Co-Expression of Bacterial Aspartate Kinase and Adenylylsulfate Reductase Genes Substantially Increases Sulfur Amino Acid Levels in Transgenic Alfalfa (<i>Medicago sativa</i> L.)

<div><p>Alfalfa (<i>Medicago sativa</i> L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial <i>AK</i> and <i>APR</i> genes, modified <i>AK</i>, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.</p></div

PCR and Southern blot analysis of transgenic alfalfa plants.

<p>A. PCR analysis of <i>AK</i> and <i>APR</i> genes in the transgenic alfalfa plants. Lane WT: wild-type line; Lane1-6: Line1-6 transgenic alfalfa lines; Lane 7: water; Lane 8: pDESAK-APR vector; Lane M: GeneRuler TM 1kb DNA ladder (MBI Fermentas, Maryland, USA). Arrows on the left indicate the standard marker bands: 800 bp, 1,000 bp and 1,500 bp. B. Southern blot analysis of <i>AK</i> and <i>APR</i> genes in the transgenic alfalfa plants. Lane L2-1,L2-2,L6-1,L6-2,L8-1,L8-2: two replicates of each transgenic line (Line 2, Line 6, Line 8), Lane WT: wild-type line. M: M: GeneRuler TM 1 kb DNA ladder. Arrows on the right indicate the standard marker bands: 3,000 bp, 5,000 bp, 6,000 bp, 8,000 and 10,000 bp.</p

Analysis of total amino acids and forage products in wild-type and transgenic plants.

<p>A. Total amino acid content in leaves of transgenic and wild-type plants. m/m, % represented mass ratio of total amino acids from dry weight; WT: wild-type plant; 1–12: 12 lines of transgenic alfalfa plants; the bar represented ±SE. B. Weight of up-ground part and leaves products and height of wild-type and transgenic plants. WT : wild-type plant; OE: the average number of 12 lines of transgenic alfalfa plants, the bar represented ±SE.</p

Relative expression levels of SAT, CGS and MS in wild-type and transgenic plants.

<p>Lane WT: wild-type line; Lane 2,6,8: Line 2,6,8 transgenic alfalfa lines. SAT: serine acetyltransferase; CGS: cystathionine γ-synthase; MS: methionine synthase. * represents statistically significant differences (P<0.05). ** represents statistically significant differences (P<0.01).</p

RT-qPCR and Western blot analysis of transgenic alfalfa plants.

<p>A. <i>AK</i> relative expression level in RT-qPCR analysis, Lane WT: wild-type line; Lane 2,6,8: Line 2,6,8 transgenic alfalfa lines. Each bar represents the mean of three biological replicates±SE. ** represents statistically significant differences (P<0.01). B. <i>APR</i> relative expression level in RT-qPCR analysis, Lane WT: wild-type line; Lane 2,6,8: Line 2,6,8 transgenic alfalfa lines. Each bar represents the mean of three biological replicates±SE. ** represents statistically significant differences (P<0.01). C. Western blot assay of expression of APR protein in transgenic alfalfa lines. The total soluble protein extracted from the young leaves of the transgenic lines and non-transformed negative controls. Lane WT: wild-type line, Lane Line2, Line6, Line8: three transgenic lines; Lane +: 6×His-APR fused protein; Lane M: PageRula^r™ prestained protein ladder (Thermo scientific,USA). 26 kDa and 34 kDa indicate the standard marker bands; 37 kDa indicates the size of 6×His-APR fused protein.</p

OAS and free amino acid levels in wild-type and transgenic plants.

<p>Lane WT: wild-type line; Lane 2,6,8: Line 2,6,8 transgenic alfalfa lines. Each bar represents the mean of three biological replicates±SE. * represents statistically significant differences (P<0.05). ** represents statistically significant differences (P<0.01).</p

Construction and confirmation of pDESAK-APR.

<p>A. Schematic map of the T-DNA region of pDESAK-APR. The <i>AK</i> and <i>APR</i> genes were fused with the chloroplast transit peptide by restriction digest using <i>Xho</i>I and <i>Xba</i>I enzymes to construct the intermediate vectors pOSB108-AK and pOSB208-APR. <i>In vivo</i> Cre/loxP-mediated recombination via the loxP site to construct the co-expression vector pDESAK-APR. <i>Hind</i>III, <i>Asc</i>I and <i>I-sce</i>I: restriction endonuclease; <i>NPT</i>II: kanamycin resistance gene; 35S: cauliflower mosaic virus 35S promoter; TP: chloroplast transit peptide; OCS: octopine synthase terminater; LB and RB: left border and right border of the T-DNA. B. Enzyme digestion analysis of pDESAK-APR. Lane <i>Asc</i>I: Vector digested by <i>Asc</i>I and Lane <i>I-sce</i>I: Vector digested by <i>I-sce</i>I. Lane M: molecular mass marker is GeneRuler TM 1 kb DNA ladder (MBI Fermentas, MD, USA). Arrow on the left indicates the expected fragment of 13,000 bp, arrows on the right indicate the expected fragment of 5,000 bp and 8,000 bp.</p

Amino acid metabolic pathways and its integration into the TCA cycle.

<p>Red indicates increased amino acids; Green indicates decreased amino acids.</p

The content of crucial amino acids in leaves of wild-type and transgenic plants.

<p>Extracts were obtained from the leaves of 16-week-old non-transformed plants and transgenic plants co-expressing bacterial <i>AK</i> and <i>APR</i>. Seven kinds of crucial amino acids were list. The amounts of amino acids were calculated from dry weight of samples as detected by Hitachi L-8900 and are given inµmol/g DW. The data presented represent the mean and the bars represent ±SE of three plants per line.</p

Amino acid contents in the leaves of wild-type and transgenic alfalfa plants over-expressing <i>AK</i> and <i>APR.</i>

<p>The data are presented as the means ±SE obtained from three independent measurements.</p><p>* represents statistically significant differences (P<0.05). ** represents statistically significant differences (P<0.01). The amounts of amino acids were calculated from dry weight of samples as detected and are given inµmol/g of dry weight (DW) of leaves samples. The total amino acid content did not differ significantly between wild-type and transgenic lines. Three plantlets of each type were analyzed.</p