UBQ10 and GAPDH as double reference genes for rice stripe virus (RSV)-infected plant gene function analysis and use thereof
An internal reference gene, a technology for infecting plants, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as stability and errors
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Embodiment 1
[0018] Embodiment 1, the acquisition of internal reference gene combinations for gene function analysis in RSV-infected plants
[0019] 1. Plant material, virus analysis and infection method
[0020] Rice seeds (Nipponbare) were first treated with 0.1% HgCl 2 Disinfect for 1 hour; then rinse with deionized water, soak the seeds at 25°C for one day, and germinate for one day; the seeds after germination are cultured in Kimura B culture medium, with 14 hours of light and 10 hours of darkness at a temperature of 28±1°C.
[0021] From April to May 2005, the nymphs of SBPH were collected from the seriously ill area in Hai'an, Jiangsu Province, and raised on the susceptible variety Wuyujing No. 3. After mating, a single female worm lay eggs alone, and then the female was detected by dot immunocombination method In the case of poisoning, keep the offspring of infected females, and obtain a colony of SBPH after 2-3 generations, select groups with a virus-carrying rate greater than 50...
Embodiment 2
[0037] Embodiment 2, the comparison of multi-internal reference gene and single internal reference gene correction
[0038] In order to verify the effectiveness of the above screened internal reference gene correction fluorescent quantitative PCR data, two virus response-related genes OsPR1b and OsWRKY were used as target genes ( Figure 5 ). In RSV-infected rice, UBQ 10+GAPDH was used as multiple internal reference genes, and TIP41-Like was used as a single internal reference gene for calibration and comparison experiments. The results showed that OsPR1b gene was rapidly expressed under virus infection with multiple internal reference gene correction, while OsWRKY was rapidly expressed in the early stage of virus infection and then down-regulated. In comparison, when TIP41-Like was used as a single internal reference, the expression levels of OsPR1b and OsWRKY were relatively low, and their recognition was significantly lower than that of the double internal reference system...
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