Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter

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• 作者：Pritu Pratibha^a ; Sunil Kumar Singh^a ; Isha Sharma^a ; Ravi Kumar^b ; Ramamurthy Srinivasan^b ; Shripad Ramachandra Bhat^b ; ^{srbhat@nrcpb.org} ; ^{srbhat22@rediffmail.com} ; Paramvir Singh Ahuja^a ; Yelam Sreenivasulu^a ; ^{sree_yelam@yahoo.com} ; ^{sreenivasulu@ihbt.res.in}
• 关键词：CTAB ; cetyl trimethyl ammonium bromide ; GFP ; green fluorescent protein ; RT-PCR ; reverse transcriptase-polymerase chain reaction ; GUS ; ¦Â-glucuronidase
• 刊名：Gene
• 出版年：2013
• 出版时间：15 July, 2013
• 年：2013
• 卷：524
• 期：1
• 页码：22-27
• 全文大小：1306 K

文摘

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461 bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461 bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461 bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461 bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond ? 461 nucleotides were not detected in the wild type suggesting that this 461 bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.