Recombinant vector, transformant, primer for amplifying AtNAC58 gene as well as preparation method and application of primer

A technology for recombinant vectors and transformants, which is applied in the field of AtNAC58 transformants and their preparation, and primers for amplifying the AtNAC58 gene, which can solve the problems of death, low tissue culture efficiency, and low success rate of tissue culture

Active Publication Date: 2021-04-30
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in many cases, it is difficult to obtain the young tissue of plants due to various reasons, and the young tissue is often fragile and often dies after being damaged, which leads to a low success rate of tissue culture.
[0004] In addition, the prior art requires at least one month for the differentiation of tissue culture, making the efficiency of tissue culture relatively low

Method used

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  • Recombinant vector, transformant, primer for amplifying AtNAC58 gene as well as preparation method and application of primer
  • Recombinant vector, transformant, primer for amplifying AtNAC58 gene as well as preparation method and application of primer
  • Recombinant vector, transformant, primer for amplifying AtNAC58 gene as well as preparation method and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Cloning of AtNAC58 gene

[0071] Generate its corresponding cDNA sequence according to the Arabidopsis AtNAC58 transcription factor protein sequence obtained on the Tair database (https: / / www.arabidopsis.org / ), and design a specific primer pair according to the cDNA sequence, and the specific primer See Table 1 for the sequence of the pair.

[0072] Table 1 Amplification primer list

[0073]

[0074]In Table 1, AtNAC58-sense is the forward primer of the AtNAC58 gene, and its gene sequence is cggatccAGAGAGAGCGAGGAGAAACC, where the first base c is the protective base, and the second to seventh bases ggatcc are the restriction sites , the name of the restriction site is BamHI, and the rest are the forward primer sequence; AtNAC58-anti is the reverse primer of the AtNAC58 gene, and its gene sequence is cactagtAAAGGACGTCGAGATGCAT, wherein the first base c is a protected base, and the first The base actagt from the second to the seventh bases is a restriction...

Embodiment 2

[0081] Example 2 Preparation of AtNAC58 recombinant vector and AtNAC58 transformant

[0082] (1) Preparation of AtNAC58 recombinant vector

[0083] Using the enzyme cutting sites BamHI and SpeI in the primers, the empty vector pCanG was digested and ligated with the target fragment by T4 ligase. The ligation system was as follows:

[0084]

[0085] Mix the materials in the above connection system and centrifuge, incubate at 22°C for 1 hour, transform the incubated system into E. coli competent DH5α, use the plasmid mini-extraction kit to extract the recombinant plasmid from E. coli, and use double Enzyme digestion and colony PCR for verification, the results are as follows image 3 shown. exist image 3 In the figure, lanes 1 and 2 are the electrophoresis results of the digested product of the recombinant vector; M is DL2 000bp DNA marker. Depend on image 3 It can be seen that the target gene segment is cut off by the restriction endonuclease, which proves that the ...

Embodiment 3

[0090] Example 3 Application of AtNAC58 Transformant in Tissue Culture

[0091] Generally, for plant leaf tissue culture, the young leaves that are not stretched out at the top of the plant are generally selected, such as Figure 4A As shown, Qin Jingyuan. Plant tissue culture technology [M]. Chongqing University Press, 2014.

[0092] In this embodiment, the selected plant material is more mature (tissue differentiation ability is weaker) compared with the conventional selection material, and it is a leaf of Nicotiana benthamiana grown for about 3 months, and the part taken is a 9-11 node leaf, such as Figure 4B The circled leaves.

[0093] In this application, the buds that germinate at the beginning of tissue differentiation are called adventitious buds, and if more adventitious buds germinate, all the adventitious buds are called clustered buds.

[0094] The method adopted in this embodiment is the blisk method.

[0095] Specifically, the AtNAC58 transformant obtained...

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Abstract

The invention provides a plant tissue culture method, which uses an AtNAC58 gene transformant to promote rapid differentiation of plant tissues, can break through the limitation of explant material selection in a traditional scheme, can use a mature plant material as an explant, and can shorten the plant tissue differentiation time, Therefore, the difficulty of plant tissue culture is reduced, and the success rate and efficiency of plant tissue culture are improved.

Description

technical field [0001] The application belongs to the field of biotechnology, and in particular relates to a primer for amplifying AtNAC58 gene, AtNAC58 recombinant vector, AtNAC58 transformant and its preparation method and application. Background technique [0002] Plant tissue culture is a method of inoculating isolated explants on artificially prepared medium through aseptic operation, and cultivating them under artificially controlled conditions to make them a complete plant. Plant tissue culture is a plant cell engineering technology developed on the basis of plant physiology in the early 20th century. It is one of the most important and active fields in agricultural technology. It is known as the fourth green revolution in the history of agricultural development, and it has strategic significance for solving major problems such as the shortage of agricultural resources and environmental pollution. [0003] The selection of plant tissue culture materials is very impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/84C12N5/10A01H5/00A01H6/20A01H6/82
CPCC07K14/415C12N15/8261
Inventor 李国婧红格日其其格许可齐力旺朱木兰高仙灵王瑞刚
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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