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Application of rice alpha-isopropyl malate synthase gene

A technology of malate synthase and isopropyl, which is applied in the fields of application, genetic engineering, acyltransferase, etc., and can solve the problem of few varieties or resource applications

Inactive Publication Date: 2018-10-30
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of rice seed vigor genes to identify rice varieties or resources with high vigor is rarely used

Method used

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  • Application of rice alpha-isopropyl malate synthase gene
  • Application of rice alpha-isopropyl malate synthase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Gene Cloning

[0035] The sequence of the OsIPMS1 gene was cloned by PCR using the japonica rice variety Nipponbare cDNA as a template. The sequence of the upstream primer is shown in SEQ ID NO.3 in the sequence table, and the sequence of the downstream primer is shown in SEQ ID NO.4 in the sequence table. The nucleotide sequence and amino acid sequence of the rice OsIPMS1 gene are obtained, the nucleotide sequence is shown in SEQ ID NO.1 in the sequence table, and the amino acid sequence is shown in SEQ ID NO.2.

Embodiment 2

[0036] Example 2: Mutant construction

[0037] Analyze the target gene, find NGG (N is A, T, C or G) in the CDs region sequence of the gene, preferably AGG, take the 20bp in front of NGG as the target sequence, clone the target fragment, and the upstream primer sequence As shown in the sequence listing SEQ ID NO.5, the downstream primer sequence is shown in the sequence listing SEQ ID NO.6. T4 ligase was used to connect the 20 bp target fragment in the above OsIPMS1 gene to the CH vector to obtain the CH-Target recombination vector. The pCAMBIA1300 and CH-target vectors were digested with EcoRI HF and Hind III respectively, and the digested products were detected by 1.2% agarose gel, and the vector pCAMBIA1300 and fragment CH-target were recovered by cutting the gel respectively; p1300-CH-Target recombination was obtained by using T4Ligase carrier.

[0038] The constructed rice OsIPMS1CRISPR / Cas9 mutant gRNA target sequence is shown in the sequence table as SEQ ID NO.7; the ...

Embodiment 3

[0039] Example 3: Phenotype Analysis of Gene Mutants

[0040]Seed germination experiments were carried out using the constructed OsIPMS1CRISPR / Cas9 mutant osipms1a, osipms1b seeds, and wild-type Nipponbare (WT) rice varieties. The specific method is as follows: each time repeatedly select 50 healthy and plump seeds, sterilize the surface with 0.1% mercuric chloride solution for 5 minutes, rinse with distilled water for 3 times, dry the surface of the seeds, and place them on a petri dish with two layers of filter paper (diameter 9 cm), add 10 mL of distilled water, place at 30°C under light / dark conditions for 12 hours and culture for 5 days, and finally count the seedling rate. The experiment was repeated 3 times. The results showed that compared with the control seeds, the germination speed of the mutant seeds was slow and uneven, and the seedling growth was significantly weaker ( figure 1 ). It can be seen that the gene plays an important role in improving the germinatio...

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Abstract

The invention discloses application of a rice alpha-isopropyl malate synthase gene. The nucleotide sequence of gene OsIPMS1 is shown as SEQ ID NO.1 and the encoded corresponding protein amino acid sequence is shown as SEQ ID NO.2 in a sequence table. According to the application disclosed by the invention, the OsIPMS1 gene can regulate the vitality of rice seeds, which is firstly reported in the rice field; the experiment shows that the mutation of the gene affects the germination speed and seedling growth of seeds under normal conditions. The experiment proves that the OsIPMS1 gene disclosedby the invention regulates the vitality of the rice seeds; the screening and the cultivation of rice varieties with high seed vigor as well as the production of direct-seeding rice are facilitated byutilizing the gene.

Description

technical field [0001] The invention belongs to the field of seed biotechnology and relates to the application of rice α-isopropylmalate synthase gene. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in the world. In recent years, with the development of the economy and the shortage of rural labor force, direct-seeding rice production has become more and more common, and the cultivation of high-vigorance rice varieties is of great significance. In rice direct seeding production, unfavorable conditions such as low temperature, flood, and drought are often encountered during sowing, and low-vigorous seeds often show slow germination speed, low emergence rate, and poor resistance in the field, which eventually cause field seedlings to rot, necrosis and death. Phenomena such as overgrown weeds are unfavorable to direct-seeding rice production. Breeding rice varieties with high vigor can not only improve the germination speed and uni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/54C12N15/82A01H5/00A01H6/46
CPCC12N9/1025C12N15/8213C12Q1/6895C12Y203/03013
Inventor 张红生王州飞程金平何永奇唐海娟
Owner NANJING AGRICULTURAL UNIVERSITY
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