Expression Analysis Of Pre-miR171 And ETM171 During Somatic Embyogenesis And Vector Construction In Lilium

Lilies?Lilium spp.?is one of the famous flowering bulbs in world.It has ornamental medical uses.However,there are still many problems in bulb production of our country.For example,we still have difficulties in germplasm conservation which lead to many precious resources face the extinction,the bulb production has not been home-produced and the domestic varieties with independent intellectual property remain limited.Somatic embryogenic is vital to the germplasm preservation,genetic traits improvement and bulb propagation in Lilium.But the molecular mechanism of Lilium is not clear.It has been reported that miR171 plays an important role in the regulation of Lilium somatic embryogenic.This research will use a precursor of miR171 Lilium pumilum DC.Fisch and qRT-PCR technology is to discuss the expression pattern of pre-miR171 and eTM171 of Lilium brownie's somatic embryo in different developmental stages.And establish overexpression vector of mi R171 and also the silent and deletion vector by using small series simulated target technology and CRISPR/Cas9 gene editing technology.In order to deeply research the molecule regulatory mechanism of eTM-miR171-SCL6 in the process of Lilium brownie's somatic embryo,here are some major experiment results:1.Clone and expression analysis of lpu-pre-miR171a/bAccording to miRNA precursor's highly conservatism,we made a clone and acquired precursor's sequences of lpu-miR171a and lpu-miR171b successfully.We predicted the secondary structure of precursor's sequences of lpu-miR171a/b.The result shows that lpu-pre-miR171a/b both have typical stem ring structure.The minimal folding free energy is-44.0 kal·mol^-11 and-36.8 kal·mol^-1.The expressions of pre-miR171a are very different in the process of Lilium brownie's somatic embryo.Its highest expression quantity occurred in the non-embryogenic callus stage and the quantity reduced rapidly in embryogenic conserving stage.However,the expression quantity of pre-miR171b reduced rapidly from non-embryogenic stage to embryogenic callus stage,and it rose a lot in the late stage of Lilium brownie's somatic embryo such as torpedo embryo stage and cotyledon embryo stage.2.Expression analysis of eTM171 in the process of Lilium brownie's somatic embryoWe have made sure that the endogenous trap target of miR171a is c40819_g1_i1 and c46484_g1_i1.And we found that in the process of Lilium brownie's somatic embryo,c40819_g1_i1 has the highest expression quantity in the embryo genic-conserving stage,while c46484_g1_i1 has the highest expression quantity in the embryogenic callus stage.c40253_g1_i1 is the endogenous trap target of miR171b and it has the highest expression quantity in globular embryo stage and lowest expression quantity in torpedo embryo stage.3.Vector establishment of miR171a/bThe clone has acquired the EST sequence of pre-miR171a/b and we successfully established overexpression vector pRI101-miR171a and pRI101-miR171b of miR171a and miR171b.In this experiment we designed and artificially synthesized sequences of miR171aSTTM and miR171bSTTM,and we also successfully established overexpression vector of pRI101-miR171a STTM and pRI101-miR171bSTTM of miR171aSTTM and miR171bSTTM.We looked for target locus from miR171a/b EST sequence obtained by clone,and we used CRISPR/Cas9 gene editing technology in Lilium brownie for the first time to establish the deletion vector pBUE411-miR171a and pBUE411-miR171b of miR171a/b.