Application of rice OsARF6 and OsARF17 genes in regulation and control of laodelphax striatellus resistance

Abstract

The invention discloses rice OsARF6 and OsARF17 genes and application of the rice OsARF6 and OsARF17 genes in regulation and control of resistance of laodelphax striatellus. The protein provided by the invention is a protein as shown in (a) or (b): (a) a protein consisting of a sequence 1 in a sequence table and an amino acid sequence as shown in the sequence 1; and (b) a protein which is formed by substitution and / or deletion and / or addition of one or more amino acid residues of the sequence 1 and the amino acid sequence of the sequence 2, is related to plant insect resistance and is derived from the sequence 1 and the sequence 2. The plant insect-resistant related protein provided by the invention affects the insect resistance of plants. Insect-sensitive plants can resist insects by improving the expression of the protein coding gene, so that insect-resistant transgenic plants can be cultivated. The protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention relates to the application of rice OsARF6 and OsARF17 genes in regulating resistance in planthoppers. Background Technology

[0002] The Small Brown Planthopper (SBPH) belongs to the order Hemiptera and family Plantipodidae. It has a wide host range, damaging various crops including rice, barley, wheat, and corn. The planthopper primarily feeds on rice sap by piercing and sucking its sap. It congregates in groups on the upper leaves and panicles of rice plants, and severe infestations can cause rice to wither and turn yellow. The Small Brown Planthopper is distributed throughout my country, but is more prevalent in the middle and lower reaches of the Yangtze River and North China. Besides direct feeding damage, the Small Brown Planthopper is also a vector for major food crop viral diseases such as Rice Stripe Virus (RSV), Rice Black-streaked Dwarf Virus (RBSDV), and Maize Rough Dwarf Virus (MRDV), posing a serious threat to food security.

[0003] Auxin (IAA), an important hormone, participates in regulating plant growth, development, and many other physiological processes. The auxin signaling pathway comprises three core components: Transport Inhibitor Response 1 / Auxin Signaling F-boxes (TIR1 / AFBs), auxin / indole-3-acetic acid (Aux / IAA), and auxin response factor (ARF). Under low auxin conditions, Aux / IAA binds to and inhibits the activity of ARF transcription factors. When auxin levels increase, TIR1 / AFBs directly interact with the transcriptional repressor Aux / IAA protein, promoting its degradation and relieving the inhibition of downstream ARF transcription factors, thereby activating the auxin signaling pathway.

[0004] Recent studies have found that auxins not only regulate plant growth and development but also play an important role in stress responses. Identifying genes that can resist rice planthoppers in rice is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a plant-related protein for resistance to planthoppers, its encoding gene, and its application.

[0006] The plant resistance-to-planthopper-related proteins OsARF6 and OsARF17 provided by this invention are derived from common rice varieties. OsARF6 and OsARF17 can be synthesized artificially or obtained by first synthesizing their encoding genes and then expressing them biologically.

[0007] This invention provides a protein OsARF6 that regulates the resistance of planthoppers, the protein sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a gene encoding the resistance protein OsARF6 of the planthopper, said gene being designed according to conventional methods. In a preferred embodiment, said gene is a DNA molecule as shown in 1) or 2) or 3) or 4) below:

[0009] 1) The DNA molecule shown in SEQ ID NO.3;

[0010] 2) The DNA molecule shown in SEQ ID NO. 5;

[0011] 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein described in SEQ ID NO. 1;

[0012] 4) Having more than 90% homology with the DNA sequence defined in 1), 2), or 3), and encoding a DNA molecule that regulates the resistance-related protein of the planthopper.

[0013] The stringent conditions may be hybridization and washing of the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0014] This invention provides a protein OsARF17 that regulates the resistance of planthoppers, the protein sequence of which is shown in SEQ ID NO.2.

[0015] The present invention also provides a gene encoding the regulatory protein OsARF17 of the planthopper resistance protein, said gene being designed according to conventional methods. In a preferred embodiment, said gene is a DNA molecule as shown in 1) or 2) or 3) or 4) below:

[0016] 1) The DNA molecule shown in SEQ ID NO.4;

[0017] 2) The DNA molecule shown in SEQ ID NO. 6;

[0018] 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 2;

[0019] 4) DNA molecules that have more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encode proteins related to endosperm development.

[0020] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0021] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0022] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the lip synthase gene Nos) and plant genes (such as the soybean storage protein gene).

[0023] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing promoter or constitutive promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter, can be added before the transcription initiation nucleotide. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0024] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0025] Primer pairs for amplifying the full length or any fragment of the genes OsARF6 and OsARF17 are also within the scope of protection of this invention. In one specific example, the primer pair for amplifying the gene OsARF6 is shown in SEQ ID NO.11 and SEQ ID NO.12; in another specific example, the primer pair for amplifying the gene OsARF17 is shown in SEQ ID NO.13 and SEQ ID NO.14.

[0026] The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or recombinant bacteria, or the method can all be applied to rice breeding.

[0027] The primer pairs and knockout vectors for knocking out genes OsARF6 and OsARF17 are also within the scope of protection of this invention. In one specific example, the primer pair for knocking out gene OsARF6 is shown in SEQ ID NO.7 and SEQ ID NO.8; in another specific example, the primer pair for knocking out gene OsARF17 is shown in SEQ ID NO.9 and SEQ ID NO.10.

[0028] The present invention also provides the application of the gene, or the knockout primer or knockout vector of the gene, in the breeding of rice varieties resistant to planthoppers, specifically knocking out or silencing the gene OsARF6 or the gene OsARF17.

[0029] A method for developing a rice variety resistant to the planthopper involves knocking out or silencing the gene OsARF6 or the gene OsARF17 in rice.

[0030] This invention also provides a method for targeted gene knockout using CRISPR-Cas9 technology. Specifically, the recombinant knockout vector can be introduced into rice to specifically knock out the OsARF6 or OsARF17 gene.

[0031] This invention also provides the application of the aforementioned gene, protein, recombinant knockout / expression vector, expression cassette, transgenic cell line, or recombinant bacteria in the cultivation of rice varieties susceptible to planthoppers. Specifically, it involves overexpressing gene OsARF6 or gene OsARF17 in rice to obtain rice varieties susceptible to planthoppers. Specifically, the gene can be introduced into rice via the recombinant overexpression vector to increase the expression level of gene OsARF6 or gene OsARF17.

[0032] This invention also provides a method for breeding rice varieties susceptible to planthoppers, which involves overexpressing the gene OsARF6 or the gene OsARF17 in rice to obtain planthopper-susceptible rice varieties. Specifically, the gene can be introduced into rice through the recombinant overexpression vector to increase the expression level of the gene OsARF6 or the gene OsARF17.

[0033] By using any vector capable of guiding the expression of exogenous genes in plants, the gene encoding the stated protein can be introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the stated gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The plant host being transformed can be either monocotyledonous or dicotyledonous, such as tobacco, birdsfoot, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0034] The plant insect resistance-related protein of this invention affects plant disease resistance responses. Introducing the gene encoding this protein into plants can yield transgenic plants with enhanced insect resistance. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0035] Figure 1 OsARF6 overexpression and knockout families' resistance to planthoppers. A, Mutation type of knockout families; B, Resistance performance of knockout and overexpression families in planthoppers; C, Seedling mortality statistics of knockout and overexpression families.

[0036] Figure 2 OsARF17 overexpression and knockout families' resistance to planthoppers. A, Mutation type of knockout families; B, Resistance performance of knockout and overexpression families in planthoppers; C, Seedling mortality statistics of knockout and overexpression families. Detailed Implementation

[0037] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0038] Example 1: Discovery of plant insect resistance-related proteins and their encoding genes

[0039] I. Origins of OsARF6 and OsARF17

[0040] In our previous work, we cloned a planthopper resistance gene, SBPH5, which encodes an Aux / IAA family protein, OsIAA16. SBPH5 positively regulates rice resistance to planthoppers. Using a yeast two-hybrid assay, we screened for two ARF transcription factors, OsARF6 and OsARF17, that interact with SBPH5. We then constructed their transgenic materials. The amino acid sequence encoded by gene OsARF6 is shown in SEQ ID NO.1, its cDNA sequence in SEQ ID NO.3, and its genomic DNA sequence in SEQ ID NO.5. The amino acid sequence encoded by gene OsARF17 is shown in SEQ ID NO.2, its cDNA sequence in SEQ ID NO.4, and its genomic DNA sequence in SEQ ID NO.6.

[0041] Example 2: Obtaining and Identifying Transgenic Plants

[0042] I. Construction of the knockout vector

[0043] Using the CRISPR-P online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), primers with PAM sites were designed in the CDS region of the predicted gene. Primers with high specificity, low off-target rate, and proximity to ATG were selected to construct CRISPR knockout vectors for OsARF6 and OsARF17, respectively.

[0044] The knockout primer sequences are as follows:

[0045] CRISPR-ARF6 F:

[0046] 5'-GGCATCCTCAAGGCCACAGTGAGC-3'; (SEQ ID NO.7)

[0047] CRISPR-ARF6 R:

[0048] 5'-AAACGCTCACTGTGGCCTTGAGGAC-3'; (SEQ ID NO.8)

[0049] CRISPR-ARF17 F:

[0050] 5'-GGCAAGGCAACGAAACAAGGGGAC-3'; (SEQ ID NO.9)

[0051] CRISPR-ARF17 R:

[0052] 5'-AAACGTCCCCTTGTTTCGTTGCCTC-3'; (SEQ ID NO.10)

[0053] II. Construction of cDNA expression vectors containing OsARF6 and OsARF17 coding regions

[0054] The hygromycin resistance expression vector Pcambia1305.1-GFP was digested with XbaⅠ and BamHI, and the vector fragments were recovered for later use. Using the cDNA of the rice variety Nipponbare as a template, PCR amplification was performed to obtain the OsARF6 and OsARF17 genes. At the same time, recombination adapters were added to both ends of the cDNA sequences of OsARF6 and OsARF17 and recombinated into the pCUbi1390 vector (Duan et al., (2019). OsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in Rice. The Plant Cell, 31(5), 1026-1042), and overexpression vectors of OsARF6 and OsARF17 were constructed, and sequenced to confirm their existence.

[0055] The PCR primer sequences are as follows:

[0056] OE-OsARF6 F (the underlined sequence is the XbaⅠ recombination site):

[0057] 5'- CGGAGCTAGCTCTAGA ATGAAGCTCTCGCCGTCGGCCGG-3'; (SEQ ID NO.11)

[0058] OE-OsARF6 R (the underlined sequence is the BamHI recombination site):

[0059] 5'- TGCTCACCATGGATCC GAACTCAACTGAGCCCACCGACG-3'; (SEQ ID NO.12)

[0060] OE-OsARF17 F (the underlined sequence is the XbaⅠ recombination site):

[0061] 5'-CGGAGCTAGCTCTAGAATGAGGCTTTCGTCGTCGTCCG-3'; (SEQ ID NO. 13)

[0062] OE-OsARF17 R (the underlined sequence indicates the BamHI recombination site):

[0063] 5'- TGCTCACCATGG ATCCGAATTCAACTGAGCCGACAGATG-3'; (SEQ ID NO.14)

[0064] III. Agrobacterium-mediated transformation

[0065] Using Agrobacterium strain EHA105 (purchased from American Ingenuity), the knockout vectors and overexpression vectors of the OsARF6 and OsARF17 genes constructed above were introduced into the insect-susceptible species Nipponbare (NIP).

[0066] (1) Agrobacterium containing recombinant OsARF6 and OsARF17 was cultured at 28℃ for 16 hours. The bacterial cells were collected and diluted in N6 liquid medium containing 100 μmol / L to a concentration of OD100. 600 ≈0.5, to obtain bacterial culture;

[0067] (2) Mix the mature embryonic callus tissue of rice cultured for one month with the above bacterial solution and infect for 30 min. After the bacterial solution is dried with filter paper, transfer it to co-culture medium (N6 solid co-culture medium, purchased from Sigma) and co-culture at 24℃ for 3 days.

[0068] (3) The above callus was inoculated on N6 solid screening medium containing 150 mg / L hygromycin B (purchased from Sigma) for the first screening for 16 days;

[0069] (4) Select healthy callus and transfer them to N6 solid selection medium containing 200 mg / L hygromycin B for a second selection. Subculture every 15 days.

[0070] (5) Select resistant callus and transfer it to a differentiation medium containing 150 mg / L hygromycin B for differentiation;

[0071] (6) The regenerated rice plants that differentiate into seedlings are the transgenic plants with the OsARF6 and OsARF17 genes obtained.

[0072] IV. Expression level analysis of transgenic plants

[0073] mRNA was extracted from OsARF6 and OsARF17 transgenic plants with NIP background, reversed to cDNA, and ARF gene expression analysis was performed by qRT-PCR using this cDNA as a template.

[0074] The qRT-PCR primer sequences are as follows:

[0075] OsARF6 RT-F:

[0076] 5'-GAACCGTTGGAAGGTTGCTC-3';

[0077] OsARF6 RT-R:

[0078] 5'-TCTTCCAACTGGCCCTCAAG-3';

[0079] OsARF17 RT-F:

[0080] 5'-TTTACAAATCGGGAACCTATGG-3';

[0081] OsARF17 RT-R

[0082] 5'-TTTATGCAGGAGACGCTATTCA-3';

[0083] III. Identification of Insect Resistance in Transgenic Plants

[0084] Materials from the knockout and overexpression families OsARF6 and OsARF17, along with the background variety NIP, were soaked and germinated separately, then sown in small black square pots (70mm x 70mm x 80mm) filled with nutrient soil. These pots were placed in 65cm x 44cm x 14cm plastic containers (maintaining a water level of approximately 2cm). 25 sprouting seeds were sown per pot. Four days before inoculation, seedlings were thinned, discarding diseased and weak seedlings, leaving approximately 20 uniform, healthy seedlings per pot for inoculation with planthopper larvae. This was repeated three times. Inoculation was performed when the seedlings reached the one-leaf stage. Each seedling was inoculated with 15-20 2nd-3rd instar planthoppers. Inoculated seedlings were placed in greenhouse containers or transplanted to the field for growth, with careful water and fertilizer management.

[0085] OsARF6 results are as follows Figure 1 As shown, when the mortality rate of the background parents was approximately 52%, the mortality rate of the OsARF6 knockout families was 23%-35%, while the mortality rate of the OsARF6 overexpression families was 77%-80%; the results for OsARF17 are as follows... Figure 2 As shown, when the seedling mortality rate of the background parent was about 66%, the seedling mortality rate of the OsARF17 knockout family was 11%-20%, while the seedling mortality rate of the OsARF17 overexpression family was 93%-95%. These results indicate that both OsARF6 and OsARF17 negatively regulate rice planthopper resistance.

Claims

1. A modulating resistance protein to Laodelphax striatellus OsARF6 or OsARF17, wherein the OsARF6 protein sequence is shown as SEQ ID NO. 1; the OsARF17 protein sequence is shown as SEQ ID NO.

2.

2. A gene encoding the modulating resistance protein to Laodelphax striatellus OsARF6 of claim 1, preferably the gene is a DNA molecule shown as 1) or 2) or 3) or 4) below: 1) a DNA molecule shown as SEQ ID NO. 3; 2) a DNA molecule shown as SEQ ID NO. 5; 3) a DNA molecule hybridizing to the DNA sequence defined in 1) or 2) under stringent conditions and encoding the protein shown as SEQ ID NO. 1; 4) a DNA molecule having more than 90% homology to the DNA sequence defined in 1) or 2) or 3) and modulating the resistance protein to Laodelphax striatellus.

3. A gene encoding the protein OsARF17 of claim 1, preferably the gene is a DNA molecule shown as 1) or 2) or 3) or 4) below: 1) a DNA molecule shown as SEQ ID NO. 4; 2) a DNA molecule shown as SEQ ID NO. 6; 3) a DNA molecule hybridizing to the DNA sequence defined in 1) or 2) under stringent conditions and encoding the protein shown as SEQ ID NO. 2; 4) a DNA molecule having more than 90% homology to the DNA sequence defined in 1) or 2) or 3) and encoding the endosperm development related protein.

4. A recombinant expression vector, expression cassette or recombinant bacteria containing the gene of claim 2 or 3; preferably the recombinant expression vector is a recombinant plasmid obtained by inserting the gene of claim 2 or 3 between the Xba I and BamH I of the pCUbi1390 vector.

5. A primer pair amplifying the full length of the gene of claim 2 or 3 and any fragment thereof; preferably the primer pair amplifying the gene OsARF6 is shown as SEQ ID NO. 11 and SEQ ID NO. 12; preferably the primer pair amplifying the gene OsARF17 is shown as SEQ ID NO. 13 and SEQ ID NO.

14.

6. A primer pair or a knockout vector knocking out the gene of claim 2 or 3; preferably the primer pair knocking out the gene OsARF6 is shown as SEQ ID NO. 7 and SEQ ID NO. 8; preferably the primer pair knocking out the gene OsARF17 is shown as SEQ ID NO. 9 and SEQ ID NO.

10.

7. Use of the protein OsARF6 or OsARF17 of claim 1, the gene of claim 2 or 3, the recombinant expression vector, expression cassette or recombinant bacteria of claim 4 or the primer pair of claim 5 in breeding a Laodelphax striatellus susceptible rice variety, preferably overexpressing the gene of claim 2 or claim 3 in rice to obtain a Laodelphax striatellus susceptible rice variety.

8. A method for breeding a Laodelphax striatellus susceptible rice variety, which comprises overexpressing the gene of claim 2 or claim 3 in rice to obtain a Laodelphax striatellus susceptible rice variety. ​ ​ ​ ​ ​ ​ ​ ​ 9. Use of the protein OsARF6 or OsARF17 of claim 1, the gene of claim 2 or 3, the primer pair or knock-out vector of claim 6 in breeding a rice variety resistant to the white-backed planthopper, preferably knocking out or silencing the gene of claim 2 or 3; more preferably targeting the gene of claim 2 or 3 for knock-out by CRISPR-Cas9 technology.

10. A method of breeding a rice variety resistant to the brown planthopper, characterized in that, knocking out or silencing the gene of claim 2 or 3 in rice; preferably targeting the gene of claim 2 or 3 for knock-out by CRISPR-Cas9 technology.

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