Selection Of Reliable Reference Genes And Preliminary Analysis Of Eutipi、Eutpsy Function In Autumn Olive

Lycopene is a carotenoid pigment and was found in tomatoes and other red fruits. It is one of the strongest antioxidant, which existes in nature. It plays an important role in anticancer, prevention and controlling of certain chronic diseases, and protection the skin and cardiovascular.Autumn olive (Elaeagnus umbellate Thunb.) belongs to the genus Elaeagnus in the Elaeagnaceae family. The red-ripe fruit is rich in lycopene. Its content is 5-18 times higher than that of ordinary tomato. Therefore, the function analysis of the lycopene synthesis related genes and the high-yielding mechanism of lycopene in autumn olive, is vital to expand the sources of lycopene.Our previous results showed that the expression of EutPsy and other key genes were up-regulated, and EutIPI protien was accumulated during fruit ripening, indicating that EutPsy and EutIPI were related with high yielding of lycopene in autumn olive fruit. Meanwhile, the EutPsy will be over-expressed in tomato, which is designed to obtain high yield of transgenic tomato. In this paper, we preliminary analyzed the function of EutIPI and EutPsy genes. The main results are as follows:1. Ten housekeeping gene fragments were cloned from autumn olive. The reliable reference genes for autumn olive under different conditions were screened out from 12 selected ones:(1) eIF4A, RPL7 and UBCE were identified as realiable reference genes under all condintions; (2) UBCE and RPL7 were suitable reference genes in all organs from autumn olive; (3) eIF4A and UBCE as reliable reference genes in fruit ripening; (4) eIF4A, UBCE as reliable reference genes for autumn olive under exogenous hormone (ABA, GA3) treatment; (5) Actin, EF1-α, and eIF4A as reliable reference genes for autumn olive under abiotic stresses including salt and dehydration.2. The expression of EutIPI in leaf, stem, root, flower and during fruit-ripening process were analyzed by using quantitative real-time PCR:(1) EutIPI expression did not change significantly in yellow and red fruit, but significantly increased in dark-yellow fruit compared with the green fruit by using the eIF4A and UBCE as refernence genes; (2) EutIPI mRNA was abundant in leaf, moderate in root, but very low in stem and flower by using the UBCE as a refernence gene.3. Colour complementary experiments of EutIPI and EutPsy genes were carried out by co-transformation. EutIPI increased the levels of β-carotene produced by DH5a (pAC-EutIPI+pAC-BETA), indicated that EutIPI could provide more substrates for engineering bacteria to synthetic β-carotene. EutPsy allowed TOP10F (pUC18EutPsy+pAC-85b) to produce β-carotene, which indicated that EutPsy had phytoene synthase activity. The results demonstrated that EutIPI and EutPsy were involved in carotenoid synthesis.4. The genomic DNAs of EutIPI and EutPsy were cloned, and the promoter sequences of them were obtained. The genomic sequence of EutIPI was 4025bp, and contained 6 exons and 5 introns. The genomic sequence of EutPsy was 1311bp, and was not interrupted by any intron. The sequence of 758bp 5’-upstream of EutPsy promoter and 484bp 5’-upstream of EutIPI promoter were cloned by useing TAIL-PCR, respectively. Both promoters contained some common sequences known as TATA element, CAAT box and other elements.