NDLI: Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum

Content Provider	PubMed Central
Author	Chen, Helong Hu, Caiping Yi, Kexian Huang, Guixiu Gao, Jianming Zhang, Shiqing Zheng, Jinlong Liu, Qiaolian Xi, Jingen
Editor	Stevenson, Brian
Copyright Year	2014
Abstract	Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens’ AGL-1 concentration (OD600), 0.8; concentration of Colletotrichum conidium, 1×106 conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25°C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300–400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant’s pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).
Related Links	http://dx.doi.org/10.1371/journal.pone.0111172
Starting Page	111172
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	10
Volume Number	9
Language	English
Publisher	Public Library of Science
Publisher Date	2014-10-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Comprehensive MALDI-TOF Biotyping of the Non-Redundant Harvard Pseudomonas aeruginosa PA14 Transposon Insertion Mutant Library

Transposon Mutagenesis in Bifidobacterium breve: Construction and Characterization of a Tn5 Transposon Mutant Library for Bifidobacterium breve UCC2003

An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells

Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Construction of High-Density Linkage Maps of Populus deltoides × P. simonii Using Restriction-Site Associated DNA Sequencing

Ultra-Low Background DNA Cloning System

Expression of HA of HPAI H5N1 Virus at US2 Gene Insertion Site of Turkey Herpesvirus Induced Better Protection than That at US10 Gene Insertion Site

Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum

Similar Documents

Comprehensive MALDI-TOF Biotyping of the Non-Redundant Harvard Pseudomonas aeruginosa PA14 Transposon Insertion Mutant Library

Transposon Mutagenesis in Bifidobacterium breve: Construction and Characterization of a Tn5 Transposon Mutant Library for Bifidobacterium breve UCC2003

An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells

Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Construction of High-Density Linkage Maps of Populus deltoides × P. simonii Using Restriction-Site Associated DNA Sequencing

Ultra-Low Background DNA Cloning System

Expression of HA of HPAI H5N1 Virus at US2 Gene Insertion Site of Turkey Herpesvirus Induced Better Protection than That at US10 Gene Insertion Site

Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum