Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (<i>Penaeus monodon</i>)

<div><p>Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified <i>E2F-2</i> genes from yeasts to humans, but few reports investigated <i>E2F-2</i> gene from black tiger shrimp. In the present study, we cloned <i>E2F-2</i> gene from black tiger shrimp (<i>Penaeus monodon</i>). Full-length <i>PmE2F-2</i> complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete <i>PmE2F-2</i> genomic sequence (17,305 bp) of <i>P</i>. <i>monodon</i> contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that <i>PmE2F-2</i> is highly expressed in hepatopancreas and ovaries of <i>P</i>. <i>monodon</i>. Highest <i>PmE2F-2</i> expression levels were observed in stage III ovarian development of <i>P</i>. <i>monodon</i>. <i>PmE2F-2</i> expression levels were significantly augmented in ovaries of <i>P</i>. <i>monodon</i> after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine <i>PmE2F-2</i>, <i>PmCDK2</i>, and <i>PmCyclin E</i> expression profiles. <i>PmE2F-2</i> was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, <i>PmE2F-2</i> expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, <i>PmE2F-2</i> may be involved in ovarian maturation in <i>P</i>. <i>monodon</i>.</p></div

Relative expression levels of <i>PmE2F-2</i> in various tissues detected by quantitative real-time PCR analysis using <i>EF-1α</i> as an internal reference.

<p>Vertical bars represented mean ±SD (n = 3). Significant different letters above vertical bars indicate difference (<i>P</i> <0.05).</p

The gonadosomatic index (GSI, ovarian weight/body weight × 100) of shrimp after dsRNA-GFP-, dsRNA-E2F-2- and PBS injection.

<p>The gonadosomatic index (GSI, ovarian weight/body weight × 100) of shrimp after dsRNA-GFP-, dsRNA-E2F-2- and PBS injection.</p

Comparison of the genomic DNA sequence encoding E2F-2 in <i>P</i>. <i>monodon</i>.

<p>Green-shaded rectangles indicate exons, gray horizontal lines represent introns, and the numbers indicate exon and intron length (in bp).</p

Relative expression levels of <i>PmE2F-2</i> in ovary and hepatopancreas of shrimps after treatment with dsRNA-RBL.

<p>a. Relative expression level of <i>PmE2F-2</i> in the ovary. b. Relative expression level of <i>PmE2F-2</i> in the hepatopancreas. Ovary and hepatopancreas tissues collected from shrimps injected with dsRNA-RBL were compared with respect to <i>PmE2F-2</i> mRNA expression (relative to EF-1α) using Students t-tests. Vertical bars represented mean±SD (n = 3). Significant differences from controls were indicated: **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Thermally Induced [3 + 2] Cycloaddition of Alkynyl-Tethered Diazoamides: Synthetic and Mechanistic Insights

A general and unprecedented thermally induced formal [3 + 2] cycloaddition has been developed that provides a general access to fused lactam derivatives in high to excellent yields with broad substrate scope. In comparison with the reported metal-catalyzed carbene/alkynyl metathesis, this is the only example in this area under catalyst-free conditions with excellent selectivity. Mechanistic studies indicate that the 3<i>H</i>-pyrazole is the key intermediate in this cascade reaction, which is confirmed spectroscopically for the first time

Relative expression levels of <i>PmE2F-2</i> in ovary and hepatopancreas of shrimps after treatment with dsRNA-E2F-2.

<p>a. Relative expression level of <i>PmE2F-2</i> in the ovary. b. Relative expression level of <i>PmE2F-2</i> in the hepatopancreas. Vertical bars represented mean±SD (n = 3). Significant differences from controls were indicated: **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

In situ detection of PmE2F-2 hybridization.

<p>The ovary and hepatopancreas were collected after dsRNA-E2F-2 injection. Nuclei were stained; the blue points indicate positive reactions (indicated by arrows). B and C represented the collected ovaries after dsRNA-GFP and dsRNA-RBL injections, respectively. A represented the negative control. E and F represented the collected hepatopancreas after dsRNA-GFP and dsRNA-RBL injections, respectively. D represented the negative control. Scalebar = 30.</p

<i>PmE2F-2</i> mRNA expression profiles after stimulated by 5-HT.

<p><i>PmE2F-2</i> mRNA relative expression level in ovary tissue post-treatment with 5-HT. Vertical bars represented the mean ± SD (n = 3). Significant different letters above vertical bars indicate difference (P < 0.05).</p

Multiple alignment of the deduced amino acid sequences of E2F-2 from <i>P</i>. <i>monodon</i> and other species.

<p>Sequence logo representing the similarity is shown at the top of alignments and numbers of amino acid are listed on the right side of alignments. The GenBank numbers of E2F-2 are listed as follows: <i>P</i>. <i>monodon</i>: KY628943; <i>Apis mellifera</i>: XP_006561713.1; <i>Eufriesea mexicana</i>: OAD58031.1; <i>Linepithema humile</i>: XP_012219394.1; <i>Lasius niger</i>: KMQ93879.1; <i>Polistes canadensis</i> XP_014607688.1. E2F-TDP domain and coiled coil domain are indicated with black and green lines, respectively.</p