Application of rice gene oslat3 in regulation of uptake and accumulation of diquat
A technology of diquat and rice, applied in the field of plant genetic engineering, can solve the problems of low efficacy, genetic pollution, time-consuming and labor-consuming, etc., and achieve the effects of reducing content, improving resistance, and reducing health risks
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Embodiment 1
[0041] Example 1: CRISPR knockout construction of OsLAT3 mutant plants
[0042] 1. Use the CRISPR / Cas9 system to select the target sequence according to the exon sequence of OsLAT3
[0043] Using the simple and efficient CRISPR / Cas9 system, select the specific target sequence according to the OsLAT3 exon sequence, the target sequence: 5'-GCGCCGAGCAGGCCGTGAGCG-3'. The target sequence is directed against the OsLAT3 gene and specifically inactivates the OsLAT3 protein.
[0044] 2. Construct the pCRISPR / Cas9 recombinant vector containing the above target sequence fragment
[0045] 1) According to the target sequence, design adapter primers with sticky ends
[0046] Add the designed target sequence to the pCRISPR / Cas9 system's specific sticky end adapter, and synthesize the complete adapter primer.
[0047] F1: 5'-TGTGTGCGCCGAGCAGGCCGTGAGC-3';
[0048] R1: 5'-AAACGCTCACGGCCTGCTCGGCGCA-3'.
[0049] 2) Annealing the adapter primer with sticky ends to complement each other to for...
Embodiment 2
[0077] Embodiment 2: Rice resistance test to diquat
[0078] In order to detect the resistance of the functional loss mutant rice obtained in Example 1 to diquat, select plants grown in a rice incubator for 30 days, cut leaves of equal length and soak them in 20mL 0.1% (v / v) Silwet L- 77 (Beijing Boao Tuoda Technology Co., Ltd.) solution (containing 5 mmol / L diquat), and cultured in an artificial climate incubator for 36 hours. After 36 hours, the rice was taken out, and the leaves were washed 4 times with pure water to ensure that the drug attached to the surface of the leaves was completely removed, and the surface moisture was dried with filter paper, and the phenotype was observed and photographed for subsequent experiments.
[0079] The experimental conditions and methods for measuring chlorophyll content are in accordance with the literature (Zhou Yong, Fan Xiaolei, Lin Yongjun, Chen Hao. (2018). Determination of chlorophyll content in rice. Bio-101e1010147.).
[0080] ...
Embodiment 3
[0082] Example 3 Real-time Fluorescent Quantitative Test of Diquat Treated Paddy Rice
[0083] Prepare 0.1% (v / v) Silwet L-77 (Beijing Boao Tuoda Science and Technology Co., Ltd.) solution (containing 5mmol / L diquat), and spray the 25-day-old wild-type species Zhonghua 11 plants with 0.1 % (v / v) Silwet L-77 solution (without drug) was used as control. After the treatment, RNA (OMEGA Cat#R6827-02) of aerial parts (stems and leaves) was extracted at 0, 2, 4, and 9 hours respectively, and real-time fluorescent quantitative PCR was performed after reverse transcription (Takara, PrimeScript RT reagent Kit with gDNA Eraser). The expression of OsLAT3 gene was detected. The experiment was repeated three times, and the results were averaged.
[0084]Real-time fluorescent quantitative 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), and the specific ...
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