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A method for increasing rice resistant starch content through genome editing and its dedicated sgRNA

A technology of resistant starch and gene editing, applied in the direction of recombinant DNA technology, DNA/RNA fragments, genetic engineering, etc., can solve the problems of non-improved agronomic traits, to reduce blood sugar, prevent hyperglycemia, prevent hyperglycemia and obesity Effect

Active Publication Date: 2019-04-23
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a new targeted gene modification technology, CRISPR / Cas9 has been applied in the research of targeted gene modification in rice, wheat, Arabidopsis and Nicotiana benthamiana, but there is no research on improving the target agronomic traits of important crops.

Method used

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  • A method for increasing rice resistant starch content through genome editing and its dedicated sgRNA
  • A method for increasing rice resistant starch content through genome editing and its dedicated sgRNA
  • A method for increasing rice resistant starch content through genome editing and its dedicated sgRNA

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, preparation recombinant plasmid

[0035] Artificially synthesized recombinant plasmid pCXUN-Cas9-gRNA1 (circular plasmid). The recombinant plasmid pCXUN-Cas9-gRNA1 is shown in Sequence 1 of the sequence listing. In sequence 1 of the sequence listing, the 109-361 nucleotides are reverse complementary to the NOS terminator, the 386-4516 nucleotides are reverse complementary to the coding gene of the Cas9 protein, and the 4537-6527 nucleotides are reverse complementary to the Cas9 protein encoding gene. The Ubi promoter is reverse complementary, nucleotides 6751-7154 are the U3 promoter, nucleotides 7155-7257 are the coding gene of sgRNA1 (the nucleotides 7155-7174 are the target sequence recognition region). The recombinant plasmid pCXUN-Cas9-gRNA1 expressed sgRNA1, and the target sequence of sgRNA1 was located in the first exon of rice SBEI gene. The rice SBEI gene is shown in sequence 3 of the sequence table (genomic DNA), the 1875-1958th nucleotide in t...

Embodiment 2

[0037] Embodiment 2, use recombinant plasmid to prepare transgenic plant

[0038] 1. Obtaining of transgenic plants

[0039] 1. Introduce the recombinant plasmid pCXUN-Cas9-gRNA1 into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0040] 2. Using rice kitaake as the starting plant, using the recombinant Agrobacterium obtained in step 1, performing Agrobacterium-mediated genetic transformation to obtain T 0 generation of regenerated plants.

[0041] The specific steps of genetic transformation are as follows:

[0042] (1) Resuspend the recombinant Agrobacterium obtained in step 1 with AAM medium to obtain OD 600nm =0.3-0.5 bacterial suspension.

[0043] AAM medium (pH5.2): 4.3g / L MS salts&vitamins+68.5g / L sucrose+0.5g / LMES+36g / L glucose+500mg / L casamino acids+100ml 10×AA amino acids solution+40mg / L acetosyringone. 10×AA amino acids solution: 8.76g / L L-glutamine, 2.66g / L L-aspartic acid, 1.74g / LL-arginine and 75mg / L glycine.

[0044] (2) Take rice kitaake se...

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Abstract

The invention discloses a method of increasing the content of resistant starch in rice through genome editing and sgRNA special for the same. The method provides a method of increasing the content of resistant starch and / or amylase in rice seeds. The method includes the following steps that expression of SBEIIb genes in the rice is inhibited; the SBEIIb genes are genes of encoding SBEIIb protein. The invention further provides a method of increasing the content of resistant starch in rice and includes the step of inhibiting the activity of the SBEIIb protein in the rice. By using the CRISPR / Cas9 technology, the rice SBEIIb genes are edited at fixed points, the rice SBEIIb genes are knocked off by causing frameshift mutation, and the new generation of new rice germplasm with the content of amylase and resistant starch obviously increased is obtained.

Description

technical field [0001] The invention relates to a method for increasing rice resistant starch content through genome editing and a special sgRNA thereof. Background technique [0002] Starch is the main component of wheat and rice grains. According to the characteristics of molecular structure, starch is divided into amylose and amylopectin. The starch synthesis process is regulated by a series of enzymes. [0003] Starch branching enzyme (SBE) is a key enzyme directly involved in starch biosynthesis. It has dual catalytic functions: on the one hand, it can cut glucan chains linked by α-1,4 glycosidic bonds (including amylose and the linear region of amylopectin); on the other hand, it can connect the cut short chain to the acceptor chain through α-1,6 glycosidic bonds. -1,4 glucan chains are further extended. The molecular weight of SBE is generally in the range of 70-114kD. According to the main amino acid sequence, SBEs in higher plants are mainly divided into two fam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/29C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/113C12N15/8245C12N2310/10C12N2800/80
Inventor 夏兰琴孙永伟
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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