Unlock instant, AI-driven research and patent intelligence for your innovation.

Detection method for transgenic-S6RNAi black-streaked-dwarf-resistant rice line WLJ1-US6-11-5

A technology for black-streaked dwarf disease and rice strains, applied in the biological field, can solve problems such as no transgenic rice

Inactive Publication Date: 2020-05-19
YANGZHOU UNIV
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

WLJ1-US6-11-5 is a newly developed transgenic rice line resistant to black-streaked dwarf disease. There are no reports or patents on the flanking sequence of the foreign gene insertion fragment of related transgenic rice WLJ1-US6-11-5.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Detection method for transgenic-S6RNAi black-streaked-dwarf-resistant rice line WLJ1-US6-11-5
  • Detection method for transgenic-S6RNAi black-streaked-dwarf-resistant rice line WLJ1-US6-11-5
  • Detection method for transgenic-S6RNAi black-streaked-dwarf-resistant rice line WLJ1-US6-11-5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Transgenic rice WLJ1-US6-11-5 foreign gene insertion site flanking sequence cloning

[0038] The reverse PCR method is a very effective method for cloning the flanking sequence of the T-DNA insert fragment. We used the reverse PCR method to clone the flanking sequence of the foreign gene insertion site in transgenic rice WLJ1-US6-11-5. Including the following specific steps:

[0039] 1. Extraction of rice DNA

[0040] Using the CTAB method to extract the total DNA of rice leaves, the specific steps are as follows:

[0041] 1. Take 1-2g of fresh rice leaves, cut them into a 2mL centrifuge tube, add steel balls, put the centrifuge tube into liquid nitrogen to freeze, then use a vibrating grinder to grind the leaves into powder, and quickly pour out the steel balls.

[0042] 2. Add 600 μL of 1.5% CTAB preheated at 65°C, take a water bath at 65°C, shake it every 5 minutes, take it out after 30 minutes to cool, and add 600 μL of chloroform solution.

[0043] 3. ...

Embodiment 2

[0066] Example 2 Based on transgenic rice line WLJ1-US6-11-5 foreign gene insertion flanking sequence-specific PCR detection

[0067] In order to verify the detection effect of primers US-W1, US-W2 and US-W3 in transgenic rice WLJ1-US6-11-5 and its derivatives, we tested the F of WLJ1-US6-11-5 / Wulingjing 1 2 10 individuals were randomly selected from the first-generation segregation population materials, and US-W1 US-W2 primer combination M and US-W3, US-W2 primer combination N were used for PCR amplification and electrophoresis detection. Including the following specific steps:

[0068] 1. Acquisition of Segregated Groups

[0069] F 2 groups, from F 2 10 plants were randomly selected from the population, and WLJ1-US6-11-5 and Wulingjing 1 were used as controls for the following operations.

[0070] Two, the extraction of plant genomic DNA is the same as "rice DNA extraction" in Example 1.

[0071] 3. PCR detection based on the flanking sequence of the foreign gene insert...

Embodiment 3

[0078] The examples of this group provide a kit for homozygous identification of the transgenic rice line WLJ1-US6-11-5. The kit includes US-W1, US-W2 and US-W3 primers described in Example 1.

[0079] The kit also includes conventional reagents for PCR amplification, and / or conventional reagents for electrophoretic detection. Those skilled in the art, according to the records of this application, for the purpose of "homozygous identification of transgenic rice line WLJ1-US6-11-5", rationally select reagents and / or electrophoresis reagents conventional in the art for PCR amplification .

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a detection method for a transgenic-S6RNAi black-streaked-dwarf-resistant rice line WLJ1-US6-11-5 and belongs to the technical field of biology. The method is a specific PCR amplification identification technology established based on a sequence shown in SEQ ID NO: 1 through inserting a fragment sequence and a fragment RB end side specific sequence shown in the SEQ ID NO: 1to rice chromosomes by using a T-DNA expression frame containing an exogenous gene S6RNAi in a transformant WLJ1-US6-11-5. A forward vector primer US-W1 employed by PCR is designed according to sequences of 1-1178 in the SEQ ID NO: 1, a reverse genome primer US-W2 is designed according to sequences of 1179-2000 in the SEQ ID NO: 1, and a forward genome primer US-W3 is designed according to a 528bp position of the upstream of a T-DNA insertion position. Whether a plant line contains the exogenous gene S6RNAi or not is judged according to whether target fragments of a US-W1 US-W2 primer combination M and a US-W3 US-W2 primer combination N are obtained or not through amplification, and the PCR method can serve as an effective measure for identifying the transgenic rice line WLJ1-US6-11-5 andderived lines thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method for identifying the rice line WLJ1-US6-11-5 resistant to black-streaked dwarf disease of the S6RNAi gene and its derivative lines and the specific sequence of the transformant on which it depends. Background technique [0002] Rice is one of the main food crops in the world, and it is the main food crop for nearly half of the world's population whose staple food is rice. Rice is also one of the most important food crops in my country. The safety of rice production plays a very important role in ensuring the safety of food production in my country. Rice disease is one of the important factors affecting the goal of stable, high and excellent rice yield in my country. Rice black-streaked dwarf disease is a rice virus disease that is mainly transmitted by the small planthopper, and it is caused by rice black-streaked dwarf virus. Once rice is infected by ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/6895C12Q2600/13
Inventor 左示敏冯志明张亚芳陈宗祥邹捷崔傲李明友杜海波潘学彪
Owner YANGZHOU UNIV