A library of Arabidopsis 35S-cDNA lines for identifying novel mutants

A library of Arabidopsis 35S-cDNA lines for identifying novel mutants We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation. We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter. The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis and selected over 30 000 transformants. A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta. About 90% of T1 plants in the collection have inserts, the average insert size is ca. 1.1 kb, and ca. 43% of these inserts appear to encode full-length proteins. T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study. This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 1.18.1.2). This construct co-suppresses the endogenous petH transcript. We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis. Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected. As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

A library of Arabidopsis 35S-cDNA lines for identifying novel mutants

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2001 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1011699722052
Publisher site
See Article on Publisher Site

Abstract

We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation. We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter. The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis and selected over 30 000 transformants. A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta. About 90% of T1 plants in the collection have inserts, the average insert size is ca. 1.1 kb, and ca. 43% of these inserts appear to encode full-length proteins. T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study. This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 1.18.1.2). This construct co-suppresses the endogenous petH transcript. We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis. Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected. As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 3, 2004

References

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