Application of oscpy and its encoded protein in improving rice quality
A quality and rice technology, applied in the field of biogenetic engineering, to achieve the effect of improving quality and improving rice quality
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Embodiment 1
[0073] Embodiment 1: Construction and genetic transformation of RNA interference carrier
[0074] (1) Obtaining the forward interference fragment: extract the total RNA of the young panicle of Zhonghua 11, reverse it into cDNA, and use the designed primer pair 3 according to the Oscpy gene sequence: SEQ ID NO.3CPY-SIF22: 5'-CGCGGATCCTCCATCGTGGAGCCGGAGAA-3' (The underline is the restriction site of BamHI) and SEQ ID NO.4CPY-SIR22: 5'-GCCTAAGCTTCCCTGTAGAAACCTCCTGCTCCAG-3' (the underline is the restriction site of HindIII). The positive interference fragment (SEQID NO.5) of Oscpy was amplified by RT-PCR, and was used for the construction of the interference vector after sequencing verification.
[0075] (2) Construction of RNA interference vector: forward interference fragment (SEQ ID NO.5) and pYL vector (see figure 1 ) were subjected to double enzyme digestion, purification, and recovery with BamH I and Hind III enzymes, and then ligated with T4 DNase. The ligated product was ...
Embodiment 2
[0086] Example 2 Detection of Transgenic Positive Rice Plants and Obtaining of Oscpy Expression Down-regulated Plants
[0087] After the DNA of transgenic rice leaves was extracted by CTAB method, hygromycin phosphotransferase gene-specific primers (SEQ ID NO.9:5'-CTGAACTCACCGCGACGTCTGTC-3'; SEQ ID NO.10:5'-TAGCGCGTCTGCTGCTCCATACA-3' ) The rice that was amplified positive by PCR was determined to be the transgenic positive plant of the T0 generation; using the fluorescent quantitative PCR method, with Oscpy gene-specific primers SEQ ID NO.11:5'-AGGGCACTGATGCTAGAGGA-3'; SEQ ID NO.12:5 '-CTCATTGGCGCCGGATTTTC-3', using rice OsActin as an internal reference gene, its primer sequence is SEQ ID NO.13:5'-ACCTTCAACACCCCTGCTAT-3' and SEQ ID NO.14:5'-CACCATCACCAGTCC AAC-3' to detect the down-regulated expression of Oscpy , and obtain Oscpy gene down-regulated expression RNAi rice plants and T-DNA insertion mutant plants, such as image 3 As shown, compared with wild-type Zhonghua 11, w...
Embodiment 3
[0088] Example 3 The chalky grain rate and chalkiness degree of Oscpy down-regulated rice are significantly reduced
[0089] The Oscpy gene down-regulated rice (RNAi interference and T-DNA insertion mutant) and wild-type rice were harvested, dried in a blast drying oven at 45°C for one week, and placed at room temperature for at least two months to remove the chaff. Utilize Ministry of Agriculture NY / T83-2017 to measure chalky grain rate, chalkiness degree, the result shows, the chalky grain rate of T-DNA mutant (cpy) is 32% ± 0.04, RNAi interference plant is 35.67% ± 0.12 and wild The chalky kernel rate of type Zhonghua 11 was 51.33%±0.05, the chalkiness of cpy and RNAi interference plants were 3.7% and 4%, respectively, and the chalkiness of wild type Zhonghua 11 was 5.5%. Compared with the wild-type control, the chalky grain rate and chalkiness degree of rice with down-regulated Oscpy gene expression were significantly reduced. We used the regulations of NY / T2639 to detect...
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