UBQ10 and GAPDH as double reference genes for rice stripe virus (RSV)-infected plant gene function analysis and use thereof

Abstract

The invention discloses a reference gene composition for rice stripe virus (RSV)-infected plant gene function analysis. Expression stability of fluorescent quantitative PCR candidate reference genes in the RSV-infected plant is analyzed by geNorm and NormFinder, a paired difference V value of a normalized factor is computed by geNorm program after introduction of a novel reference gene, and the most suitable reference gene composition is determined according to V value change so that the optimal reference gene composition of UBQ10 and GAPDH for RSV-infected plant gene function analysis is obtained. RSV-infected rice plant is used as an object, and compared with a single reference, the reference gene composition of UBQ10 and GAPDH can produce an accurate and reliable gene expression result. The reference gene composition can be used for research on an expression level and functions of RSV-infected rice related genes and a RSV invasion mode.

Description

Technical field:

[0001] The present invention uses geNorm and NormFinder software to identify the internal reference gene combination of fluorescent quantitative PCR in plants susceptible to rice stripe virus (Rice strip virus, RSV). Based on the correction effect of a single internal reference gene, it can be applied to the study of gene function in RSV-infected plants, and belongs to the field of molecular biology. Background technique:

[0002] Rice stripe leaf blight, the pathogen of which is rice stripe virus, is a persistent ovum-transmitted virus by the vector insect SBPH. Infection of rice with RSV causes dead seedlings and severe yield reduction. In addition to rice, it also harms crops such as wheat. In the late 1960s and 1990s, the disease broke out in North China, East China and other places. In recent years, the widespread planting of susceptible varieties, the promotion of rice-wheat rotation, and changes in environmental conditions such as winter climate warm...

Examples

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...

Technical field:

[0001] The present invention uses geNorm and NormFinder software to identify the internal reference gene combination of fluorescent quantitative PCR in plants susceptible to rice stripe virus (Rice strip virus, RSV). Based on the correction effect of a single internal reference gene, it can be applied to the study of gene function in RSV-infected plants, and belongs to the field of molecular biology. Background technique:

[0002] Rice stripe leaf blight, the pathogen of which is rice stripe virus, is a persistent ovum-transmitted virus by the vector insect SBPH. Infection of rice with RSV causes dead seedlings and severe yield reduction. In addition to rice, it also harms crops such as wheat. In the late 1960s and 1990s, the disease broke out in North China, East China and other places. In recent years, the widespread planting of susceptible varieties, the promotion of rice-wheat rotation, and changes in environmental conditions such as winter climate warm...

Examples

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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