Rice transcription factor OsHRS1 mutant construction method and application thereof in rice drought stress
A rice transcription factor, rice technology, applied in the field of crop genetics and breeding
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Embodiment 1
[0040] Example 1: Obtaining the Nucleotide Sequence and Amino Acid Sequence of the Rice Transcription Factor OsHRS1
[0041] Log in to the homepage of the rice database center (http: / / www.ricedata.cn / gene / ), and search for the nucleotide sequence and amino acid sequence of OsHRS1 according to its LOC_Os03g55590 number. Reference sequence such as figure 1 shown.
Embodiment 2
[0042] Example 2: Subcellular Localization of Rice Transcription Factor OsHRS1
[0043] Through the multiple cloning site of the pEXT06 / g empty vector sequence, design forward primers and reverse primers with pEXT06 / g vector sequences at both ends of the CDS region of the target gene OsHRS1. The pEXT06 / g vector has a GFP tag. Green under fluorescence. The primers are as follows (the bold is the vector sequence, and the following is the first and last ends of the CDS region of the target gene):
[0044]
[0045] The fragment of OsHRS1 carrying the vector sequence was obtained by PCR, and a DNA fragment of about 1259 bp was recovered using a DNA recovery kit, and this fragment was connected with the corresponding pEXT06 / g vector by homologous recombination. The obtained vector was named pEXT06 / g-HRS1. Agrobacterium GV3101 was transformed after sequencing.
[0046] (1) Pick a single colony of transformed Agrobacterium GV3101 in 3ml LB+rifampicin+gentamycin liquid medium for...
Embodiment 3
[0053] Example 3: Identification of OsHRS1 knockout material T0 generation positive plants
[0054] The full-length cDNA (open reading frame) of the OsHRS1 gene was amplified from the total cDNA of rice Nipponbare by RT-PCR. First-strand synthesis was performed according to the instructions of Roche's Revert Aid TM First Strand cDNA Synthesis Kit. The following primers were designed according to the sequence of the OsHRS1 gene, F: 5'-ACCCTTCCTCCAGTTCTTG-3' (SEQ ID NO.1) and R: 5'-TCCACGAGCACGATCTTTC-3' (SEQ ID NO.2) were RT-PCR primers, using RT -PCR for cDNA amplification, the amplification conditions are: 95°C preheating for 3 minutes; 95°C for 10s, 60°C for 30s, 72°C for 2min, a total of 35 cycles; 72°C for 10min. After PCR, electrophoresis analysis was performed, and the amplified fragment of about 1700 bp was recovered using the gel recovery kit of Kangwei Century Company. Connect the recovered gel fragments to T vectors to transform competent cells, pick white colonies...
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