Primers, kits and applications for controlling Arabidopsis leaf senescence

A technology of leaf senescence and Arabidopsis thaliana, applied in the field of plant genetic engineering, can solve problems such as difficult to be used and poor agronomic traits

Active Publication Date: 2021-11-09
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the existing studies on delayed senescence mutants in many crops have poor agronomic traits and are difficult to exploit

Method used

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  • Primers, kits and applications for controlling Arabidopsis leaf senescence
  • Primers, kits and applications for controlling Arabidopsis leaf senescence
  • Primers, kits and applications for controlling Arabidopsis leaf senescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cloning of TaNACS gene and construction of overexpression vector

[0050] Using Chinese spring wheat cDNA as template, PCR amplification was carried out with SEQ ID NO.1 as upstream primer and SEQ ID NO.2 as downstream primer. The template concentration was 50ng / ml, and the amplification procedure was as follows:

[0051] 95°C for 3min; 33 cycles of 95°C for 30s, 59°C for 30s, 72°C for 1min; 72°C for 10min.

[0052] The PCR amplified product was connected to the pCambia3301 overexpression vector (ubi-driven overexpression) by infusion recombinase, finally forming the recombinant vector pCambia3301-TaNACS.

[0053] The connection method is:

[0054] (1) Digest the pCambia3301 vector with EcoRI and BamHI, and recover the fragments for later use;

[0055] (2) Establish the ligation system: 1.5 μL of the digested pCambia3301 vector; 2.5 μL of PCR fragments; 1 μL of infusion enzyme:

[0056] (3) Establish reaction conditions: 50°C for 15 minutes, place on ice for 5 minute...

Embodiment 2

[0059] 1. Obtain TaNACS gene induction expression vector

[0060] Using the pCambia3301-TaNACS vector as a template, using SEQ ID NO.3 as an upstream primer, and SEQ ID NO.4 as a downstream primer for PCR amplification, the amplification procedure:

[0061] 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 1min, 35 cycles; 72°C for 10min.

[0062] After the amplified fragment was purified and recovered according to the kit instructions, it was connected to the intermediate vector pDonor207 by BP enzyme. After sequencing, the positive clone was selected to extract the plasmid, named TaNACS-in-pDonor207, and the vector was recombined with the final vector pER8 by LR enzyme. , forming an inducible expression vector named TaNACS-pER8; transforming the inducible expression vector into Escherichia coli DH5a for positive identification and sequencing;

[0063] 2. Obtain a 35S-driven TaNACS gene constitutive overexpression vector

[0064] Using the aNACS-in-pDonor207 vector obtain...

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Abstract

The invention discloses a primer for controlling Arabidopsis thaliana leaf senescence, a kit and an application thereof, which belong to the technical field of plant genetic engineering and can solve the problem that among many crop senescence-delaying mutants, the crops have poor agronomic traits and are difficult to be detected. problem of utilization. The technical solution includes designing specific upstream and downstream primers based on the wheat gene TaNACS, integrating the TaNACS gene into the Arabidopsis genome through transgenic technology, and promoting the senescence of Arabidopsis leaves by overexpressing the TaNACS gene. The invention uses the wheat TaNACS gene to control the senescence process of Arabidopsis thaliana leaves, and at the same time, the overexpression of the gene does not affect other agronomic traits of the Arabidopsis thaliana.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a primer, a kit and an application thereof for controlling the senescence of Arabidopsis leaves. Background technique [0002] Senescence is the last stage of plant development. It is not only affected by external factors such as temperature, disease, and stress, but also regulated by genes and affected by hormone levels in the body. Leaf senescence is closely related to crop yield and quality. Premature or late senescence of crops is not conducive to completing its normal life cycle, resulting in reduced yield or quality of crops, which seriously affects agronomic traits. Therefore, the discovery and application of leaf senescence regulators is one of the effective ways to achieve high yield and high quality of crops. [0003] Arabidopsis is one of the model organisms in plant science, including genetics and plant development research. The research...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 张增林郭永峰李伟高晓明徐萌萌
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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