Application of histone deacetylase gene to regulation and control on development of rice seed starch

A deacetylase, rice seed technology, applied in application, genetic engineering, plant genetic improvement and other directions to achieve the effect of alleviating food shortages

Inactive Publication Date: 2014-01-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of starch synthesis genes detected by Realtime-PCR found that the expression of these genes was obviously suppressed in the suppressed plants

Method used

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  • Application of histone deacetylase gene to regulation and control on development of rice seed starch
  • Application of histone deacetylase gene to regulation and control on development of rice seed starch
  • Application of histone deacetylase gene to regulation and control on development of rice seed starch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Amplification of the full-length cDNA of the OsSRT1 gene

[0036] For the gene OsSRT1 (gene accession number LOC_Os04g20270) required by the present invention, mainly by RT-PCR method (see: J. Experiment Guide (Third Edition), Beijing, Science Press, 2002 Edition) was amplified to obtain the full-length sequence of the OsSRT1 gene. The specific operation is as follows:

[0037] 1) Extract the RNA from the seedling leaves of the rice variety Huanghui 63. The reagent for RNA extraction is the Trizol extraction kit from Invitrogen (see the kit’s instruction manual for specific steps);

[0038] 2) The steps of reverse transcription to synthesize the first strand of cDNA in RT-PCR are as follows:

[0039] ① Prepare mixed solution 1: 4 μg of total RNA, 2U of DNaseI, 1 μl of 10×DNaseI buffer, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase) treated water (0.01% DEPC) to 10 μl, mix well and mix Solution 1 was placed at 37°C for 20 minutes to remove DN...

Embodiment 2

[0051] Example 2: Construction of OsSRT1 double-strand suppression vector

[0052] For the genes required by the present invention, by RT-PCR method (see: J. Sambrook, EF Fritsch, T Mani Artis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 edition) to amplify to obtain a specific sequence of OsSRT1. The specific steps are: by using the full-length cDNA of OsSRT1 obtained by self-amplification as a template, find the specific sequence of OsSRT1 to design primers, and PCR amplifies the RNAi inhibitory fragment. The amplified product was connected to pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., ie Promega, USA) by T / A cloning for sequencing verification.

[0053] The primers used to clone the RNAi suppressor fragment of OsSRT1 are as follows:

[0054] ds-F:5'-GGGACTAGTGGTACCAGTCCTGCAAGAGTTGCAAC-3'

[0055] ds-R:5'-GGGGAGCTCGGATCCCCAGCTTTCACATGCACTAG-3'

[0056] Specific steps ar...

Embodiment 3

[0061] Example 3: Transformation of binary Ti plasmid vector and detection of positive and expression levels of transgenic plants

[0062] 1) The newly constructed suppression expression vector pDS1301-OsSRT1-2 (from Example 2) was introduced into Agrobacterium EHA105 (purchased Among the strains from CAMBIA Laboratory in Australia, the strain transformed with pDS1301-OsSRT1-2 was named pDS1301-OsSRT1-2-EHA105.

[0063] 2) Transform the pDS1301-OsSRT1-2-EHA105 obtained in the previous step into the rice variety Minghui 63. The transformation method refers to the method reported by Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6:271-282.). The obtained T0 transgenic plants were named Rn, where n=1, 2, 3...represent different transgenic families.

[0064] 3) Total DNA was extracted from the leaves of the transformed plants of the T0 generation. The DNA ...

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Abstract

The invention belongs to the field of plant transgenes and relates to application of a histone deacetylase OsSRT1 gene to regulation and control on development of rice seed starch. H3K9 acetylation of an OsSRT1 inhibited plant is found to be obviously enhanced, which indicates that the gene is histone H3K9 deacetylase gene. According to research on the transgenic plant which inhibits the gene, the fertility of the plant is obviously reduced, and most of seeds stop developing three days after fertilization and are withered gradually. According to observation, the endosperm starch content of the seeds is reduced two days after the seeds are fertilized. According to Realtime-PCR detection on the change of starch synthesis genes, in the seeds which are fertilized for one day and two day, the expression quantity of the starch synthesis genes of the OsSRT1 inhibited plant is obviously reduced compared with that of the wild type. The OsSRT1 is confirmed to regulate and control starch synthesis by influencing histone acetylation.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the functional verification and application of the histone deacetylase gene OsSRT1 related to the regulation of rice seed starch development. Background technique [0002] Rice plays an important role in my country's grain production. Endosperm accounts for more than 90% of polished rice, and is the part of rice that is directly eaten. The development of endosperm directly affects the yield and quality of rice. Therefore, it is of great significance to study the genes related to rice endosperm development in actual production. [0003] Current research has found that the main sites of rice starch synthesis are amyloplasts and chloroplasts, and photosynthesis provides the raw materials for its synthesis. Starch biosynthesis in rice grains is a very complex biochemical process. The products of leaf photosynthesis are first transported in the form of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/80A01H5/00
Inventor 周道绣张华赵毓
Owner HUAZHONG AGRI UNIV
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