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Rice gene directional editing method based on pollen tube channel introduction

A technology of pollen tube passage and rice, applied in genetic engineering, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of long transformation period, difficult transformation, and increasing the difficulty and complexity of genetic transformation.

Inactive Publication Date: 2021-03-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Agrobacterium-mediated method is highly selective for genotypes, indica rice is more difficult to transform than japonica rice, and the transformation cycle is long, and this method requires a complex artificial tissue culture process, which increases the cost of genetic transformation. difficulty and complexity

Method used

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  • Rice gene directional editing method based on pollen tube channel introduction
  • Rice gene directional editing method based on pollen tube channel introduction
  • Rice gene directional editing method based on pollen tube channel introduction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction process of pYLCRISPR / Cas9-MT vector includes the following steps:

[0035] (1) Target design

[0036] The bases A and G are the transcription start bases of the U3 and U6 promoters, respectively, so if the 20th base upstream of the target gene NGG is A, use the U3 promoter, and if it is G, use the U6 promoter.

[0037] If the 20th base upstream of NGG is not A or G, 20 bases can be selected as the form of the target sequence synthetic linker. That is, if the transcription initiation point of the promoter used is the same as the 20th base upstream of NGG, the target site is 19 bases, and if the target site is different, it is 20 bases.

[0038] The case where the transcription start point is the same as the 20th base upstream of NGG is as follows:

[0039]

[0040] The transcription start point is different from the 20th base upstream of NGG as follows:

[0041]

[0042] The 19-base target sequence synthesis linker form (linked to the U3 promote...

Embodiment 2

[0109] The rice genetic transformation process mediated by Agrobacterium is as follows:

[0110] (1) Preparation of Agrobacterium Competent Cells

[0111]A. On the YEB plate, inoculate Agrobacterium tumefaciens EHA105, and culture it upside down at 28°C for 24-48 hours.

[0112] B. Pick a single colony from the plate and dilute it with sterilized water, apply the plate again (to reduce the amount of glycerol), and incubate it upside down at 28°C for 24-48 hours.

[0113] C. Pick a single colony from the plate and inoculate it in YEB liquid medium, culture at 28°C and 200rpm for 14-16h.

[0114] D. Take 2ml and transfer to fresh 100ml YEB liquid medium, 28℃, 220rpm rapid shaking culture for 3~4h to OD 600 When =1.0~1.5, the bacterial cells were collected.

[0115] E. Transfer the bacterial solution to two pre-cooled sterile centrifuge tubes, and place in an ice bath for 20 minutes to lower the bacterial solution to 0°C. Centrifuge at 3000 g for 5 min at 4°C to collect the c...

Embodiment 3

[0144] The pollen tube channel genetic transformation method comprises the following steps:

[0145] (1) Before the rice blooms, select an ear with most of the florets in the flowering period, and cut off the florets that have bloomed. Open small flowers. After about an hour, cut off a part of the glume, and cut off the position where the style is close to the ovary without hurting the ovary.

[0146] (2) 1.5 to 3 hours after rice flowering, select the florets that have bloomed that day, remove other florets that have bloomed and have not bloomed, use tweezers to open from the seam of inner and outer glumes from top to bottom, and fix them by hand to avoid glomerulation. The shell is closed, and the stigma is cut off (note that the ovary cannot be injured), and the concentration of 1 μg / μl is injected into 15 μl with a micro-injector. .

[0147] According to the statistics of injected rice florets, there are about 2546 Cas9-tms5 and 1303 Cas9-SBE3. After the rice was fully...

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Abstract

The invention discloses a rice gene directional editing method based on pollen tube channel introduction, and belongs to the technical field of biology. The method is characterized in that by utilizing a pollen tube channel, a CRISPR / Cas9 vector is introduced into a rice embryonic sac, and directional editing of a tms5 target gene is realized. The gene directional editing method base on the pollentube channel does not depend on genetic transformation of calluses, target gene mutation can be obtained, and the operation process is simple and reliable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rice gene-directed editing method based on pollen tube channel introduction. Background technique [0002] Rice is one of the most important food crops in the world. At present, with the increase of population, decrease of arable land, environmental pollution and frequent occurrence of extreme natural disasters, rice food security is facing severe challenges. Traditional breeding technology relies on hybrid breeding, which cannot meet the directional improvement of traits, and it is difficult to achieve targeted mutation in mutation breeding. Although the artificial modification of rice genes at the genome level is of great significance to the improvement of crops, scientists have not found an effective plant genome editing technology. In recent years, the newly discovered CRISPR / Cas9 technology has been widely used in genome editing of rice and other crops due to its ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8206C12N15/8218C12N15/8289
Inventor 郭涛王加峰陈淳孟庆彬黄翠红周丹华黄明肖武名刘永柱杨瑰丽王慧陈志强
Owner SOUTH CHINA AGRI UNIV
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