MOLECULAR BASIS OF STRUCTURAL STUDIES, PHYTOCHEMICAL CHARACTERIZATION, AND NUTRITIONAL EVALUATION OF NEW MODIFIED ALFALFA DEVELOPED THROUGH DIFFERENT GENE TRANSFORMATION AND GENE MODIFICATION TECHNIQUES IN RUMINANT LIVESTOCK SYSTEMS
Date
2019-08-29
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-7175-4542
Type
Thesis
Degree Level
Doctoral
Abstract
Alfalfa is one of the most important forage crops in the world due to its high nutritive value and good adaptability. However, alfalfa contains relatively high lignin that hinders its nutrients availability. Recently, genetic engineering has been used in alfalfa breeding and scientists from Agriculture Agri-Food Canada (AAFC) have developed several new genotypes of genetic-modified alfalfa. To reduce lignin content of alfalfa, transcriptional factor genes of HB12 and TT8 were silenced. In addition, overexpression of miR156 (miR156 OE) has been shown to delay flowering onset of alfalfa thereby increasing forage quality. Moreover, alfalfa with silenced miR156-targeting SPL6 and SPL13 (Squamosa promoter binding like protein, SPL) genes were generated to determine their roles in miR156 OE event. To date, little is known about the comprehensively nutritional values of these genetic modified alfalfa genotypes. This research combined conventional nutritional analysis with molecular structural analysis to assess nutritional profiles of genetic modified alfalfa and explored the relationship between spectral parameters and nutritional profiles of alfalfa.
Results showed that both HB12-silenced (HB12i) and TT8-silenced (TT8i) alfalfa had higher fiber and endogenous protein loss, but lower protein, dry matter (DM) degradation and microbial protein synthesis compared with wild type (WT). In addition, HB12i had higher lignin content, but lower energy, productions of gas, volatile fatty acids (VFA) and ammonia, protein effective degradation (EDCP), total available protein and feed milk value compared with other alfalfa genotypes. Molecular structure of HB12i and TT8i were different from WT in carbohydrate and lipid regions and all genotypes were different in amide region. As for miR156 OE and SPL6/13-silenced alfalfa, miR156 OE had lower fiber and endogenous protein loss, but higher insoluble true protein, energy, DM degradation and microbial protein synthesis compared with other genotypes. In addition, overexpression of miR156 also improved protein degradation profiles of alfalfa. Molecular structures were similar between miR156 OE and SPL6/13-silenced alfalfa, which were different from WT in carbohydrates and lipid regions. Both projects found differences between transgenic alfalfa genotypes and WT in molecular structures and chemical localization of alfalfa leaves. Furthermore, significant correlations were found between molecular structures and nutritional profiles of alfalfa, providing good predictions of nutrient availability of alfalfa from spectral parameters.
In summary, TT8i provided equivalent energy and protein with improved nutrient balance compared with WT, making it a promising grazing variety. In addition, miR156 OE had improved forage quality that was more similar to SPL6 RNAi alfalfa, implying SPL6 plays a more important role in miR156 OE event than SPL13. Meanwhile, there might be more SPL genes involved in miR156 OE event indicated by the nutritional differences between miR156 OE and SPL6/13-silenced alfalfa genotypes. Molecular structures of alfalfa forage were closely correlated with its nutritional profiles, which made it possible to predict alfalfa nutrient availability from its structural parameters with ATR-FTIR spectroscopy.
Description
Keywords
alfalfa, genetic modification, TT8, HB12, miR156, SPL6, SPL13, ATR-FTIR spectroscopy, Synchrotron FTIR microspectroscopy
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Animal and Poultry Science
Program
Animal Science