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Rice XIOsPR10 gene and use thereof

A rice and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem that RNase activity has not been confirmed

Inactive Publication Date: 2010-07-21
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although other rice PR10 proteins have been confirmed to have transcriptional activity under pathogen infection or other abiotic stress (drought, salt stress, etc.), whether they also have RNase activity has not been confirmed.
Similarly, there is no report on the application of rice XIOsPR10 gene to improve the resistance of transgenic plants to bacterial stripe disease.

Method used

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  • Rice XIOsPR10 gene and use thereof
  • Rice XIOsPR10 gene and use thereof
  • Rice XIOsPR10 gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Prokaryotic expression of rice XIOsPR10 gene and purification of expression product

[0075] 1. Prokaryotic expression of rice XIOsPR10 gene

[0076] Rice XIOsPR10 gene primers were designed according to the open reading frame of rice XIOsPR10 gene, the underlined restriction enzyme sites:

[0077] OsPR10-E-F: 5′-CGC GAA TTC ATG GCT CCG GTC AGC ATC-3′

[0078] EcoR I

[0079] OsPR10-N-R: 5′-GGC GCG GCC GC A ATT TTT AAG CAT ACT CG-3′

[0080] Not I

[0081] The XIOsPR10 gene was amplified from rice by RT-PCR method. Reaction conditions: 42°C / 30min; 94°C / 7min, 30 cycles of 94°C / 30sec+55°C / 45sec+72°C / 45sec, 72°C / 7min.

[0082] After purification, the PCR product was digested with EcoR I and Not I, and connected to the expression vector pET28a(+), to obtain the recombinant expression plasmid pET28a-XIOsPR10( figure 1 ). The pET28a-XIOssPR10 was transformed into Escherichia coli BL21(DE3), and a transformed single clone was obtained...

Embodiment 2XI

[0086] The RNase activity identification of embodiment 2XIOsPR10 protein

[0087] Take 10 μg of yeast total RNA, add 8 μg of purified XIOsPR10 protein, bathe in water at 37°C for 0-4 hours, take samples at 0 hours, 2 hours, and 4 hours, and extract the reaction product with an equal volume of phenol-chloroform to remove the protein in the sample and denature it with formaldehyde Gel electrophoresis analysis and detection, the results showed ( Figure 4 ), XIOsPR10 protein has RNase activity.

Embodiment 3

[0088] Example 3 Construction of Rice XIOsPR10 Gene Overexpression Vector

[0089] The pCMVFL3-XIOsPR10 plasmid and pBPFΩ7 were digested with EcoR I and Xba I to obtain XIOsPR10 cDNA and pBPFΩ7 enzyme fragments, and the target fragments were recovered respectively, and ligation was carried out to construct a rice XIOsPR10 gene overexpression chimeric gene--pBPF-XIOsPR10; the ligation product DH5α competent cells were transformed, and the LB plates coated with Ampr were cultured overnight. Single clones were picked for PCR identification. Plasmid DNA of positive strains was extracted and identified by double digestion with EcoR I and Xba I.

[0090] The pBPF-XIOsPR10 and pCAMBIA 1301 vectors were respectively digested with Hind III, and the target fragments digested by pCAMBIA 1301 were dephosphorylated by CIAP, and then ligated with the recovered fragments of pBPF-XIOsPR10 to construct the plant overexpression vector p1301-35S-XIOsPR10-Tnos (abbreviated as p1301-XIOsPR10); a...

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Abstract

The invention discloses a rice XIOsPR10 gene and use thereof, which relate to the field of plant genetic engineering. The invention also provides a rice XIOsPR10 gene sequence, a gene encoding protein sequence, use of the gene in RNA enzyme digestion degradation, a method for constructing an over expression vector of the gene, a method for rice genetic transformation and the use of the gene in over expression in transgenic rice. After-infection expression up-regulating protein PR10 is obtained by analyzing rice leave expressing differential proteins before and after the infection with xanthomonas oryzicola fangetal bacteria. The product of the prokaryotic expression of the rice XIOsPR10 gene has RNase activity and can be used in RNA enzyme digestion degradation; by the genetic transformation of rice, transgenic rice plants with XIOsPR10 gene over expression are obtained and the xanthomonas oryzicola fangetal resistance of rice is improved, so the rice XIOsPR10 gene can be used in the prevention and control of xanthomonas oryzicola fangetal.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a rice XIOsPR10 gene and its application, including the application of prokaryotic expression product and the application of plant transgene. Background technique [0002] PR10 (process-related protein 10) proteins are acidic proteins with a molecular weight of 16-19 kDa in plants and located in the cytoplasm. PR10 family proteins have a conserved region P-loop, namely GxGGxG. At present, it is generally believed that the RNase activity of PR10 protein works through the nucleic acid binding site of this conserved region. [0003] PR10 protein is regulated by different factors in different plant tissues and organs. During plant growth and development, plant flowers and vegetative organs usually have constitutive expression of PR10 protein. At the same time, most plants often induce high expression of PR10 gene in response to pathogen infection and trauma. In addition, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/22C12N15/82C12P19/30A01H4/00A01H5/00
Inventor 陈亮郭迟鸣毛倩王丽娟李东霄郭立佳
Owner XIAMEN UNIV
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