMDR negatively regulates the drought-related abilities of rice.

[0074] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. The application of rice drought-resistance-related transcription factor OsMDR in regulating rice drought resistance, characterized by: The amino acid sequence of the rice drought-resistance-related transcription factor OsMDR is shown in SEQ ID No:

1. Overexpression of the gene encoding the rice drought-resistance-related transcription factor OsMDR in rice is used for: Reduce the drought resistance of genetically modified rice during the seedling and grain-filling stages; Reduce chlorophyll content, proline content, and the ratio of completely closed stomata under stress in transgenic rice under PEG treatment conditions; Increase malondialdehyde content and water loss rate in genetically modified rice.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the rice drought-related transcription factor OsMDR is shown in SEQ ID No:2.

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