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Seed specific interference vector containing pOsGluB-4 promoter and application of seed specific interference vector

A posglub-4, posglub-4-rnai technology, applied in the application, the use of vectors to introduce foreign genetic material, angiosperms/flowering plants, etc., can solve the problem of unclear direct regulation, decreased plant fertility and yield, and no known OsMADS1 gene rice protein content application research report and other issues, to achieve the effect of improving rice quality and increasing rice yield

Active Publication Date: 2021-05-11
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at the same time, the fertility and yield of interfering plants are extremely significantly reduced, making it difficult for practical application (Liao PF, Ouyang JX, Zhang JJ, et al. OsDCL3b affects grain yield and quality inferior[J]. Plant Molecular Biology, 2019, 99( 3): 193-204.)
[0007] At the same time, although the OsMADS1 gene has an important regulatory function on rice flower development, its direct regulation on rice seed development and rice quality development is still unclear, and there are no research reports on the application of OsMADS1 gene in improving rice protein content. and invention patents

Method used

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  • Seed specific interference vector containing pOsGluB-4 promoter and application of seed specific interference vector
  • Seed specific interference vector containing pOsGluB-4 promoter and application of seed specific interference vector
  • Seed specific interference vector containing pOsGluB-4 promoter and application of seed specific interference vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Construction of pOsGluB-4-RNAi vector

[0073] Construct the pOsGluB-4-RNAi vector, specifically using the homologous recombination directional cloning method to forward clone the promoter of the rice seed-specific expression gene OsGluB-4 into a plant constitutive RNA interference binary vector with a hygromycin selection marker pLHRNAi, and replace the original Ubiquitin constitutive expression promoter pUbi in the pLHRNAi vector, so as to obtain the pOsGluB-4-RNAi seed-specific interference vector. Wherein, the pOsGluB-4 is the promoter of the OsGluB-4 gene. The construction flowchart and vector map of the pOsGluB-4-RNAi vector are as follows: figure 1 As shown, the specific construction method is as follows:

[0074] 1. OsGluB-4 gene expression profile analysis

[0075]1. OsGluB-4 gene expression profile analysis based on gene chip database

[0076] Expression profile analysis of OsGluB-4 gene in Nipponbare (Nipponbare, rice variety, Japonica subspeci...

Embodiment 2

[0141] Example 2: Construction of pOsGluB-4::OsMADS1-RNAi vector

[0142]Schematic diagram of the structure of pOsGluB-4::OsMADS1-RNAi vector and the main construction process as follows Figure 7 shown. The specific operation method is as follows:

[0143] 1. OsMADS1 gene expression profile analysis

[0144] 1. Transcriptional profiling analysis of OsMADS1 gene based on gene chip database

[0145] First, the expression profile of the OsMADS1 (gene number: Os03g0215400) gene in Nipponbare was analyzed using the RiceXPro rice gene chip expression database, as shown in Figure 8 As shown (according to the picture, you need to enter the URL http: / / ricexpro.dna.affrc.go.jp / category-select.php, enter Os03g0215400 in the Search options on the web page, then click search, and then click FeatureNum in the new web page After downloading the serial number, check the Spatio-temporal profile on the new webpage, you can find this graph, and click Downloadselected graph on the webpage t...

Embodiment 3

[0236] Example 3: Specific application and effect demonstration of pOsGluB-4::OsMADS1-RNAi seed-specific interference vector

[0237] 1. The pOsGluB-4::OsMADS1-RNAi and pUbi::OsMADS1-RNAi plasmids constructed in Example 2 and Comparative Example were sent to Hangzhou Baige Biotechnology Co., Ltd. to transform the rice variety Nipponbare respectively. The conversion process is as follows:

[0238] 1. Callus induction and subculture of mature rice seeds

[0239] Select mature rice seeds, peel off the glumes, pour them into a 50ml centrifuge tube, add 75% ethanol to sterilize for 1 minute, pour off the ethanol, rinse with sterile water once, pour it out, then add 30% sodium hypochlorite to disinfect for 20 minutes, pour out the sodium hypochlorite, and use Rinse with sterile water 5-6 times. Use a pipette to absorb excess water, and transfer the seeds to the induction medium, 20-25 seeds per dish. After the callus grows, the original embryo can be directly used for transformat...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a seed specific interference vector containing a pOsGluB-4 promoter and application of the seed specific interference vector. The seed specific interference vector is a pOsGluB-4-RNAi vector, and the DNA sequence of the pOsGluB-4-RNAi vector is shown as SEQ ID NO 9. By utilizing the pOsGluB-4-RNAi vector, the expression of a target gene in rice seeds can be specifically interfered without influencing the normal expression of the target gene in other tissues and organs so that the direct regulation effect of the target gene on the seed development can be effectively researched, the purposes of improving rice grain shape, yield and rice quality can be achieved, and an effective technical means and solution is provided for molecular precise design and breeding of rice yield and rice quality. Meanwhile, the invention also provides an OsMADS1 seed specific interference vector pOsGluB-4::OsMADS1-RNAi based on the pOsGluB-4-RNAi vector, and the DNA sequence of the OsMADS1 seed specific interference vector pOsGluB-4::OsMADS1-RNAi is as shown in SEQ ID NO 10. By utilizing the OsMADS1 seed specific interference vector pOsGluB-4::OsMADS1-RNAi, the protein content in rice can be increased, and the nutritional value of the rice is increased. According to the seed specific interference vector containing the pOsGluB-4 promoter and the application of the seed specific interference vector, the important scientific research significance and application value are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a seed-specific interference vector containing a pOsGluB-4 promoter and an application thereof. Background technique [0002] Rice is the main ration of more than half of the world's population, and it is also the basic source of human starch and protein. The growth and development of rice seeds not only controls the thousand-grain weight, which is one of the three elements of rice yield, but also is an important factor that directly determines the nutritional quality of rice such as rice protein content. The morphological development of rice seeds and rice protein content are regulated by a series of important genes. Therefore, studying the functions of these important regulatory genes and their regulatory mechanisms on seed morphological development and rice protein content is the basis for using these important genes to carry out precise seed design breeding and genet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/10A01H6/46
CPCC12N15/8218C12N15/8234C12N15/8251C12N15/8261C07K14/415
Inventor 李辉李鹏慧黄维藻涂升斌汪松虎蒋海波庄勇刘阳轩魏明刘志坚古袁扬唐钰莹乐露胡永志路龙祥喻兰
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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