Double reference genes (UBC and Actinl) for gene function analysis on RBSDV (rice black-streaked dwarf virus) infected plant and application of double reference genes (UBC and Actinl)

A technology for dwarfing black stripes and actin1 in rice, which can be used in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., and can solve problems such as errors and stability problems.

Inactive Publication Date: 2015-09-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Application Information

AI Technical Summary

Problems solved by technology

The internal reference gene used for correction and standardization must be continuously and stably expressed under any conditions, otherwise it will lead to erroneous results. Most of the existing studies use a single gene such as TIP41-Like as the internal reference gene, which may be different in different treatments. There will be stability problems, it is necessary to establish a suitable internal reference gene system as soon as possible to solve this problem

Method used

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  • Double reference genes (UBC and Actinl) for gene function analysis on RBSDV (rice black-streaked dwarf virus) infected plant and application of double reference genes (UBC and Actinl)
  • Double reference genes (UBC and Actinl) for gene function analysis on RBSDV (rice black-streaked dwarf virus) infected plant and application of double reference genes (UBC and Actinl)
  • Double reference genes (UBC and Actinl) for gene function analysis on RBSDV (rice black-streaked dwarf virus) infected plant and application of double reference genes (UBC and Actinl)

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1, Obtaining the combination of internal reference genes for gene function analysis in RBSDV-infected plants

[0019] 1. Plant material, virus analysis and infection method

[0020] Rice seeds (Nipponbare) were first sterilized with 0.1% HgCl for 1 hour; then rinsed with deionized water, soaked at 25°C for one day, and accelerated germination for one day; the seeds after the acceleration were cultivated in Kimura B culture medium, illuminated for 14 hours, and dark for 10 hours. The temperature is 28±1°C.

[0021] Collect rice diseased plants showing RBSDV symptoms (Zhejiang Agricultural Sciences, 1984, (4): 185-192.) in Yancheng, Xuzhou, Lianyungang and other rice black-streaked dwarf disease areas in Jiangsu, and use RT-PCR method (Jiangsu Agricultural Science Journal , 2009, 25(6): 1263-1267) to detect whether the diseased plants carry RBSDV, and use the primers (CP-F: ATGGGTACCAACAAGCC, CP-R: CTAGTCATCTGCACCTT) corresponding to the coat protein gene of rice...

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 RBSDV-infected rice, UBC+Actin1 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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Abstract

The invention discloses combinations of reference genes for gene function analysis on an RBSDV (rice black-streaked dwarf virus) infected plant. The combinations are obtained according to the following steps: analyzing the expression stability of a fluorescent quantitative PCR candidate reference gene in the RBSDV infected plant by utilizing geNorm and NormFinder, then the paired difference value V of a normalized factor is calculated after a new reference gene is introduced according to a geNorm program, and determining optimal reference gene combinations according to the variation of V, so as to obtain the optimal reference gene combinations for gene function analysis on the gene function analysis, wherein the optimal reference gene combinations are UBC and Actinl, and when the RBSDV infected plant is taken as a target, the optimal reference gene combinations (UBC and Actinl) can be used for obtaining more accurate and reliable gene expression results as compared with a single reference gene. The combinations can be used for researching the expression level and function of related genes of the RBSDV infected plant and the infecting mode of RBSDV.

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 black-streaked dwarf virus (RBSDV), and UBC and Actin1 are used as the dual genes for gene function analysis in RBSDV-infected plants. The internal reference gene is obviously better than the correction effect of a single internal reference gene, and can be applied to the research on gene function of RBSDV-infected plants, belonging to the field of molecular biology. Background technique: [0002] Rice black-streaked dwarf disease, the pathogen is rice black-streaked dwarf virus, is a vicious viral disease transmitted by persistent non-oval means by the small planthopper. Most of the diseased rice plants infected with RBSDV can't head, and the yield is seriously reduced. In addition to rice, it also harms crops such as wheat, corn, barley and sorghum. In the late 1960s and 1990s, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70C12N15/11
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 周彤周益军方鹏杜琳琳兰莹孙枫刘之洋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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