Down-Regulation of CYP79A1 Gene Through Antisense Approach Reduced the Cyanogenic Glycoside Dhurrin in [ Sorghum bicolor (L.) Moench] to Improve Fodder Quality

Abstract

A major limitation for the utilization of sorghum forage is the production of the cyanogenic glycoside dhurrin in its leaves and stem that may cause the death of cattle feeding on it at the pre-flowering stage. Therefore, we attempted to develop transgenic sorghum plants with reduced levels of hydrogen cyanide (HCN) by antisense mediated down-regulation of the expression of cytochrome P450 CYP79A1, the key enzyme of the dhurrin biosynthesis pathway. CYP79A1 cDNA was isolated and cloned in antisense orientation, driven by rice Act1 promoter. Shoot meristem explants of sorghum cultivar CSV 15 were transformed by the particle bombardment method and 27 transgenics showing the integration of transgene were developed. The biochemical assay for HCN in the transgenic sorghum plants confirmed significantly reduced HCN levels in transgenic plants and their progenies. The HCN content in the transgenics varied from 5.1 to 149.8 μg/g compared to 192.08 μg/g in the non-transformed control on dry weight basis. Progenies with reduced HCN content were advanced after each generation till T₃. In T₃ generation, progenies of two promising events were tested which produced highly reduced levels of HCN (mean of 62.9 and 76.2 μg/g, against the control mean of 221.4 μg/g). The reduction in the HCN levels of transgenics confirmed the usefulness of this approach for reducing HCN levels in forage sorghum plants. The study effectively demonstrated that the antisense CYP79A1 gene deployment was effective in producing sorghum plants with lower HCN content which are safer for cattle to feed on.

Keywords: CYP79A1; HCN; Sorghum bicolor; antisense; dhurrin; forage quality; transgenics.

Figure 1

Partial map of CYP79A1 Antisense RNA construct (pJS108-CYP79A1-AS) Vector backbone from plasmid pJS108 (courtesy: Dr. Ray Wu, Cornell University, USA); Act1- Promoter of rice actin (Act 1) gene; A.S CYP79A1- cDNA of CYP79A1 in antisense orientation; 35S—CaMV35S promoter; bar encodes the enzyme Phosphinothricin acetyl transferase that confers Basta resistance; PIN and NOS—termination sequences.

Figure 2

Different steps of Sorghum transformation to generate putative transgenices. (A) Shoot meristem explants on induction medium; (B) Growth and bulging of explants on induction medium after 12–14 days of incubation; (C) Survival of transformed explants in selection; (D) Explants with shoot formation in elongation medium; (E) Well-developed plants in the rooting medium; (F) Putative transgenic plants acclimatized in the glasshouse.

Figure 3

Identification of transgene presence in T₀ plants by Polymerase Chain Reaction (PCR) with primers specific to bar gene shows amplification of 294 bp product. Ingel Lane 1 signifies 100 bp DNA ladder (Fermentas). Lane 2 and 3 designate negative control and positive control. Plasmid DNA of construct employed in transformation was used as positive control and genomic DNA of untransformed plant was used as negative control. Lanes 4–15 indicate the line number of respective T₀ plants (S18, S28, S13, S6, S14, S32, S17, S31, S27, S25, S29, S9, S11).

Figure 4

Southern blot of T₀ Transgenics lines for gene integration using pJS108-CYP79A1-AS gene as probe. The genomic DNA of T₀ plants was digested with HindIII enzyme and blot was probed with radiolabelled pJS108-CYP79A1-AS gene. Lane 1- GeneRuler 1Kb (Fermentas), Lane 2- Wild type genomic DNA (negative control), Lane 3- Plasmid DNA of pJS108-CYP79A1-AS construct used for transformation, Lanes 4–15 indicate the line number of respective T₀ plants (S28, S13, S6, S35, S4, S32, S15, S22, S25, S2, S9, S7). The presence of 1.3 Kb band indicates the presence of transgene (pJS108-CYP79A1-AS gene).

Figure 5

Southern blot of T₀ Transgenics lines for sites of transgene integration. The genomic DNA of T₀ plants was digested with Xba1 enzyme and blot was probed with radiolabelled bar gene. Lane 1- GeneRuler 1Kb DNA ladder (Fermentas), Lanes 2–13 indicate the line number of respective T₀ plants (S13, S18, S4, S32, S17, S6, S35, S15, S25, S8, S16, S34), Lane 14- Wild type genomic DNA (Negative control), Lane 15 Plasmid DNA of pJS108-CYP79A1-AS construct used for transformation as positive control.

Figure 6

Reverse transcriptase PCR (RT-PCR) of T₀ Transgenics lines for bar gene RT-PCR with primers specific to bar gene shows amplification of 294 bp product. Lane 1 signifies 100 bp DNA ladder (Fermentas). Lane 2 designate negative control. Wild type RNA of untransformed plant was used as negative control, Lanes 3–15 indicate the line number of respective T₀ plants (S18, S28, S13, S6, S35, S4, S32, S17, S22, S11, S25, S9, S16). The presence of 294 bp band indicates the expression of bar gene in transgenics plants.

Figure 7

Reverse transcriptase PCR (RT-PCR) of T₀ Transgenics lines for antisense gene. RT-PCR analysis of To Transgenics lines using antisense CYP79A1 gene-specific primers shows amplification of 1.67 Kb product. Lane 1 signifies GeneRuler 1 kb DNA ladder (Fermentas), Lane 2- designate negative control. Wild type RNA of untransformed plant was used as negative control; Lanes 3–15 indicate the line number of respective T₀ plants. (S18, S28, S13, S6, S35, S4, S32, S17, S22, S11, S25, S9, S16).

Figure 8

Fold reduction (on log scale) of CYP79A1 gene expression as determined by quantitative Real time PCR in selected progenies of two promising events S4 and S32. The plants from the event S4 and S32 showed reduced expression of the CYP79A1.