The constructs of GUS expression driven by <i>AhLEC1B</i> promoter and schematic representation of the different length promoters with 5′ or 3′ terminal deletion.

<p>Q1-Q6 indicates the different promoters with 5′ or 3′ terminal deletion. The white and gray rectangles show the upstream promoter region from TSS and 5′ UTR region respectively.</p

Localization of transcription start sites of the peanut <i>AhLEC1B</i> gene using 5′ RACE.

<p>P1 –Product of the first round PCR; P2 –Product of the second round PCR</p

Effects of <i>AhLEC1B</i> promoter deletions on the expression profile of <i>GUS</i> gene in transgenic Arabidopsis lines.

<p>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.</p

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

<div><p>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 <i>AhLEC1B</i> gene, a homolog of <i>Arabidopsis LEC1</i>, 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 <i>Arabidopsis</i> lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of <i>AhLEC1B</i> contain the key motifs required for the essential promoting activity. Moreover, <i>AhLEC1B</i> is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding <i>cis</i>-regulatory elements.</p></div

Phylogenetic tree for peanut AhLEC1A and AhLEC1B, and the <i>Arabidopsis</i> NF-YB family.

<p>Phylogenetic tree for peanut AhLEC1A and AhLEC1B, and the <i>Arabidopsis</i> NF-YB family.</p

The sequence of 5′ flanking regulation region of peanut <i>AhLEC1B</i> gene and some major elements harbored in this region.

<p>The bold capital letter “A” represents the transcription start site (TSS), and other capital letters show different regulatory elements.</p

The primers used in this study.

<p>The primers used in this study.</p

Major elements in 5′UTR and 300bp promoter region.

<p>^aN = G/A/C/T; R = A/G; S = C/G; W = A/T; Y = T/C</p><p>^bThe symbol ‘+’ or ‘-’ in the bracket represents the DNA strand in which the element is situated.</p><p>^cThe positive number indicates the location of element in 5′UTR, while the negative represents that in promoter.</p><p>Major elements in 5′UTR and 300bp promoter region.</p

The second round of PCR amplification products of 5′ flanking regulation regions of peanut <i>AhLEC1B</i> gene by chromosome walking.

<p>The arrow indicates the target band.</p

Table_1.docx

<p>Peanut (Arachis hypogaea L.) is one of the major oil crops and is the fifth largest source of plant oils in the world. Numerous genes participate in regulating the biosynthesis and accumulation of the storage lipids in seeds or other reservoir organs, among which several transcription factors, such as LEAFY COTYLEDON1 (AtLEC1), LEC2, and WRINKLED1 (WRI1), involved in embryo development also control the lipid reservoir in seeds. In this study, the AtLEC1 gene was transferred into the peanut genome and expressed in a seed-specific manner driven by the NapinA full-length promoter or its truncated 230-bp promoter. Four homozygous transgenic lines, two lines with the longer promoter and the other two with the truncated one, were selected for further analysis. The AtLEC1 mRNA level and the corresponding protein accumulation in different transgenic overexpression lines were altered, and the transgenic plants grew and developed normally without any detrimental effects on major agronomic traits. In the developing seeds of transgenic peanuts, the mRNA levels of a series of genes were upregulated. These genes are associated with fatty acid (FA) biosynthesis and lipid accumulation. The former set of genes included the homomeric ACCase A (AhACC II), the BC subunit of heteromeric ACCase (AhBC4), ketoacyl-ACP synthetase (AhKAS II), and stearoyl-ACP desaturase (AhSAD), while the latter ones were the diacylglycerol acyltransferases and oleosins (AhDGAT1, AhDGAT2, AhOle1, AhOle2, and AhOle3). The oil content and seed weight increased by 4.42–15.89% and 11.1–22.2%, respectively, and the levels of major FA components including stearic acid, oleic acid, and linoleic acid changed significantly in all different lines.</p