Promoter Element Mining And Genome Editing In Larch

Larch(Larix kaempferi)is a type of tree that has the following advantages: growing fast in the early stage,strong stress resistance,excellent disease resistance,and good ecological benefits.It is important for afforestation in northern China.It is widely used in conversion of farmland to forests.However,due to larch has long growth cycle growth cycle and low efficiency of asexual reproduction,the improvement process of its genetic trait is largely restricted.With the development of modern molecular biology and genome editing technology,genetic improvement in larch meets new opportunities and challenges.However,the reference genome sequencing of larch is still incomplete,the genetic transformation and regeneration technology is not mature,and the core elements for genome engineering are limited,making it difficult to achieve efficient genetic modification for larch in the short term.In view of the above problems,this study firstly constructs the larch transient expression system based on the protoplast transformation,secondly based on the "genome-transcriptome" analysis strategy,the highly active promoters are explored and identified,Furthermore,according to the expression characteristics of larch genome,an effective over-expression and genome editing system at exogenous site is constructed.The main results of this study are as follows:1.The key parameters of isolation,extraction,purification and transformation of larch protoplast were confirmed,including enzymolysis time,the concentration of mannitol and the ratio of enzyme concentration in the enzymolysis solution,the concentration of PEG 4000,and the speed of centrifugation.Results showed that when the enzymolysis time was 6 h,the mannitol concentration was 0.4 M,the centrifugation speed was 60×g,and the concentration of PEG 4000 was 20%,the number of active cells in larch protoplasts was 90% and the transformation efficiency reached to 40%.2.By "genome-transcriptome" sequencing analysis strategy,the candidate promoter with high transcriptional activity was confirmed: 41 mRNA transcripts with high gene expression level in larch needle and calli were identified.The 2000 bp sequence upstream of these 41 transcripts was selected as the potential promoter sequence of larch.The primers were designed to construct the report vector using pLB41 as the backbone.After larch protoplast transient transformation,a total of six larch endogenous promoters were found that can initiate transient expression of green fluorescent reporter gene in larch protoplast cells,with the name of LarPE004,LarPE005,LarPE051,LarPE055,LarPE057 and LarPE058 promoter.Among all the promoters,the activity of LarPE004 was the best and significantly better than the ZmUbi1 and CaMV 35 S promoter,which commonly used in plant genome engineering.3.The activity of LarPE004 was further confirmed: LarPE004 promoter had outstanding transient expression activity.For futher application,rice was used as the test material for transient and stable transformation.The results showed that the LarPE004 promoter could effectively drive the expression of GFP and GUS reporter genes in rice protoplasts,regeneration callus,and rice leaves and roots.4.Constructing larch genome editing vector to achieve endogenous site genome editing of larch: Based on the STU-Cas9 genome editing system,the larch STU-Cas9 genome editing vector with LarPE004 and ZmUbi1 promoter was constructed,and the sgRNA was designed targeting to the LarActin and LarNAC gene.After larch protoplasts transient transformation,the genome editing efficiency was calculated by highthroughput sequencing.The results showed that the genome editing vector constructed in this study can be used to edit the genome of larch at the endogenous sites.