Application of osnrt2.3b in Improving Yield and Rice Quality
A high-quality rice technology, applied in the field of genetic engineering, can solve the problems of poor yield increase effect, large application amount, and high grain production cost
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Embodiment 1
[0016] Example 1 Construction of OsNRT2.3b gene overexpression vector
[0017] According to the full-length cDNA sequence of the OsNRT2.3b gene (obtained from the NCBI website), primers for the amplification of the full-length ORF were designed with the software Primer 5.0, and two different enzyme cleavage sites, SpeI and BglII, were designed at the 5' ends of the primers, respectively. For a different expression vector pCAMBIA1302, the sense strand was overNRT2.3b-F: 5'-caACTAGTGCTACCACGTGTTGGAGATG-3', the antisense strand overNRT2.3b-R: 5'-gaAGATCTGAGCAAACCACCAACAAG-3', purchased from the National Institute of Agrobiological Sciences ( The NIAS) OsNRT2.3b gene cDNA full-length cloning plasmid was used as the template to amplify the open reading frame (ORF) sequence. The PCR product containing the target gene was recovered and purified, and then connected to pMD19-Tcloning Vector, verified by enzyme digestion and sequenced. The correctly sequenced plasmids were digested wit...
Embodiment 2
[0018] Example 2 OsNRT2.3b overexpression plant acquisition and molecular biology identification
[0019] GM T 0 After the substitute seedlings were screened by hygromycin or GUS staining, 50 transgenic positive lines of OsNRT2.3b-Ox (35S promoter) were obtained, and the obtained transgenic seedlings were transplanted into the field for breeding to obtain T 0 Substitute seeds. Among the positive lines, we obtained 18 OsNRT2.3b-Ox transgenic lines whose phenotype was significantly different from that of the wild type. In order to further determine whether the target gene was enhanced in these lines, the T 0 The total RNA was extracted from the leaves of the transgenic seedlings for preliminary RT-PCR identification, and several lines with enhanced expression of the target gene were obtained.
[0020] To verify the genetic stability of the transgenic lines, the T 0 Substitute seeds and wild-type seeds were hydroponic and seedlings were germinated for 2-3 days and then transfe...
Embodiment 3
[0021] Example 3 Experimental Design
[0022] The experiment was set up with three fertilizer application levels (M: organic fertilizer treatment, NPK: inorganic fertilizer treatment, NPKM: organic fertilizer), and two fertilizer gradients (1: low, 2: high), namely organic low (M1) and organic high (M2) ), inorganic low (NPK1), inorganic high (NPK2), inorganic fertilizer + organic fertilizer combined application low (NPKM1), inorganic fertilizer + organic fertilizer combined application high (NPKM2), a total of 6 treatments, each treatment set 3 replicates, A total of 18 cells, arranged in random blocks, each cell has an area of 3.75m 2 (3m×1.25m), the depth of the plot is 1.2 m, and the plots are separated by bricks and concrete to prevent the exchange of water and fertilizer between plots. Nitrogen fertilizer was applied according to base fertilizer: tiller fertilizer: ear fertilizer = 5: 2: 3, and urea (containing N 46%) was applied. Among them, N ≥ 10% in organic ferti...
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