Application of Rice osrhd1-1 Gene in the Breeding of Rice Male Male Sterile Line
A technology of male sterile line, rice, applied in application, genetic engineering, plant genetic improvement and other directions
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Embodiment 1
[0022] Example 1: Construction of Rice OsRHD1-1 Gene Knockout Vector and Rice Genetic Transformation
[0023] The base sequence of the rice OsRHD1-1 gene is shown in SEQ ID NO.1.
[0024] (1) According to the "http: / / skl.scau.edu.cn / targetdesign / " website, design the specific knockout target of OsRHD1-1 gene, the sequence is as follows:
[0025] Knockdown target: TCCTCCAGCTTCTCCAACT.
[0026] According to the knockout target sequence, the expression sequence that expresses the corresponding sgRNA sequence is designed.
[0027] (2) Use NEB's BsaI endonuclease to double-digest (two positions with the same enzyme) pYLCRISPR / Cas9Pubi-H vector
[0028] The system is as follows:
[0029] buffer 2 μL; vector 1 μL; BsaI endonuclease 1 μL; ddH 2 O was added to a total volume of 10 μL.
[0030] The digestion conditions are: 37°C for 30 minutes.
[0031] (3) Knockout target synthesis
[0032] The target sequences of the synthesized primers are as follows:
[0033] 595100-aFP: 5...
Embodiment 2
[0050] Example 2: Gene editing of rice OsRHD1-1 gene knockout lines
[0051] In order to detect the pollen development in the florets of the rice OsRHD1-1 gene knockout line, the DNA of the gene-edited seedlings was extracted, and the amplified fragment was sent to Hangzhou Shangya Company for sequencing.
[0052] Such as figure 1 As shown in the results of sequencing, it was found that the two DNA single strands of the three sequenced strains were mutated.
Embodiment 3
[0053] Example 3: Observation of anther traits in florets of rice OsRHD1-1 gene knockout lines
[0054] The anthers in the florets of rice OsRHD1-1 gene knockout lines became smaller, lighter in color and stunted.
[0055] Observation of the floret traits of the rice OsRHD1-1 gene knockout line obtained in Example 2, such as figure 2 As shown, the anthers in the florets of the rice OsRHD1-1 gene knockout line became smaller, lighter in color, and stunted.
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