WO2016165243A1 - 水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记 - Google Patents

水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记 Download PDF

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WO2016165243A1
WO2016165243A1 PCT/CN2015/086207 CN2015086207W WO2016165243A1 WO 2016165243 A1 WO2016165243 A1 WO 2016165243A1 CN 2015086207 W CN2015086207 W CN 2015086207W WO 2016165243 A1 WO2016165243 A1 WO 2016165243A1
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rice
gene
bph6
brown planthopper
resistance
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何光存
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武汉大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention belongs to the field of plant genetic engineering, and particularly relates to a rice brown planthopper resistance gene Bph6 and a closely linked molecular marker thereof.
  • Rice is an important food crop, with more than half of the world's staple foods.
  • the fine genetic map and physical map of the rice genome have been completed, the transgenic technology is relatively easy and is collinear with other gramineous crop genomes, and thus is regarded as a model plant.
  • humans began to enter the post-genome era.
  • Comprehensive functional genomics research has become a frontier in life sciences. Therefore, the study of rice functional genes is of great significance for socio-economic development and biological research.
  • brown planthopper has become the first major pest in rice production in China, and it has become a serious threat to China's current food security.
  • the control of brown planthopper has mainly relied on the application of chemical pesticides. Since the brown planthopper bloom mostly occurs in the mature grain filling stage of rice, the rice plant grows vigorously at this time, and it is very difficult to apply the insecticide to the base of the rice plant. In fact, due to the large-scale application of chemical pesticides in recent years, the resistance of brown planthoppers has multiplied, which has limited effect on the prevention and treatment of chemicals.
  • the use of chemical pesticides to control brown planthoppers increases the production costs of farmers.
  • chemical pesticides also cause environmental and ecological problems such as poisoning of non-target organisms, environmental and food pollution.
  • Bph1 (Athwal et al., 1971; Hirabayashi and Ogawa, 1995; Sharma et al., 2003; Cha et al., 2008)
  • Bph2 (Athwal et al., 1971; Murata et al., 1998; Murai et al 2001, Bph3 (Lakshminarayana and Khush, 1977; Jairin et al., 2007)
  • Bph4 (Kawaguchi et al., 2001), bph5 (Khush et al., 1991), Bph6 (Kabir and Khush, 1988; Qiu Et al., 2010), Bph7 (Kabir and Khush, 1988), Bph8 (Nemoto et al., 1989), Bph9 ((Nemoto et al., 1989; Muruta and Fujiwara, 2001), Bph10 (Ishii et al., 1994), Bph11 (Takita,
  • the Bph14 gene has been successfully cloned, which is the first time in the world to use map cloning.
  • the first rice insect-resistant gene isolated by the method (Du et al 2009).
  • the Bph26 and Bph3 genes were also cloned in 2014 (Tamura et al. 2014; Liu et al. 2014).
  • Map-based cloning also known as positional cloning, is a gene cloning technique developed with the development of genetic linkage maps for molecular markers.
  • the map cloning step includes genetic mapping, physical localization, sequence analysis and genetic transformation verification of the target gene.
  • any gene that can be mapped can be isolated by map cloning.
  • the map cloning method is generally suitable for relatively small genomes, such as the monocotyledonous plant rice, which has the characteristics of small genome, small ratio of genomic physical distance to genetic distance and rich labeling.
  • rice is one of the concentric circles of the genomes of seven gramineous plants such as wheat and sorghum.
  • map-based cloning multiple genes that have been cloned in rice have been cloned by map-based cloning, such as Bacterial blight resistance gene Xa-21 (Song WY et al 1995, A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21. Science, 270: 1804-1806), Xa-1 (Yoshimura et al 1998, Expression of Xa-1, a bacterial blight-resistance gene in rice, is induced bybacterial inoculation.
  • Bacterial blight resistance gene Xa-21 Short WY et al 1995, A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21. Science, 270: 1804-1806
  • Xa-1 Yoshimura et al 1998, Expression of Xa-1, a bacterial blight-resistance gene in rice, is induced bybacterial in
  • the object of the present invention is to provide a gene Bph6 against rice brown planthopper and its application.
  • the invention adopts a genetic method to construct an isolated population of rice brown planthopper, and uses the method of map cloning to isolate the rice brown planthopper resistance gene Bph6.
  • the co-segregation marker showed that the gene was co-segregated with the resistance to brown planthopper, and the function of the gene was confirmed by genetically transforming Bph6 gene to make the phenotype of susceptible rice brown locust.
  • the invention provides a rice brown planthopper resistance gene Bph6, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO. 1, the gene is 7836 bp in length, has two introns and three exons, and the CDS is a region respectively.
  • the segment is 3841-6786 and the cDNA is 4140 bp in length.
  • cDNA sequence of the gene is as shown in SEQ ID NO. 2 and encodes 981 amino acids.
  • the invention also provides a protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.
  • a polynucleotide encoding the above protein can be exemplified in its coding region, without changing the amino acid sequence, or in its non-coding region without affecting protein expression.
  • the sequence is modified.
  • the present invention also encompasses the substitution, addition and/or deletion of one or more nucleotides to a polynucleotide sequence encoding the above protein, having a nucleotide sequence having a functionally identical protein as described above.
  • the above polynucleotide fragments provided by the present invention are operably linked to a homologous or heterologous promoter sequence.
  • the present invention also includes a sense sequence or an antisense sequence based on the polynucleotide, including a cloning vector or expression vector containing the polynucleotide sequence or a fragment thereof, a host cell containing the vector, and the nucleotide Transformed plant cells and transgenic plants of the sequences or fragments thereof.
  • the invention also provides the use of the gene in rice breeding, which is selected to increase the resistance of rice to brown planthopper.
  • the present invention also provides a molecular marker closely linked to the rice brown planthopper resistance gene Bph6, the molecule Labeled as H or Y37; the molecule is labeled H or Y37;
  • the primer pairs used to amplify the molecular marker H are as follows:
  • Reverse primer HR ATTCCAGCATCGATTGCTTC
  • the primer pairs used to amplify the molecular marker Y37 are as follows:
  • Forward primer Y37F TCGACCCACGCGGTTTTTCTGTA;
  • Reverse primer Y37R TAGATTCCTTCGGTTCCCATT.
  • the invention also provides the use of the molecular marker in breeding rice against brown planthopper.
  • the present invention also provides a molecular detection method for rice brown planthopper resistance gene Bph6, which amplifies the genomic DNA of the rice to be detected by the primer pair, and detects the amplification product: if the amplified fragment of 1654 bp is amplified by using primers HF and HR, or Amplification of the 174 bp amplified fragment using primers Y37F and Y37R marked the presence of the rice brown planthopper resistance gene Bph6.
  • the invention also provides a method for screening rice against brown planthopper, wherein the genomic DNA of the rice to be tested is amplified by the primer pair, and the amplified product is detected: if the amplified fragment of 1654 bp is amplified by using primers HF and HR, or Primers Y37F and Y37R amplified a 174 bp amplified fragment, which indicated the presence of resistance to the rice cultivar.
  • the present invention provides a method of cultivating a plant having resistance to brown planthopper, comprising:
  • the polynucleotide comprises a rice brown planthopper resistant Bph6 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1 or SEQ ID NO. 2;
  • the present invention also provides a method of producing a plant having resistance to brown planthopper, which comprises crossing a plant having the brown planthopper resistance gene Bph6 with other plants to produce a progeny plant having resistance to brown planthopper.
  • the plant is rice.
  • the present invention clones the Bph6 gene by the following steps:
  • the seedling period group method identifies the resistance of the targeted population to brown planthopper. Seeds were harvested from F 2 plants, and seeds harvested from each F 2 plant were sown in a tray of 20 seedlings (referred to as a family). In the 2 leaf 1 heart stage, 2-4 years old brown planthopper nymph (10 heads/strain) was placed, and the damage of each family was recorded, and each experiment was repeated 3 times. According to the results of the insect resistance identification, the insect-resistant grades of the localized population lines were classified.
  • Candidate gene determination The genomic DNA of the resistant parental rice material (Swarnalata) containing Bph6 gene was amplified by PCR and spliced to obtain the genomic sequence of the region where Bph6 was located. The gene was predicted by RiceGAAS software and compared with the Nipponbare and 9311 sequences. Analysis to determine candidate genes for Bph6.
  • the Bph6 gene can be amplified from the brown planthopper resistant rice genome by designing an appropriate PCR primer according to the nucleotide sequence of Bph6 disclosed in the present invention.
  • RNAi vector construction Based on the ORF sequence of the Bph6 gene, Blast analysis was performed on NCBI, and the most conserved 500 bp was selected for RNAi vector construction. A fragment of a 500 bp conserved sequence complementary to both sides of the intron was constructed by overlapping PCR, and the fragment was added to A and ligated into XcmI-cut PCXUN vector. After sequencing verification, the resulting vector was the RNAi vector of Bph6 gene, which was electroporated into Agrobacterium EHA105.
  • the overexpression vector was introduced into the normal indica variety Nipponbare by Agrobacterium tumefaciens EHA105-mediated genetic transformation, and finally 32 Bph6 positive plants were obtained.
  • the T1 generation plants were first tested for insect resistance.
  • the seedling stage was identified by the group method. All the rice Nipponbare died, and the transgenic positive plants survived, and the insect resistance level was 5-7.
  • Bph6-positive plants homozygous for T2 generation were identified for insect resistance.
  • the seedling stage was identified by the group method. All the rice Nipponbare died, and the transgenic positive plants survived.
  • the insect resistance level was 3-5.
  • the Agrobacterium tumefaciens EHA105-mediated genetic transformation method was used to introduce the RNAi vector into the near-isogenic line of the normal indica rice variety Nipponbare background, and finally 4 RNAi-positive plants were obtained. Infection-resistant identification was performed using T2 homozygous RNAi-positive plants. The seedling stage was identified by the group method. All the rice Nipponbare died, the near-isogenic plants survived, the RNAi transgenic plants died, and the insect resistance level was 7-9. It was confirmed that the Bph6 gene has a function against brown planthopper. Therefore, the brown planthopper resistance gene Bph6 can be applied in rice or in rice seeds to breed rice varieties with resistance to brown planthopper.
  • the successful cloning of this gene further confirmed the reliability of the cloning of important genes in rice by map cloning.
  • the cloned gene has clear function and good effect.
  • the cloned Bph6 gene of the present invention has obvious resistance to brown planthopper, which is important for comprehensive understanding of the diversity of rice gene types of brown planthopper.
  • Bph6 greatly improved the resistance of rice to brown planthopper, and applied Bph6 to rice breeding through genetic transformation or hybridization, which could improve the brown planthopper resistance of rice varieties, thereby reducing the damage of brown planthopper and achieving the goal of increasing yield and stable yield.
  • Figure 1 shows the distribution frequency of the brown planthopper resistance value.
  • the genotypes of SSR marker RM6997 in the F2 population are shown; the black, white and gray columns represent Swarnalata, 9311 and heterozygous marker genotypes; the seedlings of 140 rice F2 lines (3 leaf stage or 11d growth) are 10 brown planthoppers per plant were treated for 9-10 days.
  • Figure 2 shows the preliminary positioning results of Bph6.
  • the left side of the chromosome is the name of the marker, and the value is the genetic distance (cM) between the markers.
  • the QTL scan has a maximum LOD value of 43.8 between the molecular markers RM6997 and RM5742, which indicates a browning phenotype value of about 77.5%.
  • Figure 3 shows the results of Bph6 fine positioning.
  • A the value between the molecular markers indicates the number of recombinant cells with Bph6, the 18 kb region between Bph6 and molecular markers H and Y37, and the B: 18.1 kb region contains two predicted genes; the gene shown in ORF1 The box is an exon, where black is the CDS area, white is the UTR area, and the horizontal line is the intron.
  • Figure 4 shows the results of identification of transgenic rice plants against brown planthopper.
  • OE was a transgenic overexpressing plant
  • RNAi was a transgenic plant in which Bph6 was inhibited
  • the above is a phenotype, and the following is an insect resistance level.
  • the transgenic lines expressing Bph6 gene were all significantly resistant to brown planthopper, and the transgenic lines with inhibited expression of Bph6 gene were not resistant to brown planthopper.
  • Figure 5 is an example of a functional molecular marker H developed according to the Bph6 genomic sequence, the amplified fragment of which is 1654 bp in length; in the figure, Swarnalata is an insect-resistant parent carrying the brown planthopper resistance gene Bph6, and Yangdao 6 (93-11) is a susceptible insect.
  • Figure 6 is a molecular marker-assisted selection map for breeding a brown planthopper resistant rice with the Bph6 gene.
  • 9311 represents the susceptible variety Yangdao 6 (93-11), NIL.
  • -9311 represents Xiangyang No. 6 (Yangdao 6 genetic background carrying the brown planthopper resistance gene Bph6). After the larvae, the inductive receptor parent Yangdao 6 apparently died, while the ⁇ Yang 6 (Yangdao 6 genetic background carrying the brown planthopper resistance gene Bph6) survived and the plants grew healthy.
  • the anti-BPH group Swarnalata (containing BELL6) was crossed with the brown planthopper rice variety Yangdao 6 (93-11) to construct the F 2 population containing Bph6.
  • 93-11 and Swarnalata were from the National Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences.
  • the crop germplasm preservation center was used to extract the genomic DNA of each of the parental and F 2 populations by the CTAB method (Murray MG & Thompson, 1980 Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8: 4321-4325).
  • Each F 2 individual was obtained by self-crossing to obtain the corresponding F 2:3 family.
  • the seedling stage group method was used to investigate the resistance performance of each individual F2:3 family, and the F 2:3 family resistance level represented F 2 single plant. Resistance to brown planthopper phenotype.
  • all test materials were soaked and germinated separately before sowing.
  • Each of the family (variety) 60 seeds was sown in a bread box of 58 cm in length, 38 cm in width, 9 cm in height, and containing 7 cm of nutrient soil. Three replicates of each material were seeded per box, with 3 replicates of each of the randomly seeded parent and TN1 (sensible control).
  • the results of the seedling group method showed that the insect resistance levels of Swarnalata and 9311 were 2.9 and 8.7, respectively, indicating that Swarnalata is resistant to brown planthopper and 9311 is brown planthopper.
  • the frequency distribution of insect resistance levels of 140 F 2:3 families to brown planthopper was continuous, and some of the resistance levels of individual plants showed super-parent phenomenon.
  • the F 2:3 families were divided into three phenotypes: insect resistance, anti-infective isolation and susceptibility, while the corresponding F 2 single genotypes were recorded as RR (homozygous insect resistance). , Rr (heterozygous insect resistance) and rr (homozygous insect) three.
  • the genomic sequences of rice variety 9311 and Nipponbare were compared, and the SSR search tool SSRIT (http://www.gramene.org/db/markers/ssrtool) was used to find the SSR motif. Primers are designed based on their flanking sequences as an alternative marker. Among them, the SSRIT setting parameters are: the maximum motif length is 4 mer, the minimum number of repetitions is 5, and all SSR motifs are searched. All SSR motifs greater than 15 bases (motif length x repeat number) were selected.
  • PowerBlast software was used to find the insertion/deletion sites in the genomic sequence, and the InDel marker was developed by Primer Premier 5.0 software.
  • the analysis of the SSR markers is based on the method of Temnykh (Temnykh S et al, 2000. Mapping and genome organization of microsatellite sequences in rice. Theor Appl Genet 100: 697-712).
  • the 10 ⁇ l reaction system included: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 50 ⁇ M dNTPs, 0.2 ⁇ M primer, 0.5 U Taq polymerase and 20 ng DNA template.
  • the amplification reaction was carried out on a PTC-100 PCR machine: 94 ° C for 2 min; 94 ° C for 15 sec, 55 ° C for 30 sec, 72 ° C for 1.5 min, 35 cycles; 72 ° C for 5 min.
  • the amplified product was separated by 6% non-denaturing PAGE gel and developed by silver staining (Zhu et al, 2004. Identification and characterization of a new blast resistance gene located on rice chromosome 1 through linkage and differential analyses. Phytipathology 94: 515-519 ).
  • the amplified DNA bands were observed using a light box equipped with a fluorescent lamp. Recording results, polymorphic primers between parents were analyzed in the F 2 population to obtain population genotype data.
  • the DNA of 10 extreme insect-resistant plants and 10 extreme insect-splitting plants were selected to construct an anti-inductive pool.
  • the anti-sensitive pools were screened by primers with polymorphism between the parents, and molecular markers with polymorphism between the anti-sensitive pools were obtained.
  • the polymorphic markers indicated that they were linked to resistance.
  • primers with polymorphism between the parents on the chromosome are selected to screen each individual of the F 2 isolated population, and the PCR procedure is the same as above to obtain the population genotype data.
  • the partial genotypic data of the population were constructed using the software JoinMap 3.0 to obtain the genetic distance of each molecular marker.
  • the QTL locus of target chromosome was scanned by MapQTL 5.0 software composite interval mapping method. The results indicated that there was a maximum peak with a LOD value of 43.8 between the SSR markers RM6997 and RM5742 on chromosome 4, which accounted for approximately 77.5% of the brown planthopper phenotype value (as shown in Figure 2). It can be seen that there may be a major gene controlling the brown planthopper resistance at this site.
  • the present invention screened 4300 BC2F2 populations by PCR (polymerase chain reaction) and polyacrylamide gel electrophoresis with SSR markers RM16994 and RM17008 on both sides. For each plant, 41 individuals were recombined between the two markers.
  • the SSR search tool SSRIT http://www.gramene.org/db/markers/ssrtool
  • Primers are designed based on their flanking sequences as an alternative marker.
  • PowerBlast software was used to find the insertion/deletion sites in the genome sequence, and the InDel marker was developed using Primer Premier 5.0 software.
  • the disease resistance protein encoded by the first gene of the insect-resistant rice was significantly different from Nipponbare. At present, it is considered that the inhalation process of sucking insects and rice pathogens is similar to that of rice, and the mechanism of rice sucking insects may be the same as that of pathogens. Therefore, the gene was identified as Bph6.
  • the primers were designed according to the results of the gene prediction, and a cDNA sequence of the candidate gene was amplified.
  • the primers were designed by this sequence, and the 5' and 3'Full RACE kits of TaKaRa were used to obtain the 5' and 3' end sequences of the candidate gene.
  • the transcription initiation site and termination site of the candidate gene were identified and spliced.
  • the full-length cDNA sequence of the gene is derived.
  • the full-length cDNA of Bph6 was amplified by re-synthesizing the primer according to the full-length cDNA sequence, and the nucleotide sequence thereof is shown in SEQ ID NO. 2 of the Sequence Listing (Fig. 3B).
  • the physical distance between the markers H and Y37 is approximately 18 kb. Therefore, the use of molecular markers within the linkage map is used:
  • the primer H can be used to amplify a 1654 bp amplified fragment, or the primer Y37 can amplify a 174 bp amplified fragment, all of which mark the main target locus of rice against brown planthopper
  • the existence of Bph6 Therefore, the use of the above molecular marker method provided by the present invention to identify the presence of the Bph6 resistance gene has a very high efficiency, can predict the brown planthopper resistance of rice plants, and accelerate the breeding process of rice varieties resistant to brown planthopper.
  • the ORF was directly amplified by PCR, and the vector was added after adding A.
  • the resulting vector was the Bph6 gene overexpression vector, which was electroporated into Agrobacterium EHA105. Pick up the monoclonal expansion culture, and after PCR verification, add an equal volume of 50% glycerin and mix at -70 °C for later use.
  • the carrier is pCXUN (provided by Professor Wang Guoliang of Ohio State University, USA)
  • the pcmXUN vector was digested with XcmI, and the exogenous fragment was directly added after adding A. Blast analysis was performed based on the obtained full-length cDNA sequence, and the most conserved 500 bp was selected for amplification. A fragment of a 500 bp conserved sequence complementary to both sides of the intron was constructed by overlapping PCR. The fragment was recovered by gelatinization and added to A and then ligated into the PCXUN vector. After the sequencing verification was correct, the resulting vector was the Bph6 gene RNAi vector, which was electroporated into Agrobacterium EHA105. Pick up the monoclonal expansion culture, and after PCR verification, add an equal volume of 50% glycerin and mix at -70 °C for later use.
  • Agrobacterium EHA105-mediated genetic transformation method Hiei et al., 1994, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271-282
  • the above Bph6 gene overexpression vector was introduced into Nipponbare, a common rice variety susceptible to the brown planthopper.
  • the above Bph6 RNAi vector was introduced into the near isogenic line of the Nipponbare background of the common rice variety by the same method.
  • the Bph6 gene over-expression transformation vector obtained 32 positive transgenic plants. After harvesting the seed, by substituting Bph6 T 2 transgenic plants were identified by insect seedbox method. As shown in Figure 3, positive transgenic lines OE1, OE2 were identified for resistance to brown planthopper. The results of the identification are shown in Fig. 3. After 7 days of access to the brown planthopper, the whole plant of the susceptible insect variety Nipponbare died, and the near-isogenic plants containing the Bph6 gene, which is resistant to the brown planthopper gene, grew healthy and had no leaf damage.
  • the Bph6 gene over-expression transformation vector obtained the positive transgenic plant OE1, and OE2 also grew healthy, no leaf damage, and the insect resistance level was 2-4, which confirmed that the Bph6 gene has the function of resisting brown planthopper. Therefore, the brown planthopper resistance gene Bph6 can be applied in rice or in rice seeds to breed rice varieties with resistance to brown planthopper.
  • the Bph6 gene RNAi vector obtained 4 positive transgenic plants.
  • the homozygous T 2 generation transgenic plants were identified for brown planthopper resistance. The results of the identification are shown in Fig. 4. After 7 days of access to the brown planthopper, the whole plant of the susceptible species Nipponbare died, and the near-isogenic material grew healthy without leaf damage, while the material of RNAi died, confirming that the Bph6 gene has resistance to brown planthopper. The function.
  • Swarnalata including BELL6 resistance to brown planthopper
  • the indica rice cultivar Yangdao 6 93-11
  • the F 23 family constructed by Swarnalata and Yangdao 6 were constructed.
  • Molecular marker primers H and Y37, the nucleotide sequences of which are shown in SEQ ID No. 4-5, respectively.
  • the genomic DNA of rice samples was extracted by CTAB extraction. Sample DNA was amplified using primers H and Y37, respectively. 10 ⁇ l system.
  • the 10 ⁇ l reaction system included: 10 ⁇ PCR buffer, 1.0 ⁇ l; 10 mM dNTPs, 0.1 ⁇ l; 10 ⁇ M primer, 0.4 ⁇ l; 5 U/ ⁇ l Taq DNA polymerase, 0.2 ⁇ l and 50 ng DNA template.
  • the amplification reaction was carried out on a Bioer PCR machine: 94 ° C for 4 min; 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 90 s, 30 One cycle; 72 ° C for 5 min.
  • the amplification product of H was separated by 1% agarose gel and directly analyzed after electrophoresis.
  • the amplified product of Y37 was separated by 8% non-denaturing PAGE gel, and after electrophoresis, it was stained with silver nitrate silver staining and then read and analyzed.
  • the molecular marker method provided by the invention can accurately screen out the Bph6 containing the brown planthopper resistance gene, thereby greatly improving the breeding efficiency.
  • the above-mentioned molecular markers were used for molecular marker-assisted selection to breed the BPH6-resistant rice brown planthopper rice variety Yangyang No.6, and the specific implementation was as follows: the anti-Brown planthopper parent Swarnalata (containing the brown planthopper resistance gene Bph6) and the brown planthopper susceptible rice variety Yangdao 6 No. (93-11) hybrid F 1, backcross with Yangdao No.
  • BC 4 F 1 is added, using a linkage with Bph6 Molecular markers (RM5742, RM6997) were screened for Yangdao 6/Swarnalata//Yangdao 6 BC 4 F 2 homozygous for the Bph6 locus, which was homozygous for the brown planthopper gene, and was subjected to additional breeding.
  • the material was agronomically phenotyped and raised in rice. No. 6 is indistinguishable, but it contains Bph6, a brown planthopper resistance gene, and this material is temporarily named as Yangyang No. 6.
  • the brown planthoppers in Wuhan field were identified by seedling group method identification, near tillering stage identification and maturity identification to confirm their resistance to brown planthopper (see Figure 1).
  • the biotypes P, S and Y of the brown planthopper biotype I, II, III and the laboratory were used for the identification of the seedling group method.
  • the results showed that the control traits TN1 and the recipient parent Yangdao 6 were 7 days after the larvae. Obviously died, while the cultivar No. 6 (Yangdao 6 genetic background carrying the brown planthopper resistance gene Bph6) survived and the plants grew healthy (Fig. 6). At present, the number of rice varieties has been declared on the 6th.
  • the banding pattern of a single plant is 1 when it is consistent with the anti-Brown-flying host Swarnalata, and 2 when it is consistent with Yangdao 6 (93-11), and the band type with two parents at the same time.
  • the hybrid belt type is recorded as 3.
  • the results of molecular marker screening indicated that BC4F1 only differed from Yangdao 6 in the Bph6 locus (RM5742 and RM6997) of the brown planthopper resistance gene, and the other loci were replaced by Yangdao 6 No. Genetic background.
  • the rice brown planthopper resistance gene Bph6 disclosed in the invention can significantly improve the resistance of rice to brown planthopper, and the application of Bph6 to rice breeding through genetic transformation or hybridization can improve the brown planthopper resistance of rice varieties, thereby reducing the damage of brown planthopper and increasing yield. And the purpose of stable production.

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Abstract

提供了水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记H和Y37，所述基因Bph6定位于所述分子标记H和Y37之间。所述基因Bph6的核苷酸序列如SEQ ID No.1所示，其cDNA序列如SEQ ID No.2所示。利用分子标记H和Y37可筛选含有所述基因Bph6的水稻。通过遗传转化和杂交，将所述Bph6基因转入普通水稻品种，能够提高水稻对褐飞虱的抗性。

Description

水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记 技术领域
本发明属于植物基因工程领域，具体涉及一种水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记。
背景技术
水稻是一种重要的粮食作物，世界上有超过一半的人以其为主食。同时，由于水稻基因组精细遗传图和物理图谱已完成，其转基因技术相对容易，并且与其它禾本科作物基因组具有共线性，因而被视做模式植物。随着包括水稻在内的多种生物基因组测序的完成，人类开始进入后基因组时代。全面开展功能基因组研究已成为生命科学的前沿领域。因此水稻功能基因的研究对社会经济发展和生物学研究具有重大意义。
粮食安全问题，是全世界人民面临的挑战。50、60年代的矮化育种和70年代的杂交水稻培育两次科技革命使水稻产量大幅度提高。但近几十年来水稻受到大面积的病虫为害，使水稻生产受到威胁。褐飞虱是我国水稻生产中的主要害虫，其成虫和若虫以口针刺吸水稻汁液，引起黄叶或枯死，导致减产或绝收。据中国农业年鉴记载，1966、1969、1973、1977、1983和2003年全国性大发生，1987、1991、2005、2006和2007年全国性特大发生，褐飞虱危害面积达到水稻总面积的50％以上，给我国水稻生产造成了严重的损失。。目前我国每年水稻褐飞虱发生面积为2000万公顷以上，每年因褐飞虱危害造成的直接产量损失达280万吨以上。褐飞虱已对我国水稻生产安全形成严重威胁。
目前，褐飞虱已经成为我国水稻生产中的第一大虫害，对我国当前粮食安全已形成严重威胁。长期以来，褐飞虱的防治主要是依靠施用化学杀虫剂。由于褐飞虱爆发多发生在水稻成熟灌浆期，此时稻株长势旺盛，将杀虫剂施到稻株基部的操作非常困难。事实上由于化学杀虫剂的连年大量施用，褐飞虱抗药性成倍增加，使药剂防治的效果有限。同时使用化学杀虫剂防治褐飞虱，一方面增加了农民的生产成本，另一方面化学杀虫剂还造成对非目标生物的毒杀、对环境和粮食污染等环境和生态问题。
利用抗褐飞虱基因培育抗虫水稻品种是褐飞虱综合防治中最为经济有效的方法。国际水稻研究所(IRRI)的研究结果和东南亚的水稻生产实践证明，即使是只有中等抗性水平的水稻品种，也足以将褐飞虱的群体控制在造成危害的水平以下，不至于对水稻造成实际的危害和产量损失。因此，挖掘水稻抗褐飞虱基因并在水稻育种项目中应用是防治水稻褐飞虱的根本措施。
水稻抗褐飞虱基因的研究始于上世纪70年代初。至今已经在普通栽培稻和野生稻资源中鉴定和定位了二十多个水稻抗褐飞虱的主效抗虫基因(具体综述见Jena等，2010.Current status of Brown Planthopper(BPH)resistance and  genetics.Rice 2010(3),161-171)。如Bph1(Athwal et al.,1971；Hirabayashi and Ogawa,1995；Sharma et al.,2003；Cha et al.,2008),Bph2(Athwal et al.,1971；Murata et al.,1998；Murai et al.,2001)，Bph3(Lakshminarayana and Khush,1977；Jairin et al.,2007)，Bph4(Kawaguchi et al.,2001),bph5(Khush et al.,1991)，Bph6(Kabir and Khush,1988；Qiu et al.,2010),Bph7(Kabir and Khush,1988),Bph8(Nemoto et al.,1989),Bph9((Nemoto et al.,1989；Muruta and Fujiwara,2001),Bph10(Ishii et al.,1994),Bph11(Takita,1996)，Bph12(Hirabayashi et al.,1998，1999)，Bph13(t)(Liu et al.,2001)，Bph14(Wang et al.,2001；Du et al.,2009)，Bph15(Huang et al.,2001；Yang et al 2004)，Bph17(Renganayaki et al.,2002)，Bph18(t)(Jena et al.,2006)，Bph19(t)(Chen et al.,2006),Bph20(t)、Bph21(t)、Bph22(t)、Bph23(t)、Bph24(t)(Li et al.,2006；李容柏等，2008),Bph20、Bph21(Rahman et al 2009),Bph22(t)、Bph23(t)(Ram et al 2010),Bph 24(t)(Deen et al 2010),Bph22(t)、Bph23(t)(Hou et al 2011),Bph25(t)、Bph26(t)(Myint et al.2005；Yara et al.2010；Myint et al.2012)。其中，Bph14基因已经成功克隆，这是国际上首次利用图位克隆法分离得到的第一例水稻抗虫基因(Du et al 2009)。Bph26和Bph3基因也于2014年分别得到了克隆(Tamura et al.2014；Liu et al.2014)。
图位克隆(map-based cloning)又称为定位克隆(positional cloning)，是随着分子标记遗传连锁图谱的发展而发展起来的一种基因克隆技术。图位克隆法步骤包括对目标基因进行遗传定位、物理定位、序列分析及遗传转化验证功能。从理论上讲，任何一个能定位的基因都可用图位克隆法分离。图位克隆法一般适合于基因组比较小的物种，如单子叶模式植物水稻，具有基因组小、基因组物理距离与遗传距离之比小而且标记丰富的特点。水稻作为禾本科模式植物，其基因组是麦、高梁等七种禾本科植物基因组组成的同心圆的圆心，是最适合应用图位克隆法分离目的基因的作物之一。水稻中已经克隆的多个基因都是通过图位克隆法克隆的，如抗白叶枯病基因Xa-21(Song WY等1995,A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene,Xa21.Science,270:1804-1806)、Xa-1(Yoshimura等1998,Expression of Xa-1,a bacterial blight-resistance gene in rice,is induced bybacterial inoculation.PNAS,95:1663-1668)和Xa-26(Sun等2004,Xa26a gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice,encodes an LRR receptor kinase-like protein.Plant Journal,37:517-527)，抗稻瘟病基因Pi-b(Wang等1999,The Pi-b gene for rice blast resistance belongs to the nucleotide binding andleucine-rich repeat class of plant disease resistance genes.Plant Journal,1999,19:55-64)和Pi-ta(Bryan等2000,A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell,12:2033～2046)，我国科学家克隆的分蘖基 因(Li等2003,Control oftillering in rice.Nature 422:618-621)、耐盐基因(Ren等2005，A rice quantitative trait locus for salt tolerance encodes a sodium transporter.Nature Genetics 37(10):1141-1146)和高产基因(Weiya Xue等2008，Natural variation in Ghd7is an important regulator of heading date and yield potential in rice.Nature Genetics 40,761-767)，以及应用图位克隆法分离得到的第一例水稻抗虫基因(Du等2009，Identification and characterization of Bph14,a gene conferring resistance to brown planthopper in rice.PNAS,106:22163-22168)。
发明内容
本发明的目的在于提供一种水稻抗褐飞虱的基因Bph6及其应用。
本发明采用遗传学的方法，构建水稻抗褐飞虱的分离群体，利用图位克隆的方法，分离到水稻抗褐飞虱基因Bph6。通过共分离标记检测表明该基因与抗褐飞虱性能是共分离的，通过遗传转化Bph6基因，使感性水稻出现抗褐飞虱的表型，证实了该基因的功能。
本发明提供水稻抗褐飞虱基因Bph6，所述基因的核苷酸序列如SEQ ID NO.1所示，该基因全长7836bp，具有2个内含子和3个外显子，其CDS分别为区段为3841-6786，cDNA全长4140bp。
进一步地，所述基因的cDNA序列如SEQ ID NO.2所示，编码981个氨基酸。
本发明还提供了所述基因编码的蛋白，所述蛋白的氨基酸序列如SEQ ID NO.3所示。
应当理解，在不影响BPH6蛋白活性的前提下(即不在蛋白的活性中心)，本领域技术人员可对SEQ ID NO.3所示的氨基酸序列进行各种取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列。
此外，考虑到密码子的简并性，例如可在其编码区，在不改变氨基酸序列的条件下，或在其非编码区在不影响蛋白表达的条件下，对编码上述蛋白的多核苷酸序列进行修改。因此，本发明还包含对编码上述蛋白的多核苷酸序列进行的替换、添加和/或缺失一个或多个核苷酸，具有与上述编码具有形同功能蛋白的核苷酸序列。
本发明提供的上述多核苷酸片段其与一个同源或异源启动子序列可操纵地连接。
本发明还包括基于所述多核苷酸的正义序列或反义序列，包括含有所述多核苷酸序列或其片段的克隆载体或表达载体、含有所述载体的宿主细胞、含有所述核苷酸序列或其片段的转化的植物细胞和转基因植物。
本发明还提供了所述基因在水稻选育中的应用，所述水稻选育为提高水稻对褐飞虱的抗性。
本发明还提供了与水稻抗褐飞虱基因Bph6紧密连锁的分子标记，所述分子 标记为H或Y37；所述分子标记为H或Y37；
用于扩增分子标记H的引物对如下：
正向引物HF：AGAATTGCTGCATGCTGTTG；
反向引物HR：ATTCCAGCATCGATTGCTTC；
用于扩增分子标记Y37的引物对如下：
正向引物Y37F：TCGACCTACGGCTTTTCTGTA；
反向引物Y37R：TAGATTCCTTCGGTTCCCATT。
本发明还提供了所述分子标记在选育抗褐飞虱水稻中的应用。
本发明还提供了水稻抗褐飞虱基因Bph6的分子检测方法，通过所述引物对扩增待检水稻基因组DNA，并检测扩增产物：若利用引物HF和HR扩增出1654bp的扩增片段，或利用引物Y37F和Y37R扩增出174bp的扩增片段，则标志着水稻抗褐飞虱基因Bph6的存在。
本发明还提供了一种筛选抗褐飞虱水稻的方法，通过所述引物对扩增待检水稻基因组DNA，并检测扩增产物：若利用引物HF和HR扩增出1654bp的扩增片段，或利用引物Y37F和Y37R扩增出174bp的扩增片段，则标志着水稻品种褐飞虱抗性的存在。
本发明提供一种培育具有褐飞虱抗性的植物的方法，包括：
1)用多核苷酸转化植物细胞；所述多核苷酸含有水稻抗褐飞虱Bph6基因，其核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；
2)将被转化的植物细胞再生为植物；
3)培养再生的植物并使上述多核苷酸得到表达。
本发明还提供一种产生具有褐飞虱抗性的植物的方法，所述方法包括将具有褐飞虱抗性基因Bph6的植物与其他植物杂交产生具有褐飞虱抗性的子代植物。
其中所述植物是单子叶植物。
优选地，所述植物是水稻。
本领域技术人员应能理解，根据本发明公开的序列来设计或产生分子标记可用于抗褐飞虱水稻的选育工作。
本发明通过如下步骤克隆得到Bph6基因：
1.创建定位群体。利用抗褐飞虱水稻与普通水稻品种杂交，F1代自交获得F2群体，作为抗褐飞虱基因定位群体。
2.抗褐飞虱鉴定。苗期集团法鉴定定位群体的抗褐飞虱性能。从F2代植株上收获种子，每份F2代植株收获的种子于秧盘中播种20棵苗(称为1个家系)。2叶1心期，放入2-4龄的褐飞虱若虫(10头/株)，记录各家系的受害情况，每份材料重复3次实验。根据抗虫鉴定结果，对定位群体株系的进行了抗虫级别的 划分。
3.抗褐飞虱基因定位。用PCR(polymerase chain reaction)和聚丙烯酰胺凝胶电泳，以及RFLP标记和Southern杂交的方法，检测F2各个单株的SSR和RFLP分子标记的分离情况，结合相应各家系的抗虫级别，应用JoinMap3.0和MapQTL5.0软件，构建水稻第12染色体的分子标记遗传连锁图，将Bph6定位在分子标记RM5742和RM6997之间。
4.加密目标区段分子标记与精细定位。根据国际水稻基因组计划公布的水稻基因组序列，设计第4染色体Bph6所在目标区段的SSR标记和InDel标记的引物，用PCR和聚丙烯酰胺凝胶电泳方法检测标记在BC2F2大群体中的分离，通过筛选得到的重组单株的表型与基因型的关系，将抗褐飞虱基因Bph6定位在分子标记H和Y37之间，与标记Y37紧密连锁。
5.候选基因确定。通过PCR方法分段扩增含抗褐飞虱基因Bph6的抗性亲本水稻材料(Swarnalata)的基因组DNA并拼接得到Bph6所在区段的基因组序列，应用RiceGAAS软件预测基因，并通过与日本晴和9311序列作对比分析，确定Bph6的候选基因。
6.全长cDNA克隆。根据预测的cDNA序列设计引物，从抗褐飞虱水稻的cDNA中扩得2900bp片段，以该段序列设计引物，通过RACE(cDNA末端快速扩增技术)得到了cDNA的3’端和5’端序列，最终获得Bph6的全长cDNA。
然而，本领域技术人员应当理解，根据本发明公开的Bph6的核苷酸序列，通过设计恰当的PCR引物，即可从抗褐飞虱水稻基因组中扩增得到Bph6基因。
7.遗传转化Bph6基因验证其功能。根据Bph6基因的ORF序列，用PCR的方法分别扩增出含有Bph6基因的ORF的2946bp的片段经加A后连入经XcmI酶切的PCXUN载体内。测序验证无误后，所得载体即为Bph6基因遗传转化载体，将其电转入农杆菌EHA105中。
根据Bph6基因的ORF序列，在NCBI上进行Blast分析，选择最保守的500bp进行RNAi载体的构建。通过重叠PCR的方法构建出中间为内含子两边为互补的500bp保守序列的片段，将该片段加A后连入经XcmI酶切的PCXUN载体内。测序验证无误后，所得载体即为Bph6基因的RNAi载体，将其电转入农杆菌EHA105中。
采用农杆菌EHA105介导的遗传转化方法，将过表达载体导入正常粳稻品种Nipponbare中，最后获得Bph6阳性植株32株。先用T1代的植株进行了抗虫鉴定。苗期采用集团法鉴定，对照水稻Nipponbare全部死亡，转基因阳性植株存活，抗虫级别为5-7级。用T2代纯合的Bph6阳性植株进行了抗虫鉴定。苗期采用集团法鉴定，对照水稻Nipponbare全部死亡，转基因阳性植株存活，抗虫级别为3-5级。
采用农杆菌EHA105介导的遗传转化方法，将RNAi载体导入正常粳稻品种Nipponbare背景的近等基因系中，最后获得RNAi阳性植株4株。用T2代纯合的RNAi阳性植株进行了抗虫鉴定。苗期采用集团法鉴定，对照水稻Nipponbare全部死亡，近等基因系植株存活，RNAi转基因阳性植株死亡，抗虫级别为7-9级。证实Bph6基因具有抗褐飞虱的功能。因此，抗褐飞虱基因Bph6可以在水稻中应用也可以在水稻种子中应用，培育具有抗褐飞虱性能的水稻品种。
本发明的优点和效果：
1.本基因的成功克隆进一步证实了图位克隆法克隆水稻重要基因的可靠性，该方法克隆的基因其功能明确、效果好。
2.本发明克隆的Bph6基因具有明显的抗褐飞虱性能，这对全面理解水稻对褐飞虱基因类型的多样性有重要意义。
3.除Bph14基因和Bph26、Bph3分别在2009年和2014年得到克隆外，国际上尚未克隆出其它的水稻抗褐飞虱基因，对水稻抗褐飞虱的分子机理仍不清楚。而本发明克隆的Bph6基因能够显著提高水稻对褐飞虱的抗性，这对水稻抗褐飞虱的分子机理研究将有极大的推动作用。
4.Bph6使水稻抗褐飞虱性能大大提高，通过遗传转化或杂交将Bph6应用于水稻育种中，可以改善水稻品种的抗褐飞虱性，从而减轻褐飞虱的为害，达到增产和稳产的目的。
5.刺吸式昆虫是农业生产中的一大类虫害，Bph6基因克隆和抗褐飞虱功能证实，对于其他植物的抗刺吸式昆虫研究具有重要参考作用。
附图说明
图1为褐飞虱抗性值的分布频率。
图示为SSR标记RM6997在F2群体中的基因型；黑色，白色和灰色柱子分别代表Swarnalata，9311和杂合的标记基因型；140个水稻F2株系的幼苗(3叶期或生长11d)按每株10头褐飞虱处理9～10d。
图2为Bph6初步定位结果。
染色体左边为标记名称，数值是标记间的遗传距离(cM)，QTL扫描结果在分子标记RM6997和RM5742之间有一个最大的LOD值43.8，该位点解释的抗褐飞虱表型值约77.5％。
图3为Bph6精细定位结果。
其中：A：分子标记之间数值表示标记与Bph6重组单株数，Bph6与分子标记H和Y37之间18kb的区域；B：18.1kb的区域内含有2个预测的基因；图示为ORF1的基因，方框是外显子，其中黑色为CDS区，白色为UTR区，横线是内含子。
图4为转基因抗褐飞虱水稻的鉴定结果。
转基因植株放虫后14天的结果；OE为转基因超量表达植株，RNAi为Bph6被抑制的转基因植株；上面为表型，下面为抗虫级别。Bph6基因表达的转基因株系均对褐飞虱有明显抗性，Bph6基因被抑制表达的转基因株系均对褐飞虱无抗性。
图5为根据Bph6基因组序列开发的功能性分子标记H示例，其扩增片段长度为1654bp；图中Swarnalata为携带抗褐飞虱基因Bph6的抗虫亲本，扬稻6号(93-11)为感虫水稻，利用功能性分子标记H从Swarnalata与扬稻6号回交后代中筛选出的携带抗褐飞虱基因Bph6的材料(该材料抗褐飞虱)。
图6为分子标记辅助选择图培育带有Bph6基因的抗褐飞虱水稻。
苗期抗褐飞虱鉴定，虫源为褐飞虱生物型I、II、III和本实验室饲养的生物型P、S、Y；图中9311代表感虫对照品种扬稻6号(93-11)，NIL-9311代表珞扬6号(扬稻6号遗传背景携带抗褐飞虱基因Bph6)。放虫后感性受体亲本扬稻6号明显死亡，而珞扬6号(扬稻6号遗传背景携带抗褐飞虱基因Bph6)仍然存活，植株生长健康。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 Bph6基因的定位克隆及连锁分子标记开发
1.Bph6初步定位结果
抗褐飞虱亲本Swarnalata(含抗褐飞虱基因Bph6)与褐飞虱感性水稻品种扬稻6号(93-11)杂交构建了含Bph6的F2群体，93-11和Swarnalata均来自中国农业科学院作物科学研究所国家农作物种质保存中心，并用CTAB法(Murray MG&Thompson，1980Rapid isolation of high-molecular-weight plant DNA.Nucleic Acids Res 8:4321-4325)提取亲本及F2群体各单株的基因组DNA。每个F2单株通过自交收种获得相应的F2:3家系。为了鉴定F2定位群体中每个单株的抗褐飞虱表型，采用了苗期集团法考察F2:3家系各个单株的抗性表现，以F2:3家系抗性级别代表F2单株的抗褐飞虱表型。为确保亲本和F2:3群体中的每个家系生长一致，所有供试材料在播种前分别浸种催芽。每个家系(品种)各60粒种子播种于一个长58cm、宽38cm、高9cm，且盛有7cm厚营养土的面包盒中。每盒每个材料播种3个重复，其中随机播种亲本和TN1(感性对照)各3个重复。播种7天后间苗，淘汰病弱苗。待苗长到两叶一心期时，按8头/苗的比例接种2-3龄褐飞虱若虫，最后罩上尼龙纱网。当感虫品种TN1(台中本地1号)全部死亡时，参照 Huang等(Huang Z et al，2001Identification and mapping of two brown planthopper resistance genes in rice.Theor.Appl.Genet.102,929–934)的方法对每个单株进行0、1、3、5、7或9级的抗性评价(表1)，对亲本材料和群体的每个家系通过加权平均计算该家系的抗性级别，并根据抗性级别推测此单株基因型。
表1 抗感褐飞虱鉴定分级标准
Figure PCTCN2015086207-appb-000001
苗期集团法鉴定结果显示Swarnalata和9311的抗虫级别分别为2.9和8.7，这表明Swarnalata抗褐飞虱而9311感褐飞虱。140个F2:3家系对褐飞虱的抗虫级别频率分布呈连续分布，并有部分单株的抗性级别表现出超亲现象。根据对褐飞虱的抗虫级别将F2:3家系分为抗虫、抗感分离和感虫三种表型，而相应的F2单株的基因型则分别记为RR(纯合抗虫)、Rr(杂合抗虫)和rr(纯合感虫)三种。抗性单株与感性单株的株数比为108:32，卡方分析表明抗、感单株数目符合3:1的分离比(χc2＝0.24<χ20.05,1＝3.84)(图1)。可见，在抗性亲本Swarnalata中存在单个显性基因控制着F2群体中各单株的抗性分布。根据Gramene网站(http://www.gramene.org/)公布的标记按照较均匀的遗传距离在每条染色体上选择一定数目SSR分子标记。另外，基于该基因最后的定位区段，比较水稻品种9311及日本晴相应的基因组序列，利用SSR搜索工具SSRIT(http://www.gramene.org/db/markers/ssrtool)来寻找SSR基序，并根据其侧翼序列设计引物，为备选标记。其中，SSRIT设置参数为：最大基序长度为4聚体，最小重复数为5，搜索所有的SSR基序。选择所有大于15个碱基(基序长度×重复数)的SSR基序。同时根据公共数据库中水稻品种9311及日本晴相应的基因组序列，利用PowerBlast软件，寻找基因组序列中存在的插入\缺失位点，利用Primer Premier 5.0软件开发InDel标记。
SSR标记的分析参照Temnykh的方法(Temnykh S et al，2000.Mapping and genome organization of microsatellite sequences in rice.Theor Appl Genet 100：697-712)。10μl反应体系包括：10mM Tris-HCl pH8.3，50mM KCl，1.5mM MgCl2，50μM dNTPs，0.2μM引物，0.5U Taq polymerase和20ng DNA模板。扩增反应在PTC-100PCR仪上进行：94℃2min；94℃15sec，55℃30sec，72℃1.5min， 35个循环；72℃5min。扩增产物用6％的非变性PAGE胶分离，通过银染显色(Zhu et al,2004.Identification and characterization of a new blast resistance gene located on rice chromosome 1through linkage and differential analyses.Phytipathology 94:515-519)。扩增的DNA条带利用装有荧光灯的灯箱进行观察。记录结果，亲本间有多态的引物在F2群体中进行分析，获取群体基因型资料。
根据F2:3家系的抗虫级别，分别选择10个极端抗虫单株和10个极端感虫单株的DNA混合构建抗感池。同时，利用在亲本间有多态性的引物分别筛选抗感池并获得在抗感池之间有多态性的分子标记，该类多态性标记表明与抗性是连锁的。然后，根据连锁标记所在的染色体，选择该染色体上在亲本间有多态性的引物筛选F2分离群体的各个单株，PCR程序同上，获得群体基因型资料。根据连锁交换规律，利用软件JoinMap 3.0将群体基因型资料构建水稻的部分遗传图谱并获得各分子标记的遗传距离。最后，结合F2群体各个单株的分子标记基因型资料和相应的褐飞虱抗性鉴定的抗虫级别，利用MapQTL 5.0软件复合区间作图法，对目标染色体进行QTL位点扫描。结果表明在第4染色体的SSR标记RM6997和RM5742之间有一个LOD值为43.8的最大峰值存在，该位点解释的抗褐飞虱表型值约77.5％(如图2所示)。可见，在该位点处可能存在控制抗褐飞虱的主效基因。
2.Bph6的精细定位
由于RM6997和RM5742之间物理距离较大。为了寻找与Bph6连锁更紧密的标记，根据QTL的定位结果，本发明用PCR(polymerase chain reaction)和聚丙烯酰胺凝胶电泳的方法，以两侧的SSR标记RM16994和RM17008筛选了4300株BC2F2群体单株，获得两个标记间发生重组的单株41棵。
根据SSR标记RM16994和RM17008区段公共数据库中水稻品种9311及日本晴相应的基因组序列，利用SSR搜索工具SSRIT(http://www.gramene.org/db/markers/ssrtool)来寻找SSR基序，并根据其侧翼序列设计引物，为备选标记。同时利用PowerBlast软件，寻找基因组序列中存在的插入\缺失位点，利用Primer Premier 5.0软件开发InDel标记。用这些新开发的SSR标记、InDel标记对RM16994和RM17008两个标记间发生重组的41棵单株进行分子标记加密，构建饱和连锁图谱，并结合重组单株的抗虫鉴定结果，最后将Bph6定位于H和Y37之间(图3)。
3.H-Y37区段候选基因的分析
根据定位区间在日本晴和9311之间进行比对分析，选择保守序列每隔2-3Kb进行引物设计，在Bph6抗性亲本DNA上进行扩增、测序和拼接。用RiceGAAS进行基因预测和注解，同样用ClustalW进行比较分析，结果显示该区段含有两个ORF，分别命名为ORF1和ORF2(图3A)。
比较两者预测的基因，发现抗虫水稻的第1个基因编码的抗病蛋白与日本晴差异较大。目前均认为刺吸式昆虫吸食水稻与病原菌对水稻的侵染过程类似，水稻抗刺吸式昆虫的机理就有可能和抗病原菌相同。因此，将该基因确定为为Bph6。
4.RACE获得全长cDNA
以抗褐飞虱亲本Swarnalata叶鞘总RNA反转录产物为模板，根据基因预测结果设计引物，扩增出候选基因的一段cDNA序列。通过该序列设计引物，使用TaKaRa公司5’与3’Full RACE试剂盒，获得了该候选基因5’末端及3’末端序列，确定了候选基因的转录起始位点及终止位点，并拼接出该基因的全长cDNA序列。根据全长cDNA序列重新合成引物，扩增获得Bph6的全长cDNA，其核苷酸序列如序列表SEQ ID NO.2所示(图3B)。
5.抗褐飞虱基因Bph6连锁分子标记
在抗性亲本中，标记H和Y37之间的物理距离约为18kb。因此，利用连锁图谱内分子标记即是用：
H标记引物：
正向引物序列AGAATTGCTGCATGCTGTTG
反向引物序列ATTCCAGCATCGATTGCTTC
或Y37标记引物：
正向引物序列TCGACCTACGGCTTTTCTGTA
反向引物序列TAGATTCCTTCGGTTCCCATT
扩增水稻抗褐飞虱品种或者育种材料DNA，如果用引物H能够扩增出1654bp的扩增片段，或者用引物Y37能够扩增出174bp的扩增片段，均标志着水稻抗褐飞虱主效基因位点Bph6的存在。因此，利用本发明提供的上述分子标记方法来鉴定Bph6抗性基因的存在具有非常高的效率，可以预测水稻植株的褐飞虱抗性，加快抗褐飞虱水稻品种育种进程。
实施例2 Bph6基因的功能验证和应用
1.遗传转化载体的构建
(1)Bph6基因过表达载体的构建。所用载体为pCXUN(由美国Ohio State University的王国梁教授提供)，采用XcmI酶切pCXUN载体，将外源片段加A后可以直接连入。
根据RACE的结果，采用PCR的方法直接扩增ORF，经加A后连入载体。测序验证无误后，所得载体即为Bph6基因过表达载体，将其电转入农杆菌EHA105中。挑取单克隆扩大培养，进行PCR验证无误后，加等体积的50％甘油混匀，－70℃保存备用。
(2)Bph6基因RNAi载体构建
用载体为pCXUN(由美国Ohio State University的王国梁教授提供)，采用 XcmI酶切pCXUN载体，将外源片段加A后可以直接连入。根据获得的cDNA全长序列进行Blast分析，选择最保守的500bp进行扩增。利用重叠PCR的方法构建出中间为内含子两边为互补的500bp保守序列的片段。片段经切胶回收和加A后连入PCXUN载体。测序验证无误后，所得载体即为Bph6基因RNAi载体，将其电转入农杆菌EHA105中。挑取单克隆扩大培养，进行PCR验证无误后，加等体积的50％甘油混匀，－70℃保存备用。
2.遗传转化
采用农杆菌EHA105介导的遗传转化方法(Hiei等，1994，Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant Journal 6:271-282)将上述Bph6基因过表达载体导入褐飞虱感性的普通水稻品种Nipponbare。利用同样的方法将上述的Bph6RNAi载体导入普通水稻品种Nipponbare背景的近等基因系中。
3.Bph6基因转基因功能验证
Bph6基因过表达转化载体获得阳性转基因植株32株。收获种子后，用T2代Bph6转基因植株，采用苗期集团法进行了抗虫鉴定。如图3所示，对阳性转基因株系OE1、OE2进行了抗褐飞虱鉴定。鉴定结果如图3所示，接入褐飞虱7天后，感虫对照品种Nipponbare整株死亡,含有抗褐飞虱基因Bph6的近等基因系植株生长健康，无叶片受害。Bph6基因过表达转化载体获得阳性转基因植株OE1，OE2也生长健康，无叶片受害，抗虫级别为2-4级，证实Bph6基因具有抗褐飞虱的功能。因此，抗褐飞虱基因Bph6可以在水稻中应用也可以在水稻种子中应用，培育具有抗褐飞虱性能的水稻品种。
Bph6基因RNAi载体获得阳性转基因植株4株。对纯合T2代转基因植株进行了褐飞虱抗性鉴定。鉴定结果如图4所示，接入褐飞虱7天后，感虫对照品种Nipponbare整株死亡，近等基因系材料生长健康，无叶片受害，而RNAi的材料则整株死亡，证实Bph6基因具有抗褐飞虱的功能。
实施例3 分子标记的验证
1、材料和方法
1.1 材料：抗褐飞虱亲本Swarnalata(含抗褐飞虱基因Bph6)、褐飞虱感性水稻品种扬稻6号(93-11)及Swarnalata与扬稻6号杂交构建的F23家系。
分子标记引物：H和Y37，其核苷酸序列分别如SEQ ID No.4-5所示。
1.2 方法
CTAB抽提法提取水稻样品基因组DNA。分别用引物H和Y37扩增样品DNA。10μl体系。10μl反应体系包括：10×PCR缓冲液，1.0μl；10mM dNTPs，0.1μl；10μM引物，0.4μl；5U/μl Taq DNA聚合酶，0.2μl和50ng DNA模板。扩增反应在Bioer PCR仪上进行：94℃4min；94℃30s，55℃30s，72℃90s，30 个循环；72℃5min。H的扩增产物用1％的琼脂糖胶分离，电泳后可以直接分析。Y37的扩增产物用8％的非变性PAGE胶分离，电泳后用硝酸银银染法染色后读图分析。
2、结果：用上述方法，分别对水稻品种Swarnalata、扬稻6号、Swarnalata与扬稻6号杂交构建的24份F23家系进行扩增。结果表明，在Swarnalata与扬稻6号杂交构建的F23家系中，利用H和Y37分子标记引物能分别扩增出相应的1654片段和174bp片段的F23家系对褐飞虱均表现出抗虫性。而不能扩出上述特异性片段的Swarnalata与扬稻6号杂交构建的F23家系对褐飞虱均表现出感虫性(图5)。
由此说明，本发明提供的分子标记方法能够准确筛选出含有抗褐飞虱基因Bph6，从而大大提高育种效率。
实施例4 分子标记辅助选择带有Bph6基因的抗褐飞虱水稻
本例中利用上述分子标记进行分子标记辅助选择选育出携带Bph6基因的抗褐飞虱水稻珞扬6号，具体实施如下：抗褐飞虱亲本Swarnalata(含抗褐飞虱基因Bph6)与褐飞虱感性水稻品种扬稻6号(93-11)杂交F1，用扬稻6号进行回交，利用与Bph6连锁的分子标记(RM5742、RM6997)对BC1F1进行筛选，筛选出含抗褐飞虱基因Bph6的BC1F1用扬稻6号进行回交，得到BC2F1；利用与Bph6连锁的分子标记(RM5742、RM6997)对BC2F1进行筛选，筛选出含抗褐飞虱基因Bph6的BC2F1用扬稻6号进行回交，得到BC3F1；利用与Bph6连锁的分子标记(RM5742、RM6997)对BC3F1进行筛选，筛选出含抗褐飞虱基因Bph6的BC3F1用扬稻6号进行回交，得到BC4F1；，并利用分子标记进行全基因组扫描，选择只在抗褐飞虱基因Bph6位点与扬稻6号存在差异，其余位点均被替换为扬稻6号背景的BC4F1进行加代，利用与Bph6连锁的分子标记(RM5742、RM6997)筛选出抗褐飞虱基因Bph6位点纯合的扬稻6号/Swarnalata//扬稻6号BC4F2，进行加代繁殖，该材料在农艺性状表型上与扬稻6号无区别，但其含有抗褐飞虱基因Bph6,将此材料暂命名为珞扬6号。利用武汉田间褐飞虱群体进行苗期集团法鉴定、近分蘖期鉴定及成熟期鉴定确认其抗褐飞虱(见图1)。同时利用褐飞虱生物型I、II、III和本实验室饲养的生物型P、S、Y进行苗期集团法鉴定，结果显示放虫后7天，对照感性品种TN1与受体亲本扬稻6号明显死亡，而珞扬6号(扬稻6号遗传背景携带抗褐飞虱基因Bph6)仍然存活，植株生长健康(图6)。目前，珞扬6号已申报水稻品种权。
对于H和Y37任何一个分子标记，单株的带型与抗褐飞虱亲本Swarnalata一致时记为1，与扬稻6号(93-11)一致时记为2，同时具有两个亲本的带型即杂合带型记为3。分子标记筛选结果表明BC4F1只在抗褐飞虱基因Bph6位点(RM5742和RM6997区间)与扬稻6号存在差异，其余位点均被替换为扬稻6 号遗传背景。
工业实用性
本发明公开的的水稻抗褐飞虱基因Bph6能够显著提高水稻对褐飞虱的抗性，通过遗传转化或杂交将Bph6应用于水稻育种中，可以改善水稻品种的抗褐飞虱性，从而减轻褐飞虱的为害，达到增产和稳产的目的。
Figure PCTCN2015086207-appb-000002
Figure PCTCN2015086207-appb-000003
Figure PCTCN2015086207-appb-000004
Figure PCTCN2015086207-appb-000005
Figure PCTCN2015086207-appb-000006
Figure PCTCN2015086207-appb-000007
Figure PCTCN2015086207-appb-000008
Figure PCTCN2015086207-appb-000009

Claims (9)

  1. 水稻抗褐飞虱基因Bph6，其特征在于，所述基因的核苷酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的基因，其特征在于，所述基因的cDNA序列如SEQ ID NO.2所示。
  3. 权利要求1或2所述基因编码的蛋白，其特征在于，所述蛋白的氨基酸序列如SEQ ID NO.3所示。
  4. 含有权利要求1或2所述基因的载体。
  5. 权利要求1或2所述的基因在水稻选育中的应用，其特征在于，所述水稻选育为提高水稻对褐飞虱的抗性。
  6. 与水稻抗褐飞虱基因Bph6紧密连锁的分子标记，其特征在于，所述分子标记为H或Y37；
    用于扩增分子标记H的引物对如下：
    正向引物HF：AGAATTGCTGCATGCTGTTG；
    反向引物HR：ATTCCAGCATCGATTGCTTC；
    用于扩增分子标记Y37的引物对如下：
    正向引物Y37F：TCGACCTACGGCTTTTCTGTA；
    反向引物Y37R：TAGATTCCTTCGGTTCCCATT。
  7. 权利要求6所述的分子标记在选育抗褐飞虱水稻中的应用。
  8. 水稻抗褐飞虱基因Bph6的分子检测方法，其特征在于，通过权利要求6中所述引物对扩增待检水稻基因组DNA，并检测扩增产物：若利用引物HF和HR扩增出1654bp的扩增片段，或利用引物Y37F和Y37R扩增出174bp的扩增片段，则标志着水稻抗褐飞虱基因Bph6的存在。
  9. 一种筛选抗褐飞虱水稻的方法，其特征在于，通过权利要求6中所述引物对扩增待检水稻基因组DNA，并检测扩增产物：若利用引物HF和HR扩增出1654bp的扩增片段，或利用引物Y37F和Y37R扩增出174bp的扩增片段，则标志着水稻品种褐飞虱抗性的存在。
PCT/CN2015/086207 2015-04-13 2015-08-06 水稻抗褐飞虱基因Bph6及其紧密连锁的分子标记 WO2016165243A1 (zh)

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