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Method for living-body whole-plant detection of transgenic plant by luciferase and application thereof in transgenic maize

A luciferase and plant technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of complex operation and high cost, and achieve the effects of simple operation, reducing uncertainty and increasing accuracy

Pending Publication Date: 2020-04-10
CROP SCI RES INST SHANXI ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It solves the technical "bottleneck" of using a series of complex, time-consuming and high-cost in vitro molecular detection in the current study of plant functional genomics

Method used

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  • Method for living-body whole-plant detection of transgenic plant by luciferase and application thereof in transgenic maize
  • Method for living-body whole-plant detection of transgenic plant by luciferase and application thereof in transgenic maize
  • Method for living-body whole-plant detection of transgenic plant by luciferase and application thereof in transgenic maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Construction of expression vector

[0028] 1. Construction of pMD18T-Nos: Using the pCAMBIA1305.1 vector (GenBank: AF354045) containing the Nos terminator as a template, use primers NOS-F and NOS-R to amplify by PCR reaction to obtain Nos with restriction sites added at both ends Terminator fragment (the restriction site added at the 5' end is Spe I, and the restriction site added at the 3' end is Sac I ), and connected to the pMD18-T vector, the insertion direction was opposite to the Lacz direction, and the sequence verification was correct, and the intermediate vector pMD18T-Nos was obtained.

[0029] 2. Construction of pMD18T-Luc-Nos: Using the pCAMBIA1305-Luc vector (Genebank: QEG14167.1) containing the Luc luciferase gene as a template, use primers Luc-F (SEQ ID No: 4) and Luc-R (SEQ ID No: 5), the Luc gene fragment with restriction sites added at both ends was amplified by PCR reaction (the restriction site added at the 5' end is Bam H I, xho ...

Embodiment 2

[0032] Example 2: Ultrasonic-assisted pollen-mediated transformation of maize Transformation of maize

[0033] 1. Preparation of transformed plasmids: extract the plasmids by alkaline lysis, dissolve them in TE buffer, and detect the concentration of the plasmids with a micro-ultraviolet spectrophotometer. Adjust the plasmid concentration to 200 µg / ml. A 260 / A ２８０ The value is around 1.8 for backup.

[0034] 2. Prepare pollen suspension: 9% sucrose + 0.9% NaCl solution (PH7.0), pre-cool at 4°C, and add oxygen to increase its oxygen content.

[0035] 3. Ultrasonic-assisted pollen-mediated maize transformation: collect 3 g of maize pollen at the full flowering stage from 10:00 to 12:00 on the day of transformation, put it in 10 ml of pollen suspension, add 200 g of plasmid DNA, and use an ultrasonic pulverizer (JY92-Ⅱ type) for ultrasonic treatment. Ultrasonic treatment parameters are: power 180 W, working time 6 s, interval 6 s, working times 6 times. Then apply the tre...

Embodiment 3

[0036] Example 3: Optimizing the conditions for live whole plant detection of transgenic maize by live imaging system

[0037] (1) Determine the parameters of the in vivo imaging system: After spraying the substrate on the young corn sprouts, put them into the dark box of the in vivo imaging system, and after standing for 2 minutes, compare the exposure time of 2 minutes, the pixel binning value binning4×4 and the exposure time of 5 minutes, and the pixel Merge value binning 8×8 imaging effect. It was found that the highest signal value of the former was 124 cps, while the signal value of the latter was 1736 cps, which was more sensitive and suitable for the detection of maize sprouts ( figure 2 ).

[0038] (2) Determine the size of maize plants: Under the above exposure conditions, the fluorescence signals of maize buds and two-leaf stage seedlings were compared. It was found that the fluorescent signal could not be detected when the shoot length was less than 1cm; a clear...

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Abstract

The invention belongs to the technical field of molecular biology and gene engineering to solve problems of using a series of in-vitro molecular detection technologies which are complex in operation,time-consuming and high in cost in current transgenic plant detection. The invention provides a method for living-body whole-plant detection of transgenic maize by using luciferase reporter molecules.The nucleotide sequence of a luciferase reporter gene is shown as SEQ ID No. 1. A luciferase gene is introduced into maize by using an ultrasonic-mediated pollen transformation method; a plant living-body imaging technology is used for detecting a fluorescence signal of luciferase in transgenic maize in a living-body, whole-plant and real-time manner; and a high-expression transgenic plant can beidentified by introducing a signal-to-noise ratio and applying the signal-to-noise ratio. A large number of non-transgenic plants or strains with low expression are eliminated visually and accuratelyin real time with low cost; and an identification method which is simple to operate, accurate, rapid and low in cost is provided for identification of transgenic plants in plant functional genomics research.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a method for detecting transgenic plants in live whole plants using luciferase as a reporter molecule and its application in detecting transgenic corn. Background technique [0002] In recent years, the rapid development of genomics and bioinformatics has accelerated the number and speed of people obtaining genes from crops. Although a variety of molecular biology methods can be used to study the functions of these genes in vitro, the biological function verification through transgenic crops is the most direct proof. Because the genetic transformation efficiency of crops is not high at present, it is necessary to obtain a large number of contemporary inspected plants, and the number of inspected progeny plants of transgenic plants is even greater. Usually, the identification of transgenic plants is usually through PCR and Southern h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/52A01H5/00A01H6/46C12Q1/6895C12Q1/686
CPCC12N15/8209C12Q1/6895C12Q1/686C12Q2563/107
Inventor 白建荣闫蕾李锐常利芳郝曜山杨瑞娟康晨
Owner CROP SCI RES INST SHANXI ACADEMY OF AGRI SCI
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