Paddy hpfr gene expression, product and application thereof
A technology for expressing products and genes, which is applied in the field of agricultural biology and can solve problems such as unreported applied research
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Embodiment 1
[0046] 1. Discovery and cloning of rice hpfr gene based on Xanthomonas oryzae bacterial blight pathogenic variety hrfA Xoo Gene open reading frame design rice hpfr gene primers:
[0047] 5'-GGAATTCCATATGAACTCTTTGAACACACAATTGG-3' and
[0048] 5'-CGGGATCCTATTACTGCATTGATGCGCTTCC-3', the hpfr gene derived from rice was amplified from rice by PCR method. PCR amplification conditions: 1 cycle of 94°C / 5min, 35 cycles of 94°C / 45sec+60°C / 45sec+72°C / 45sec, 1 cycle of 72°C / 7min. The gene fragment was named hpfr gene (Fig. 1). The rice hpfr gene was used as a probe to hybridize with the total DNA of the rice genome. The hybridization result showed that there was a positive hybridization signal in the rice genome ( FIG. 2 ).
[0049] 2. In vitro expression of rice hpfr gene
[0050] After purification, the PCR product was digested with NdeI and BamHI, and connected to the expression vector pET30a(+) to obtain the recombinant plasmid pETR[pET30a(+)::hpfr] (Figure 5). Transform pETR int...
Embodiment 2
[0053] The direct use of embodiment 2 rice hpfr gene expression product hpfr protein
[0054] The rice hpfr gene expression product hpfr protein is sprayed on the leaves of the plants at the concentration of 15, 30, and 60 μg / ml.
[0055] 1. Inducing the resistance phenotype of Xanthi tobacco to TMV
[0056] Combine rice hpfr and harpin Xoo The protein CFEP mother liquor was diluted to concentrations of 15, 30, and 60 μg / ml, respectively, and sprayed with a micro-sprayer on Shanxi tobacco at the adult plant stage, and each treatment was repeated 5 times; 16 hours later, the activated TMV was inoculated on the tobacco leaves by friction; 4 Days later, observe the results and count the number of lesions. The results of the study showed that rice hpfr and harpin Xoo All treatments of tobacco can obviously induce the ability of tobacco to resist TMV (decrease in the number of scabs) ( FIG. 8 ). Multiple comparison analysis by LSD method (F 189.464 ) showed that BL21(DE3) / pETR...
Embodiment 3
[0063] Cloning and expression of hpfw gene homologous to rice hpfr gene in wheat in embodiment 3
[0064] 1. Design primers for wheat hpfw gene cloning and nucleotide sequence:
[0065] 5'-GGAATTCCATATGAACTCTTTGAACACACAATTGG-3' and 5'-CGGGATCCTATTACTGCATTGATGCGCTTCC-3' were used to amplify the homologous gene of rice hpfr gene from wheat and named as hpfw gene by PCR method (the same method as in Example 1). The nucleotide sequence of the hpfw gene is SEQ ID NO.3 (Fig. 11), the size is 414bp, and the homology with the rice hpfr gene is 93%. The deduced amino acid sequence of hpfw gene is SEQ ID NO.4 (Fig. 12).
[0066] 2. In vitro expression of wheat hpfw gene and small quantity production hpfw protein (method is the same as embodiment 1)
[0067] The wheat hpfw gene was connected to the expression vector pET30a(+) to obtain the recombinant expression vector pETW9[pET30a(+)::hpfw], and the product expressed by Escherichia coli BL21(DE3) was named hpfw protein. After 12% SDS...
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