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A seed-specific interference vector comprising posole18 promoter and its application

An interference carrier and specific technology, applied in the biological field, can solve problems such as the decline of plant fertility and yield, unclear direct regulation, and no application research report on the protein content of OsMADS1 gene in rice, so as to increase rice yield and improve rice. quality effect

Active Publication Date: 2022-07-12
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A seed-specific interference vector comprising posole18 promoter and its application
  • A seed-specific interference vector comprising posole18 promoter and its application
  • A seed-specific interference vector comprising posole18 promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Construction of pOsOle18-RNAi vector

[0076]The pOsOle18-RNAi vector was constructed. Specifically, the promoter of the rice seed-specific expression gene OsOle18 was cloned into the plant constitutive RNA interference binary vector pLHRNAi with the hygromycin selection marker by using the homologous recombination directional cloning method, and The original Ubiquitin constitutive expression promoter pUbi in the pLHRNAi vector was replaced to obtain the pOsOle18-RNAi seed-specific interference vector. Wherein, the pOsOle18 is the promoter of the OsOle18 gene. The construction flow chart and vector map of the pOsOle18-RNAi vector are as follows: figure 1 As shown, the specific construction method is as follows:

[0077] 1. OsOle18 gene expression profile analysis

[0078] 1. OsOle18 gene expression profile analysis based on gene chip database

[0079] Expression profiling of OsOle18 gene in Nipponbare (Nipponbare, rice variety, subsp. japonica). First us...

Embodiment 2

[0144] Example 2: Construction of pOsOle18::OsMADS1-RNAi vector

[0145] The schematic diagram of the structure of pOsOle18::OsMADS1-RNAi vector and the main construction process are as follows Figure 7 shown. The specific operation method is as follows:

[0146] 1. OsMADS1 gene expression profile analysis

[0147] 1. Transcriptional profiling of OsMADS1 gene based on gene chip database

[0148] Firstly, the expression profile of OsMADS1 (gene number: Os03g0215400) gene in Nipponbare was analyzed using RiceXPro rice gene chip expression database. Figure 8 As shown (you need to enter the URL http: / / ricexpro.dna.affrc.go.jp / category-select.php according to the picture, enter Os03g0215400 in the Search options in the webpage, then click search, and then click FeatureNum in the new webpage After entering the number, check the Spatio-temporal profile on the new web page, you can query this graph, and click Downloadselected graph on the web page to get this graph). OsMADS1 wa...

Embodiment 3

[0239] Example 3: Specific application and effect display of pOsOle18::OsMADS1-RNAi seed-specific interference vector

[0240] 1. The pOsOle18::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:

[0241] 1. Callus induction and subculture of rice mature seeds

[0242] Pick mature rice seeds, peel off the glumes, pour them into a 50ml centrifuge tube, add 75% ethanol for sterilization for 1 min, pour off the ethanol, rinse with sterile water, and then pour out, add 30% sodium hypochlorite for disinfection for 20 minutes, pour out the sodium hypochlorite, use Rinse with sterile water 5 to 6 times. Remove excess water with a pipette and transfer the seeds to induction medium, 20 to 25 seeds per dish. After the callus grows, the original embryo can be used for direct transformation; the small ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a seed-specific interference vector comprising a pOsOle18 promoter and its application. Specifically, a seed-specific interference vector is provided, and the vector is a pOsOle18-RNAi vector, and its DNA sequence is shown in SEQ ID NO 9. The pOsOle18-RNAi vector can specifically interfere with the expression of the target gene in rice seeds without affecting the normal expression of the target gene in other tissues and organs, thereby not only effectively studying the direct regulation of the target gene on seed development It can also achieve the purpose of improving rice grain shape, yield and rice quality, and provide an effective technical means and solution for molecular precision design and breeding of rice yield and rice quality. At the same time, an OsMADS1 seed-specific interference vector pOsOle18::OsMADS1-RNAi based on the pOsOle18-RNAi vector is also provided, the DNA sequence of which is shown in SEQ ID NO 10; the use of the vector can improve the protein content in the rice and increase the protein content of the rice. nutritional value. The invention has important scientific research significance and application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a seed-specific interference vector comprising a pOsOle18 promoter and its application. Background technique [0002] Rice is the staple food for more than half of the world's population and a basic source of starch and protein for humans. 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 is also an important factor that directly determines the nutritional quality of rice such as rice protein content. The morphological development and protein content of rice seeds 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 genetic engineering breedi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/10A01H6/46
CPCC12N15/8218C12N15/8234C12N15/8251C12N15/8261C07K14/415Y02A40/146
Inventor 李辉李鹏慧涂升斌黄维藻汪松虎蒋海波刘阳轩古袁扬庄勇刘志坚魏明唐钰莹乐露路龙祥胡永志喻兰
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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