| Camelina sativa(L.)Crantz is an annual herb plant in Crucifer family(Brassicaceae).This plant species is regarded as a renewed important oilseed crop because of high seed oil,enrichment in healthy fatty acids such as α-linolenic acid(ω-3 type fatty acids),and excellent agronomic traits including low input for water and nutrients,adaptability to adverse environmental conditions and resistance to pests and pathogens.Camelina seed oil is increasingly and widely used in commercial production of edible oil,medicine and nutraceuticals,biofuels and high-valued chemicals.With the short life cycle and an established system of a simple and effective genetic transformation,camelina is currently also being developed as an ideal model crop.Therefore,it is so important in scientific theory and application value to investigate the biosynthesis and regulation mechanism of storage oil and proteins in camelina seeds.A number of new findings are obtained in this study,including the followings:1.For comprehensive understanding of camelina oil synthesis in seed development under the natural environments in Shanxi,we characterized the morphological changes,total oil content,and the fatty acid composition of developing camelina seeds.In the early stages of seed development(1～16DAF,days after flowering),higher levels of palmitic,stearic and oleic acids were formed,but total oil is lower.During the middle developing period(16～24 DAF),the fatty acid composition was progressively changed,and many kinds of long-chain unsaturated fatty acids were produced,oil content increased up to 26.8%,and 0.47 mg/seed at 24 DAF.At the final developmental stages(24～36 DAF),seed oil content continued to increase due to desiccation.Compared to the vegetable organs of roots,stems and leaves,seed at 36 DAF accumulated much higher levels of linoleic acid(up to 14.8%)and linolenic acid(up to 41.3%).The present data provide valued scientific references for camelina genetic improvement and developing a cultivation system in such newly expanded areas for this important oilseed.2.Gene expression in camelina developing seeds is analyzed by transcriptome sequencing.6.96 Gb clean data were generated,and a total of 95649 genes were assembled.75.84%reads were matched in the camelina genome,and 87803 genes were annotated.Among these,5653 genes were identified to involve in lipid synthesis and metabolism,and 73 genes in seed storage proteins synthesis.Additionally,a total of 1009 new gene without matching in camelina genome were identified in camelina seeds,and 895 new genes were functionally annotated by BLAST with NR,Swiss-Prot,GO,COG,KOG,Pfam and KEGG databases.Functional annotations showed that 34 genes were identified as lipid biosynthesis genes.Notably,these transcriptomic data will be useful for further elucidating biosynthesis and regulatory mechanism of camelina seed oil and proteins.3.MicroRNAs(miRNAs)are important regulators of plant development and seed formation.They can regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in plants.We systematically examined the microRNA profiles of developing camelina seeds at five stages by deep sequencing,and 93.20 M clean reads were generated.Expression analysis revealed a number of miRNAs with varied expression levels at different developmental stages.A total of 237 new miRNA and 340 miRNA target genes were identified in camelina seeds by miRNA sequencing.These CsmiRNAs and their targets were involved in a broad range of physiological functions including lipid metabolism.In order to verify the CsmiRNA target genes and their cleavage site,degradome library were constructed from developing camelina seeds at five stages for deep sequencing.A total of 70 miRNA target genes were identified in camelina seeds.Comprehensive analysis revealed a group miRNAs and their targets related to lipid synthesis and metabolism in camelina seeds.These data provides new information for the miRNA-mediated regulatory network of camelina seed oil and protein biosynthesis,and further advances our understanding of miRNA functions in seed development and the related metabolisms.4.To enhance seed oil content for meeting an increasing market demand of camelina oil,a cDNA clone(VgDGAT1)encoding type 1 diacylglycerol acyltransferase with the higher DGAT enzymatic activity was introduced into camelina by Agrobacterium-mediated floral dip infiltration.Seed-specific over-expression of VgDGAT1 significantly enhanced the DGAT activity in the transgenic seeds by 30 folds more compared to the wild-type control,resulting in seed oil content increase from 37%(dry weight)in the wild-type seeds up to 46%-51%in the transgenics.However,seed protein level was not significant difference between the transgenic and the wild type,indicating that genetic modification on DGAT can break up the negative genetic linkage of oil and protein contents in seeds.Moreover,the seed-specific high expression of VgDGATl did not adversely affect seed weight,seed germination and other agronomic traits.Such novel engineered camelina lines with higher seed oil content and no reduction of protein accumulation could be used for breeding new camelina varieties with multiple excellent agronomic traits for commercialization.5.Camelina sativa is an important oilseed crop with high value in food nutrition and industrial application.In addition to high content of unsaturated fatty acids,camelina seeds contain lots of storage proteins.2S albumin/napin,a major storage protein in seeds of oil crops including camelina,is detected as an allergen to partial human populations,particularly for children.This protein negatively affects the nutrition value of camelina seeds used as food.EMS treating seeds is an efficient mutation method to induce point mutation of plant tissue or organ.In this study,camelina seeds were treated for mutation by EMS(ethyl methane sulphonate).The treatments were conducted with different EMS concentrations(0,0.2%,0.4%,0.6%,0.8%,1.0%,1.2%)and various treating times(0,8 h,12 h and 16 h).An optimized mutagenesis system by EMS was developed for camelina seeds.One mutant showing 2S albumin deficiency in seed proteins was obtained,and designated as 2SP1.Except for this feature,the mutant exhibited no significant difference from the wild-type camelina for other agronomic traits including seed oil and protein contents,plant morphology and seed germination.This special mutant of 2SP1 could be used as a new excellent germplasm for investigating the mechanism of seed storage protein biosynthesis and accumulation,for breeding new camelina varieties with high nutrition quality,and also for further functional genomics research.Such knowledge and techniques developed in this study will advance our comprehensive understanding of biosynthesis and regulatory mechanism of camelina seed oil and proteins,and expand camelina functional genomics and industrialization of camelina seeds.Such findings obtained here also provide new gene elements and the corresponding technology for efficient assembly of desirable oil biosynthesis and genetic improvements in other oil crops including Brassica,sesame,and soybean. |
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