Validation of internal control for gene expression study in soybean by quantitative real-time PCR-0

Sample.<p><b>Copyright information:</b></p><p>Taken from "Validation of internal control for gene expression study in soybean by quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1471-2199/9/59</p><p>BMC Molecular Biology 2008;9():59-59.</p><p>Published online 23 Jun 2008</p><p>PMCID:PMC2443375.</p><p></p

Validation of internal control for gene expression study in soybean by quantitative real-time PCR-1

Fferent tissues at the same developmental stage (B), developmental series (C), photoperiod treatments (D), different cultivar (E), different time of the day series (F), leaves located on different nodes on the main stem (G). A lower average expression stability M indicates more stable expression.<p><b>Copyright information:</b></p><p>Taken from "Validation of internal control for gene expression study in soybean by quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1471-2199/9/59</p><p>BMC Molecular Biology 2008;9():59-59.</p><p>Published online 23 Jun 2008</p><p>PMCID:PMC2443375.</p><p></p

Validation of internal control for gene expression study in soybean by quantitative real-time PCR-5

Sample.<p><b>Copyright information:</b></p><p>Taken from "Validation of internal control for gene expression study in soybean by quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1471-2199/9/59</p><p>BMC Molecular Biology 2008;9():59-59.</p><p>Published online 23 Jun 2008</p><p>PMCID:PMC2443375.</p><p></p

Validation of internal control for gene expression study in soybean by quantitative real-time PCR-4

He most variable HKGs selected by geNorm as internal controls. The geometric average of , and , and were used as internal controls for developmental stage (A), the geometric average of and , and were used as internal controls for photoperiod treatment (B). a, SD 1-day leaves; b, SD 25-day leaves; c, SD 1-day shoot tips; d, SD 25- day shoot tips; e, LD 25-day leaves; f, LD 25-day shoot tips.<p><b>Copyright information:</b></p><p>Taken from "Validation of internal control for gene expression study in soybean by quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1471-2199/9/59</p><p>BMC Molecular Biology 2008;9():59-59.</p><p>Published online 23 Jun 2008</p><p>PMCID:PMC2443375.</p><p></p

Validation of internal control for gene expression study in soybean by quantitative real-time PCR-3

Centile (boxes) and ranges (whiskers) for all the 21 sample pools. A. Comparisons of the completely possible sets of HKGs which included , , , , ; B. Comparisons of the completely possible sets of HKGs, which included , , , and .<p><b>Copyright information:</b></p><p>Taken from "Validation of internal control for gene expression study in soybean by quantitative real-time PCR"</p><p>http://www.biomedcentral.com/1471-2199/9/59</p><p>BMC Molecular Biology 2008;9():59-59.</p><p>Published online 23 Jun 2008</p><p>PMCID:PMC2443375.</p><p></p

Sequence analysis of GmACSL2.

<p>(A) Multiple amino acid sequences alignment of <i>Glycine max</i> GmACSL2 and GmLACS sequence with <i>Arabidopsis thaliana</i> AtLACS1 to AtLACS9. The <b>I</b> and <b>II</b> blocks of shadows indicate the highly conserved acid residues in AMP-binding protein. Black frames indicate the higher conservation amino acids, dark grey frames indicate the conservation amino acids, and light frames indicate the lower conservation amino acids. The protein follows: AtLACS1 (AAM28868), AtLACS2 (AAM288689), AtLACS3 (AAM28870), AtLACS4 (AAM28871), AtLACS5 (AAM28872), AtLACS6 (AAM28873), AtLACS7 (AAM28874), AtLACS8 (AAM28875), and AtLACS9 (AAM28876). (B) Phylogenic analysis between GmACSL2 with other ACSL enzymes and FATP proteins from plant, mammalian and yeast. The ACSL enzymes include <i>G.max</i> GmLACS, <i>Arabidopsis thaliana</i> AtLACS1-9, <i>Gossypium hirsutum</i> GhACS1 (ABA00144), <i>Brassica napus</i> BnACS6 (CAC19877), <i>Ricinus communis</i> RcACSL1 (XP_002520618) and RcACSL2 (XP_002520615), <i>Aegilops tauschii</i> AeACSL1 (EMT11835), <i>Triticum urartu</i> TuACSL (EMS60031), <i>Homo sapiens</i> HsACSL1 (NP_001986), HsACSL3 (NP_004448), HsACSL4 (NP_004449), HsACSL5 (NP_057318), and HsACSL6 (NP_056071), <i>Mus musculus</i> MmACSL1 (NP_032007), MmACSL3 (XP_129894), MmACSL4 (NP_062350), MmACSL5 (AAH31544), and MmACSL6 (NP_659072), <i>Rattus norvegicus</i> RnACSL1 (NP_036952), RnACSL3 (NP_476448), RnACSL4 (NP_446075), RnACSL5 (NP_446059), and RnACSL6 (NP_570095), and <i>Saccharomyces cerevisiae</i> ScFAA1 (P30624), ScFAA2 (P39518), ScFAA3 (P39002), and ScFAA4 (P47912). FATP proteins include <i>Homo sapiens</i> HsFATP1 (NP_940982), HsFATP3 (NP_077306), HsFATP4 (Q6P1M0), HsFATP5 (Q9Y2P5), and HsFATP6 (NP_001017372), <i>Mus musculus</i> MmFATP1 (NP_036107), MmFATP2 (AAC40186), and MmFATP4 (XP_130079), <i>Rattus norvegicus</i> RnFATP1 (NP_036119), and <i>Saccharomyces cerevisiae</i> (EWH19453). The bars stand for evolutionary distance. Bar = 0.2.</p

Overexpression of <i>GmACSL2</i> in yeast <i>pep4</i>.

<p>(A) Expression analysis of <i>GmACSL2</i> in different transformed lines by RT-PCR. Numbers 1 to 5 represents the five lines, GmACSL2-1, GmACSL2-2, GmACSL2-3, GmACSL2-4, and GmACSL2-5, transformed with pYES2-GmACSL2. The actin gene was used as the control for equal gel loading. Control represents the line transformed with pYES2 empty vector. (B) Sudan black B staining. The cells of three transformed lines GmACSL2-1, GmACSL2-3, and GmACSL2-4 were stained with Sudan Black B. The absorbance was measured at 580 nm and the line transformed with pYES2 vectors as control. (C) Fatty acids analysis comparisons between three transformed lines GmACSL2-1, GmACSL2-3 and GmACSL2-4 and the control line. The four major fatty acid species C16∶0, C16∶1, C18∶0, C18∶1 and the total fatty acids content in the yeast were detected by gas chromatography-mass spectrometry. (D) β-oxidation assay comparisons between three transformed lines GmACSL2-1, GmACSL2-3 and GmACSL2-4 and the control line. Oleate β-oxidation measurements in cells were followed by quantification of [¹⁴C] CO₂ and ¹⁴C-labelled β-oxidation products in a liquid scintillation counter. The β-oxidation activity in control cells in each experiment was taken as reference (100%). Data are presented as the mean ± SEM of the three experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

<div><p>As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (<i>Glycine max</i> (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (<i>bar</i>) and six sgRNAs that targeted different sites of two endogenous soybean genes (<i>GmFEI2</i> and <i>GmSHR</i>). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.</p></div

Yeast complementation test and subcellular localization.

<p>(A) Yeast complementation test. Right: culture of yeast strain YB525 cells containing the pYES2 empty plasmids; Left: culture of yeast strain YB525 cells containing the pYES2-GmACSL2 plasmids. Bar = 1 cm. (B) ACS enzyme activities. YB525 cells carrying the pYES2 or pYES2-GmACSL2 plasmids were harvested after galactose induction for 18 h. 1-[¹⁴C] oleic acid was used as a substrate. Enzyme activities were measured based on the [¹⁴C] label incorporated into the acyl-CoA fraction per assay. Values are means of triplicate with standard deviation (SD). (C) Growth rate of the transformed line in different fatty acid culture medium. YB525 was transformed with the pYES2-GmACSL2 and empty PYES2 plasmids, which were cultured in liquid medium with various fatty acids (12∶0, 14∶0, 16∶0, 18∶0, 18∶1, and 22∶1) as the sole carbon source. (D) Subcellular localization. Fluorescence signals of eGFP were detected in cells expressing GmACSL2-eGFP fusion protein by Leica TCS scanning confocal microscope (left panels). Fluorescence signals of dsRFP were detected in cells expressing SSE1-dsRFP fusion protein (middle panels). Right panels were merged by left and middle panels. The upper panels are low-resolution pictures and the lower panels were high-resolution pictures. The immunofluorescence was done with tobacco leafs. Bar = 10 µm. Data are presented as the mean ± SEM of three experiments.</p

Overexpression of <i>GmACSL2</i> in transgenic hairy roots.

<p>(A) Hairy roots were induced by <i>A. rhizogenes.</i> (a) Soybean seeds were germinated in germination medium; (b) Explants were co-cultivated with <i>A. rhizogenes in</i> MCC medium; (c) Hairy roots were induced in 1/2 MS medium with hygromycin B as selection; (d) Hairy roots appeared at the wounding sites of the explants in 1/2 MS medium; (e) Hairy roots were grown in 1/2 MS solid medium; (f) Hairy roots were propagated in 1/2 MS liquid medium. (B) GUS histochemical staining and GFP fluorescence signal detection. After extended culture, the hairy roots were stained by GUS (upper panel) and signals of fluorescence GFP observation (lower panel). WT represents the wild-type soybean hairy roots hairy roots transformed with the K599 strain only; Trans represents the soybean hairy roots transformed with the control vector pGFPGUS and pGFPGUS-GmACSL2. (C) PCR analysis of hairy roots DNA using the primers to amplify the fragment of the GmACSL2 gene to further identify the transgenic line of GmACSL2. M, Marker; “+”, positive control, GmACSL2 plasmid was used as template; “−”, negative control, soybean hairy roots were transformed with the control vector pGFPGUS as template; 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33, individual lines transformed with the binary vector pGFPGUS-GmACSL2. (D) Expression analysis of <i>GmACSL2</i> in different transformed lines by RT-PCR. 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33 represent different transformed hairy roots overexpression with <i>GmACSL2</i>. Soybean actin gene was used as an internal control. (E) Analysis of lipid contents in the transgenic lines by soxhlet extraction. Control, transgenic hairy roots induced by pGFPGUS control vector; GmACSL2-1-8, GmACSL2-2-4, GmACSL2-3-5, GmACSL2-3-14, and GmACSL2-3-33, the five transgenic hairy root lines, induced by the pGFPGUS-GmACSL2 vector. (F) Fatty acid analysis comparisons between five <i>GmACSL2</i> transgenic lines and the control. The five major fatty acid species C16∶0, C18∶0, C18∶1, C18∶2, C18∶3 and the total fatty acids content in hairy roots were detected by gas chromatography-mass spectrometry. Data are the mean ± SEM of the three experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p