Creation Of Autotetraploid And The Function Analysis Of Regulationg Drought Resistent In Lilium Distichum Nakai

Lilium distichum Nakai is one of the unique wild lilies in Northeast China.It grows only in mountainous areas with humid soil and air environment.As an ornamental lily with both flowers and leaves,it is more meaningful to become a courtyard plant or greening plant.In Northeast China,is dry and rainless all year round.If we obtain drought resistant germplasm,its ornamental value and economic value can be improved.Polyploid breeding is an important method for germplasm improvement,which can improve the stress resistance of plants.However,the tolerance and response of polyploid lilies to abiotic stress remains to be explored.In this study,autotetraploid Lilium distichum Nakai was obtained by using somatic embryos;Screen the lines with excellent apparent characters to explore the physiological,biochemical and resistance advantages of different genotypes;Transcriptome and miRNA sequencing techniques were used to further investigate the response mechanism of drought stress;Constructed the miRNA-m RNA regulatory network in autotetraploid lily under drought stress;Exploring the function of ldi-miR396b-LdPMaT1 under drought stress in autotetraploid lily.In order to lay a foundation for further understanding of the molecular mechanisms of polyploid high stress resistance.The main results in this study are presented as follows:1.The autotetraploid lilies successfully obtained by somatic embryos were soaked in0.05% colchicine for 48 hours,the mutation rate was 23.5%.The morphological characteristics of autotetraploid showing larger bulbs,thicker leaves and stronger roots.Autotetraploid lily had high content of endogenous substances,low stomatal density,low content of proline and malondialdehyde and stronger root activity.After PEG(5%,10%,20%,30%)simulated drought stress for 7 days,the drought damage index(DDI)showed an upward trend with the increase of stress intensity.Under the same stress conditions,the DDI degree of diploid was always higher than that of autotetraploid;Compared with diploid lily,autotetraploid lily had more relative water content,stronger photosynthetic capacity,increased soluble sugar content,lower MDA content and higher antioxidant enzyme(SOD,CAT)activity under drought stress,which proved that autotetraploid has stronger drought resistance.2.The transcriptome sequencing of diploid and autotetraploid lily after drought stress was carried out.The results showed that carbohydrate metabolism played an important role in diploid and tetraploid,but there were obvious differences in response to stress.The tetraploid function was mainly concentrated in the function of stress.In the process of hydrogen peroxide metabolism and decomposition,the diploid function was mainly concentrated in galactosidase activity,pigment body and photosynthesis.Four key genes SUS,PP2 C,PMa T1 and CYP71A9 were screened by WGCNA analysis of 14278 differentially expressed genes.Its key genes were closely related to MYB,b HLH and other transcription factors(ERF,ARF,NAC,GATA,etc.).The ability of scavenging reactive oxygen species is important to the regulation after stress,PMa T1 as an active oxygen scavenger may play a key role in the regulation of drought resistance of autotetraploid lily.3.The target gene d LdPMaT1 was cloned and identifie.The total length of LdPMaT1 sequence is 1047 bp,encoding 348 amino acids.LdPMaT1 belongs to nuclear protein.After infecting Arabidopsis inflorescence with Agrobacterium mediated flower dipping method,overexpression of LdPMaT1 transgenic plants were obtained.The identification and drought tolerance analysis of overexpressing LdPMaT1 transgenic plants showed that LdPMaT1 gene had a certain enhancing effect on plant drought tolerance.4.Identified 370 miRNAs belonging to 69 miRNA families,and no novel miRNAs were detected in Lilium distichum Nakai.Among them,5 miRNAs(miR395 a,miR395 b,miR396 b,miR2118 r,miR5059)and their target genes play a key role in drought resistance in autotetraploid lily.According to the combined analysis of transcriptome and miRNA,we found that the resistance of Autotetraploid lily to drought stress is mainly divided into three aspects:(1)ABA biosynthesis and signal transduction;(2)Carbohydrate metabolism;(3)Reactive oxygen species mediated oxidative stress response.5.Through the combined analysis of transcriptome and miRNA,the regulation module ldi-miR396b-LdPMaT1,which played a key role under drought resistance in autotetraploid.The interaction between ldi-miR396b-LdPMaT1 was determined by RLM 5’Race,the STTM396 b was transferred by stable genetic transformation system.According to the results,four STTM396 b silencing lines were obtained.It was found that the expression of STTM396 b silencing lines decreased significantly,while the expression of LdPMaT1 increased significantly,indicating that ldi-miR396 b had a targeted negative regulation relationship on LdPMaT1,and the increase of LdPMaT1 content is conducive to plants’ resistance to abiotic stress,Therefore,it was preliminarily proved that ldi-miR396b-LdPMaT1 can regulate the drought tolerance of autotetraploid lily.In conclusion,this study obtained new autotetraploid germplasm with excellent apparent characters,explored the mechanism of response and resistance to drought stress of autotetraploid lily,and preliminarily proved the molecular mechanism of ldi-miR396b-LdPMaT1 regulating drought tolerance of tetraploid lily.