Rice-derived insect-resistance-related gene oslrr6 and its encoded products and applications
A rice and genetic technology, applied in the direction of climate change adaptation, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of resistance to multiple or newly evolved biological types of rice planthoppers, etc.
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Embodiment 1
[0030] Example 1. Acquisition and sequence analysis of OsLRR6 gene
[0031] 1) Extraction, quality inspection and first-strand cDNA synthesis of total RNA from rice leaf sheaths
[0032] 2) Using the first strand of the total cDNA as a template, carry out a PCR reaction to obtain the full-length gene sequence of OsLRR6
[0033] OsLRR6-F1:5'-CATTGTGCCAACTGCCAAGG-3';
[0034] OsLRR6-R1: 5'-ACTCCCATCTCCCAAGGTGT-3';
[0035] PCR amplification conditions: 95℃×4min→(98℃×15sec→60℃×40sec→68℃×50sec)×35 cycles→68℃×5min, see the specific PCR amplification product figure 1 .
[0036] 3) OsLRR6 gene sequence analysis
[0037] The obtained PCR product was sent to Nanjing GenScript Company for sequencing, and the sequencing result was the sequence SEQID No.1 in the sequence table. We named this gene OsLRR6.
Embodiment 2
[0038] Example 2. Analysis of OsLRR6 expression characteristics before and after brown planthopper egg-laying female adults
[0039] 1) Brown planthopper treatment
[0040] A cylindrical glass cover (4cm in diameter, 8cm in height, with 24 air holes with a diameter of 0.8mm evenly distributed on the wall) was fixed at the base of the leaf sheath of rice. The top is sealed with a sponge. After inoculation with N. lugens, the outer leaf sheaths of the damaged parts were clipped at different time points, immediately immersed in liquid nitrogen, and stored at -80°C for later use. Healthy rice leaf sheaths sheathed in an empty glass cover were used as controls.
[0041] 2) RNA extraction and expression analysis
[0042] The total RNA was extracted with the MiniBEST Plant RNA Extraction Kit (TaKaRa), and the concentration, purity and quality of the obtained RNA were detected and evaluated with an ultra-trace protein nucleic acid spectrophotometer (BioDrop) and formaldehyde gel de...
Embodiment 3
[0050] Example 3. Acquisition of OsLRR6 transgenic lines
[0051] 1) Synthesize the sense strand and antisense strand of the target sequence described in claim 3 (adding the linker sequence respectively), anneal and synthesize the double strand of the target sequence, and use the DNA subcloning method to connect it into the experiment by Professor Shu Qingyao of Zhejiang University. The pHun4c12-Beli vector was transformed into the chamber to obtain the pHun4c12-Beli-koLRR6 mutant expression plasmid. The pHun4c12-Beli-koLRR6 plasmid was transferred into Agrobacterium LBA4404 by electric shock method for subsequent plant transformation. The artificially synthesized target sequences for adding linkers are as follows:
[0052] OsLRR6-F2: 5'-ggcaCCAGGTGCAGGGATTTACA-3'
[0053] OsLRR6-R2: 5'-aaacTGTAAATCCCTGCACCTGG-3'
[0054] 2) Infect rice callus with Agrobacterium containing pHun4c12-Beli-koLRR6 vector plasmid. The callus after co-infection was screened and cultured on NBDS ...
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