Coding gene for enhancing tolerance of plant to cadmium toxicity and reducing cadmium content of plant and application of coding gene
A technology encoding gene and tolerance, applied in the field of bioengineering, can solve the problems of response to heavy metal stress that have not been studied, yield decline, etc.
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Embodiment 1
[0027] Embodiment 1, WRKY47 Acquisition of its coding gene
[0028] We screened heavy metal cadmium-tolerant mutants using Arabidopsis mutant seeds obtained from the American Arabidopsis Germplasm Bank. Use containing 200 μM CdCl 2 The Arabidopsis thaliana seed mutant library was screened on ½ MS medium, and a mutant strain tolerant to cadmium stress was obtained, and the mutant was found to be WRKY47 loss-of-function mutants, named wrky47- 1 . for further study WRKY47 Genes play a role in plant response to cadmium stress, we obtained another one from the American Arabidopsis Germplasm Bank WRKY47 loss-of-function mutants, named wrky47-2. The sequencing results were compared with the NCBI database Blast, and the T-DNA insertion site information was obtained through comprehensive analysis, see figure 1 . Using qRT-PCR, the wrky47-1 with wrky47-2 Transcript level identification was carried out and it was found that in the two mutants WRKY47 Genes were expressed...
Embodiment 2
[0030] Embodiment 2, cultivating cadmium-tolerant Arabidopsis thaliana
[0031] 1, WRKY47 Gene Overexpression Transgenic Lines WRKY47-OE3, OE6 the acquisition
[0032] To further verify the function of this gene in the regulation of heavy metal cadmium stress in plants, we constructed WRKY47 Gene overexpression vector ( 35S:WRKY47 ). Firstly, the target fragment is amplified. The wild-type Arabidopsis was normally cultured on ½ MS medium for two weeks, the total RNA of the plant was extracted, reverse-transcribed to synthesize cDNA, and the synthesized cDNA was used as a template for PCR to amplify a sufficient amount of the target product. Using the PCR product as a template, the second amplification was carried out for the purpose of introducing enzyme cutting sites. The PCR product and vector pCAMBIA1301 were digested and recovered. Then, the recovered and purified target DNA fragment and the vector were ligated overnight with T4 DNA ligase. The above connection s...
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