Stem mustard stress resistance gene bjefh1 and its plant expression vector and application
A technology of plant expression vector and stem mustard, which is applied in the field of stress-resistant gene BjEFh1 and its plant expression vector, can solve the problems of few reports on molecular mechanism research, less research on calmodulin-like and calcium-dependent protein kinase functions, and achieve Variety improvement, the effect of improving stress tolerance characteristics
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experiment example 1
[0023] Experimental Example 1: Cloning of Stress Resistance Gene BjEFh1 in Stem Mustard
[0024] 1 Main reagents: column type small amount plant total RNA extraction kit (W7021) was purchased from Shanghai Huashun Biotechnology Co., Ltd.; DNA purification and recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.; 5'-Full RACE Kit kit , M-MLV Reverse Transcriptase, Premix Ex Taq, DL2000DNA Marker, and pMD19-T vectors were all purchased from Dalian Bao Biological Engineering Co., Ltd.
[0025] 2 Experimental methods and steps
[0026] Acquisition of BjEFh1 gene sequence fragment SEQ ID NO.4: use column type small amount plant total RNA extraction kit to extract the total RNA of the tuber mustard stem, and take a small part of the total RNA to detect OD260 / OD280 by ultraviolet spectrophotometer The ratio of 1.93, the brightness of 28S by agarose gel electrophoresis is about 2 times that of 18S, indicating that the RNA has high purity and no degradation, which ...
experiment example 2
[0058] Experimental Example 2: Construction of the recombinant plant expression vector pCAMBIA1302-BjEFh1 of the stem mustard stress-resistant gene BjEFh1
[0059] 1 Main reagents: common plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; T4DNA ligase, restriction endonuclease Bgl II and BstE II were purchased from Dalian Bao Biological Engineering Co., Ltd.; strains containing plasmid pCAMBIA1302 were purchased from our laboratory save.
[0060] 2 Construction of recombinant plant expression vector pCAMBIA1302-BjEFh1
[0061] 2.1 Primer design: Design upstream and downstream primers according to the sequence of the coding region of BjEFh1 gene SEQ ID NO.1, and respectively introduce restriction site Bgl II (AGATCT) and BstE II (GGTAACC) sequences, the primers are:
[0062] Upstream primer BjEFh1-Bgl: 5'-AGATCTATGGAGAAGAGCTTATTG-3'
[0063] Downstream primer BjEFh1-Bst: 5'-GGTTACCTTAGCAGAAGCTACTC-3'
[0064] 2.2 PCR amplification: use the fi...
experiment example 3
[0070] Experimental Example 3: Genetic Transformation of Arabidopsis
[0071] 1 Main reagents: Fluorescence quantitative PCR kit SYBR Premix Ex Taq TM Kit was purchased from Dalian Bao Biological Engineering Co., Ltd.; W7021 Column Small Plant Total RNA Extraction Kit was purchased from Shanghai Huashun Biotechnology Co., Ltd.; DNase was purchased from Promega.
[0072] 2 Cultivation of Arabidopsis thaliana and Agrobacterium infection experiment
[0073] ① Take wild-type Arabidopsis seeds, sterilize with 75% ethanol for 1 min, and then wash with sterile water; then sterilize with 5% NaClO for 10 min, wash with sterile water for 5 times, and remove the residual NaClO. Add a certain amount of sterile water, and vernalize at 4°C for 3-4 days;
[0074] ②Cultivate Arabidopsis thaliana seeds that have been vernalized for 3-4 days on the MS blank medium for 7 days under light, and after the seedlings grow, transplant them to the vermiculite medium (vermiculite: nutrient soil: perl...
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