Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs

A bacterial blight bacteria and library construction technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical libraries, etc., can solve the problems of no patents and achieve high sensitivity

Inactive Publication Date: 2012-09-12
云南省农业科学院生物技术与种质资源研究所
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Most of the above studies have focused on the application of SSH technology to study differential gene expression under stress or induction conditions, and many genes involved in specific expression induced by specific conditions have also been found. However, so far, SSH technology has been used to study common wild rice ( Oryza rufipogon Griff.) The differential expression of genes under bacterial blight stress has not been reported yet, and there is no related patent

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  • Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs
  • Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs
  • Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 A method for constructing a common wild rice SSH library under bacterial blight stress, comprising the following steps:

[0048] (1) Inoculation treatment of common wild rice leaf material

[0049] ① Streak the strains C1 and Y8 of bacterial blight on the NA medium plate, and incubate at 28°C for 2-3 days for activation. Turbidimetric method prepared to 6 × 10 8 ·ml -1 the bacterial liquid;

[0050] ② After 15:00 in the afternoon, use the scissors dipped in the bacterial solution to subtract 2-3 cm from the tip of the leaf to inoculate the leaves of common wild rice with bacterial blight in the greenhouse, and each strain is inoculated with 3 pier , 30 leaves, the control is to inoculate common wild rice leaves with sterile water instead of bacterial solution, and inoculate with sterile water as a control;

[0051] ③Samples were taken every 24 hours after inoculation, that is, at 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 144 hours. Quick-fr...

Embodiment 2

[0081] Example 2 Detection of the size of the inserted cDNA fragment in the common wild rice SSH library under bacterial blight stress constructed by the present invention

[0082] (1) 34 positive clones were randomly selected from the constructed Oryza sativa SSH library, inoculated in LB liquid medium containing AMP antibiotics, and cultured overnight at 28°C with shaking at 180 rpm;

[0083] (2) Extract plasmids from 34 positive clones by alkaline lysis;

[0084] (3) Use the plasmid extracted in step (2) as a template to establish a PCR reaction system: 5.0 μl of 10×PCR buffer, MgCl 2 3 μl, dNTP 4 μl, Nested PCR primer F 1 μl, Nested PCR primer R 1 μl, Taq polymerase 0.5 μl, plasmid template 1 μl, sterile water to make up the reaction volume of 50 μl;

[0085] (4) PCR reaction program: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 35 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, a total of 35 cycles, and a final extension at 72°C f...

Embodiment 3

[0088] Example 3 Application of the common wild rice SSH library constructed in Example 1 under bacterial blight stress

[0089] The steps are as follows:

[0090] (1) Inoculate all the single clones from the SSH library constructed in Example 1 in LB liquid medium containing AMP antibiotics, culture overnight at 28°C with shaking at 180 rpm;

[0091] (2) Send the bacterial liquid to Huada Gene Company for sequencing. The DNAStar software is used to remove the carrier of the sequencing results, and the ContigExpress software is used for sequence splicing to remove repetitive sequences;

[0092] (3) Compare and analyze the non-redundant sequences in the protein database and nucleic acid database in NCBI (http: / / www.ncbi.nlm.nih.gov), the National Center for Biological Information of the United States, respectively, and the judgment standards refer to rice, In the EST research of Arabidopsis, the consistency of blastx results is greater than 40%, and the score is greater than 8...

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Abstract

The invention discloses a method for constructing a suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs. The method includes steps of: sample processing, total ribonucleic acid (RNA) extraction, mRNA purification, suppression subtractive hybridization and polymerase chain reaction (PCR) amplification of testing parts and driven elements, PCR products and polarization mode dispersion 18-T (pMD18-T) carrier connection restraining, connection product conversion, monoclonal storing and the like. The SSH library of oryza rufipogon threatened by bacterial blight germs constructed by the method is high in sensitivity, even length of insertion elements of the library is 450bp, a large number of difference expressed sequence tags (ESTs) of the oryza rufipogon threatened by the bacterial blight germs can be obtained fast in one step, partial sequence information is provided for separating overall-length cDNA sequence of clone bacterial blight resisting related genes, simultaneously important theoretical basis for improvement of biology is provided and new bacterial blight resisting related genes can be obtained.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to artificially inoculating the leaves of common wild rice with bacterial blight to cause stress induction conditions, and constructing a common wild rice SSH library under the stress of bacterial blight. Background technique [0002] In the past, SSH (suppression subtravtive hybridization, SSH) library has been widely used in plants. Such as isolating differentially expressed genes at different developmental stages of plants, or isolating genes related to plant flower development, leaf senescence and rhizome development (Kim JY et al, Biochimicaet Biophysica Acta, 1489:389-392,1999; Kim JY et al, Molecular Cell , 9: 392-397,1999; Hinderhofer K and Zentgraf U, Planta, 213:469-473,2001). In recent years, there have been more and more studies on the differential gene expression of SSH technology under various stress conditions, such as pest stress, low temperature t...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/70C12N1/21C12R1/19
Inventor 程在全黄兴奇蒋春苗孙正文李定琴李维蛟殷富有张敦宇钟巧芳余腾琼陈玲王玲仙付坚李娥贤蒋聪罗红梅王波
Owner 云南省农业科学院生物技术与种质资源研究所
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