Function Analysis Of GLIP Promoter And Localization Of Pyrethins In Pyrethrum

Pyrethrum(Tanacetum cinerarrifolium)is an ornamental plant of Composite family and plant material of natural pyrethrins.Currently,there has a large-scale cultivation system in many countries and areas around the world.However,the content of natural pyrethrins is low and its supply has not been adequate to the demand..Recent molecular technique provides the way to improve the production of pyrethrins.Currently,a number of key genes involved in the pathways of pyrethrins biosynthesis have been identified,but the regulation of the gene expression of esterification and the location of pyrethrins are unclear.GLIP gene of Pyrethrum(GDSL lipase-like protein),belongs to GDSL family,which is the key esterification enzyme of the last step of pyrethrin Ⅰ synthesis.It catalyzes the esterification reaction of chrysanthemyl Co A and pyrethrolone to form pyrethrin Ⅰ,but the place of this reaction is not clear.In this study,we investigate the expression of GLIP gene in different developmental stages of pyrethrum and the function and location of GLIP gene promoter.These results can be used for the reveal of biosynthesis and transport of pyrethrins.Besides the transformation of tobacco,GLIP promoter was also fused with GFP and transformed to pyrethrum.Then we optimized the transformation system of pyrethrum by changing the explants type and transformation conditions.The main results of this study are as follows:1.Differences in the Expression of GLIP gene in PyrethrumThe expression of GLIP gene in different developmental stages showed that expression of GLIP gene vary among different tissues in the growth and flowering period of pyrethrum.In growth stage,the expression level of GLIP gene in stems is higher than in leaves.In flowering stage,the expression levels of GLIP are as follows: flowers > peduncles > leaves > stems,which is consistent with the highest content of pyrethrins in dryed flowers.The GLIP gene does not expressed in roots.2.Tissue Specificity and Functional Analysis of GLIP promoterThe GLIP promoter with GUS gene was transformed to early flowering tobacco.The results showed that GLIP promoter was specifically expressed in the stem,petiole and veins of T0 and T1 generation,which showed it was a vascular-specific promoter.Drought stress applying on transgenic tobacco improved the promoter activity in 6 hours showed that the promoter could response to drought stress.3.Identification of Core Functional region in GLIP promoterThe 5’ sequential deletion fragments of the GLIP promote-GUS fusions were constructed and transformed to tobacco.The GUS staining revealed that the blue color is the deepest in the veins and stem of the 1822-length GLIP promoter and the expression rate of GUS was also the highest,reach to 66.7%.The GUS activities of sequentially deletion fragments of the GLIP promoter show significant differences among different length of the promoter,GUS activity was significantly decreased in-507 bp fragments.4.The histochemical staining of terpenoids and pyrethrins in flower of pyrethrumThe staining of NADI and Sudan Red on different stages of flowers showed that there are obvious blue oil which were putatively identified as terpenoids in receptacle,ray flower and disk flower.And there were obvious positive-sudan red stained oils between embryo and the seed coat in the ovary,which may be the pyrethrins.The content and distribution of such oils have been varied along with the development of ovary.The oil transported from seed coats to the space between seed coats and embryo.