Construction Of CRISPR/Cas9 Targeted Editing Vectors For LpCKX1 Gene And The Establishment Of Genetic Transformation System In Lolium Perenne L.

Perennial ryegrass(Lolium perenne L.)is a high-valued forage grass with the advantages of fast growth,multiple tillers,good grazing tolerance and fast regrowth rate.However,its seeds are usually small and not fully filled,which seriously reduces the harvest index and the seed quality.This is a worldwide long existing limitation in large scale seed production of perennial ryegrass.CRISPR/Cas9 gene editing technology might be a potential powerful tool for enhancing the seed yield and quality of perennial ryegrass through genetic engineering.However,the genetic transformation system in perennial ryegrass is not mature and lagged far behind other crop species,which makes it difficult to carry out targeted improvement through molecular techniques such as gene editing.Therefore,it is of great significance to establish a high efficient genetic transformation system and to enhance the genetic potential of seed yield through CRISPR/Cas9technology tarwards the the sustainable seed production and mechanical harvest in perennial ryegrass.In this study,a callus induction and regeneration system in Lolium perenne L.cultivar NUI was established,the full length of LpCKX1 gene sequence was cloned,and the CRISPR/Cas9 gene editing vector targeting LpCKX1 gene was constructed.A transient protoplast expression system was optimized to verify the efficiency of the editing vector.The main results are as follows:1.Callus induction and regeneration system in perennial ryegrassThe mature seeds of perennial ryegrass cultivar NUI were used as explants,and the efficient regeneration system was established.The cross cut seeds inoculated on the induction medium containing 5 mg/L of 2,4-D gave the best result on callus induction,which was used later as the genetic transformation system.Meanwhile,the callus induced from seeds unshelled through grinding were easily browning and unbale to regenerate viable plants.Callus differentiated best on the medium when 1 mg/L of 6-BA was added.2.Cloning of full length LpCKX1 gene sequence in perennial ryegrassGenomic DNA of perennial ryegrass cultivar NUI was extracted,and primers were designed according to the LpCKX1 gene fragments determined previously in our lab.The full-length of LpCKX1 gene sequence was cloned using two-stage PCR.3.Construction of CRISPR/Cas9 editing vector targeting LpCKX1 geneAccording to the genome sequence of LpCKX1,five CRISPR/Cas9 editing vectors targeting LpCKX1 were constructed.4.Protoplast isolation and establishment of gene transient expression system in perennial ryegrassThe conditions of protoplast preparation and transformation were optimized via orthogonal experiment,and a trantient protoplast expression system was established.The optimal conditions for protoplast preparation were 1%cellulase concentration,0.3%isolated enzyme concentration,0.5 mol/L mannitol concentration,4 h enzymolysis time.The optimal conditions for protoplast transformation were 2×10~6 protoplast/ml,0.7?g/?L plasmid concentration,5 min transformation time,40%PEG4000 concentration.5.Activity verification for CRISPR/Cas9 editing vectorThe constructed vector was transferred into protoplasts for transient expression.One target site with a T-G substitution mutation occurred at the target site was verified using T7 Endonuclease I and Sanger sequencing.Results of the current work would provide useful information for functional genomics studies and gemplasm development in perennial ryegrass.