GENE XA7 IN RICE CONFERS A RESISTANCE TO XANTHOMONAS ORYZAE PV. ORYZAE

Abstract

The invention refers to the function and application of a disease-resistance gene Xa7 highly resistant to the bacterial blight of rice, belonging to the field of plant genetics. The invention discloses a gene Xa7 with high resistance to rice bacterial blight. The nucleotide sequence of gene Xa7 is shown in SEQ ID No: 1. The invention also provides the usage of the gene Xa7 to improve the resistance of rice to bacterial blight.

Claims

1. A recombinant vector comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

2. The recombinant vector of claim 1, wherein the recombinant vector comprises the nucleotide sequence of SEQ ID NO: 1.

3. The recombinant vector of claim 2, wherein the recombinant vector comprises the nucleotide sequence of SEQ ID NO: 3.

4. The recombinant vector of claim 1, wherein the recombinant vector is a plasmid.

5. A transgenic rice plant comprising the recombinant vector of claim 1.

6. A method for increasing resistance of a rice plant against the bacterial-blight disease, comprising: transforming a recombinant vector comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 into the rice plant, resulting a transgenic rice plant capable of expressing a protein having the amino acid sequence of SEQ ID NO: 2.

7. The method of claim 6, further comprising amplifying the nucleotide sequence of SEQ ID NO: 1 through polymerase chain reaction.

8. The method of claim 7, further comprising introducing the amplified nucleotide sequence into a gene expression vector.

9. The method of claim 7, wherein amplification is performed through a pair of primers as set forth in SEQ ID NOs: 14 and 15.

10. The method of claim 6, wherein the recombinant vector is plasmid.

11. The method of claim 6, wherein the recombinant vector comprises the nucleotide sequence of SEQ ID NO: 1.

12. The method of claim 11, wherein the recombinant vector comprises the nucleotide sequence of SEQ ID NO: 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The specific embodiments of the invention are described in detail below with reference to the accompanying drawings.

[0027] FIG. 1. PCR identification of Xa7 transgenic plants of Zhong-hua 11.

[0028] In FIG. 1:

[0029] M refers to DNA marker DL5000; Zhen-hui 084 is the positive control; Zhong-hua 11 is the negative control; T-1 to T-4 plants are different positive transgenic plants; T-5 and T-6 plants are non-transgenic negative plants.

[0030] FIG. 2. The sequences of knockout targets to Xa7 gene by CRISPR/Cas9 and the mutation types of the knockout transgenic plants;

[0031] In FIG. 2:

[0032] A refers to the sequences and sites of the two target sites Target-1 (SEQ ID NO. 4) and Target-2 (SEQ ID NO. 5) to knockout the Xa7 gene by CRISPR/Cas9

[0033] B refers to the two types of Xa7 gene mutants obtained using Target-1 of knockout vector; among, WT refers to the wild-type sequence unedited; in the Xa7 gene locus, the ko-1 knockout plants contain one-base (C) insertion on a chromosome (SEQ ID NO. 6) and two-bases (TT) deletion on the other chromosome (SEQ ID NO. 7); in the Xa7 gene locus, the ko-2 knockout plants contain one-base (T) insertion on a chromosome (SEQ ID NO. 8) and 14-bases deletion on the other chromosome (SEQ ID NO. 9);

[0034] C refers to the two types of Xa7 gene mutants obtained using Target-2 of knockout vector; among, WT refers to the wild-type sequence unedited; in the Xa7 gene locus, the ko-3 knockout plants contain one-base (C) insertion on a chromosome (SEQ ID NO. 10) and 30-bases deletion on the other chromosome (SEQ ID NO. 11); in the Xa7 gene locus, the ko-4 knockout plants contain four-bases deletion on a chromosome (SEQ ID NO. 12) and one-base (T) insertion on the other chromosome (SEQ ID NO. 13).

[0035] FIG. 3. Bacterial-blight resistance identification of the Xa7-transgenic plants of Zhong-hua 11.

[0036] Zhong-hua 11 is a control variety susceptible to bacterial blight and no Xa7 gene. T-1, T-2, T-3 and T-4 are different lines of Xa7 positive transgenic plants. The infected leaves were taken pictures and measured two weeks after inoculation with Xoo strain PXO86. A refers to the pictures of lesion length of inoculated leaves; B refers to the measurement and statistics of lesion length of inoculated leaves.

[0037] FIG. 4. Bacterial-blight resistance identification of the Xa7-knockout plants of Zhen-hui 084.

[0038] Zhen-hui 084 is a bacterial-blight resistance variety with the Xa7 gene. ko-1, ko-2, ko-3 and ko-4 are different types of Xa7-knockout plants. The infected leaves were taken pictures and measured two weeks after inoculation with Xoo strain PXO86. A refers to the pictures of lesion length of inoculated leaves; B refers to the measurement and statistics of lesion length of inoculated leaves.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1. Introducing the Xa7 Gene into Zhong-Hua 11, a Bacterial-Blight Susceptible Rice Variety

(1) PCR Amplification of the Xa7 Gene

[0039] PCR primers were designed and synthesized as the following sequences:

TABLE-US-00001 forward: (SEQ ID NO. 14) 5′-cggccagtgccaagcttGTTCGGCTGCGGCCTGCAG-3′, reverse: (SEQ ID NO. 15) 5′-tgaccacccggggatccAATCAGTTGTGAGGCCGGTT-3′.

[0040] The sequence underlined are used for homologous recombination into the vector. The genomic DNA of rice variety Zhen-hui 084 was used as template to amplify the Xa7 gene by PrimeSTAR® HS DNA Polymerase (Takara, Japan). Total 50-μl PCR solution contained 25 μl Primestar HS (Premix), 1 μl forward and 1 μl reverse primers (10 μm), 2 μl template DNA (<200 ng) and 21 μl sterile water. PCR procedure was as follows: pre-denaturation at 95° C. for 5 min; denaturation at 98° C. for 10 sec; annealing at 58° C. for 15 sec; extension at 72° C. for 90 sec, 30 cycles; extension at 72° C. for 5 min.

[0041] The sequence of SEQ ID No: 3 was obtained including the Xa7 gene (SEQ ID NO: 1).

(2) Construction of Xa7-Expression Vector

[0042] The pCAMBIA-1300 vector was obtained from Abcam (Shanghai, China) through commercial purchase, and the link is https://www.abcam.cn/. This vector was digested by two restriction endonuclease Hind III and BamH I (Takara, Japan). The reaction system was as follows: Hind III 1 μl, BamH I 1 μl, 4 μl buffer (coming with the products), 15 μl pCAMBIA-1300 vector plasmid, ddH.sub.2O was added up to 40 μl, under 37° C. incubating for 4 h. The digested products were purified by the PCR Cleaning Kit (Axygen, USA) according to the manufacturer's instructions. The PCR products of Xa7 gene were purified by the same method. Then, the ligation reaction was performed by the homologous recombination Kit of TransGen company (pEASY®-Uni Seamless Cloning and Assembly Kit), and the reaction solution contains 1.25 μl vector products, 3.75 μl PCR products, 5 μl Assembly mix, incubating at 50° C. for 15 min. The reaction product was transformed into competent cells JM109 by Heat-Shock method, and the positive clones were obtained. Then, the positive clones were sent to biotechnology company for sequencing to verify the sequence of the inserted fragment (SEQ ID No: 3) in the vector, and the expression vector of Xa7 gene was obtained.

(3) Rice Genetic Transformation and Identification of Positive Transgenic Plants

[0043] The expression vector of Xa7 gene obtained above was transformed into a bacterial-blight susceptible rice variety Zhong-hua 11 to obtain the transgenic rice plants by the method reported by Nishimura et al. (Nishimura et al, Nat Protoc, 2006).

[0044] Genomic DNA was extracted from the leaves of transgenic plants according to the method of Panaud et al. (1996). The primers were synthesized as follows: forward: 5′-GTTCGGCTGCGGCCTGCAG-3′ (SEQ ID NO. 16) and, reverse: 5′-AATCAGTTGTGAGGCCGGTT-3′ (SEQ ID NO. 17). PCR amplification was performed with 2×Taq PCR Master Mix (TianGen, China).

[0045] Total 20 μl PCR solution contains 2×Taq PCR Master Mix 10 μl, forward and reverse primers (10 μm) 1 μL each, template DNA 1 μl, sterile water 7 μl. PCR procedure was as follows: pre-denaturation at 94° C. for 5 min; denaturation at 94° C. for 30 sec, annealing at 58° C. for 30 sec, extension at 72° C. for 90 sec, 35 cycles; extension at 72° C. for 5 min. The PCR products were detected by electrophoresis in 1% (w/w) agarose gel for 30 min, and then stained by ethidium bromide. The electrophoretic bands were observed on the UV transmittance instrument. A 1325-bp fragment (SEQ ID NO: 3) was obtained from the transgenic positive plants by PCR, such as T-1, T-2, T-3 and T-4. No PCR products were amplified by the primers from non-transgenic plants, such as T-5 and T-6 (FIG. 1).

Example 2. Knockout of the Xa7 Gene in Bacterial-Blight Resistance Rice Variety Zhen-Hui 084

(1) Construction of CRISPR/Cas9 Knockout Vector of the Xa7 Gene

[0046] According to the nucleotide sequence of the Xa7 gene (SEQ ID NO: 1), the CRISPR target site design website (http://www.rgenome.net/cas-designer/) was used to design the gene editing target, and one of the target sequences is Target-1: 5′-CGTATGCCCGTTGCAGTTGCAGG-3′ (SEQ ID NO. 4). The primers sequences were synthesized according to the sequence of Target-1: Target-1 F: 5′-GGCACGTATGCCCGTTGCAGTTGC-3′ (SEQ ID NO. 18) and, Target-1 R: 5′-AAACGCAACTGCAACGGGCATACG-3′ (SEQ ID NO. 19). T4 PNK enzyme was used to carry out 5-hydroxyl terminal phosphorylation (NEB, USA). The 50 μl solution contained 10 μl Target-1 F primer (10 μM), 10 μl Target-1 R primer (10 μM), 1 μl T4 ligase, 5 μl Buffer (coming with the products) and 24 μl ddH.sub.2O, and was incubated at 37° C. for 1 h. After that, 2.5 μl (1M) NaCl was add, incubated at 95° C. for 5 sec and naturally annealed for 2-3 h to obtain the Target-1 annealing probe.

[0047] The pCAMBIA1300-pYAO-cas9 vector was obtained from Zhili Zhongte Biotechnology company (Wuhan, China) through commercial purchase, and the link is http://www.zlzt.com/plasmid/z1-040598.html. This vector was digested by restriction endonuclease BsaI (NEB, USA) according to the manufacturer's instructions. The linearized vector fragment was recovered from by gel cutting and purified by AxyPrep DNA Gel Recovery Kit (Axygen, USA). The Target-1 probe annealled above and the vector fragment digested be enzyme were ligated with T4-ligase reagents (NEB, USA) and transformed into DH5a competent cells. The positive clones obtained were sent to biotechnology companies for sequencing. If the inserted fragment in the vector was 5′-CGTATGCCCGTTGCAGTTGCAGG-3′ (SEQ ID NO. 4), the construction of the vector was judged to be successful, and the Target-1 knockout vector of Xa7 gene was obtained. Namely, the Target-1 knockout vector of Xa7 gene can be directed to edit the Target-1 site in Xa7 gene (SEQ ID NO: 1) by CRISPR/Cas9 technology, resulting in mutations.

[0048] According to the same method above, another gene-editing target sequence was designed in the different site of Xa7 gene as Target-2: 5′-CCAGTTCCCGCGCGCCGCTGGGG-3′ (SEQ ID NO. 5). The primers sequences were synthesized according to the sequence of Target-2: Target-2 F: 5′-GGCACCAGTTCCCGCGCGCCGCTG-3′ (SEQ ID NO. 20) and, Target-2 R: 5′-AAACCAGCGGCGCGCGGGAACTGG-3′ (SEQ ID NO. 21). The Xa7-gene knockout vector for Target-2 was constructed. If the sequencing result shows that the inserted fragment in the vector is 5′-CCAGTTCCCGCGCGCCGCTGGGG-3′ (SEQ ID NO. 5), the construction of the vector is judged to be successful. The Target-2 knockout vector of Xa7 gene can be directed to edit the Target-2 site in Xa7 gene (SEQ ID NO: 1) by CRISPR/Cas9 technology, resulting in mutations.

(2) Rice Genetic Transformation and Identification of Knockout Plants

[0049] The two knockout vectors of the Xa7 gene obtained above were respectively transformed into the bacterial-blight resistance rice variety Zhen-hui 084 by the method reported by Nishimura et al. (Nishimura et al, Nat Protoc, 2006) to get the transgenic rice plants.

[0050] Genomic DNAs were extracted from the leaves of transgenic plants according to the method of Panaud et al. (Mol Gen Genet, 1996). The primers were synthesized as follows: forward: 5′-ATGGCGGCCGCTGATCATCC-3′ (SEQ ID NO. 22) and reverse: 5′-TTAATTGCCACCGATGAGGTAATC-3′ (SEQ ID NO. 23). The Xa7 gene sequences (SEQ ID NO: 1) of very transgenic plants were amplified by PCR using the proof-reading Taq enzyme PrimeSTAR® HS DNA Polymerase (TaKaRa, Japan), and the mutations of Target-1 and Target-2 sites in the gene were analyzed. Total 50 μl PCR solution contains PrimeSTAR HS (Premix) 25 μl, forward and reverse primers (10 μm) 1 μl, template DNA 2 μl (<200 ng), sterile water 214 PCR procedure was as follows: pre-denaturation at 95° C. for 5 min; denaturation at 98° C. for 10 sec, annealing at 60° C. for 15 sec, extension at 72° C. for 30 sec, 30 cycles; extension at 72° C. for 5 min.

[0051] The PCR products from transgenic plants were connected into T-Vector PMD18 (TaKaRa, Japan), and the ligation solutions and reaction conditions were operated according to the manufacturer's instructions. The ligation products of 10 μl were transformed into JM109 competent cells by the heat-shock method, and the positive clones were sent to biotechnology companies for sequencing.

[0052] Serval editing types were found at the knockout sites of the Xa7 gene in transgenic plants, as shown in FIG. 2, all of which led to frameshift mutation or fragment deletion of the Xa7 gene. Finally, four kinds of Xa7-knockout mutants were obtained named as ko-1, ko-2, ko-3 and ko-4.

Example 3. Bacterial-Blight Resistance Identification of the Two Kinds of Transgenic Rice Plants

[0053] The Xa7 transgenic plants obtained above (the transgenic positive plants obtained from Example 1) and the control variety Zhong-hua 11, Xa7-knockout plants (ko-1, ko-2, ko-3, ko-4) and the control variety Zhen-hui 084 were planted in summer field at temperatures of 28° C. to 37° C. At tillering stage of rice, the fully expanded new leaves of the plants were selected to inoculate with Xoo (Xanthomonas campestris pv. Oryzae) strain PXO86 using leaf-cutting method (Kauffman et al., Plant Dis Rep, 1973). The PXO86 strain contains an avirulent factor AvrXa7 which can activate the resistance of Xa7 gene. Two weeks after inoculation, the lesion lengths of inoculated leaves were measured downward from the cut sites of the inoculated leaves, and evaluated according to the standard: resistant (≤3.0 cm), moderately resistant (3.1 to 6.0 cm), moderately susceptible (6.1 to 9.0 cm), and susceptible (>9.0 cm).

[0054] The identification results are as follows: the susceptible variety Zhong-hua 11 is highly susceptible to PXO86 (FIG. 3), the transgenic positive plants of Zhong-hua 11 with Xa7 gene are highly resistant to PXO86 (FIG. 3); the resistant variety Zhen-hui 084 is highly resistant to PXO86 (FIG. 4), the mutant plants of Zhen-hui 084 knockout the Xa7 gene are highly susceptible to PXO86 (FIG. 4). The results indicated that rice plants containing the Xa7 gene or being transformed with an exogenous Xa7 gene can obtain highly resistant to bacterial blight. It has a great application value in breeding.

[0055] Finally, it is important to note that the above description is only specific embodiments of the present disclosure. Obviously, the disclosure is not limited to the above embodiments, but can also have a lot of deformation. All the deformation that the general technical personnel in this field can directly derive or associate with the contents disclosed in this field should be considered as the scope of protection of the disclosure.