Abstract
Gene silencing with RNA interference (RNAi) technology may be capable of modifying internal structure at a molecular level. This structural modification could affect biofunctions in terms of biodegradation, biochemical metabolism, and bioactive compound availability. The objectives of this study were to (1) Detect gene silencing-induced changes in carbohydrate molecular structure in an alfalfa forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced changes in nutrient bioutilization and bioavailability in the alfalfa forage (Medicago sativa); and (3) Quantify the correlation between gene silencing-induced molecular structure changes and the nutrient bioutilization and bioavailability in animals of ruminants. The experimental treatments included: T1 = Non-transgenic and no-gene silenced alfalfa forage (code “NT”); T2 = HB12-RNAi forage with HB12 gene down regulation (code “HB12”); T3 = TT8-RNAi forage with TT8 gene down regulation (code “TT8”). The HB12 and TT8 gene silencing-induced molecular structure changes were determined by non-invasive and non-destructive advanced molecular spectroscopy in a middle infrared radiation region that focused on structural, non-structural and total carbohydrate compounds. The nutrient bioutilization and bioavailability of the modified forage were determined using NRC-2001 system in terms of total digestive nutrient (TDN), truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid (tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy, metabolizable energy, net energy) for ruminants. The carbohydrate subfractions were evaluated using the updated CNCPS 6.0 system. The results showed that gene silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8); p = 0.009). The gene-silencing also affected carbohydrate CA4 (7.4 (NT) vs. 4.2 (HB12) and 4.4% carbohydrate (CHO) (TT8), p = 0.063) and CB1 fractions (5.3 (NT) vs. 2.0 (HB12) and 2.6% CHO (TT8), p = 0.006). The correlation study showed that the structural CHO functional group peak area intensity at ca. 1315 cm−1 was significantly correlated to the TDN1x (r = −0.83, p = 0.042) and the tdNFC (r = −0.83, p = 0.042), the structural CHO functional group height intensity at ca. 1370 cm−1 was significantly correlated to the tdNDF (r = −0.87, p = 0.025). The A_Non-stCHO to A_StCHO ratio and A_Non-stCHO to A_CHO ratio were significantly correlated to the tdFA (r = 0.83–0.91, p < 0.05). As to carbohydrate fractions, both CA4 and CB1 correlated with carbohydrate spectral intensity of the H_1415 and the H_1315 (p = 0.039; p = 0.059, respectively), CB3 tended to correlate with the H_1150, H_1100 and H_1025 (p < 0.10). In conclusion, RNAi-mediated silencing of HB12 and TT8 modified not only inherent CHO molecular structure but also the biofunctions. The CHO molecular structure changes induced by RNAi gene silencing were associated with biofunctions in terms of the carbohydrate subfractions and nutrient digestion.
Highlights
Gene silencing through RNA interference (RNAi) technology is capable of modifying internal structure at a molecular level
Jonker et al [1] reported that insertion of the Lc gene into three winter-hardy alfalfa varieties in western Canada induced production of proanthocyanidin (PA) and anthocyanidin (AC) compounds, which are not produced in non-transgenic alfalfa plants
The hypothesis of this study was that silencing HB12 and TT8 genes in alfalfa would induce molecular structure changes, which would result in biological changes in terms of carbohydrate subfractions and nutrient availability
Summary
Gene silencing through RNAi technology is capable of modifying internal structure at a molecular level. The PA and AC compounds are capable of bonding with highly soluble forage protein in the rumen, preventing soluble protein from being degraded in the rumen and shifting protein from the rumen to the small intestine to be digested by internal enzymes released from the small intestine This genetic modification was shown to reduce incidences of bloating and digestive disorders, resulting in improvement of nutrient availability in ruminants [2,3,4]. Two novel RNAi alfalfa genotypes were developed by down-regulating the expression of the alfalfa TT8 and HB12 genes [6] These two genes are expected to modify the lignin biosynthesis pathway resulting in reducing the forage lignin level and affecting lignin structural conformation [6]. The hypothesis of this study was that silencing HB12 and TT8 genes in alfalfa would induce molecular structure changes, which would result in biological changes in terms of carbohydrate subfractions and nutrient availability
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
