Cloning And Function Analysis Of ?-12 Fatty Acid Desaturases GhFAD2 Gene In Upland Cotton (Gossypium Hirsutum L.)

Objective: Objective: The ?12-desaturase enzyme gene FAD2 encodes the key enzyme that catalyzes the conversion of oleic acid to linoleic acid during plant fatty acid synthesis and metabolism.In oil crops,oleic acid(C18:1)and linoleic acid(C18:2)are the main fatty acid components in seed oil.The relative proportion of C18:1 and C18:2 in the seed of oil crops determines the nutritional properties and oxidation stability of edible vegetable oil.Cotton is not only the most important fiber crop,but also an important oil crop.The study of Gh FAD2,that of specific and highly expression in cottonseed would be great significance for the regulation and directional modification of fatty acids in cottonseed oil by gene engineering technology.Methods:(1)The GhFAD2 family genes were obtained from the cotton genome database based on the known GhFAD2 amino acid sequence(Gen Bank X97016),and were analyzed by the bioinformatics.(2)The expression characteristics of the GhFAD2 gene family during different development stages and tissues were analyzed by qRT-PCR technology.(3)Soxhlet extraction(petroleum ether)extraction was used to extract the total oil from cottonseeds at different developmental stages,and the samples of cottonseed in 5-60 days(DPA)were measured by GC-MS.The GhFAD2 mRNA expression level and C18:1/C18:2 ratio of during the different developmental cottonseed were analyzed.(4)566 cotton varieties(lines)with high(>20%),medium(17%-19%)and low(11%-16%)oil content were screened by the NIR scanner without damage,and the relative content of each fatty acid component was determined.The correlation between GhFAD2 expression level(the relative proportion of oleic acid / linoleic acid)and the oil content of cottonseed was analyzed.(5)GhFAD2-1 gene that highly expressed in the seed was obtained from cotton by homologous cloning technique.The plant interference expression vector 35HK-GhFAD2-1 was constructed by Gateway technology,and the plant overexpression vector p BI121-GhFAD2-1 was constructed by DNA recombinant technology,respectively.Arabidopsis and the Cotton variety “Xinluzao33” was transformed by Agrobacterium mediated genetic transformation.And then the function of GhFAD2-1 was further analyzed.Results and conclusions: 1.Bioinformatics method was used to obtain GhFAD2 family genes from the cotton genome database.The GhFAD2 gene has 4 copies in the A genome and the D genome,named GhFAD2-1A,GhFAD2-1D,GhFAD2-2A,GhFAD2-2D,GhFAD2-3A,GhFAD2-3D,GhFAD2-4A,GhFAD2-4D.The sequence analysis shows that there is no intron sequence in the Gh FAD2 gene coding region,and the same FAD2 gene of cotton is highly consistent with the sequence of the A genome and the D genome.The results of 2.qRT-PCR showed that the GhFAD2-1/2/3/4 gene was different in different tissues,and the GhFAD2-1 gene was highly expressed in the developing seeds,but the expression in other tissues was very low(Figure 3-4A).GhFAD2-2 gene is expressed in all tissues of upland cotton,but the expression level is low.The homology of GhFAD2-3 and GhFAD2-4 gene is 98.44%,which is expressed in all tissues,including roots,stems,leaves,flowers and seeds.3.,using qRT-PCR technology analysis,we found that GhFAD2-1 gene increased and then decreased in cottonseed development.40 DPA was the peak of its expression.The fatty acid composition was determined by GC-MS gas chromatography-mass spectrometry.Results showed that the ratio of oleic acid to linoleic acid was negatively correlated with FAD2-1 gene expression.Through the analysis of fatty acid components of upland cotton materials,it was found that the content of seed oil content was not significantly correlated with the relative percentage content of palmitic acid,stearic acid,oleic acid and linoleic acid.The expression level of GhFAD2-1 gene in seed determines the relative proportion of oleic acid / linoleic acid,but it has no obvious correlation with the oil content of cottonseed.4.the GhFAD2-1 gene obtained cotton variety Xinluzao 33 homologous cloning technology ".The coding region of the gene is 1158 bp,which encodes 385 amino acids.The 35HK-GhFAD2-1 interference expression vector was constructed to convert the wild Arabidopsis thaliana to the pattern plant,and the overexpression vector p BI121-GhFAD2-1 transformed the FAD2 mutant of Arabidopsis(CS8041),and the GhFAD2-1 transgenic homozygote was obtained.The content of oleic acid decreased and the content of linoleic acid increased.The interference expression of GhFAD2-1 gene in wild Arabidopsis inhibited the expression of FAD2-1 gene in Arabidopsis thaliana,so it showed a similar character with mutant.5.the pBI121-GhFAD2-1 overexpression vector and interference carrier 35HK-GhFAD2-1 were used to infect the hypocotyl of cotton Xin Lu Zao 33 by Agrobacterium mediated method and obtain cotton positive plants for further study on the expression and regulation mechanism of GhFAD2-1 at the molecular level,thus laying the foundation for the improvement of the quality of cottonseed oil.