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High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns.

Xiao YL, Redman JC, Monaghan EL, Zhuang J, Underwood BA, Moskal WA, Wang W, Wu HC, Town CD - Plant Methods (2010)

Bottom Line: A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened.More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis.

View Article: PubMed Central - HTML - PubMed

Affiliation: J, Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. cdtown@jcvi.org.

ABSTRACT

Background: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.

Results: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.

Conclusions: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

No MeSH data available.


Related in: MedlinePlus

Outcome of generation of promoter reporter (GFP) transgenic lines. The figure shows the numbers of genes for which primers were designed, the success rate of high throughput cloning, transformation and the outcome of transgenic lines.
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Figure 2: Outcome of generation of promoter reporter (GFP) transgenic lines. The figure shows the numbers of genes for which primers were designed, the success rate of high throughput cloning, transformation and the outcome of transgenic lines.

Mentions: To handle the relatively large number of genes to be studied, we modified our original protocols [29,31] to produce a robust high throughput pipeline that included batch primer design, high throughput cloning and transformation, and a project-tracking LIMS implemented in a MySQL database (Figure 1). In this study, the Gateway® Gene Cloning strategy [32] was used to make promoter-reporter constructs. PCR amplification of promoters and Gateway cloning were performed in 96-well plate format. Two colonies from each Gateway BP cloning reaction (from each promoter) were picked and sequenced to confirm target identity. DNA isolated robotically from the sequence-confirmed clones was used in Gateway LR reactions. The triparental mating method [33] was used to transfer promoter-reporter constructs from E. coli to Agrobacterium (GV3101). Bypassing the step of destination clone DNA isolation from E. coli made this step economical and large scale. In addition, we cultured only 50 ml of Agrobacterium for plant transformation and did the floral dip in 50 ml Falcon tubes. As shown in Figure 2, a total of 627 candidate genes were put through the construction and transformation pipeline. Gateway entry clones were obtained from 469 genes and sequence confirmed. Of these, 442 were successfully transferred into the destination vector, pYXT2 containing the GFP reporter gene [34]. Subsequently, Agrobacterium clones from 353 genes were obtained by tri-parental mating, and then transformed into Arabidopsis by the floral dip method [35]. Three independent transformations (floral dips with separate plants) were performed for each construct and 3 seedlings from each kanamycin selection plate (whenever possible) were transferred into soil for maximum possible set of 9 transformed plants per construct. Positive transgenic plants from 266 constructs representing promoters from 266 genes were obtained and GFP expression patterns have been observed from 150 constructs in at least one of the 4 developmental stages examined. This newly developed cloning and transformation pipeline greatly improved the throughput and lowered the effort compared with our previous work.


High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns.

Xiao YL, Redman JC, Monaghan EL, Zhuang J, Underwood BA, Moskal WA, Wang W, Wu HC, Town CD - Plant Methods (2010)

Outcome of generation of promoter reporter (GFP) transgenic lines. The figure shows the numbers of genes for which primers were designed, the success rate of high throughput cloning, transformation and the outcome of transgenic lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927586&req=5

Figure 2: Outcome of generation of promoter reporter (GFP) transgenic lines. The figure shows the numbers of genes for which primers were designed, the success rate of high throughput cloning, transformation and the outcome of transgenic lines.
Mentions: To handle the relatively large number of genes to be studied, we modified our original protocols [29,31] to produce a robust high throughput pipeline that included batch primer design, high throughput cloning and transformation, and a project-tracking LIMS implemented in a MySQL database (Figure 1). In this study, the Gateway® Gene Cloning strategy [32] was used to make promoter-reporter constructs. PCR amplification of promoters and Gateway cloning were performed in 96-well plate format. Two colonies from each Gateway BP cloning reaction (from each promoter) were picked and sequenced to confirm target identity. DNA isolated robotically from the sequence-confirmed clones was used in Gateway LR reactions. The triparental mating method [33] was used to transfer promoter-reporter constructs from E. coli to Agrobacterium (GV3101). Bypassing the step of destination clone DNA isolation from E. coli made this step economical and large scale. In addition, we cultured only 50 ml of Agrobacterium for plant transformation and did the floral dip in 50 ml Falcon tubes. As shown in Figure 2, a total of 627 candidate genes were put through the construction and transformation pipeline. Gateway entry clones were obtained from 469 genes and sequence confirmed. Of these, 442 were successfully transferred into the destination vector, pYXT2 containing the GFP reporter gene [34]. Subsequently, Agrobacterium clones from 353 genes were obtained by tri-parental mating, and then transformed into Arabidopsis by the floral dip method [35]. Three independent transformations (floral dips with separate plants) were performed for each construct and 3 seedlings from each kanamycin selection plate (whenever possible) were transferred into soil for maximum possible set of 9 transformed plants per construct. Positive transgenic plants from 266 constructs representing promoters from 266 genes were obtained and GFP expression patterns have been observed from 150 constructs in at least one of the 4 developmental stages examined. This newly developed cloning and transformation pipeline greatly improved the throughput and lowered the effort compared with our previous work.

Bottom Line: A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened.More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis.

View Article: PubMed Central - HTML - PubMed

Affiliation: J, Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. cdtown@jcvi.org.

ABSTRACT

Background: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.

Results: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.

Conclusions: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

No MeSH data available.


Related in: MedlinePlus