OsPEAMT2 gene for increasing heading stage maturing rate of rice under high-temperature stress as well as protein and application of OsPEAMT2 gene

A technology of high temperature stress and heading time, applied in the field of genetic engineering, can solve the problems of high temperature research difficulties and lagging rice research

Active Publication Date: 2020-12-08
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty of high temperature research and environmental limitations, the research on high temperature in rice is still relatively lagging behind.
Although a large number of high-temperature-related genes have been reported in rice, there is still a big gap in how plants perceive high-temperature and conduct downstream signal transduction. Therefore, studying the functions of high-temperature-related genes, revealing the molecular mechanism, and mining and utilizing some excellent genes are of great help in ensuring high temperature. High and stable rice yield under stress has important practical significance

Method used

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  • OsPEAMT2 gene for increasing heading stage maturing rate of rice under high-temperature stress as well as protein and application of OsPEAMT2 gene
  • OsPEAMT2 gene for increasing heading stage maturing rate of rice under high-temperature stress as well as protein and application of OsPEAMT2 gene
  • OsPEAMT2 gene for increasing heading stage maturing rate of rice under high-temperature stress as well as protein and application of OsPEAMT2 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Homologous Alignment Analysis of PEAMT Family Protein Sequences in Rice

[0027] 1. Homologous gene sequence acquisition

[0028] The protein sequence of rice is from the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu). The codes of the obtained protein sequences are as follows: OsPEAMT1 (LOC_Os01g50030), OsPEAMT2 (LOC_Os05g47540).

[0029] 2. Creation of evolutionary tree

[0030] Using DNAMAN7, the protein sequences of OsPEAMT1 and OsPEAMT2 were loaded into different lanes, and then the protein homology was analyzed by double-sequence alignment. The sequence analysis results are as figure 1 As shown, there are two PEAMT genes in rice, which are named OsPEAMT1 and OsPEAMT2 respectively. The data show that the homology of the two genes is as high as 86.46%. This application conducts research on OsPEAMT2, and explores the biological function of this type of gene in rice by designing a knockout experiment.

[0031] 3. Expression profiling ...

Embodiment 2

[0036] Example 2 OsPEAMT2 gene editing vector construction

[0037] 1. Gene editing site design

[0038] In this study, according to the existing CRISPR / Cas9-related experimental methods, ACTAATGTACCTTTCAGATGAGG, which contains AGG as the recognition site, was selected on the fifth exon of OsPEAMT2 as the knockout target site of OsPEAMT2 ( image 3 , Figure 4 ), it is expected to obtain a loss-of-function transgenic plant to clarify the biological function of the OsPEAMT2 gene.

[0039] 2. CRISPR / Cas9 vector construction

[0040] Take 5 μL of 10 μM primers before and after the target site, mix them, anneal at 99 °C for 5 min, and cool naturally to obtain a double-stranded sequence containing the knockout target site, then use Bas1 (NEB company) to digest and ligate (37 °C), and target site The dot sequence was connected to the intermediate vector pYLsgRNA-OsU6a, and then the universal amplification primers CTCCGTTTTACCTGTGGAATCG (UF) and CGGAGGAAAATTCCATCCAC (gR-R) were us...

Embodiment 3

[0047] Example 3 OsPEAMT2 gene editing under the background of Nihonbare

[0048] 1. Detection of transgenic seedlings

[0049] A total of 10 seedlings were obtained by Agrobacterium infection and transformation. First, the G418 detection primer was used to detect the transformation of the vector, and then the sequence in the positive individual plants was amplified with the amplification primers U1028 (TTCTGTAGAATGAGAACAT) + U1029 (ACTAGCTTCTCGACCTACAGTA) across the target site. Sent to Qingke Sequencing Company for sequencing, the results are as follows Figure 4 As shown, the comparison analysis of the obtained sequences showed that the transgenic plant ospeamt2, which was prematurely terminated in protein translation, was co-acquired.

[0050] 2. Phenotype Analysis

[0051] In the T1 generation planting, different lines were divided into 3 repeated plantings in field experiments, and 2 rows were planted for each repeated planting. First, the presence of stable mutations ...

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Abstract

The invention discloses an OsPEAMT2 gene for increasing the heading stage maturing rate of rice under high-temperature stress as well as a protein and application of the OsPEAMT2 gene. The nucleotidesequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The invention provides a new rice ear stage high temperature related gene, and provides a new gene resource for improvement of rice yield traits and breeding utilization of a rice thermo-sensitive sterile line.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an OsPEAMT2 gene and its protein for improving the seed-setting rate of rice at the heading stage under high temperature stress and its application. Background technique [0002] In recent years, the trend of global warming has intensified and extreme high temperatures have occurred frequently, posing serious challenges to world food security (Pittelkow C M et al., 2015). As a high-temperature and short-day crop, rice is more vulnerable to high temperature stress during its growth. However, due to the difficulty of high temperature research and environmental limitations, research on high temperature in rice is still relatively lagging behind. Although a large number of high-temperature-related genes have been reported in rice, there is still a big gap in how plants perceive high-temperature and conduct downstream signal transduction. Therefore, studying t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00A01H6/46C12Q1/6895C12Q1/6837C12N15/11C40B40/06
CPCC12N9/1007C12Y201/01103C12N15/8271C12Q1/6895C12Q1/6837C40B40/06C12Q2600/13C12Q2600/158
Inventor 陈薇兰黄开来樊世军刘艺胥磊袁华涂斌马炳田王玉平钦鹏李仕贵
Owner SICHUAN AGRI UNIV
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