A Fusion Gene Moderately Delaying Plant Senescence and Improving Stress Resistance and Its Application

A technology of plant aging and gene fusion, applied in the field of plant genetic engineering, can solve problems affecting crop yield and quality

Active Publication Date: 2017-10-27
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] On the other hand, a variety of adversity stresses in the environment, such as drought, flood, frost damage, high temperature and disease, will seriously affect the yield and quality of crops.

Method used

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  • A Fusion Gene Moderately Delaying Plant Senescence and Improving Stress Resistance and Its Application
  • A Fusion Gene Moderately Delaying Plant Senescence and Improving Stress Resistance and Its Application
  • A Fusion Gene Moderately Delaying Plant Senescence and Improving Stress Resistance and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: A fusion gene that moderately delays plant senescence and improves stress resistance—— WX02 the acquisition

[0043] Using Arabidopsis cDNA as template to amplify 1206 bp WX02 The gene was fused, and the amplified product was recovered and subjected to TA cloning.

[0044] (1) PCR amplification of the target fragment

[0045] Arabidopsis RNA was extracted by Trizol method and reverse-transcribed into cDNA, using cDNA as a template, using specific primers for PCR amplification WX02 The downstream part of the fusion gene is also introduced in the upstream primer WX02 The upstream part of the fusion gene, thus making WX02 Fusion gene fragments.

[0046] designed in it WX02 The PCR amplification primers of the fusion gene are as follows, first use the upstream primer 2 and the downstream primer 2 to amplify, and then use the amplified product as a template to amplify using the upstream primer 1 and the downstream primer 1 to obtain WX02 Fusion gene ...

Embodiment 2

[0071] Embodiment 2: Utilize pBI121 carrier (containing CaMV 35S promoter) constructed by CaMV 35S promoter drive WX02 Binary expression vector for fusion gene expression.

[0072] (1) Extract vector pBI121 plasmid from Escherichia coli (the strain can be purchased from biological company or Clontech), use Xba I / Sac Recover the large carrier fragment (which contains CaMV 35S promoter sequence).

[0073] (2) Extract the plasmid from the TA clone prepared in Example 1, and use Xba I / Sac I double digestion, recovery by agarose gel electrophoresis (with step (2) in Example 1) WX02 Fusion gene fragments.

[0074] (3) The above two fragments were ligated overnight at 16 °C under the catalysis of ligase to complete the pBI121 carrier by CaMV35S promoter drive WX02 Construction of binary expression vectors for fusion gene expression.

[0075] Connection system:

[0076] Reagent

Amount added

pBI121 vector recovery fragment

1mL

...

Embodiment 3

[0082] Embodiment 3: Preparation of transgenic Arabidopsis

[0083] (1) Constructed by Example 2 CaMV 35S promoter drive WX02 The binary expression vector expressed by the fusion gene was transformed into Arabidopsis thaliana. The specific transformation method used the flower bud soaking method mediated by Agrobacterium (obtained in Example 2) (Clough and Bent, 1998). The obtained seeds were subjected to 25 mg / L kana For mycin resistance selection, normal growing plants were transferred to soil for culture.

[0084] (2) PCR detection of transgenic plants: Cut the leaves of transgenic plants and wild-type plants respectively, and extract genomic DNA from leaves as a template by referring to the "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002), using the example For the upstream primer 1 and downstream primer 1 described in step (1) in 1, perform PCR reaction according to the same system and PCR amplification conditions:

[0085] The PCR pr...

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Abstract

A fusion gene that moderately delays plant senescence and improves stress resistance and its application. In the present invention, a fusion gene is artificially spliced ​​using molecular biology technology, named WX02, and its nucleic acid sequence is shown in SEQ ID No.1. At the same time, a binary expression vector driven by the CaMV 35S promoter to express the WX02 fusion gene was constructed, and the bud soaking method was used to transform Arabidopsis thaliana. The research on the obtained 35S-WX02 transgenic Arabidopsis proved that the expression of the fusion gene had a positive effect on the transgene expression. The growth and development of the plants will not be affected in any way, and at the same time, the leaves of the transgenic plants will be delayed, and their drought resistance will be significantly improved. It shows that the WX02 gene has the functions of moderately delaying leaf senescence and improving plant stress resistance. By transferring it into major economic crops, it is possible to obtain new varieties of transgenic crops with delayed leaf senescence, enhanced stress resistance, and improved yield and quality traits. important application value.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically, artificially obtains a fusion gene moderately delaying plant aging and improving stress resistance by using molecular biology technology and its application in transgenic plants. Background technique [0002] my country is a large agricultural country, but with the increase of population, the continuous reduction of arable land and the increasing shortage of agricultural resources, the food supply is facing a huge challenge. Therefore, increasing and stabilizing food production and improving food quality have become my country's current national policy. The need for major strategic development (Zhou Jianjun et al., 2014). [0003] Delaying senescence is closely related to crop yield increase and quality improvement. For food crops and most commercial crops, along with the development of yield organs and the gradual formation of quality traits, assimilated products in func...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
Inventor 王宁宁熊莉郭朝霞纳苏拉王丹
Owner NANKAI UNIV
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