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Nondestructive screening system for naked eye visible plant genetic transformation under visible light, construction method and application thereof

A technology of genetic transformation and construction method, applied to the construction and application of the above-mentioned reporter system, the new field of plant genetic transformation reporter system, which can solve the problems of insensitivity, escape of non-resistant tissues, and great differences in antibiotic sensitivity

Inactive Publication Date: 2020-12-11
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] But antibiotic or herbicide screening methods have some drawbacks
First, the susceptibility of different species of plants to different antibiotics varies greatly
A few plants, such as Arabidopsis, rice, corn, wheat, rape and other plants are sensitive to hygromycin or neomycin or glyphosate, which can be used to screen transgenic plants, but many horticultural plants or forest trees are sensitive to antibiotics or commonly used Herbicides are less sensitive and cannot be screened effectively
Secondly, some plants are more prone to non-resistant tissue escape during the genetic transformation process, resulting in a high chimerism rate of genetically transformed plants, and it is impossible to effectively screen out homozygous transgenic plants
Third, antibiotic or herbicide screening strategies cannot be well screened in in-planta transformation (direct genetic transformation of a certain part of a complete plant)
Fourth, antibiotic or herbicide screening has the risk of increasing environmental pollution
These factors increase the detection cost of these reporting systems and limit the scope of use

Method used

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  • Nondestructive screening system for naked eye visible plant genetic transformation under visible light, construction method and application thereof
  • Nondestructive screening system for naked eye visible plant genetic transformation under visible light, construction method and application thereof
  • Nondestructive screening system for naked eye visible plant genetic transformation under visible light, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1. Construction of the RUBY gene-terminator fragment to obtain the vector pRUBY-tHsp

[0071] In this embodiment, the RUBY gene adopts the nucleotide sequence 2A capable of encoding 2A peptide as a DNA linking unit, and is sequentially connected in the order of CYP76AD1 gene-2A1-DODA gene-2A2-GT (glucosyltransferase) gene ( figure 1 B), wherein the nucleotide sequence of 2A1 is shown in SEQ ID NO.8, the nucleotide sequence of 2A2 is shown in SEQ ID NO.9, and both 2A1 and 2A2 are nucleosides encoding the 2A peptide shown in SEQ ID NO.4 Acid sequence, with the terminator tHsp of the Arabidopsis gene as the terminator.

[0072] The build method is:

[0073] (1) Synthesized separately by in vitro total gene synthesis:

[0074] (a) CYP76AD1 gene (SEQ ID NO.5) without stop codon,

[0075] (b) DODA gene (SEQ ID NO.6) without stop codon,

[0076] (c) comprising GT gene shown in SEQ ID NO.7, 3 consecutive stop codons TGA, TAG, TGA and the GT gene+stop codon+terminat...

Embodiment 2

[0123] Example 2. The promoter was connected to the pRUBY-tHsp vector obtained in Example 1 to construct the screening system p35S-RUBY-tHsp plasmid

[0124] The cauliflower mosaic virus (CaMV) 35S promoter is used for the RUBY gene expression cassette promoter in this embodiment, and the RUBY gene expression cassette terminator is the terminator tHsp of the Arabidopsis gene:

[0125] (1) Use the pCAMBIA1300 plasmid (the pCAMBIA1300 plasmid can be purchased from Australian CAMBIAhttp: / / www.cambia.org from public channels, and the pCAMBIA1300 plasmid used in this example is donated by Australian CAMBIAhttp: / / www.cambia.org) as a template, Use primer pairs:

[0126] 35S-F:5'-CTGTCAAACACTGATAGTTTTGAGACTTTTCAACAAAGGG-3' (SEQ ID NO.21)

[0127] 35S-R: 5'-CGCATGATCCATACTAGTTTTCAGCGTGTCCTTCCAAAT-3' (SEQ ID NO.22) was used as a primer for PCR amplification to obtain the PCR product of the 35S promoter.

[0128] PCR reaction system:

[0129] 2×PCR Buffer 10μl 2.5mM ...

Embodiment 3

[0134] Example 3 Linking RUBY-tHsp into the pOsActin1-UCAS9 vector plasmid (the backbone of the pOsActin1-UCAS9 vector is pCAMBIA, and the plasmid information can be found in literature: He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y.Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR / Cas9 mediated genome editing.JGenet Genomics,2017,44(9):469-472; pOsActin1-UCAS9 vector plasmid can pass through the mailbox in the literature Obtained from the author of the literature), wherein the pOsActin1-UCAS9 vector plasmid carries the target gene C9 (SEQ ID NO.13) to be transformed, and the pOsActin1-RUBY-tHsp plasmid of the screening system is constructed.

[0135] In this embodiment, the promoter of the RUBY gene expression cassette adopts the promoter of the rice constitutively expressed gene OsActin1 (SEQ ID NO.11):

[0136] (1) Obtain the RUBY-tHsp DNA with the linker sequence that can be connected into the pOsActin1-...

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Abstract

The invention relates to a nondestructive screening method for plant genetic transformation and application thereof. Plant genetic transformation depends on an efficient screening system, and a commonscreening system comprises antibiotic screening and reporting system screening. The existing reporting system screening needs special equipment and expensive chemicals or destructive treatment on biological samples. The invention constructs a new reporter gene RUBY, and the enzyme synthesized by the gene can convert tyrosine into red betacyanin. The invention utilizes RUBY to indicate the occurrence of plant genetic transformation, and successfully realizes nondestructive screening of naked eye visible plant genetic transformation under visible light.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a new plant genetic transformation reporting system and its application. The reporter system of the present invention is indicated by the pigment produced in the plant body which can be directly observed by the naked eye under visible light. The present invention also relates to the construction and application of the above reporting system. Background technique [0002] The screening of genetic transformation of plants mainly relies on antibiotics or herbicides for screening. By adding an antibiotic or herbicide resistance gene expression cassette to the genetic transformation vector, the genetically transformed plants will show resistance to specific antibiotics or herbicides. resistance. [0003] But antibiotic or herbicide screening methods have some drawbacks. First, the susceptibility of different species of plants to different antibiotics varies greatly. ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N15/53C12N15/54A01H5/00A01H6/20A01H6/46
CPCC12N15/8212C12N15/65C12N9/0079C12N9/1051C12N9/0069C12Y113/11008C12N2830/36
Inventor 和玉兵
Owner NANJING AGRICULTURAL UNIVERSITY
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