Plant stress resistance-related genes and their encoded proteins and their applications
A coding and genetic technology, applied in the field of plant stress resistance-related genes and their encoded proteins, can solve the problems of accumulation of metabolites, destabilization of cell membrane protein structure and activity, and destruction of metabolism
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Embodiment 1
[0098] Example 1 Identification of Arabidopsis agl16 deletion mutants at the DNA and RNA levels
[0099] First, a T-DNA insertion deletion mutation in the exon of the AT3G57230 gene near the stop codon was obtained from the Arabidopsis thaliana seed bank ABRC (Arabidopsis Biological Resource Center, The Ohio State University Rightmire Hall 1060 Carmack Road, Columbus, OH 43210 USA). Body Salk_104701( figure 1 A). Take seedlings or adult seedlings for plant DNA extraction to obtain a genome template, use primers LP and RP to amplify by PCR, complete genome level identification, and obtain homozygous lines ( figure 1 B). After RNA extraction, purified RNA was obtained, and then reverse transcription was performed to obtain a cDNA template. Primers P1 and P2 were used, and Ubiqutin5 gene was used as an internal reference. After qRT-PCR detection, it was found that AT3G57230 gene in all strains of agl16 Almost no expression, that is, the five lines of agl16 are all homozygous f...
Embodiment 2
[0121] Example 2 Acquisition of Arabidopsis AGL16 Overexpression Line OX
[0122] First, the primers LP and RP of AGL16 coding region (CDS) with full adapter (attB) connection were designed, and the target fragment was obtained by PCR ( figure 2 A), and recycle it. Using a high-throughput vector construction method (Gateway Technology) (Wang Zonggui, Zheng Wenling, Ma Wenli; Pathway Cloning System: New Progress in DNA Recombination Technology. Chinese Journal of Biotechnology (2003), Vol. 23, No. 7), the recovered AGL16 -CDS destination fragment, using Gateway R BP Clonase TM AGL16-CDS was recombinantly cloned into the shuttle vector (purchased from Invitrogen) of pDONR207 by IIEnzyme Mix kit (Invitrogen), and again passed through Gateway TM The intermediate shuttle vector was recombinantly cloned into the pCB2004 final vector (Xiang et al., 1999; Lei et al., 2007) between attR1 and attR2 by cloning technology, and the correct pCB2004-AGL16 final vector was obtained by r...
Embodiment 3
[0131] Example 3 Arabidopsis seed germination experiment
[0132] First, agl16 (that is, the Salk_104701 mutant purchased from the Arabidopsis seed bank ABRC, which is a homozygous mutant lacking agl16), OX (a homozygous line overexpressing AGL16 constructed in Example 2), Col-0 ( That is, wild-type Arabidopsis thaliana, as the experimental control material) seeds are divided into 2mL EP tubes, ensure a small amount of multiple tubes, and 1mL of 10% Bleach is added to the tube (that is, 100mL Bleach is composed of 10mL Blue Moon 84 disinfectant and 90mL pure water. composition), placed on a shaker at 230 rpm for cleaning for 15 minutes, and then washed 4 times with sterilized pure water on an ultra-clean workbench. The plump seed spots after vernalization were selected and placed in MS medium supplemented with 0 mM, 120 mM NaCl, 150 mM NaCl, and 250 mM mannitol, respectively. The operation process was completed on an ultra-clean workbench. After the seeding was completed, the...
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