E3 ubiquitin ligase gene ospiw regulates the function and application of rice root
A ubiquitin ligase, rice technology, applied in the field of plant genetic engineering, can solve the problems of rice root growth retardation, division frequency affecting meristems, etc.
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Embodiment 1
[0041] Embodiment 1: Cloning of OsPIW gene
[0042] The cloning (LOC_Os06g19680) of the gene OsPIW of the present invention is mainly obtained by the method of RT-PCR (method is referred to: J. Sam Brook, EF Fritsch, T Mani Artis, Huang Peitang, Wang Jiaxi etc. translation, molecular cloning experiment Guide (Third Edition), Beijing, Science Press, 2002 edition). The specific operation steps are as follows:
[0043] 1) RNA was extracted from the seedling leaves of the rice variety "Zhonghua 11" (gifted by the Institute of Crop Science, Chinese Academy of Agricultural Sciences), using Invitrogen's Trizol extraction kit for RNA extraction (see the kit's instruction manual for specific steps);
[0044] 2) The steps of RT-PCR reverse transcription to synthesize the first strand of cDNA are as follows: ①Preparation Mixture 1: Total RNA 4μg, DNaseI2U, 10DNaseI buffer 1μl, add DEPC (diethylpyrocarbonate, a strong inhibitor of RNase) for treatment Add water (0.01% DEPC) to 10 μl, mi...
Embodiment 2
[0051] Embodiment 2: Construction of double-strand suppression dsRNAi vector
[0052] According to the full-length cDNA sequence of the OsPIW gene published in the Rice Genome database (http: / / rice.plantbiology.msu.edu / index.shtml), design double-stranded suppression dsRNAi primers, and add SpeI and KpnI to the 5' end of the upstream primer Restriction endonuclease sites, Sac I and BamH I restriction endonuclease sites were added sequentially at the 5' end of the downstream primer. After the target sequence was amplified by PCR, the amplified product was connected to pGEMT-vector (purchased from Promega, USA) by T / A cloning for sequencing verification, and the amplified DNA fragment was shown in the sequence table as SEQ ID NO:3.
[0053] Specific steps are as follows:
[0054] 1) The T / A clone of the RNAi fragment with OsPIW was digested with Kpn I and BamHI restriction endonucleases, and the target band was recovered, and the vector plasmid pDS1301 (provided by Huazhong Agr...
Embodiment 3
[0060] Example 3. The functional verification of OsPIW in rice root growth and development,
[0061] The steps of this embodiment are as follows:
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