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Application of protein phosphatase OsPP74 in improvement of phosphorus absorption of rice

A technology for protein phosphatase and phosphorus absorption, applied in the fields of application, enzyme, hydrolase, etc., can solve the problems of molecular mechanism to be studied, achieve great application potential, improve phosphorus absorption, and enrich phosphorus absorption

Active Publication Date: 2021-11-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gradually we began to realize the importance of phosphorylation modification in regulating phosphorus homeostasis, but the molecular mechanism of phosphorylation regulation remains to be studied

Method used

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  • Application of protein phosphatase OsPP74 in improvement of phosphorus absorption of rice
  • Application of protein phosphatase OsPP74 in improvement of phosphorus absorption of rice
  • Application of protein phosphatase OsPP74 in improvement of phosphorus absorption of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1, Os PP74 prokaryotic expression and purification

[0024] 1. Construction of GST-OsPP74 vector

[0025] To clarify that OsPP74 has protein phosphatase activity, we expressed and purified it in prokaryotic. Using the wild-type rice Nipponbare (Oryza sativa Japonica Group (taxid: 39947)) cDNA as a template, using OsPP74 full-length amplification primers, the full-length coding sequence of OsPP74 (SEQ ID NO .1, the amino acid sequence is shown in SEQ ID NO.2), and the target fragment size is 1452bp. directly use Gel and PCR Clean-up kit (purchased from MACHEREY-NAGEL) to recover and use Clone ⅡOne Step Cloning Kit (purchased from Novizym) kit was connected to the PGEX-4T-1 vector digested by EcoRI and BamHI (purchased from Thermo Fisher Scientific) (that is, GST, purchased from GE Healthcare, the map is shown in Figure 10 A ), transform Escherichia coli DH5α (purchased from TAKARA), extract positive clones, and after the sequencing is correct, use a plasmid ex...

Embodiment 2

[0057] Example 2, Construction of OsPP74-GUS Fusion Gene Vector and GUS Staining

[0058] 1. PBI101.3-OsPP74 PRO -GUS plus vector construction: search from Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / index.shtml) LOC_Os05g11550 Gene number, obtain the genome information of the OsPP74 gene, select the promoter of the first 2500 bp of the open reading frame of the OsPP74 gene, design the following primers, and use the wild-type rice Nipponbare genome (Oryza sativa Japonica Group (taxid: 39947)) as a template, according to Example 1 The OsPP74 promoter (nucleotide sequence is shown in SEQ ID NO.3) was amplified by PCR using the method, and was directly recovered by the Gel and PCR Clean-up kit (purchased from MACHEREY-NAGEL) and then recovered by Clone ⅡOneStep Cloning Kit (purchased from Novizym) was connected to the PBI101.3-GUS plus vector (Yue et al., 2010) digested with HindIII and SalI (purchased from Thermo Fisher Scientific), and transformed into E...

Embodiment 3

[0073] Example 3, the subcellular localization of OsPP74

[0074] 1. Construction of pCAMBIA1300-35s-OsPP74-GFP expression vector

[0075] Using primers pCAMBIA1300-35s-GFP F and pCAMBIA1300-35s-GFPR, according to the method of Example 1 step 1, the amplified PP74 target fragment (shown in SEQ ID NO.1) was subjected to SalI single enzyme digestion, and used Clone Ⅱ One Step Cloning Kit (purchased from Novizym) kit was connected to the same pCAMBIA1300-35s-GFP vector (Yue et al., 2010), transformed into Escherichia coli DH5α (purchased from TAKARA), after the sequencing was correct, use the plasmid Extract the plasmid with an extraction kit (purchased from MACHEREY-NAGEL) to obtain the pCAMBIA1300-35s-OsPP74-GFP vector, which is 35s-OsPP74-GFP.

[0076] The pCAMBIA1300-35s-OsPP74-GFP was transformed into Agrobacterium EHA105 (purchased from Takara), and integrated into the wild-type rice Nipponbare genome by Agrobacterium infection (Wang et al., 2014), and the T2 generation of ...

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Abstract

The invention discloses an application of protein phosphatase OsPP74 in improvement of phosphorus absorption of rice. The amino acid sequence of the protein phosphatase OsPP74 is shown as SEQ ID NO:2. The protein phosphatase OsPP74 is found to participate in rice phosphorus absorption regulation and control for the first time, the expression of the protein phosphatase OsPP74 gene is increased in a plant body, and the phosphorus absorption and utilization efficiency of rice is improved. The research on phosphorus absorption and transport regulation and control mechanisms is enriched, a reference mechanism is provided for corresponding phosphorus deficiency stress of rice, and great application potential in molecular breeding is achieved.

Description

(1) Technical field [0001] The invention relates to a rice OsPP74 (protein phosphatase 74) gene cloned by a reverse genetics approach, and the function of the gene is identified through overexpression technology and gene editing technology; efficiency. (2) Background technology [0002] Phosphorus is an indispensable macronutrient element in the process of plant growth and development (Raghothama, 1999). It is an important component of many metabolites and macromolecules such as ATP, phospholipids, and nucleic acids in plants, and participates in gene expression, signal transduction, etc. lead to a large number of biochemical pathways (Liu et al., 2015). Phosphorus is mainly absorbed and utilized by plants in the form of orthophosphate (Pi, inorganic phosphate). Due to its chemical properties, phosphorus is easily fixed by cations or organic compounds in the soil (Hirsch et al., 2006), and thus has low mobility and low availability in soil. It is for these reasons that th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/84A01H5/00A01H5/04A01H5/06A01H6/46
CPCC12N9/16C12N15/8243C12N15/8218C12Y301/03
Inventor 毛传澡邓美菊王飞徐纪明
Owner ZHEJIANG UNIV
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