Application of Arabidopsis atdiqd Gene in Improving Plant Drought Resistance and Photosynthetic Efficiency
A technology of Arabidopsis thaliana and photosynthetic rate, applied in the application field of Arabidopsis thaliana AtDIQD gene in improving plant drought resistance and photosynthetic efficiency
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Embodiment 1
[0026] Example 1: Obtaining and identification of T-DNA insertion mutant atdiqd.
[0027] The acquisition of T-DNA insertion mutant atdiqd: on the Arabidopsis thaliana gene database website Tair website (https: / / www.arabidopsis.org / ), input the gene number of AtDIQD (AT3G49260), select and order the T-DNA insertion mutant ( SALK_047040C), T-DNA insertion mutant atdiqd material was obtained.
[0028] Identification of T-DNA insertion mutant atdiqd: Download the cDNA sequence of the gene, and design primers: AtDIQD-F: 5'-TACCAGACAGCATTCCAAT-3' and AtDIQD-R: 5'-TTCTTCGCATTCGTTTCC-3'. Real-time quantitative PCR was used to detect the expression of AtDIQD gene in T-DNA insertion mutants atdiqd and Col-0.
[0029] The specific steps are: (1) extract the RNA of the atdiqd mutant and Col-0 Arabidopsis leaves respectively; (2) reverse transcribe the RNA into cDNA, and use the obtained cDNA as a template for PCR reaction; (3) perform real-time quantification PCR reaction. The results...
Embodiment 2
[0037] Example 2: Construction and genetic transformation of AtDIQD overexpression vector.
[0038]In order to further study the role of AtDIQD gene in regulating the drought resistance of Arabidopsis, plants overexpressing AtDIQD gene were obtained through genetic transformation.
[0039] 1. Construction of AtDIQD overexpression vector
[0040] Download the CDS sequence (sequence as shown in SEQ ID NO:1) of this gene on Tair website, take AtDIQD gene CDS sequence as template (use Primer5 biological software to design primers (front and back primers add attB1 and attB2 respectively), primer is: AtDIQD -CDS-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGGGAAGAAAGGGAGTG-3', AtDIQD-CDS-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAATGATCATGCCTCCAGCC-3'; PCR amplification of the AtDIQD target fragment, the amplified product is the sequence shown in SEQ ID NO: 1 of the present invention ( 1-1416bp), the PCR reaction conditions are: 95°C pre-denaturation for 5min; 95°C for 30sec, 56°C for 30sec, 7...
Embodiment 3
[0051] Example 3: Construction and genetic transformation of a vector for GUS expression driven by AtDIQD promoter
[0052] Take AtDIQD target gene coding region initiation codon ATG upstream 1576bp as its promoter (the sequence of the promoter is shown in SEQ ID NO:3) amplification template, use Primer 5 software to design primers (before and after the primers respectively add attB1 and attB2); the specific primer sequence is: AtDIQD-P-F: 5'-GGGGACAAGTTTGTACAAAAAGCAGGCTTAAAAAACCCGAACCAGAGG-3', AtDIQD-P-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAATTTCCGGCAACACTCTT-3', the target fragment is amplified by PCR, and the amplified product is SEQ ID NO: 3 of the present invention Sequences indicated (1-1576 bp). The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; 32 cycles at 95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min; extension at 72°C for 5 min. Use the Gateway method to construct the vector, and use PEG8000 / 30mmol / L MgCl for the target fragment 2 After ...
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