Transcription factor gene OsHOX4 for controlling synthesis of rice gibberellin and use thereof
A transcription factor, gibberellin technology, applied in the genetic engineering of rice, new plant field, can solve the problem that the function needs to be further studied
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Embodiment 1
[0032] The cloning of embodiment 1 candidate gene OsHOX4:
[0033] Using Arabidopsis AtHB6 (Homeobox (HOX) class transcription factor, NCBI protein accession number AAD41726; in the REDB website ( http: / / redb.ncpgr.cn / ), select the Regularblast tool in the "RiceEST" database for tblastx analysis, and find the homologous clone in rice (numbered as EI#77-F09), from the cDNA library (see references: Chu Zhaohui, Peng Kaiman, etc., the whole growth period of rice is uniform The construction and identification of the cDNA library, Science Bulletin, 2002, 47 (21).1656-1662) obtained this clone, sequenced and obtained the full-length cDNA sequence (sequence determination was completed by the Shanghai National Gene Sequencing Center), and the candidate gene was named as OsHOX4, its cDNA nucleotide sequence is shown in SEQ NO: 1 in the sequence table. Use GenDoc software (version is selected from: GenDoc3.2) to the protein sequence analysis of the gene OsHOX4 of reported AtHB6 and th...
Embodiment 2
[0034] The construction of embodiment 2 binary Ti plasmid vector and the establishment of transforming Agrobacterium:
[0035] Specific steps are as follows:
[0036] 1) Cloning the cDNA with OsHOX4, loading the plasmid as pSPORT1 (purchased from Promega, a special plasmid vector for constructing cDNA library) digested with KpnI and BamHI, and then isolating the target gene fragment (plus the enzyme derived from pSPORT1 Cut the linker fragment, the size of the cloned OsHOX4 gene is 1560bp), and directly connect with the vector plasmid pDS1301 digested with KpnI and BamHI (the endonucleases used are all purchased from TAKARA company, and the specific usage and dosage refer to the product manual; The ligase was purchased from Invitrogen, and the usage and dosage refer to the instructions of the product);
[0037] 2) The connection product is introduced into DH10B (purchased from Promega Company) by electrotransformation (the electrotransformer is a product of eppendorf, and the...
Embodiment 3
[0040] The transformation of embodiment 3 binary Ti plasmid vectors and the positive detection of transgenic plants:
[0041] Specific steps are as follows:
[0042] 1) The transformed strain T-HOX4 obtained in Example 2 was introduced into the recipient rice variety "Nipponbare", and the transformation method was referred to the method reported by Hiei et al. (see: Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal 6:271-282). The obtained T0 transgenic rice plants were named HOX4-n, wherein n=1, 2, 3...represent different transgenic families.
[0043] Total DNA was extracted from leaves of transformed rice plants of the T0 generation. The DNA extraction method is the CTAB method (with reference to Zhang et al., the method and steps shown in genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis, 1992, Theor Appl Genet, 83, 495-499), and th...
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