Application of the rice gene oslat5 in regulating the uptake and accumulation of diquat
A technology of diquat and rice, applied in the field of plant genetic engineering, can solve the problems of long research and development cycle, low efficiency, gene escape, etc., and achieve the effect of improving transduction, reducing content and increasing resistance
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Embodiment 1
[0041] Example 1: CRISPR knockout to construct OsLAT5 mutant plants
[0042] 1. Using the CRISPR / Cas9 system, select the target sequence according to the exon sequence of OsLAT5
[0043] Using a simple and efficient CRISPR / Cas9 system, a specific target sequence was selected based on the OsLAT5 exon sequence, the target sequence: 5'-ggcccactcctagcaattgc-3'. The target sequence is directed against the OsLAT5 gene and specifically inactivates the OsLAT5 protein.
[0044] 2. Construction of pCRISPR / Cas9 recombinant vector containing the above target sequence fragment
[0045] 1) According to the target sequence, design adapter primers with sticky ends
[0046] The designed target sequences were added to the specific sticky end adapters of the pCRISPR / Cas9 system, and the complete adapter primers were synthesized.
[0047] F1: 5'-TGTGTGGCCCACTCCTAGCAATTGC-3';
[0048] R1: 5'-AAACGCAATTGCTAGGAGTGGGCCA-3'.
[0049] 2) annealing the adaptor primers with sticky ends complementary...
Embodiment 2
[0077] Example 2: Rice resistance test to diquat
[0078] In order to detect the resistance of the function-deficient mutant rice obtained in Example 1 to diquat, plants grown in a rice incubator for 30 days were selected, and leaves of the same length were cut and soaked in 20 mL of 0.1% (v / v) Silwet L- 77 (Beijing Boao Tuoda Technology Co., Ltd.) solution (containing 5 mmol / L diquat), and placed in an artificial climate incubator for 36 hours. After 36 hours, the rice was taken out, and the leaves were washed with pure water 4 times to ensure that the drugs attached to the surface of the leaves were completely removed.
[0079] The experimental conditions and methods for measuring chlorophyll content were in accordance with the literature (Zhou Yong, Fan Xiaolei, Lin Yongjun, Chen Hao. (2018). Determination of chlorophyll content in rice. Bio-101e1010147.).
[0080] Measure the content of diquat: weigh and record, then put it into a mortar and grind with liquid nitrogen, ad...
Embodiment 3
[0082] Example 3 Real-time Fluorescence Quantitative Experiment after Diquat Treating Rice
[0083] Prepare a 0.1% (v / v) Silwet L-77 (Beijing Boao Tuoda Technology Co., Ltd.) solution (containing 5 mmol / L diquat), and spray the wild-type Zhonghua 11 plants grown for 25 days. % (v / v) Silwet L-77 solution (without drug) treatment served as a control. After treatment, the aerial parts (stems and leaves) RNA (OMEGA Cat#R6827-02) were extracted at 0h, 2h, 4h, and 9h after spray treatment, respectively, and real-time fluorescence quantification was performed after reverse transcription (Takara, PrimeScript RT reagent Kit with gDNAEraser). PCR to detect the expression of OsLAT5 gene. The experiment was repeated three times, and the results were averaged.
[0084]Real-time PCR was performed using Bio-Rad CFX96. The PCR reaction system (20 μL) was carried out according to the product instruction manual SYBR Green Real-Time PCR Master Mix reagent (Takara). The specific system is as f...
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