RESUMEN
Alfalfa (Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization. Downregulation of two transcriptional factors, Transparent Testa8 (TT8) and Homeobox12 (HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8 (TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content, bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants (5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control. Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.
RESUMEN
Homeodomain-leucine zipper (HD-Zip) genes encode plant-specific transcription factors, which play important roles in plant growth, development, and response to environmental stress. These genes have not been fully studied in allopolyploid Brassica napus, an important kind of oil crop. In this study, 165 HD-Zip genes were identified in B. napus and classified into four subfamilies. If proteins belong to the same subfamily, they exhibit similarities in gene structure, motifs, and domain distribution patterns. BnHD-Zip genes were unevenly distributed in the An and Cn subgenomes. Whole genome triplication (WGT) events may be major mechanisms accounting for this gene family expansion. Orthologous gene analysis showed that the process of this gene family expansion was accompanied by domain loss. We further found three genes homologous to HB7 and three genes homologous to HB12, all induced by PEG, ABA, and NaCl treatment. HB7 could not form homodimers but could form heterodimers with HB12 based on yeast two-hybrid assays. The results of this study provide valuable information for further exploration of the HD-Zip gene family in B. napus.
Asunto(s)
Brassica napus , Brassica napus/genética , Genoma de Planta , Regulación de la Expresión Génica de las Plantas/genética , Dimerización , Proteínas de Homeodominio/genética , FilogeniaRESUMEN
Advanced synchrotron-based vibrational molecular spectroscopy (SR-IMS) has been developed to image molecular chemistry in biological tissues within cellular and subcellular dimension. However, it is seldomly used in gene-transformation and gene-silencing study. The objectives of this study were to apply synchrotron-based vibrational molecular spectroscopy (SR-IMS) to determine the molecular structural changes and chemical mapping of alfalfa leaves induced by silencing of TT8 and HB12 genes in alfalfa in comparison with wild type of alfalfa. Five alfalfa leaves from each alfalfa genotype were selected for FTIR spectra collection and chemical mapping with synchrotron-based FTIR microspectroscopy (SR-IMS). Peak heights and areas of empirical regions were analyzed, and peak areas of previous regions were mapped for each sample using OMNIC 7.3. Results showed that transformed alfalfa had higher peak height and area of carbonyl CO (CCO), compared with wild type (WT). Chemical groups maps for carbohydrate, amide and lipid-related regions were successfully obtained. HB12-silenced (HB12i) had higher carbohydrate intensity both in the mesophyll and epidermises, whereas TT8-silenced (TT8i) and WT only had higher carbohydrate spectral peak intensity in epidermises. In addition, HB12i had higher CCO intensity and lower lignin intensity compared with TT8i and WT. All alfalfa genotypes had higher intensity of amide and asymmetric and symmetric CH2 and CH3 (ASCC) area in mesophylls. In conclusion, silencing of HB12 and TT8 genes in alfalfa both increased CCO profiles of alfalfa leaves, while silencing of HB12 had more impacts on chemical localization in alfalfa leaves.
Asunto(s)
Medicago sativa , Sincrotrones , Alimentación Animal/análisis , Medicago sativa/genética , Medicago sativa/metabolismo , Estructura Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Análisis EspectralRESUMEN
BACKGROUND: Transparent Testa8 (TT8) and Homeobox12 (HB12) are two transcriptional factors in plant phenylpropanoid pathways and were reported to be positively related to lignin content. Alfalfa with silenced TT8 (TT8i) and HB12 (HB12i) was therefore generated using the RNA interference (RNAi) technique. Although lignin was found to be high in HB12i, such gene-silencing of alfalfa resulted in nutrient profiles that might be suitable for grazing. To extend the nutritional evaluation of transformed alfalfa, ground samples of 11 HB12i, 5 TT8i and 4 wild type (WT) were incubated in rumen fluid : buffer solution for 0, 2, 4, 8, 12, 24 and 48 h at 39 °C. Dry matter (DM) and neutral detergent fiber (NDF) degradations at each time point, and production of volatile fatty acids (VFA) at 4, 12, 24 and 48 h were analyzed, as well as degradation and production kinetics. The correlations and regressions between nutritive profiles and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral parameters were determined. RESULTS: Both transformed genotypes had lower DM degradation and HB12i had lower VFA production compared with WT. Structural carbohydrate (STC) parameters were found to be negatively correlated with DM degradation and VFA production. The kinetics of DM degradation and VFA production were predicted from spectral parameters with good estimation power. CONCLUSION: Silencing of HB12 and TT8 affected fermentation characteristics of alfalfa and some fermentation characteristics were predictable from spectral parameters using ATR-FTIR spectroscopy. © 2019 Society of Chemical Industry.
Asunto(s)
Ácidos Grasos Volátiles/metabolismo , Silenciador del Gen , Medicago sativa/genética , Medicago sativa/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/genética , Alimentación Animal/análisis , Animales , Bovinos , Fibras de la Dieta/metabolismo , Digestión , Proteínas de Plantas/metabolismo , Rumen/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Factores de Transcripción/metabolismoRESUMEN
This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Silenciador del Gen , Proteínas de Homeodominio/genética , Medicago sativa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rumen/metabolismo , Alimentación Animal/análisis , Animales , Bacterias/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Bovinos , Digestión , Proteínas de Homeodominio/metabolismo , Cinética , Medicago sativa/química , Medicago sativa/metabolismo , Proteínas de Plantas/química , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteolisis , Rumen/química , Rumen/microbiologíaRESUMEN
The objective of this study was to investigate the effects of silencing the TT8 and HB12 genes on the nutritive profiles and in vitro gas production of alfalfa in relation to the spectral molecular structures of alfalfa. TT8-silenced (TT8i, n = 5) and HB12-silenced (HB12i, n = 11) alfalfa were generated by RNA interference (RNAi) and grown with nontransgenic wild type controls (WT, n = 4) in a greenhouse. Alfalfa plants were harvested at early-to-mid vegetative stage. Samples were analyzed for their chemical compositions, CNCPS fractions, and in vitro gas production. Correlations and regressions of the nutritional profiles and in vitro gas production with the molecular spectral structures were also determined. The results showed that the transformed alfalfa had higher digestible fiber and lower crude protein with higher proportions of indigestible protein than WT. HB12 RNAi had lower gas production compared with those of the others. Some chemical, CNCPS, and gas-production profiles were closely correlated with spectral structures and could be well predicted from spectral parameters. In conclusion, the RNAi silencing of TT8 and HB12 in alfalfa altered the chemical, CNCPS and gas-production profiles of alfalfa, and such alterations were closely correlated with the inherent spectral structures of alfalfa.
Asunto(s)
Gases/química , Medicago sativa/química , Medicago sativa/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/química , Interferencia de ARN , Alimentación Animal/análisis , Animales , Bovinos/metabolismo , Gases/metabolismo , Medicago sativa/metabolismo , Valor Nutritivo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative impact on feed digestibility. TT8 and HB12 genes were shown to have low expression levels in low-lignin tissues of alfalfa, but to date, there has been no study on the effect of down-regulation of these two genes in alfalfa on nutrient chemical profiles and availability in ruminant livestock systems. The objectives of this study were to investigate the effect of transformation of alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO nutritive value in ruminant livestock systems. The results showed that transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded CHO fractions (RDCA4, P = 0.06; RDCB1, P < 0.01) and totally degraded CHO fraction (TRDCHO, P = 0.08). Both HB12 and TT8 populations had significantly higher in vitro digestibility of neutral detergent fiber (NDF) at 30 h of incubation (ivNDF30) compared to the control (P < 0.01). The TT8 populations had highest ivDM30 and ivNDF240. Transformation of alfalfa with TT8 and HB12 RNAi constructs induced molecular structure changes. Different CHO functional groups had different sensitivities and different responses to the transformation. The CHO molecular structure changes induced by the transformation were associated with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8 RNAi could improve forage quality by increasing the availability of both NDF and DM. Further study is needed on the relationship between the transformation-induced structure changes at a molecular level and nutrient utilization in ruminant livestock systems when lignification is much higher.