Analysis Of Peanut Microarray And Functional Verification Of Sphingolipid Δ8 Desaturase Gene

Peanut(Arachis hypogaea L.)is the main oil crop and economic crop in China,and its yield ranks first in the world.The oil content and quality of peanut seeds determine the economic and nutritional value of peanuts.In recent years,studies on peanut oil related genes has been reported.These studies will be of great significance for peanut molecular breeding,breeding of peanut varieties and improve ment of varieties.Sphingolipids are important components of plant cell membranes,and they are also involved in plant adverse stress.Sphingolipid Δ8 desaturase is a class of fatty acid desaturases that participate in plants by changing the degree of unsaturation of sphingolipids.Arabidopsis,tobacco and other plants were studied,but less research was conducted in peanuts.Based on the above background,the research in this paper mainly includes the following aspects:High-oleic medium oil peanut variety F18 and low-oleic acid low oil variety Luhua6 were used as research materials.Through microarray data analysis,it was found that there were 130,3 556,and 2 783 genes that were significantly differently expressed on the 30,40,and 60 DAP of the two varieties.There were 1 074 genes that were differentially expressed at 40 and 60 DAP by Venn analysis.The GO annotation and enrichment analys was showed that the DEGs were mainly enriched in the metabolic process(GO: 0008152)pathway;And KEGG enrichment result showed that the DEGs were mainly enriched in fatty acid synthesis and photosynthetic pathways.Based on GO annotation,64 and 62 genes involved in lipid metabolism were selected from 40 and 60 DAP.The PPI analysis revealed that,in addition to their interaction with each other,lipid metabolism genes also interact with sugar transport and redox process bases.The sphingolipid Δ8 desaturase gene was used as the research object.Four AhSLD genes were cloned from peanuts and named AhSLD1,AhSLD2,AhSLD3,and AhSLD4.Analysis of the AhSLD1/2/3/4 gene sequence revealed that the ORF of AhSLD1/2/3 was1347 bp,and AhSLD4 was 1359 bp.The protein encoded by AhSLD1/2/3/4 was analogous to that of AhSLD1/2/3/4.The similarities of the Arabidopsis AtSLD were75%,86%,85%,and 77%,respectively,and all contained b5 conserved domains and three histidine box structures.The results of the four AhSLD gene expression analysis in peanuts showed that the four AhSLDs had distinct tissue specificity and different accumulation patterns at different seed developmental stages;the expression characteristics under stress treatment showed that four AhSLDs affected drought,low temperature,high salt,and ABA treatments had different degrees of response.Among them,the most sensitive to drought stress,the expression level of AhSLD2/3/4 gene wasup-regulated by 21,14 and 42 times compared with the control respectively.The results revealed that AhSLD played an important role in peanut adverse stress.Four AhSLD genes were transferred into S.cerevisiae heterologously by genetic engineering methods,and their biological functions were verified.The result show that the yeast transformed into AhSLD produced the t18:1 sphingolipid LCB,which did not detected in the wild type.Further analysis revealed that the conversion rate of the sphingolipid t18:1 was different.In contrast,yeast transformants containing AhSLD1,AhSLD2,AhSLD3,and AhSLD4 accumulated novel(Z)-and(E)-desaturated sphingoid bases with productions of 17.63%,53.50%,23.40%,and 0.92%,respectively.The results revealed that the AhSLD2 gene is the major gene that affects peanut t18:1sphingolipid content.This study provides a theoretical basis for the molecular mechanism of peanut oil metabolism,and also contributes to genetic resources for the improvement of resistance to peanut varieties.