Preliminary Functional Analysis Of LpABCB21 And LpPILS7 In Somatic Embryogenesis Of Lilium Pumilum DC.Fisch.

Somatic embryogenesis is of great significance for the conservation and germplasm innovation of wild lily resources.The current studies show that auxin plays an important regulatory role in somatic embryogenesis,and auxin transport is the first step for exogenous auxin to play its function.However,how auxin transport regulates somatic embryogenesis is still unclear.The purpose of this study is to screen and explore the auxin transporter related genes in Lilium pumilum,and to explore their regulatory functions in the process of Lilium pumilum somatic embryogenesis,so as to lay a foundation for elucidating the molecular mechanism of auxin regulating somatic embryogenesis in Lilium.The main results are as follows:1.To explore the key factors affecting Lilium somatic embryogenesis,picloram concentration,picloram treatment duration,light condition,p H value and nutrient composition of culture media are analyzed.The results show that the appropriate picloram concentration and acid culture conditions are the key factors for Lilium pumilum somatic embryogenesis.Acid culture conditions may regulate the transport process of picloram by affecting its existing form.Nutrient elements in the media and light conditions can promote somatic embryogenesis.Shortening picloram treatment duration and light conditions can promote somatic embryo germination.The induction rate of Lilium pumilum somatic embryo is reach up to 80.0%when Lilium scales are cultured in MS media containing 1.0 mg/L picloram and p H 5.8 for 3 d and then transferred to MS media without hormone under light conditions?16/8 h?.2.The full-length transcriptome library of Lilium pumilum somatic embryo is successfully constructed by using SMART sequencing technology.Combined with error correction by using the second-generation transcriptome data previously obtained by our team,a total of 119,649 transcripts are obtained with N50 is 3406,the maximum length is 13,678 bp,and the average length is 2,017 bp.Among them,110,888 transcripts?92.68%?are successfully annotated.These annotated transcripts are mainly involved in carbohydrate metabolism?6,072,5.07%?and signal transduction?5,396,4.51%?in KEGG metabolic pathway,and mainly involved in metabolic process?35,123,29.36%?and catalytic activity?33163,27.72%?in GO classification.3.Auxin transport related gene family members of Lilium pumilum have been cloned and identified,and their expression profile has been constructed in somatic embryogenesis.Through conservative domain alignment and functional annotation analysis,sixty-three ABCs,eleven PIN/PILSs,and three AUX1/LAXs have been obtained.Among them,seven ABCBs,five PIN/PILSs,and two AUX1/LAXs participate in auxin transport process.The expression patterns analysis of the fourteen auxin transport related genes in somatic embryogenesis finds that all members,besides Lp ABCB2,show a different degree of increase expression,the most significant difference among them are Lp ABCB21 and Lp PILS7.The expression of Lp ABCB21 and Lp PILS7 are significantly different in the whole process of somatic embryogenesis.The expression trend analysis shows that Lp ABCB21 and Lp PILS7 can promote somatic embryo formation and development.4.The genetic transformation technology system previously developed by our team based on the Lilium pumilum is optimized.The results show that reducing the p H of resuspension solution and co-culture medium to 5.0 and increasing the concentration of Ca Cl₂ in the germination medium to1.32g/L are beneficial to genetic transformation.The number of positive lines is increased by 3—6-fold,and the efficiency of Agrobacterium-mediated stable genetic transformation is improved by 5.7—13.0%on the basis of the efficient genetic transformation technology system of Lilium previously established by our team.The highest genetic transformation efficiency can reach up to 46.3%.5.The overexpression vectors and CRISPR/Cas9 knockout vectors of Lp ABCB21 and Lp PILS7have been constructed.Using of transgenic technology produce three overexpression lines and seven mutant lines of Lp ABCB21,and seven overexpression lines and six mutant lines of Lp PILS7.The results of somatic embryo induction and histomorphological observation of transgenic lines preliminarily confirme that Lp ABCB21 regulates the early formation of somatic embryos,but the excessive expression level inhibit somatic embryo induction efficiency,and it also can regulates somatic embryogenesis via an exogenous pathway.Lp PILS7 mainly regulates somatic embryo induction efficiency and regulates somatic embryogenesis via an endogenous pathway.In this study,the key factors affecting Lilium pumilum somatic embryogenesis have been identified,auxin transport gene family members of Lilium pumilum have been identified,and the regulatory roles of Lp ABCB21 and Lp PILS7 in Lilium somatic embryogenesis have been preliminarily verified through overexpression and CRISPR/Cas9 gene editing technology.This study lays a foundation for elucidating the molecular mechanism of auxin transport mediating Lilium somatic embryogenesis and provides a gene reference library and technical means for the study of Lilium gene function.