| Peanut(Arachis hypogaea L.)is one of the main oil crops which are widely cultivated in the world.The planting area of peanuts in China ranks second in the world,with an annual output ranks first in the world.More than 50% of its total production is used for oil and the oil content which effects the capacity of peanut oil productivity and the processing efficiency of the lipid is up to 60% in peanut seeds.The oil content and fatty acid composition are the key indicator of seed quality of peanut.In order to reveal the molecular mechanism of lipid metabolism,in this study we analysised the lipid accumulation in seed development and degradation patterns in seed post-germination of high oil and common peanut seeds and compared the difference of oil content and the comparison of fatty acid in different stages,then explore the relationship between oil content in the seed cotyledon cells with oil bodies.On these basises,using iTRAQ technology the dynamic changes of the protein in different periods were analysised to reveal the molecular mechanism of the lipid accumulation in seed development and degradation in seed post-germination from the protein level.In order to investigate the molecular genetic mechanism of high oleic acid peanut traits,In this study,six ah FAD2 genes were cloned and analyzed,using the CRISPR-Cas9 site-directed mutagenesis system constructed of ah FAD2-1 gene to further investigate the function of ah FAD2.By Agrobacterium mediated method,the FAD2 gene was knockout through the CRISPR-Cas9 site-directed mutagenesis system to lay a foundation for further cultivating the high oleic acid peanut varieties.The main result of this study were listed as follows:1.This study was used to analysised the oil content and the composition of fatty acid in different stages of HUA U606 and HUA 17,it revealed that the oil content of both materials lay on “S”curve in the development of peanut seed and rapidly decreased in the post-germination.In the seed development stages the composition of fatty acid was consistent but the rangeability was not the same.In the early of seed development linolenic acid appeared and dispeared latter,the correlation of oil content and fatty acid composition was basically the same.In the post-germination stages fatty acid composition of U606 changed significantly,and new fatty acids appeared while in HUA17 fatty acid composition had no significant difference and no new fatty acids generated.This indicated that the use of fatty acid was different in the different oil content of the material in the germinatio stages.This study preliminary exploration the dynamic changes of the lipid and fatty acid in different stages of different peanut material to lay a foundation for cultivating high quality new peanut germplasm2.Using laser scanning confocal microscopy,with the fat synthesis,accumulation and increased oil content,we found the number of oil bodies increased gradually and gradually concentrated in the central cell,arranged more closely.While in the post germination of the seed the number of oil bodies declined and the oil bodies arranged along the wall of the cotyledon cell in 30 DAG.Further study was analysised about the cross-sectional area of the oil body,we found that cross-sectional area increased in the seed development while in the seed post-germination cross-sectional area decreased.3.In the present study,proteomic analyses of four seed developmental stages(20,40,60 80 days after pollination),followed by four germination stages(5,10,20 and 30 days after germination)in peanut seeds,were carried out by iTRAQ technology to gain insights into protein profiles that are responsible for the lipid dynamics over seed development and post germination.5712 of 8505 proteins have been identified,quantified,and divided into 23 functional groups with the largest one to be General function prediction only.The number of the protein which involved in lipid metabolism was 241 which included 88 different expression protein.These protein were divided into five categories: oil accumulation related proteins,lipid degration related proteins,lipid storage-related proteins,lipid transport and some lipid binding related proteins,respectively.Further analyses of the proteins and their pathways revealed that fatty acid accumulation was initiated at early stages of seed development while lipid degradation was mainly through lipoxygenase-dependent pathway.The protein expression patterns,which were related to lipid accumulation and degradation,were verified at transcript levels by quantitative real-time PCR.The proteome profiles of peanut seed development and post-germination constructed in this study will significantly enrich our understanding regarding the process of lipid accumulation and degradation,and the dynamic changes of the metabolic network during different developmental stages in peanut.4.FAD2 which regulated the O/L value in peanut is a large gene family,in this study we identified six full-length FAD2 gene c DNA sequence and analyzed the structure characteristics,finding that Ah FAD2-1,-2,-3 and 4 located in the endoplasmic reticulum membranes and Ah FAD2-5 and Ah FAD2-6 located in chloroplast membranes.Ah FAD2-2,amino acid sequence and Ah FAD2-4 Ah FAD2-3 has very high similarity,but were significantly different with Ah FAD2-1,Ah FAD2-1 in the development of peanut seed had highly specific expression.We used the sequence of Ah FAD2-1 to design sg RNA target point to construction of CRISPR-Cas9 vector.By Agrobacterium mediated method,we primary construction the method that don't need to go through tissue culture and directly injected the keel of the flower and the node of the first branch.This result revealed by injection method there was high conversion efficiency but CRISPR-Cas9 system of target gene editing efficiency low which may related to the guide sequence of the sg RNA.So it needed to design different guide sequence to improve the efficiency editing of the target gene. |
PDF Full Text Request