| Upland cotton(Gossypium hirsutum)is one of the most important oil crops in the world.In addition to the applications in food industry,cottonseed oil is also widely used to produce biodiesel and other oil chemical products.However,cottonseed oil with high level of polyunsaturated linoleic acid(C18:2?~9,12,about 59%)is easily apt to be oxidized.Therefore,cottonseed oil needs to be handled through hydrogenation to increase its saturation during food processing,yet it will generate trans-fatty acid,a byproduct which will increase the risk of cardiovascular disease.Compared with common oilseed crops,such as soybean and rapeseed oil,cottonseed oil contains more saturated palmitic acid(C16:0,about 25%),it is also an important limiting factor in the nutritional function of cottonseed oil.?-7 monounsaturated fatty acid(such as palmitoleic acid,C16:1?~9)is a kind of healthy fatty acid,which is of high nutrient and industrial value.However,the major oil crops,including cotton,soybean and rapeseed,contain hardly any?-7 fatty acids(FAs).Therefore,modifying the fatty acid composition of cottonseed oil,and developing high-quality cottonseed oil has become a main target in cotton breeding.In this study,we systematically characterized the accumulation patterns and regulatory features of lipid and fatty acid composition in cottonseed,identified cotton acyl ACP-?~9 desaturase(Gh SAD)and?-ketoacetyl ACP synthase ?(Gh KAS?)family members,which controlled the synthesis of monounsaturated fatty acid(C16:1?~9 or C18:1?~9)and the ratio of C16 and C18 fatty acids,respectively,and then examined their expression profiles,transformed an RNAi silencing vector targeting cotton Gh KAS? genes and overexpression vector of Macfadyena unguis-cati ACP-?~9 desaturase(Muc ACP-?~9d)gene into cotton,respectively,to explore the accumulation changes of fatty acids,combined the RNAi and overexpression vector to assembly an ?-7 fatty acid biosynthesis pathway in cotton,created a new cotton germplasm rich in ?-7 FA.Our results enriched the regulatory theory of seed oil synthesis,and developed a new metabolic assembly strategy which can be used to enrich ?-7 FA in other field oilseeds.The main results are as follows:1.Fatty acid composition analysis from four different varieties of upland cotton showed the very low content of palmitoleic acid(C16:1?~9,<1%)in mature cottonseeds.Further,we found the content and accumulation patterns of fatty acid composition between cottonseed embryos and endosperms at developing stages were very different.Though the disappearance of endosperm in mature cotton seeds,the content of palmitoleic acid in endosperm was significantly higher than that in embryo before the maturation of seeds,and it was first increased and then decreased during the seed development and reached the highest level(16.95 Mol%)in endosperm at 25 days after flowering(DAF).While the content of palmitoleic acid in embryo was very low(less than 1%),the oleic acid(C18:1?~9)content was higher than that in endosperm at all developing stages.This implied that there existed acyl-ACP desaturases with specific 16:0-ACP activity in cotton genome,which could specifically boost the accumulation of palmitoleic acid in developing endosperm,while acyl-ACP desaturases with specific 18:0-ACP activity existed in developing embryo.2.We identified 17 cotton acyl ACP-?~9 desaturase(Gh SAD)family members by genome-wide data analysis,and renamed them as Gh A-SAD1~Gh A-SAD9 and Gh D-SAD1~Gh D-SAD8.By aligning the key amino acids in protein conserved domains,10 Gh SAD members contained the same eight amino acids as typical SAD enzymes which specifically catalyzed desaturation of 18:0-ACP.In addition,these eight amino acids in six members(Gh A-SAD5/6/7,Gh D-SAD5/7/8)showed great variance with known SAD enzymes with 18:0-ACP activity,but hold the similarities with known enzymes with 16:0-ACP,such as At ADD2.Three-dimensional structure analysis showed that these eight amino acids were located at the bottom of substrate binding cavity closed to catalytic center of iron ion,these two types of SAD enzymes with different substrate specificity hold the different substrate binding cavity structure.We speculated that the six Gh SADs may have specific selectivity for 16:0-ACP,while the ten Gh SADs have specific selectivity for 18:0-ACP.3.We further found the promoter upstream sequences in these six Gh SAD genes with specific selectivity for 16:0-ACP selectivity contained cis-elements expressed in endosperm,implying their specific expressions in developing cotton endosperm,and further catalyzing 16:0-ACP to produce 16:1?~9-ACP.Expression analysis showed that these six genes were lowly-expressed in cotton roots,stems,leaves and flowers,while they tended to be highly expressed in mid-developing ovules.Further,expression analysis in developing embryos and endosperms showed that Gh A-SAD6 and Gh D-SAD8 had the highest levels in endosperm of 15-25 DAF,while a little expression of Gh A-SAD5 and Gh D-SAD7 was found in cotton embryo.The tissue-specific expression of Gh SADs showed their functional differences.4.Through the transient-expression system in tobacco and functional complementary assay in TAG synthesis deficient yeast,both Gh A-SAD6 and Gh D-SAD8 could catalyze the production of high-level palmitoleic acid,and the activity of Gh A-SAD6 was slightly stronger than Gh D-SAD8.In contrast,the host cellsheterologously expressing Gh A-SAD5 and Gh A-SAD7 produced more oleic acid than palmitoleic acid.These results indicated that Gh A-SAD6 and Gh D-SAD8 have strong substrate specificity for C16:0-ACP and they may be responsible for the synthesis of palmitoleic acid in developing endosperm,while Gh A-SAD5 and Gh D-SAD7 have specificity for C18:0-ACP,responsible for the synthesis of oleic acid in developing embryo.5.In plastid,?-ketoacyl-ACP synthase ?(KAS?)(EC: 2.3.1.179)catalyzes 16:0-ACP to form 18:0-ACP which determines the ratio of 16 C and 18 C fatty acids in plant cells.To accumulate more 16:0-ACP,thereby providing more substrates for 16:0-ACP-specific SAD to synthesize 16:1?~9-ACP,we designed one RNAi interfering target site based on conserved region of Gh KAS? coding sequences,and constructed Gh KAS? gene silencing vector(Gh KAS?-RNAi)for genetic transformation with agrobacterium-mediated method in cotton.Molecular detection showed that the expression level of Gh KAS? genes in transgenic cotton lines was significantly lower than that in the control lines.The content of palmitic acid(C16:0)in Gh KAS?-silenced transgenic cottonseed oil was increased by about 13% compared with wild type,and the corresponding stearic acid,oleic acid and linoleic acid were decreased by 1.33%,4% and 5.4%,respectively.Poor phenotypes including agronomic traits(such as total oil,starch and soluble sugar content)and germination rate were not detected in transgenic cottonseeds.6.The c DNA of acyl-ACP-?~9 desaturase(Muc ACP-?~9d)was isolated and cloned from the developing seeds of the high ?-7 fatty acids in Macfadyena unguis-cati,which could efficiently catalyze the formation of 16:1?~9-ACP from 16:0-ACP.A seed-specific expression vector(Gly-Muc ACP-DSRB)of this gene was constructed and Muc ACP-?~9d gene was seed-specifically overexpressed in transgenic lines obtained by agrobacterium-mediated genetic transformation in cotton.Biochemical tests showed that the palmitoleic acid(C16:1?~9)content in Muc ACP-?~9d transgenic cottonseed was increased from 0.47% of wild type to6.62%,and the carbon chain extension product cis-11-carbenoic acid(C18:1?11)also was increased about1.5%.Accordingly,the levels of palmitic acid and linoleic acid were significantly reduced.Compared with the control,the content of total oil in transgenic seeds was also increased by about 4%,while other physiological,biochemical indicators(such as starch and soluble sugar content)and germination rate at27 ? did not change significantly.In addition,the germination rate of transgenic seeds at low temperature(17 ?)was significantly higher than that of wild type,indicating that the stress tolerance of transgenic cottonseeds capable of synthesizing omega-7 fatty acids was significantly improved.7.Further,synchronous manipulation was conducted in cottonseeds by down-expressing Gh KAS? genes and over-expressing Muc ACP-?~9d gene.The content of ?-7 fatty acids in transgenic seeds oil was increased from less than 1% to 19.6%.Among them,the content of palmitoleic acid and cis-11-octadecanoic acid(C18:1?11)in transgenic cottonseeds was improved to 14.62% and 4.98%,respectively.Correspondingly,the linoleic acid and palmitic acid content were decreased by 13.1% and2.58%,respectively.The agronomic traits showed no negative changes in this double-gene modified cotton engineering strains.It is clear that silencing of endogenous Gh KAS? to block the 18:0-ACP production in the plastid can increase the accumulation of 16:0-ACP.Overexpression of the 16:0-ACP-specific Muc ACP-?~9d enzyme proteins to catalyze more 16:0-ACP to form 16:1?~9-ACP,ultimately achieving enrichment of ?-7 fatty acids.This successful assembly of ?-7 fatty acid biosynthesis pathways by combination of silencing endogenous KAS? and over-expressing 16:0-ACP-specific SAD in cotton developing seeds can also be used for genetic improvement of oil quality in other oil crops. |
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