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Rice OsPPR34 gene as well as encoding protein and application thereof

A technology of gene coding, rice, applied in the field of genetic engineering

Active Publication Date: 2021-05-14
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are currently more than ten PPR genes that can affect plant height, but there is no relevant report on whether they are related to the gibberellin pathway

Method used

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  • Rice OsPPR34 gene as well as encoding protein and application thereof
  • Rice OsPPR34 gene as well as encoding protein and application thereof
  • Rice OsPPR34 gene as well as encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of embodiment 1 rice OsPPR34 gene

[0045] 1. Seedling cultivation

[0046] The rice variety Nipponbare was germinated at 30°C for 48 hours, and then sowed in the greenhouse. When the rice leaves were 3-5 pieces, the DNA or RNA was ready to be extracted.

[0047] 2. RNA isolation:

[0048] RNA extraction: freeze the sample in a mortar and grind it into a powder, add 1mL TRNzol-A+ reagent to a 2mL EP tube, shake fully, place at room temperature for 5min, then add 0.2mL chloroform, shake vigorously After 15 s, place at room temperature for 3 min; centrifuge at 12,000 rpm for 10 min at 4°C, transfer the supernatant to a new 2 mL EP tube, add an equal volume of isopropanol to precipitate RNA, and add 100 μL RNase-free ddH2O to dissolve. The quality of total RNA was identified by electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0049] 3. Synthesis of first-strand cDNA by reverse transcription

[0050] (1) Before reverse transcrip...

Embodiment 2

[0061] Example 2 Protein sequence information and homology analysis of rice OsPPR34

[0062] According to the ORF of the new rice OsPPR34 (gene number: Os07g0598500 / LOC_Os07g40750) of the present invention, the amino acid sequence of rice OsPPR34 is deduced, with a total of 506 amino acids and a molecular weight of 56520 Daltons. See SEQ ID NO: 4 for the detailed sequence. According to the BLASTP program on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the OsPPR34 protein has a typical P domain and belongs to the P subfamily of the PPR protein family.

[0063] Through multiple sequence alignment of some PPR-encoded proteins in plants, we found that these proteins all contain a conserved PPR domain ( figure 1 ).

Embodiment 3

[0064] Example 3 Creation of Rice Gene OsPPR34 Knockout Mutant

[0065] 1. Using CRISPR-Cas9 technology to construct a multi-target knockout vector containing OsPPR34:

[0066] (1) Guide RNA target sequence selection and primer design

[0067] According to the genome sequence of OsPPR34 (gene number: Os07g0598500 / LOC_Os07g40750), two sgRNAs were designed. 20nt nucleotide sgRNA target sequence according to 5'-GN 19 NGG-3' (SEQ ID NO:5) sequence was designed, and the target sequence primers PPR34-gRT+ and PPR34-OsU6aT- were designed at the same time, with 15-17 nt at the 3' end paired with the sgRNA and U6a promoters respectively. The designed sgRNA target sequence was compared with the rice genome database to exclude non-specific target cutting sites. The specific target nucleotide sequence is as follows.

[0068] PPR34-1-gRT+:5'-AAGGATCACGCTGTTCACCgttttagagctagaaat-3', SEQ ID NO:6;

[0069] PPR34-1-OsU6aT-:5'-GGTGAACAGCGTGATCCTTCggcagccaagccagca-3', SEQ ID NO:7;

[0070] ...

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Abstract

The invention provides an OsPPR34 gene which is separated and cloned from rice DNA and is related to a rice plant type and drought resistance. The protein coded by the gene contains a conservative P structural domain of a PPR (pentatricopeptide repeat) family, and participates in RNA editing of a mitochondrial gene. The OsPPR34 gene regulates and controls the plant height of the rice through a gibberellin way, meanwhile, the root tropism is changed, the deep root ratio of a knockout mutant of the OsPPR34 gene is remarkably increased, and the OsPPR34 gene shows high drought resistance in a root canal experiment. The OsPPR34 gene regulates and controls the plant height and the drought resistance of the rice, the plant height of a knockout mutant of the OsPPR34 gene is reduced, and the OsPPR34 gene has a high depth-to-root ratio and can be applied to improvement of the plant type of the cultivated rice and improvement of the drought resistance of the cultivated rice.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a gene related to rice production and stress resistance, in particular to a new gene OsPPR34 related to rice plant height and drought avoidance and its application. Background technique [0002] Rice is the most important food crop in my country and even in the world, providing food for more than 50% of the world's population, and it is also the most important ration in my country. Therefore, how to improve the yield and stress resistance of rice under the situation of global environmental change is an effective means to ensure my country's food security. Plant height is one of the important agronomic traits of rice. In the domestication history of cultivated rice, it is very important to reduce the plant height of rice, reduce the damage of rice lodging, and improve the harvest index. The application of rice semi-dwarf genetic resources during the "Green Revolution" in the 1960...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/83C12N15/84A01H5/00A01H6/46
CPCC07K14/415C12N15/8261C12N15/8273C12N15/8216
Inventor 夏辉熊杰侯桂花罗志李天菲罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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