Plant Stress Tolerance Related Transcription Factors and Their Encoding Genes and Applications
A plant stress tolerance and transcription factor technology, which is applied to plant stress tolerance-related transcription factors and their encoded genes and applications, can solve the problems of lack of systematic theoretical research on stress resistance mechanism of pear rootstocks, inability to fully develop and utilize them, and improve the The effect of salt tolerance and drought tolerance
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Embodiment 1
[0033] Example 1 Screening of PbHB12 gene encoding gene PbHB12 related to stress tolerance in Du pear and its cDNA clone
[0034] Using Arabidopsis thaliana ATHB7 (NM_130233.4) as a probe, NCBI homology alignment was carried out, and a primer pair (upstream primer PbHB12_F1 and downstream primer PbHB12_F1 and downstream Primer PbHB12_F2) was used to amplify stress tolerance-related transcription factor PbHB12 gene from Du pear.
[0035] The rooted seedlings of Du pear were treated with 150mM NaCl aqueous solution, and the leaves of Du pear were cut after 24 hours, and the total RNA of the stress-treated leaves of Du pear was extracted by conventional methods. The cDNA first-strand synthesis kit (M-MuLV First Strand cDNASynthesis Kit, Shanghai Sangon Bioengineering Co., Ltd.) was used to synthesize the first-strand cDNA, and then using the cDNA as a template, the upstream primer PbHB12_F1, the downstream primer PbHB12_F2 and the linker primer pair ( OUTER and INNER) for nested...
Embodiment 2
[0046] Example 2, PbHB12 gene tissue expression characteristics and stress expression research
[0047] In order to confirm whether the PbHB12 gene is involved in the regulation of plant stress tolerance, the present invention uses semi-quantitative RT-PCR and real-time fluorescent quantitative RT-PCR techniques to analyze the expression patterns of the PbHB12 gene in different tissues of Duli pear and under different stress conditions.
[0048] 1. Analysis of tissue expression of PbHB12 gene under normal growth conditions
[0049] Under normal growth conditions, the roots, stems, leaves, flowers, and fruits of Duli pear were collected and quickly frozen in liquid nitrogen. About 200 mg of each part was taken, and ground into powder in liquid nitrogen. RNA was extracted using TRIzon kit (Shanghai Life Technologies Corporation). Using the RNA extracted from each part as a template, reverse transcription kit ( PrimeScript TM RTreagent Kit with gDNA Eraser, Treasure Bioengin...
Embodiment 3
[0066] Embodiment 3, transformation PbHB12 escherichia coli growth curve and gene expression analysis
[0067] The prokaryotic expression vector pET-22b(+) and the recombinant plasmid p-MD18-PbHB12 obtained in Example 1 were digested with EcoR Ⅰ and HindIII (Thermo Scientific, USA) respectively, and TaKaRa MiniBESTAgarose Gel DNAExtraction Kit Ver4.0 was used to (Treasure Bioengineering (Dalian) Co., Ltd.) for gel recovery, and the recovered product was ligated with T4 DNA Ligase (Thermo Scientific, USA) and then transformed into Escherichia coli E.coli DH5α competent cells (Treasure Bioengineering (Dalian) Co., Ltd.). The recombinant plasmid was picked on the ampicillin (Amp) resistant LB plate, identified by enzyme digestion and sequenced, and the expression vector successfully fused with the target gene was named pET-22b(+)-PbHB12.
[0068] The empty vectors pET-22b(+) and pET-22b(+)-PbHB12 were respectively transformed into BL21(DE3) to obtain recombinant bacteria BL21-pET...
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