Genetic Transformation Of EPA Synthase Gene In Cotton

PUFAs played a very important role in human nutrition and health, which mainly come fromthe fish oil. Nowadays, the production of fish oil was difficult to meet the market demand becauseof the restrictions of deep-sea fish dwindling resources. Some marine microalgae and fungi cansynthesize such PUFAs in large quantities, but commercial exploitation of it had low yield andhigh cost, it is difficult to apply in large-scale applications. As the reveals of PUFAs metabolicpathway and the identification of its corresponding key enzyme, the research of fish oilproduction through integrating the EPA/DHA metabolic pathway into higher plants (especially oilseed crops) has become a hot topic,and rapid progress has been achieved in rapessed,soybeanand showed a bright future. China is the major cotton producing country in the world, which had ahigh refined cottonseed oil production (75to85tons/year) and edible oil ratio (for vegetable oilfourth to fifth). Cotton seed oil has a higher linoleic acid rate (49.5%to57.3%) than that of otheroil crops and a higher unsaturated fatty acid synthesis to provide sufficient substrate, so it is anideal receptors crop for synthesis of EPA/DPA genetic engineering. But there is no reports aboutEPA/DHA expression vectors were successfully transformed into cotton.In this study, three transform methords, including agrobacterium-mediated spraying method,stigma drip method and ovary injection method were used to transform EPA synthetic expressionvector into four cotton varieties (Lumianyan21, Lumianyan22, Shannong1018and Hanwu198).Moreover, three different transformation methods were compared, and the copy number oftransgenic plants was determined.1. A total of29PCR positive plants of four recepter varieties were identified. Among them,10plants came from agrobacterium-mediated spraying method,6plants from ovary injection methodand13plants from pollen tube pathway method. A total of809self-crossed seeds were got fromthese PCR positive plants planted in Hainan. The non GMO plants were crossed to PCR positiveplants and83seeds of four different varieties were harvested.2. Among all three transformation methods, stigma drip method showed the highest transformefficiency (conversion rate was0.54%) whose mature boll rate was28.1%while the seedgermination rate was54%. Which followed by is agrobacterium-mediated stencil printing method,whom has a mature boll rate of45.6%and the seed germination rate of59.6%and the transform rate of0.11%. The mature boll rate of ovary injection method was0.11%, the seed germinationrate was84.3%and the transform efficiency was0.23%.3. By using Sad1(stearoyl-acyl carrier protein desaturase) gene as the internal reference, thepositive plants copy numbers obtained for the first batch of planting were detected by the real-timefluorescence quantitative PCR. Estimation of exogenous gene copy number relative to thereference gene using standard curve. The results showed that objective gene copy number ofpositive plants is1-3, in which8strains have a single copy (53.3%),2strains have2copies(13.3%),2strains have3copies (13.3%),3strains were false positive. Which have laid thematerial foundation for the further study of EPA synthase gene in transgenic cotton and EPAsynthesis of cotton.