| Perilla(Perilla frutescens) is a new oil crop. Perilla seed oil contains large amount of α-linolenic acid (one kind of healthy ω-3fatty acids), which is considered to be a good substitution for the deep sea fish oil rich in ω-3fatty acids. Investigation on the biosynthesis mechanism of oil and various fatty acids in perilla seeds can create novel knowledge for the high accumulation of seed oil, particularly α-linolenic acid. Such study will provide a basis to develop new ways for perrila seed oil quality improvement and breeding of new germplasm of the established oil crops with high level of α-linolenic acid in seed oil.The present study was conducted to explore the mechanism of perrila seed oil and α-linolenic acid biosynthesis using seven selective perrila varieties as the experimental germplasm materials. The objectives included analysis of total oil and various fatty acid biosynthesis and accumulation in the seed development, examination of expression profiles in perrila various tissues and developing seed of the gene encoding diacylglycerol acyltransferase (PfDGAT1), a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis, and optimization of perrila tissue culture and regeneration system so as to developing an effective way for perrila genetic transformation. The main findings obtained in this study are described as the followings.1. Gas chromatography was employed to test the contents of oil and various fatty acids in the mature seeds of seven perrila varieties and the dynamic changes of oil and fatty acid accumulation in the developing seeds so as to investigate the physiological-biochemical features of perrila seed oil and fatty acid biosynthesis. Perrila seed oil mainly consists of five fatty acids including palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and α-linolenic (18:3) acids. The ratio of α-linolenic acid to linoleic acid is9:1. Unsaturated fatty acids (oleic, linoleic and α-linolenic acids) made up90%of total seed oil. Following seed developing, seed fresh weight and total fatty acid content were increased, and seed morphology was also changed from aqueous and transparent forms to milky white or dark purple and finally brown matured seeds. During seed development and maturation, linoleic (18:2) content was gradually reduced while the level of α-linolenic acid (18:3) was correspondingly increased up to more than60%of the total fatty acids in the mature seeds. Of seven perrila varieties tested, variety Jinsul contained the highest level of α-linolenic acid (456.6mg/g). Therefore, Jinsul is a high-quality perrila variety commercially producing health fatty acids.2. α-linolenic and other fatty acids synthesized in plant cell are subjected to a series of emzymatic reactions, and finally transferred into glycerol-3phosphate (G3P) molecule to form triacylglycerol (TAG) stored in oil body. DGAT catalized the final step of acylation to form TAG. Semi-quantitative PCR were used to examine PfDGAT1expression profiles in perrila various tissues/organs. Compared to other tissues/organs tested, PfDGAT1expressed highly in leaf and flower organs. Real-time PCR analysis showed that small expression of PfDGAT1was in root and stems while large expression of the gene was in leaves and seeds. Following seed development, PfDGAT1expression increased slowly at the earlier stages, and then increased vastly at mid stages and reduced slowly at late stages, with the peak expression level in the seed at30days after flowering. The PfDGAT1highly expression stage was just ahead of the high accumulation stage of α-linolenic acid and total oil in seed development, indicating that PfDGAT1function importantly in perrila seed oil and α-linolenic acid accumulation.2. Over-expressing or reduced expressing gene are important methods for gene functional characterization. However, perrila genetic transformation system was not reported yet. The current study took leaves from the aseptic perrila seedlings as the explants for tissue culture. The mediums and factors were optimized for perrila callus induction and differentiation of root and bud. Finally a perrila regeneration system was developed, providing a basis for perrila transgenic improvements and function analysis of the related genes involved in lipid metabolism pathway. The perrila tissue culture and regeneration system mainly include the followings:For seed sterilization, the best method is to treat seeds with75%alcohol for20S, and then0.1%HgCl23for8min. For callus induction from the leaf explant, the best medium is MS+6-BA2.5mg/L+2,4-D1mg/L+AgNO34mg/L. The optimal culture medium for adventitious bud differentiation is MS+6-BA2.0mg/L+2,4-D0.5mg/L+AgNO34mg/L. The desirable medium for proliferation is MS+6-BA2.0mg/L+NAA0.2mg/L. The best medium for root differentiation is1/2MS containing IBA lmg/L and NAA0.4mg/L. |
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