Application of oscct6 gene in controlling rice yield, flowering period and plant height
A gene, rice technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that the CCT gene has not been functionally verified.
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Embodiment 1
[0022] Embodiment 1: Isolation of OsCCT6 gene gDNA sequence
[0023] The OsCCT6 gene (LOC_Os06g44450) corresponding to the present invention has its genome sequence (http: / / rice.plantbiology.msu.edu / cgi-bin / sequence_display.edu) published in the TIGR database (http: / / rice.plantbiology.msu.edu). cgi?orf=LOC_Os06g44450.1). Total DNA was extracted from rice variety "Minghui 63" (MH63) leaves by conventional PCR method (see: Dieffenbacher C W, Devickler QS, Guide to PCR Technique Experiment, Beijing, Science Press, 1998), amplified The gDNA sequence of OsCCT6 was obtained. The specific amplification method of gDNA is as follows:
[0024] 1 Extract the total DNA from the leaves of the rice variety MH63. The DNA extraction method refers to the CTAB method (see Zhang et al., genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis, 1992, Theor Appl Genet, 83, 495-499).
[0025] 2 Then predict the ORF of the gene according to the TIGR database (http:...
Embodiment 2
[0030] Embodiment 2: Construction of OsCCT6 overexpression vector
[0031] 1 Digest the above T / A clone with OsCCT6 genome sequence with Kpn I and Xba I, recover the target band, and digest the binary overexpression vector pCAMBIA 1301S with Kpn I and Xba I (provided by Huazhong Agricultural University Crop Genetics Provided by Mr. Lian Xingming from the State Key Laboratory of Improvement, see the attached vector diagram figure 1 ) connection (the endonucleases used were all purchased from Treasure Bioengineering Dalian Co., Ltd., and the usage and dosage were in accordance with the product instructions provided by the company; the ligase was a product of Shanghai Handsome Biotechnology Co., Ltd., and the usage and dosage were in accordance with the company’s product instructions) ;
[0032] 2. The ligation product was introduced into Escherichia coli DH10B (purchased from Promega (Beijing) Biotechnology Co., Ltd., Promega, USA) by electroporation (the electroporation instru...
Embodiment 3
[0034] Example 3: Transformation of binary Ti plasmid vector and detection of positive and expression levels of transgenic plants
[0035] 1 The correct sequenced overexpression vector pCAMBIA 1301S-OsCCT6-MH63 was introduced into Agrobacterium (A. tumefaciens) EHA105 (purchased from Australia CAMBIA laboratory) strains;
[0036] 2 Transform the overexpressed Agrobacterium in step 1 into the rice recipient variety "Zhonghua 11" (a rice variety publicly reported and released by the Institute of Crop Science, Chinese Academy of Agricultural Sciences)
[0037] 3 The main steps of the genetic transformation of the present invention, the culture medium and the preparation method thereof are as follows:
[0038] 1) Reagent and solution abbreviation
[0039] The abbreviation of the plant hormone used in culture medium among the present invention is as follows: 6-BA (6-BenzylaminoPurine, 6-benzyl adenine); CN (Carbenicillin, carbenicillin); KT (Kinetin, kinetin); NAA ( Napthalene a...
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