Cloning And Analysis Of Specific Expression Promoter In Flax Seeds

Linseed flax,also known as oil flax,is one of the main oil crops in northern China with strong resistance.Linseed flax not only is the main source of industrial oil,but also can meet individual needs the flax seed contains a variety of unsaturated fatty acids which are essential for human body.The content ofα-linolenic acid in flax oil account up for 40%to 60%,which is much higher than other vegetable edible oil.Tissue-specific promoters can guide the expression of target genes in specific organs or tissues,which can effectively avoid unnecessary waste of nutrients in the process of plant growth and development,and its utilization is an important breeding method in plant genetic engineering.In this experiment,EST libraries constructed by previous study at different development stages and different tissues was analyzed.Combined with the RNA-seq results of two linseed flax varieties（Macbeth and Heiya No.14）which are constructed by our laboratory at different development stages,we screened out five candidate genes with seed expression specificity:Lus10032127,Lus10027161,Lus10031387,Lus10026220 and Lus10042449.Two known seed specific expression promoters in rape,Bn Napin and Bn OLE,were selected as controls.The main research results of this experiment are shown as follows:1.The promoters of these five genes were cloned:LuOLE1,LuOLE2,LuOLE3,LuYLS1 and LuYLS2.Bioinformatics analysis showed that these promoters all contained TATA-box and CAAT-box,cis-acting elements related to seed development and hormone response elements,etc.2.Five potential seed promoters-GUS（β-glucuronidas）plant expression vectors,were constructed respectively,and all the five promoters could regulate the expression of GUS gene in transgenic tobacco.3.Five gene promoter-GUS expression vectors were transformed into Arabidopsis thaliana.With GUS histochemical staining analysis for T₂ generation transgenic plants,it is found that all of the leaves,siliques at 5 d,10 d,20 d after flowering and seeds at 5 d,10 d,20 d of LuOLE1,LuOLE2,LuOLE3,LuYLS1,LuYLS2 transgenic Arabidopsis were dyed,which indicated the promoters not seed-specific expressed in Arabidopsis.4.The flax transgenic system initially constructed in the laboratory was improved in this study.The grafting method was adopted to replace the rooting and transplanting method of regenerated plants,which greatly improved the survival rate of regenerated plants from the original 2%to 41.17%.5.Five promoter-GUS expression vectors and two vectors derived from rape promoter were transformed into linseed flax,and two Bn Ole positive transgenic lines were obtained.The results of GUS staining showed that the flax seed gum layer on the seed outer space appeared blue,indicating that the promoters can be used for the application of seed-specific expression promoters in linseed flax.