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Rice thousand kernel weight gene TGW6 guided RNA target sequence and application thereof

A TGW6U3-T1-R, target sequence technology, applied in the field of plant biology, can solve the problem of lack of evaluation of the value of related mutants, and achieve the effect of efficient editing

Inactive Publication Date: 2015-11-18
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the CRISPR / Cas9 system has successfully achieved targeted genome editing in plants such as Arabidopsis thaliana, tobacco, sweet orange, rice, wheat, sorghum, corn, and bryophytes, but it is important for the yield and quality of rice breeding. There are few reports on the targeted editing of key genes such as , fertility, etc., and there is even a lack of evaluation of the breeding value of related mutants

Method used

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  • Rice thousand kernel weight gene TGW6 guided RNA target sequence and application thereof
  • Rice thousand kernel weight gene TGW6 guided RNA target sequence and application thereof
  • Rice thousand kernel weight gene TGW6 guided RNA target sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Creation of rice thousand-grain weight gene tgw6 deletion mutant based on CRISPR / Cas9 technology

[0042] (1) Design of guide RNA (guideRNA, gRNA) target sequence

[0043] According to the genome sequence of the rice grain length and thousand-grain weight gene TGW6 (GenBank: AB513135.1), three gRNAs targeting the thousand-grain weight gene TGW6 were designed. The 20nt oligonucleotide gRNA target sequence is designed according to the A / G(N)20NGG sequence, and the designed gRNA target sequence is compared with the rice genome database to exclude non-specific target cutting sites. The nucleotide sequence is shown in Table 1 and figure 1 .

[0044] Table 1 Oligonucleotide sequences of gRNA targets

[0045]

[0046] (2) Construction of three-target CRISPR / Cas9-gRNA vector

[0047] Referring to the method of Ma et al. (MaX, ZhangQ, ZhuQ, LiuW, ChenY, QiuR, WangB, YangZ.2015.ArobustCRISPR / Cas9systemforconvenient, high-efficiencymultiplexgenomeeditinginmonocota...

Embodiment 2

[0059] (1) Agrobacterium-mediated genetic transformation of rice callus

[0060] The CRISPR / Cas9-gRNA vector assembled in Example 1 was transformed into Agrobacterium EHA105 by electric shock. PCR detection (primers: hptF: 5'-TCCGGAGCCTCCGCTCGAAGTAG-3', hptR: 5'-CTGAACTCACCGCGACGTCTGTC-3') positive clones were used to infect rice material H447 (BC of R819 / Yuzhenxiang / / R819 2 f 7 ), the infection method was carried out with reference to the method of Hiei et al. (1994), and transgenic rice plants were obtained.

[0061] (2) Extraction of rice genomic DNA and PCR detection and sequencing analysis of mutants

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Abstract

The invention belongs to the field of plant biotechnology, and in particular relates to rice thousand kernel weight gene TGW6 guided RNA target sequences and application thereof. The oligonucleotide sequences of the rice thousand kernel weight gene TGW6 guided RNA target sequences are as shown in SEQ ID NO.1-6. The group of the target sequences is applicable to the editing of rice genome, and has the advantages of being convenient, efficient and the like. When the rice thousand kernel weight gene TGW6 guided RNA target sequences provided by the invention are used for editing the rice genome, the mutation frequency of rice thousand kernel weight gene tgw6 is 90% or above, including more than 50% of fragment-deleted homozygous mutants and around 40% of diallelic heterozygous mutants. The invention provides an important reference for the rapid establishment of high-quality new rice germplasm having an important application value on rice quality and like production, and is expected to provide a safe and efficient new way for the innovation of rice germplasm resources; and the invention has important theoretical and practical significance.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a rice thousand-grain weight gene TGW6 guide RNA target sequence and its application. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, feeding more than half of the world's population and the main source of food for nearly half of my country's population. With the continuous growth of population, people's demand for food is also increasing. The effective use of heterosis has played an important role in promoting the increase of rice yield. However, the rice yield per unit area has not been greatly improved, and with the reduction of cultivated land, the planting area of ​​rice has been declining. It is urgent to use new Genetic improvement strategies to greatly increase rice yield. In recent years, with several genes controlling grain size (GS3, SW5 and GW8), grain width and grain weight (GW2, GW5), grain l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82C12N15/66C12N15/29A01H5/00
Inventor 王加峰杨瑰丽郭涛黄明刘永柱陈志强王慧
Owner SOUTH CHINA AGRI UNIV
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