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

Tang G, Xu P, Liu W, Liu Z, Shan L - PLoS ONE (2015)

Bottom Line: We further characterized the GUS expression patterns in the transgenic Arabidopsis lines.Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity.Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

View Article: PubMed Central - PubMed

Affiliation: Bio-Tech Research Centre, Shandong Academy of Agricultural Sciences / Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, 250100, China.

ABSTRACT
LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5' flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5' terminal and/or 3' terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

No MeSH data available.


Effects of AhLEC1B promoter deletions on the expression profile of GUS gene in transgenic Arabidopsis lines.Q1-Q6 indicate the GUS expression patterns in different transgenic Arabidopsis lines containing 5′ or 3′ terminal deletion promoters, and the CK-N and CK-P showed the GUS expression profiles in non-transformed negative control and in positive control harboring 35S:GUS constructs, respectively.
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pone.0139213.g006: Effects of AhLEC1B promoter deletions on the expression profile of GUS gene in transgenic Arabidopsis lines.Q1-Q6 indicate the GUS expression patterns in different transgenic Arabidopsis lines containing 5′ or 3′ terminal deletion promoters, and the CK-N and CK-P showed the GUS expression profiles in non-transformed negative control and in positive control harboring 35S:GUS constructs, respectively.

Mentions: To identify the crucial regulatory regions that are essential for gene expression, we generated a series of constructs containing different length AhLEC1B promoter with 5' terminal deletion and 52bp 5' UTR or 3' terminal deletion fused with GUS reporter gene (Fig 5). All constructs were introduced into the Arabidopsis genome by Agrobacterium-mediated transformation. The resulting transgenic T2 lines containing a single copy homologous gene were screened for use in GUS histochemical staining studies. The results of staining in diverse tissues or organs showed that the longest fragment (Q1, 1281bp) containing 1229bp promoter region and 52bp 5' UTR, mainly regulates the GUS expression in the developing embryo. Moreover, three fragments (Q4, Q5, and Q6) with a 5' terminal deletion could drive the GUS expression in all tissues detected (Fig 6). However, the promoter fragment (Q2 and Q3) with 351bp deletion from 3' terminus lost the promoter function that had crucial activity responsive elements (Table 2). The shortest fragment (Q6, 118bp) including 66bp promoter region and 52bp 5' UTR contains the main elements that control the constitutive expression of the downstream gene (Fig 6).


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

Tang G, Xu P, Liu W, Liu Z, Shan L - PLoS ONE (2015)

Effects of AhLEC1B promoter deletions on the expression profile of GUS gene in transgenic Arabidopsis lines.Q1-Q6 indicate the GUS expression patterns in different transgenic Arabidopsis lines containing 5′ or 3′ terminal deletion promoters, and the CK-N and CK-P showed the GUS expression profiles in non-transformed negative control and in positive control harboring 35S:GUS constructs, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591277&req=5

pone.0139213.g006: Effects of AhLEC1B promoter deletions on the expression profile of GUS gene in transgenic Arabidopsis lines.Q1-Q6 indicate the GUS expression patterns in different transgenic Arabidopsis lines containing 5′ or 3′ terminal deletion promoters, and the CK-N and CK-P showed the GUS expression profiles in non-transformed negative control and in positive control harboring 35S:GUS constructs, respectively.
Mentions: To identify the crucial regulatory regions that are essential for gene expression, we generated a series of constructs containing different length AhLEC1B promoter with 5' terminal deletion and 52bp 5' UTR or 3' terminal deletion fused with GUS reporter gene (Fig 5). All constructs were introduced into the Arabidopsis genome by Agrobacterium-mediated transformation. The resulting transgenic T2 lines containing a single copy homologous gene were screened for use in GUS histochemical staining studies. The results of staining in diverse tissues or organs showed that the longest fragment (Q1, 1281bp) containing 1229bp promoter region and 52bp 5' UTR, mainly regulates the GUS expression in the developing embryo. Moreover, three fragments (Q4, Q5, and Q6) with a 5' terminal deletion could drive the GUS expression in all tissues detected (Fig 6). However, the promoter fragment (Q2 and Q3) with 351bp deletion from 3' terminus lost the promoter function that had crucial activity responsive elements (Table 2). The shortest fragment (Q6, 118bp) including 66bp promoter region and 52bp 5' UTR contains the main elements that control the constitutive expression of the downstream gene (Fig 6).

Bottom Line: We further characterized the GUS expression patterns in the transgenic Arabidopsis lines.Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity.Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

View Article: PubMed Central - PubMed

Affiliation: Bio-Tech Research Centre, Shandong Academy of Agricultural Sciences / Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, 250100, China.

ABSTRACT
LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5' flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5' terminal and/or 3' terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

No MeSH data available.