| Peony(Paeonia suffiuticosa Andr.)is a perennial woody deciduous shrub belonging to the genus Paeoniaeaceae of the peony family.the ornamental and medicinal value of which are particularly high and the cultivation history of which has lasted more than 4,000 years.The value of peony seed oil is relatively high.In recent years,it has been widely studied because of its rich unsaturated fatty acids,and it mainly range from oleic acid,linoleic acid,palmitic acid to linolenic acid.Its unsaturated acid content is significantly higher than that of saturated fatty acids,the content of which is as high as 83.42%,while the content of ?-linolenic acid accounts for the highest ratio of 66,8%.A-linolenic acid(ALA),a kind of polyunsaturated fatty acid,is an essential fatty acid in the human body,which has medical health care functions such as lowering blood sugar,blood lipids,improving vision,and preventing myocardial infarction.At present,the research on peony mainly focused on the flower color,however,the relevant research on the morphological establishment of seeds and the accumulation of main storage materials is extremely scarce,and the research on the molecular mechanism of Peony seed oil accumulation regulation is still limited.In this study,Peony cultivar 'Fengdan' was used as the experimental material to determine the physiological indexes of peony seeds at different developmental stages;The morphological characteristics and internal structure of Peony seeds were observed;The sequence of PoFAD8-1,a key gene that regulates the accumulation of a-linolenic acid,was cloned,and the characteristics and related functions of PoFAD8-1 gene were preliminary studied.The research yields the following research findings:(1)Collecting physiological indexes of peony seeds at different developmental stages:The overall trends of physiology indexes of peony seeds were consistent,they all increased with the development of seeds,and the maturation period tended to be stable.The consistence was manifested like seed weight,storage material(protein such as protein starch,total sugar content)and other physiological indicators,but the accumulative speed of storage materials in different developmental stages of Peony seeds varied.(2)Collecting Peony seeds at different developmental stages for anatomical observation:It was found that with the development of Peony seeds,the seeds gradually increased,tended to be stabled with the length being about 9 mm and the width being about 5 mm at the last 60 days.Peony seeds were composed of seed coat,embryo,and endosperm.The seed coat consisted of three parts:the outer seed coat,the middle seed coat,and the inner seed coat,corresponding to the stratum corneum,palisade cell layer and thin cell layer respectively.The seed coat color gradually changed from light yellow to dark brown or black.In the early stage of Peony seed development,only the outer seed coat and middle seed coat contained starch,with the inner seed coat being the exception.But when they grew mature,all starch disappears.In the early stage of Peony seed development,the endosperm contained a small amount of oil bodies and a large number of vacuoles.With the continuous development of the seed,the vacuole gradually disappeared,and the number of oil bodies gradually increased.When the seeds grew mature,the vacuoles disappeared and the whole endosperm was filled with the oil body,which surrounded aropund high electron dense materials.The structural change of the endosperm was mainly reflected in the gradual formation of a bouquet-like structure from the network structure,and finally the formation of an irregular network structure.(3)With respect to the screen of FAD,a key gene for a-linolenic acid synthesis:real-time fluorescence quantitative PCR was used to detect the expression patterns of several genes of the FAD family during the seed development stage of peony.The results showed that different genes of the FAD family were different in different developmental stages of peony seeds.Among which,the expression level of FAD8-1 was significantly higher than that of several other genes during development.Therefore,conclusion could be hypothesized that FAD8-1 gene played an important role in the synthesis of a-linolenic acid in peony seed oil.The full length sequence of PoFAD8-1 gene was obtained by adopting RACE cloning technology.with full-length cDNA of PoFAD8-1 being 1625 bq,the open reading frame being 1203 bp,400 amino acids being encoded and high homology with FAD sequences of other plants.In addition,It was verified that the PoFAD8-1 gene was an endoplasmic reticulum protein and that it also had some expression in the nuclear domain.In order to further study the function of this protein,cell-free expression technology was employed,which successfully expressed and purified PoFAD8-1 protein with a molecular weight of approximately being 45 KD.(4)Finally,an overexpression vector containing the PoFAD8-1 gene sequence was constructed and 7 resistant plants were obtained by using Agrobacterium-mediated leaf disk method to transformed the vector into tobacco. |
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