Improving drought-resistant property of plant by rice drought inducing gene promoter LEAP
A promoter and rice technology, applied in the field of plant genetic engineering, to achieve the effect of improving drought tolerance
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Embodiment 1
[0023] Example 1, LEAP promoter isolation and identification
[0024] Through the analysis of the drought-induced gene expression profile of the rice variety "Zhonghan 5" (a commercial variety provided by the Shanghai Academy of Agricultural Sciences), an EST (expression Sequence signature), it was found through sequence analysis that the EST was a partial sequence of the 3' end of a LEA protein gene. To isolate the promoter of the gene, the full-length cDNA of the gene was first isolated. Specific steps are as follows:
[0025] A gene-specific primer P1: 5'-ACACCCGTCAGAAA (complementary sequence) was designed based on the 3' end sequence of the EST. Using TRIZOL reagent (purchased from Invitrogen Company) to extract leaf total RNA from drought stress-treated rice variety "Zhonghan 5" (extraction method according to the above-mentioned TRIZOL reagent instruction manual), after using the kit (purchased from Invitrogen Company) to isolate complete mRNA The first strand of cDN...
Embodiment 2
[0027] Example 2, Detection of Induced Expression of Rice Endogenous Gene OsLEA1
[0028] The rice variety "Minghui 63" (a rice variety popularized in China) was used as the material, and the treatments of drought, chilling injury, high-salt stress and ABA were respectively carried out at the 3-leaf stage. Drought treatment is to soak the roots of seedlings with 20% polyethylene glycol (trade name PEG6000) and take samples once a day at noon for 5 consecutive days. The chilling injury treatment was to place the seedlings in a growth chamber at 4°C, and take samples after 0h, 1h, 3h, 6h, 12h and 24h. For high salt stress, the roots of the seedlings were soaked in 200mM / L NaCl solution and samples were taken after 0h, 1h, 3h, 6h, 12h and 24h. For ABA treatment, the roots of the seedlings were soaked in 100 μM / L ABA solution and samples were taken after 0h, 1h, 3h, 6h, 12h and 24h. After extracting the total RNA (Trizol reagent, Invitrogen) from the leaves, the RNA was transfer...
Embodiment 3
[0029] Example 3, Identification of drought-induced activity of LEAP promoter
[0030] The embodiment of the present invention is to construct the GUS gene expression vector of the LEAP promoter and transform it into the rice variety "Zhonghua 11" (commercial variety from the Crop Research Institute of the Chinese Academy of Agricultural Sciences) to quantitatively detect the drought-induced expression activity of the LEAP promoter. The specific operation is as follows:
[0031] First, the PCR product of the LEAP promoter isolated in Example 1 was ligated into the pGEM-T Easy vector (purchased from Promega), transformed into Escherichia coli DH10B (purchased from Promega), and positive clones were screened. Because the PCR product contains a Hind III restriction site inside it (see the appendix for the PCR product sequence and distribution of the restriction site for details). figure 1 ), so the 1030bp insert fragment connected to the vector can be cut into two expected frag...
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