| In plant seeds,the two fatty acid desaturases,FAD2 and FAD3,convert oleic acid into linoleic acid and convert linoleic acid into α-linolenic acid by dehydrogenation,respectively.The α-linolenic acid is an essential polyunsaturated fatty acid,being beneficial to cardiovascular health.The content of α-linolenic acid can be improved by the two steps.Our previous results show that the second reaction can be realized effectively by overexpression of FAD3.However,the attempts of improving linoleic acid content by overexpression of FAD2 have met many obstacles.Our previous results show that two kind of promoters,seed specific and non-seed specific,are needed for solving the problems.Soybean is an important oil crop,which contains 50% linoleic acid and low α-linolenic acid,about 10%.In order to improveα-linolenic acid in soybean seeds,the research was carried out from four aspects: evaluating the feasibility of engineering FAD3 in soybean;the construction of expression vector;the analysis of specific promoters and modification of the genetic transformation system of soybean.The results are listed as follows:1.The expression of FAD2 and FAD3 in different stages of soybean seed development was analyzed.Results showed that the expression level of FAD2 was about ten times that of FAD3 in the seeds,suggesting that the low content of α-linolenic acid in soybean seeds may be due to the low expression of FAD3.It will be possible to improve the content ofα-linolenic acid in soybean by overexpression of FAD3.2.The Brassica napus Bn FAD3,driven by a seed specific promoter,was constructed and transformed into Arabidopsis.Fatty acid contents of the T2 and T3 seeds of the transgenic lines were analyzed.Results showed that the seed-specific expression of FAD3 resulted in high accumulation of α-linolenic acid in Arabidopsis seeds.The α-linolenic acid level reached up to 40% in transgenic lines.It was proved that the vector worked efficiently for improvingα-linolenic acid content in plant seeds.3.Transcriptome of B.napus was analyzed and two genes,named as Bn OA03,Bn TC06,were selected for promoter study.The cis elements of the promoters were analyzed by using a promoter online analysis software.The results showed that the upstream sequences of the two genes had basic elements of promoter,such as CAAT-box,TATA-box elements.Additionally,RY-motif,which was related to seed specific expression,was found in the promoter region of Bn OA03.In the upstream sequence of Bn TC06,I-box,which is related to the root specific expression,and Pollen-box,which is related to the pollen specific expression,were identified.A 715 bp upstream sequence of Bn OA03,named as PBn OA03,and a 457 bp upstream sequence of Bn TC06,named as PBn TC06,were amplified from B.napus genomic by PCR.GUS reporter gene was used to identify the function of the two promoters in Arabidopsis thaliana.Histochemical staining of GUS activity of PBn OA03 showed that expression of GUS was not detectable in seedlings,roots,stems,leaves,flowers and seeds during early and mature development stages,but high expression levels were found in developing seed at middle and late stages.These results indicated that the promoter PBn OA03 functions in a seed-specific pattern,and will be applied to the vector of overexpressing FAD3 to improve the α-linolenic acid content in soybean.Histochemical staining of GUS activity of promoter PBn TC06 showed that GUS expression was highly detected in the seedlings,roots,stems,leaves,flowers and the seed during early development stages.These results indicated that the promoter PBn TC06 functions in a non-seed specific pattern.4.The Agrobacterium-mediated transformation of soybean was believed to be difficult with low transformation rate.In order to select a suitable soybean varieties,four genotypes,including Williams 82,a local variety,Hefeng 50 and Dongnong 50,were used to evaluate their germination,differentiation and transformation rate.A total of 1111,800 and 1270 seeds of Dongnong 50,Hefeng 50 and local veriety were germinated and 318,91 and 541 shoots were obtained,respectively.It was pity that no regenerated plants were obtained from all these three varieties.A total of 1118 seeds of Williams 82 were germinated and 202 shoots were obtained.Two regenerated plants were screened from these shoots.The results showed that Williams 82 was the best material in this study.In addition,the concentration of selection agent was evaluated by using Williams 82.The results showed that 8mg/L was suitable for screening more big buds and 3 mg/L was more conducive to bud elongation.These concentrations of selection agent will be used in future soybean genetic transformation.However,the two regenerated plants were proved to be negative transgenic plants by PCR.These results indicated that shoot regeneration rates were high except using Hefeng 50 and the difficulty of soybean transformation was the stage of shoot screening.Further efforts should be made on shoot screening,selection of soybean genotypes,modification of transformation and culture conditions in following study.In conclusion,the low content of α-linolenic acid was related to the low expression of FAD3 in soybean seeds and the overexpression of FAD3 was a worthwhile strategy for improving its α-linolenic acid;The FAD3 seed specific expression vector,can effectively improve the content of α-linolenic acid in Arabidopsis seed;A seed specific and a non-seed specific promoter,cloned from B.napus,have been characterized in Arabidopsis and they can be used for future bioengineering research;a large number of transformation of soybean by using cotyledonary node transformation system showed that the regeneration rate was high,but the negetive transgenic plants were obtained,indicating that the efficiency of soybean transformation need to be improved. |
