Genome-wide Identification And Functional Analysis Of FAD Genes And Development And Utilization Of ILP Markers In Medicago

Medicago sativa L.is one of the leguminous forages with the largest planting area in the world.It has a range of ecological adaptability,stable productivity and high nutritional value,and plays a vital role in China agricultural sector and animal husbandry production industry.In addition,the No.1 document of the Central Committee in 2015 explicitly proposed to speed up the performance of grass husbandry and support the planting of M.sativa,which reflects the central government high attention to the development of China's M.sativa industry.The M.sativa cultivation of high-quality is suggesting the first step to promote the outstanding development of China's animal husbandry.?-linolenic acid is an essential fatty acid for human body and plays an important role in human health and brain development.Since the human body cannot synthesize ?-linolenic acid itself,it must be got from external food.Therefore,an in-depth analysis of the molecular metabolic pathway mechanism of ?-linolenic acid in animals and plants is quite significance for easing the insufficient intake of polyunsaturated fatty acids,for improving human health and realizing homology of medicine food.However,the biosynthesis of ?-linolenic acid requires the introduction of unsaturated double bonds into fatty acid chain under the catalysis of fatty acid desaturase(FAD).Therefore,the identification and functional analysis of FAD gene family is the basic discovery point to study the molecular metabolic pathway of ?-linolenic acid.In this thesis,first,the FAD gene family identification,systematic evolution analysis,protein function analysis and expression analysis were carried out on Medicago truncatula and M.sativa from the whole genome level by using bioinformatics methods,and then the function research on M.sativa ?12 and ?15 FAD genes was carried out successively.In addition,molecular markers of M.sativa intron length polymorphism(ILP)were developed on the whole transcription level by using M.sativa transcriptome data and bioinformatics comparison technology.The main research results are:1.Twenty MtFADs gene family members and thirty-three MsFADs gene family members were identified from M.truncatula and M.sativa,respectively.MtFADs is divided into 6 subgroups,while MsFADs is divided into 7 subgroups.MtFADs and MsFADs genes construct the phylogenetic tree,and gene members were integrated into subgroups.MtFADs genes have different responses to temperature stress,especially MtFAD3.1 and MtFAD3.2 genes are induced by the high temperature stress,while MtFAD7.1 and MtFAD7.2 genes respond to the low temperature stress.2.The key genes MsFAD3 and MsFAD6 in ?-linolenic acid synthesis pathway of M.sativa were cloned from M.sativa leaves.Subcellular localization analysis of tobacco leaf epidermal cells showed that proteins encoded by MsFAD3 and MsFAD6 genes were on the endoplasmic reticulum and chloroplast,respectively.The MsFAD3 gene is expressed in five tissues including flower,pod,root,stem and leaf,while the MsFAD6 gene is only expressed in the pod,stem and leaf.3.One hundred and nineteen strains of MsFAD3 transgenic positive plants and 52 strains of MsFAD6 transgenic positive plants were got by transgenic means,with positive rates of 77.8% and 81.2%,respectively.Overexpression of MsFAD3 and MsFAD6 genes in M.sativa can significantly increase ?-linolenic acid content in M.sativa leaves.Nine transgenic plants with MsFAD3 gene while 8 transgenic plants with MsFAD6 gene and then high ?-linolenic acid were screened from the transgenic plants.?-linolenic acid content increased by 23.76% ~35.48% and 9.32% ~24.67%,respectively.4.A total of 502 MsILP molecular markers have been developed in alfalfa,and 100 MsILP markers have been randomly selected for transferability analysis of leguminous and non-leguminous plants.The transferability amplification rates are 40% ~83% and 21% ~22%,respectively.Twenty-one M.sativa varieties were randomly selected for genetic diversity analysis,resulting in 169 alleles,with an average of 4.7 alleles per locus.The polymorphism information content(PIC)values ranged from 0.15 to 0.87,with an average of 0.60.These molecular markers grouped 21 M.sativa varieties into four categories according to their genetic relationship.