Primers for amplifying Rrspgl, core fragment of gene and application of core fragment

A technology of core fragments and specific primers, which is applied in the field of genetic engineering and can solve problems such as not being retrieved

Inactive Publication Date: 2012-05-02
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no patent reports on the specific amplification primers for endopolygalacturonase gene of rice sheath blight, the specific primers for amplifying the core fragment of the gene, the core fragment of the gene and its application.

Method used

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  • Primers for amplifying Rrspgl, core fragment of gene and application of core fragment
  • Primers for amplifying Rrspgl, core fragment of gene and application of core fragment
  • Primers for amplifying Rrspgl, core fragment of gene and application of core fragment

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Embodiment 1

[0030] Example 1 Rrspg1 Gene acquisition

[0031] 1. Primer Design

[0032] According to the PG gene sequence (GenBank registration number: JN118556) published on the NCBI website (www.ncbi.nlm.nih.gov), use DNAMAN software for sequence alignment to find homologous sequences, and then use Primer5.0 according to the homologous sequences The software designed 24 pairs of primers.

[0033] Rrspg1 Gene Amplification Primer Screening

[0034] Using the total DNA of rice sheath blight strong pathogenic strain GD-118 as a template, the 24 pairs of primers designed above were used for PCR amplification and sequenced. The sequencing results are compared on the NCBI website, and it is judged whether the measured sequence is the required target fragment according to the homology with the corresponding enzyme gene fragment, and then the quality of the primer is judged. Poor primers were discarded. For primers with good sequencing results, further sequencing is performed af...

Embodiment 2

[0042] Example 2 rice sheath blight endo-PG gene 1 ( Rrspg1 ) Functional analysis of core fragments

[0043] 1. Construction of RNAi vector

[0044] Amplified with primers PGS10F (nucleotide sequence shown in SEQ ID NO: 3) and PGS10R (nucleotide sequence shown in SEQ ID NO: 4) Rrspg1 The core fragment PGS was obtained to obtain its sequence (the nucleotide sequence is shown in SEQ ID NO: 5). The sequences of primers PGS10F and PGS10R are as follows:

[0045] PGS10F (SEQ ID NO: 3): ATGGCTTTGATGTTTCC;

[0046] PGS10R (SEQ ID NO: 4): ACTGCGATGTTGTTGGT.

[0047] Use separately Hin dⅢ and xho Ⅰ and Kpn I and Bgl Ⅱ Two groups of restriction endonucleases are used for double digestion, and then the target fragment is Rrspg1 Part of the core fragment PGS (SEQ ID NO: 5) and the vector pSilent-1 were constructed for forward and reverse ligation reactions to obtain a new recombinant vector.

[0048] The specific operation steps are: the pSilent-1 vector ( Figure 4 -a)...

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Abstract

The invention discloses primers for amplifying Rrspgl, a core fragment of the gene and application of the core fragment. Nucleotide sequences of the specific primers for amplifying the Rrspgl gene are shown as SEQ ID NO:1 and SEQ ID NO:2; specific primers for amplifying the core fragment of the gene are shown as SEQ ID NO:3 and SEQ ID NO:4; and a nucleotide sequence of the core fragment of the gene is shown as SEQ ID NO:5. In the invention, a ribonucleic acid interference (RNAi) expression vector pSilent-PG-2 of the core fragment of the Rrspgl is also constructed; and the RNAi expression vector is obtained by performing double digestion on a vector pSilent-1 and the core fragment of the Rrspgl through two groups of restriction enzymes, namely HindIII and XhoI as well as KpnI and BglII respectively, and then performing forward and reverse connection. The function of the core fragment of the Rrspgl is simultaneously researched, so that a great guiding significance is provided for the research such as the disclosure of the pathogenic mechanism of rhizoctonia solani and the molecular mechanism of the interaction between the rhizoctonia solani and hosts and the like.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to amplification Rrspg1 (Rice sheath blight endopolygalacturonase gene 1) specific primers, specific primers for amplifying the core fragment of the gene, the core fragment of the gene and its application. Background technique [0002] Rice sheath blight is one of the three major diseases of rice worldwide, which has caused serious economic losses in our country. The causative agent of rice sheath blight is Rhizoctonia solani ( Rhizoctonia solani Kühn), which has a wide distribution range and host range, and can damage more than 260 kinds of plants such as corn, soybean and potato in addition to rice. Due to Rhizoctonia solani ( R. solani ) species members are complex, so the mycelium fusion group (anastomosis group, AG) is usually used for reclassification. Currently Rhizoctonia solani ( R. solani ) are divided into at least 14 AGs, and some AGs are further divi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/56C12N15/63C12N15/66C12N1/21C12N15/80C12Q1/68C12R1/645
Inventor 周而勋杨迎青杨媚舒灿伟郑丽
Owner SOUTH CHINA AGRI UNIV
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