Plants with enhanced tolerance to heat stress
A technology of heat stress, plants, applied in the field of plant molecular biology, can solve problems such as not showing phenotypes
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Embodiment 1
[0118] Example 1: Identification of thermotolerant mutants
[0119] Arabidopsis thalianavar.Columbia was transformed with the pSKI15 vector, which contains four 35S enhancer sequences (see the article published by Wiegel et al. in 2000). Arabidopsis seeds of the T3 generation were obtained from the Arabidopsis Biological Resource Center (ABRC) and used to prepare the T4 generation, which was used for genetic screening experiments. The Arabidopsis h138 mutant was identified which, when exposed to heat stress at about 45°C for about 30 to 60 minutes during flowering, had reduced flower abortion or no flower abortion. The initial isolation was retested by subjecting the flowering plants to a temperature gradient from 22°C to 45°C for 1 hour, followed by heat stress at 45°C for 2 hours, for flower yield, seed set and Seed development was monitored and selected for heat stress profiles.
Embodiment 2
[0120] Example 2: Identification of heat stress MYB68 gene
[0121] Genome walking was performed as follows to localize the insertion of the T-DNA activation tag. Genomic DNA was purified by phenol:chloroform extraction using 10-day-old mutant h138 seedlings. The above-mentioned isolated DNA is then digested with the restriction enzyme, such as EcoRV, PvuII, NruI, or StuI, to generate a DNA fragment with a blunt end. The above-mentioned fragments from each digestion reaction are ligated with adapters formed by annealing of two oligonucleotides (oligos), wherein the two oligonucleotides (oligos) ) are: Adapter 1 and Adapter 2. Addition of the adapters to the DNA fragments enables polymerase chain reaction (PCR) amplification using primers specific for the adapters and T-DNA insertion sites.
[0122] Two rounds of polymerase chain reaction (PCR) were performed to generate DNA fragments for further sequencing analysis. Primer HeatL1 (sequence) and primer CAP1 (sequence) wer...
Embodiment 3
[0123] Example 3: Physiological characteristics of h138 mutant (myb68)
[0124] Plants were evaluated for heat tolerance during flowering and scored based on the number of aborted flowers or pods and final seed yield. Plants were grown in a controlled environmental chamber with optimal growth conditions of 16 hours of light at 200uE and 8 hours of darkness at 22°C and 70% relative humidity. Three groups of plants were used in the experimental design: 1) a control group grown under optimal conditions; 2) a group subjected to 3 hours of heat treatment and 3) a group subjected to 4 hours of heat treatment. Heat treatment was carried out 6 days after the first flowering and the temperature was ramped from 22°C to 44°C over a period of 1 hour. Each group of plants contains the myb68 mutant and the wild-type control (myb68-blank) of the mutant, and each treatment of each item (entry) carries out 10 replicate pots (replicatepots), and in each pot (pot) Contains 5 plants. Plants ...
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