Application of HvMORF8 gene in regulation of drought stress tolerance of plants

Abstract

The invention discloses application of an HvMORF8 gene in regulation and control of drought stress tolerance of plants, and belongs to the technical field of gene engineering. The nucleotide sequence of the CDS region of the HvMORF8 gene is as shown in SEQ ID NO. 1. According to the invention, the HvMORF8 gene is cloned and analyzed, the function of the gene is verified on barley XZ5 in combination with a BSMV-VIGS technology, and the HvMORF8 gene positively regulates the tolerance of a plant to drought stress. The invention provides a theoretical basis and related genes for breeding and production of drought-stress-resistant crops, especially creation of new drought-resistant barley germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of the HvMORF8 gene in regulating plant tolerance to drought stress. Background Art

[0002] Drought is one of the most serious natural disasters that limit global crop productivity. Climate change and population expansion have led to the continuous expansion of drought areas around the world (Gupta et al., 2020). Globally, highly arid, arid, semi-arid, and sub-humid dry areas already account for 41% of the land area and are expected to exceed half of the total land area by the end of the 21st century (FAO, 2019). Therefore, understanding the underlying mechanisms of crop drought tolerance and developing strategies to enhance drought tolerance are essential to maintaining agricultural productivity and ensuring secure food production.

[0003] Barley (Hordeum vulgare L.) is a major cereal crop, and its planting area and total output rank fourth in the world. At the same time, as a diploid self-pollinating crop, barley is also an excellent model plant for studying the reproductive physiology and genetic mechanisms of other wheat crops. However, the genetic diversity of cultivated barley grown on a large scale is relatively single, and it is more sensitive to abiotic and biotic stresses. Tibetan annual wild barley (Hordeum vulgare L.ssp.Spontaneum) is considered to be the ancestor of cultivated barley. The Qinghai-Tibet Plateau has a unique climate and harsh environment. The wild barley germplasm resources grown there are more abundant, and often show better broad-spectrum stress resistance than ordinary cultivated barley (Dai et al., 2014). Therefore, Tibetan annual wild barley can become an important gene bank for barley drought-tolerant breeding. Introducing its unique drought-tolerant genes into cultivated barley to create high-yield varieties with higher yield stability under drought stress has become an important breeding approach.

[0004] RNA editing plays an indispensable role in protein metabolism, adaptation to environmental changes, signal transduction and other processes in plant cells (Wang et al., 2019). Plant RNA editing is carried out by a complex containing multiple editing factors, among which MORF is a small protein family in plants, originally discovered in Arabidopsis (Takenaka et al., 2012), which can interact with other editing factors to form RNA editing bodies (Bentolila et al., 2012). Most previous studies on MORF have focused on the role of MORF in plant organelle development and RNA editing. However, increasing evidence shows that MORF is also involved in plant responses to biotic and abiotic stresses. For example, the expression levels of all seven OsMORFs in rice change under salt stress (Zhang et al., 2019). In peach, PpMORF is downregulated under pathogen infection and UV stress (Zhang et al., 2022). However, in the annual herbaceous plant barley, the response and function of HvMORF to drought stress are still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a gene with drought stress resistance characteristics cloned from barley, so as to provide relevant genes and theoretical basis for cultivating new germplasm of drought stress resistant plants.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention uses the annual wild barley XZ5 (drought stress-resistant genotype) and XZ54 (drought stress-sensitive genotype) from the Qinghai-Tibet Plateau screened by the research team in the early stage, and the internationally recognized drought-resistant barley variety Tadmor as experimental materials, and identifies a gene HORVU.MOREX.r3.7HG0684630 that is highly expressed only in XZ5 under drought stress conditions based on the results of leaf gene expression profiles. The gene is annotated as multiple organellar RNA editing factor 8.

[0008] Cloning and analysis of HvMORF8 gene: The full-length CDS region sequence of this gene was cloned from the annual wild barley XZ5 on the Qinghai-Tibet Plateau and named HvMORF8. The CDS region of this gene is 1251 bp in length, and the nucleotide sequence is shown in SEQ ID NO.1. This gene encodes a protein sequence of 416 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] The molecular weight of HvMORF8 protein is 44.56kDa, and its isoelectric point pI is 9.06. The functional domain prediction analysis of HvMORF8 protein sequence was performed on InterPro website, and the results showed that the protein contained 1 functional domain: MORF / ORRM1 / DAG-like functional domain. The phylogenetic tree analysis of HvMORF8 protein sequence showed that the protein was most closely related to wheat TaMORF8.

[0010] The present invention uses the BSMV-VIGS system to verify the function of the HvMORF8 gene on wild barley XZ5. The results show that the expression of the HvMORF8 gene in the BSMV:HvMORF8 plant is inhibited compared with the plant inoculated with an empty vector, whether under control conditions or drought stress conditions. Under drought stress, the growth of the BSMV:HvMORF8 plant is significantly weaker than that of the plant inoculated with an empty vector, and the plant height, root length and biomass are also significantly lower than those of the plant inoculated with an empty vector, while under control conditions, there is no significant difference in the growth of the two. These results indicate that silencing the HvMORF8 gene significantly reduces the tolerance of barley to drought stress.

[0011] Therefore, the present invention provides the use of the HvMORF8 gene in regulating the tolerance of plants to drought stress. Mechanism studies show that the HvMORF8 gene positively regulates the tolerance of plants to drought stress.

[0012] Furthermore, the plant is barley. After silencing the HvMORF8 gene in barley, its tolerance to drought stress is significantly reduced.

[0013] Furthermore, the barley is the annual wild barley (Hordeum vulgare L.ssp.spontaneum) XZ5 from the Qinghai-Tibet Plateau.

[0014] Based on the positive regulatory effect of the HvMORF8 gene on plant drought stress tolerance, the present invention can use genetic engineering technology to upregulate the expression of the HvMORF8 gene in the plant to enhance the tolerance to drought stress. Specifically, the application includes: using biological technology to upregulate the expression of the HvMORF8 gene in the plant to improve the tolerance of the plant to drought stress.

[0015] Furthermore, the application includes: cloning the HvMORF8 gene with a nucleotide sequence such as that shown in SEQ ID NO.1 into a plant overexpression vector to construct a recombinant plasmid, and using Agrobacterium-mediated technology to transfer the target fragment in the recombinant plasmid into a recipient plant to cultivate a transgenic plant with enhanced tolerance to drought stress. The recipient plant may be, but is not limited to, barley.

[0016] The present invention has the following beneficial effects:

[0017] The present invention clones and analyzes the HvMORF8 gene, and verifies the gene function in barley XZ5 by combining the BSMV-VIGS technology. The results show that the HvMORF8 gene positively regulates the tolerance of the plant to drought stress. The present invention provides a theoretical basis and related genes for the breeding and production of drought-resistant crops, especially the creation of new barley drought-resistant germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The sequence alignment of three barley genotypes (XZ5, XZ54 and Tadmor), where (A) is the nucleotide sequence alignment of the HvMORF8 gene; and (B) is the amino acid sequence alignment of the HvMORF8 protein.

[0019] Figure 2 This is the predicted functional domain map of HvMORF8 protein.

[0020] Figure 3 This is a comparison chart of the amino acid sequences of HvMORF8, OsMORF8 and AtMORF8.

[0021] Figure 4 Phylogenetic tree analysis of HvMORF8 and MORF8 proteins in various plant species.

[0022] Figure 5 Schematic diagram of BSMV:γ-HvMORF8 vector.

[0023] Figure 6 The function of HvMORF8 gene was verified in wild barley XZ5 using BSMV-VIGS method, where (A) is the phenotypic identification of HvMORF8 silenced plants; (B) is the relative expression level of HvMORF8 gene in each treated plant detected by qRT-PCR; (C)-(F) are the differences in plant height, root length, aboveground fresh weight and root fresh weight of each treated plant after 7 days of treatment with 20% PEG-6000. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0025] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0026] The present invention uses the annual wild barley drought stress-resistant genotype XZ5, the drought stress-sensitive genotype XZ54 and the internationally recognized drought-resistant barley variety Tadmor screened by the research team in the early stage of the Qinghai-Tibet Plateau as the main materials, and clones the barley drought stress-resistant related gene HvMORF8, which is of great significance for clarifying the molecular mechanism of barley response to drought stress and breeding and production.

[0027] Example 1: Cloning and analysis of the CDS region of the HvMORF8 gene

[0028] 1. Barley growing conditions

[0029] Barley seeds of XZ5, XZ54 and Tadmor were sterilized with 2% H2O2 for 30 min, then rinsed with distilled water for 5-6 times, and the sterilized seeds were placed on a moist sand bed and cultured in a dark growth chamber (22°C / 18°C, day / night), supplemented with light after germination. On the 7th day, seedlings with uniform growth were selected and transferred to a 1L black plastic bucket containing a basic culture solution of barley, covered with a 5-hole plastic cover, with one seedling per hole, fixed with a sponge, and cultured in a barley culture room, and the basic culture solution of barley used a 1 / 5 Hogland formula.

[0030] 2. Cloning of the CDS sequence of HvMORF8 gene

[0031] Based on the previous research of the research group, a gene that was highly expressed in XZ5 under drought stress conditions was selected from the results of barley leaf gene chips. The full-length CDS region sequence of the gene was cloned from XZ5 (as shown in SEQ ID NO.1) and named HvMORF8.

[0032] Total RNA was extracted from leaves of annual wild barley species XZ5, XZ54, and Tadmor from the Qinghai-Tibet Plateau using a total RNA extraction kit (Takara, Japan). Genomic DNA contamination in total RNA was removed using DNaseI (Takara, Japan). TM The extracted total RNA was reverse transcribed into single-stranded cDNA using II 1st Strand cDNA Synthesis Kit (Takara, Japan). Specific primers were designed using NCBI's Primer-BLAST, and the specific primer sequences were:

[0033] HvMORF8-CDS-F: 5'-ATGGCGATGGCATCGCG-3' (SEQ ID NO. 3);

[0034] HvMORF8-CDS-R: 5'-TTACTGTTGATTATAGCCACCTGGTGC-3' (SEQ ID NO. 4).

[0035] The amplified product was connected to the pMD18-T vector (Takara, Japan), transformed into Escherichia coli DH5α, and the positive clones were selected and sent to the company for sequencing. The correct ones were extracted and stored in glycerol, and the resulting plasmid was named pMD18-T-HvMORF8 plasmid. PCR primer synthesis and gene sequencing were completed by Zhejiang Shangya Biotechnology Co., Ltd. The nucleotide sequence of the CDS region of the HvMORF8 gene in barley XZ5 is shown in SEQ ID NO.1.

[0036] The nucleotide sequence differences of the CDS region of the HvMORF8 gene in XZ5, XZ54 and Tadmor are as follows Figure 1 (A) shown.

[0037] 3. HvMORF8 protein sequence analysis

[0038] The HvMORF8 protein sequence was predicted and analyzed using the InterPro (http: / / www.ebi.ac.uk / interpro / ) website. The results showed that the protein contained a MORF / ORRM1 / DAG-like functional domain ( Figure 2 ).

[0039] The sequence similarity of HvMORF8 to rice OsMORF8 is 68.7%, and the similarity to Arabidopsis AtMORF8 is 55.7% ( Figure 3 ).

[0040] The MORF8 protein sequences in different species, including HvMORF8, were analyzed using MAFFT and MEGA-X software to construct a phylogenetic tree. The results showed that MORF8 protein only exists in seed plants, and HvMORF8 is most closely related to wheat TaMORF8 ( Figure 4 ).

[0041] Example 2: BSMV-VIGS method to verify the function of HvMORF8 gene

[0042] 1. Construction of BSMV:HvMORF8 vector

[0043] Using the pMD18-T-HvMORF8 plasmid as a template, primers were designed and amplified to obtain a 250 bp HvMORF8 gene fragment, the sequence of which is shown in SEQ ID No.5.

[0044] The primer sequences are (underlined are restriction enzyme cutting sites):

[0045] HvMORF8-γ-F:5'-GTAC GCTAGC AATGATAGGCCCCGCAACTT-3' (SEQ ID NO. 6);

[0046] HvMORF8-γ-R:5'-GTAC GCTAGC TGTATGCTGCCTGTGGGTTC-3' (SEQ ID NO. 7).

[0047] KOD enzyme PCR reaction system:

[0048]

[0049] The amplification program was: 94°C for 5 min, 34 cycles of (98°C for 10 s, 60°C for 30 s, 68°C for 1 min), and 68°C for 10 min.

[0050] The HvMORF8 gene fragment was digested with NheⅠ restriction endonuclease, and then connected with the RNAγ vector that had been digested with the same restriction endonuclease and dephosphorylated using T4 ligase. The ligation product was transformed into E. coli DH5α, and the reverse insertion was verified using the forward primer HvMORF8-γ-F of the HvMORF8 gene and the primer γ-stain-F on the RNAγ vector. The verified reverse insertion positive clone was sent to the company for sequencing. The single clone with the correct sequencing was expanded and cultured, and the plasmid was extracted, and the plasmid was named RNAγ:HvMORF8. The primer sequence on the RNAγ vector used was:

[0051] γ-stain-F: 5'-CAACTGCCAATCGTGAGTAGG-3' (SEQ ID NO. 8).

[0052] 2. BSMV vector linearization and in vitro transcription

[0053] RNAα, RNAγ and RNAγ:HvMORF8 were digested with MluⅠ restriction endonuclease, and RNAβ was digested with SpeⅠ restriction endonuclease to linearize and purify the products. TM LargeScale RNA Production System-T7 kit and Ribo m 7 In vitro transcription was performed using the GCap Analog kit (Promega, USA), and all operations were performed in an RNase-free environment.

[0054] The in vitro transcribed RNAα, RNAβ and RNAγ plasmids were mixed in a volume ratio of 1:1:1, and the in vitro transcribed RNAα, RNAβ and RNAγ:HvMORF8 plasmids were mixed in a volume ratio of 1:1:1, and diluted with two volumes of RNase-free water respectively. Then, an equal volume of 2×GKP buffer (1% bentonite, 1% diatomaceous earth, 50mM glycine and 30mM potassium dihydrogen phosphate pH 9.2) was added to the diluted products. The resulting products were named BSMV:γ and BSMV:HvMORF8, and used for inoculation after mixing.

[0055] 3. Barley seedling culture before BSMV inoculation

[0056] The annual wild barley XZ5 on the Qinghai-Tibet Plateau was disinfected with 2% H2O2 for 30 minutes, rinsed with distilled water 5-6 times, and then the seeds were placed on a moist sand bed in a dark growth chamber (22℃ / 18℃) until germination, and supplemented with light after germination. After 7 days, seedlings with consistent growth were selected and transferred to a 1L black plastic bucket, covered with a 5-hole plastic lid, with 1 seedling per hole, fixed with a sponge, and the bucket was filled with barley basic culture solution and continuously ventilated. The barley basic culture solution used a 1 / 5 Hogland nutrient solution formula. The culture solution was replaced every 3 days, and the pH of the nutrient solution was adjusted to 5.8±0.1 with NaOH or HCl.

[0057] 4. Verification of gene function by BSMV inoculation

[0058] When the barley seedlings grew to the two-leaf and one-heart stage, the second leaf was inoculated with BSMV friction in an RNase-free environment, and each plant was inoculated with 10 μL of evenly mixed BSMV:γ or BSMV:HvMORF8. The inoculated plants were immediately sprayed with a small amount of DEPC water and covered with a transparent plastic cover to keep the plants moist for 3 days. After that, the glass cover was removed and continued to be cultured in the barley growth culture room (22℃ / 18℃), and the phenotype of the plants was observed regularly.

[0059] There were 4 treatments in the experiment: inoculation of BSMV:γ+basic culture medium (BNS, pH 4.3) (treatment 1), inoculation of BSMV:γ+BNS+20% PEG-6000 (treatment 2), inoculation of BSMV:HvMORF8+BNS (treatment 3), inoculation of BSMV:HvHvMORF8+BNS+20% PEG-6000 (treatment 4). Treatment lasted for 7 days.

[0060] The results are as follows Figure 6As shown in the figure, under drought stress, the growth of the plants inoculated with BSMV:HvMORF8 (treatment 4) was significantly weaker than that of the plants inoculated with the empty vector (treatment 3), and the plant height, root length and biomass were also significantly lower than those of the plants inoculated with the empty vector, while under the control conditions, there was no significant difference in the growth of the two. These results indicate that silencing the HvMORF8 gene significantly reduces the tolerance of barley to drought stress.

[0061] 5. RT-PCR verification of HvMORF8 gene expression

[0062] Total RNA of different treated samples was extracted using total RNA extraction kit (Takara, Japan), and genomic DNA contamination in total RNA was removed using DNaseI, and total RNA of each sample was reverse transcribed into single-stranded cDNA using PrimeScriptTM RT reagent Kit reverse transcription kit (Takara, Japan). The expression of HvMORF8 gene in the corresponding samples was analyzed by fluorescence quantitative PCR (qRT-PCR) using SYBR green luciferase complex (Takara, Japan) and Light Cycler480PCR instrument (Roche, Switzerland), and the expression value was corrected using an internal reference gene Actin.

[0063] The RT-PCR primer sequences are:

[0064] HvMORF8-qRT-PCR-F: 5'-CTGGCTGGTAGTCATGGAGC-3' (SEQ ID NO.9);

[0065] HvMORF8-qRT-PCR-R: 5'-TCTTGCTCACTTCCGACGAC-3' (SEQ ID NO. 10);

[0066] Actin-F: 5'-AAGCATGAAGATACAGGGAGTGTG-3' (SEQ ID NO. 11);

[0067] Actin-R: 5'-AAATTTATTCTCGGAAGAGGTTGTACA-3' (SEQ ID NO. 12).

[0068] The qRT-PCR system is:

[0069]

[0070] The specific PCR program was: 95°C for 30 s, (95°C for 5 s, 60°C for 10 s, 72°C for 15 s) for 40 cycles. The melting curve program was: 60°C-95°C, 5 s per step, increasing by 0.5°C.-ΔΔCq The relative quantitative method was used to calculate the changes in gene expression values. Each group of experiments was repeated three times.

[0071] The results were as follows: for plants inoculated with BSMV:HvMORF8, the expression of the HvMORF8 gene was inhibited by 72.8% and 83.3% (treatment 1 vs. 3, treatment 2 vs. 4) compared with plants inoculated with the empty vector, both under control and drought conditions ( Figure 6 B).

[0072] In summary, through the cloning and analysis of barley HvMORF8, and combined with the BSMV-VIGS technology to verify the function of this gene in the annual wild barley XZ5 on the Qinghai-Tibet Plateau, it was found that the tolerance of XZ5 plants to drought stress was significantly reduced after silencing the HvMORF8 gene.

[0073] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of HvMORF8 gene in regulating plant tolerance to drought stress, characterized in that: The nucleotide sequence of the CDS region of the HvMORF8 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the HvMORF8 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The plant is barley.

4. The use according to claim 1 or 3, characterized in that: The HvMORF8 gene positively regulates the plant's tolerance to drought stress.

5. The use according to claim 4, characterized in that After the HvMORF8 gene was silenced, the tolerance of barley to drought stress was significantly reduced.

6. The use according to claim 3, characterized in that The barley is the annual wild barley (Hordeum vulgare L.ssp.spontaneum) XZ5 grown in the Qinghai-Tibet Plateau.

7. The use according to claim 1, characterized in that The application includes: utilizing biological technology to up-regulate the expression of HvMORF8 gene in plants, so as to improve the tolerance of plants to drought stress.

8. The use according to claim 6, characterized in that The application includes: cloning the HvMORF8 gene with a nucleotide sequence such as that shown in SEQ ID NO.1 into a plant overexpression vector to construct a recombinant plasmid, transferring the target fragment in the recombinant plasmid into a recipient plant using Agrobacterium-mediated technology, and cultivating transgenic plants with enhanced tolerance to drought stress.

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