Cloning And Functional Analysis Of Different U6 Promoters In Quinoa And Construction Of CRISPR/Cas9 Editing Vector Targeting CqASMT12 Gene

Chenopodium quinoa has gradually become the research object of more and more biologists because of its unique biological characteristics and comprehensive nutritional value.In addition,with the analysis of the whole genome sequence information of quinoa,more and more Quinoa functional genes have been gradually cloned,but so far the CRISPR / Cas9 genome editing system for quinoa has not been reported,which has limited the gene function research of quinoa to some extent.Acetylserotonin methyltransferase(ASMT)is the last enzyme in melatonin biosynthesis,which plays a rate-limiting role in the production of melatonin in plants.Genes encoding ASMT proteins are usually multi-gene Familial forms exist.In recent years,more and more studies have shown that increasing the expression level of ASMT gene can improve plant resistance to stress,but the phenotypic and physiological effects of ASMT gene mutation are still unclear.To this end,this research has carried out three parts of work.First,the conditions for transient expression of quinoa protoplasts were determined,which laid the foundation for screening the U6 promoter required for the construction of the quinoa CRISPR / Cas9 system.Second,the quinoa was different.The U6 promoter was cloned,and an expression vector was constructed to screen the quinoa U6 promoter with high transcription activity.Finally,the ASMT gene family of quinoa was systematically analyzed to determine the ASMT target gene,and the U6 promoter obtained from the screening was used to construct the quinoa ASMT gene.CRISPR /Cas9 targeted editing vector.The main findings are as follows:(1)Establishment of transient transformation system of quinoa protoplastsThe orthogonal test method was used to determine the conditions of the transient expression system of quinoa protoplasts.The results showed that the young leaves of quinoa were used as the material,and the MES solution containing 1.5% cellulase,0.3%isolated enzyme and 0.6M mannitol was used as the enzymatic hydrolysis.The solution was hydrolyzed for 8 hours,and the protoplasts were collected by centrifugation at 150 g for 10 minutes.The number of protoplasts was the largest,the protoplasts were full,and the protoplasts had high viability.It was suitable for the subsequent operation of protoplasts.Protoplasts were transformed with a transformation solution containing 30%PEG4000 for 15 minutes,and the transformation efficiency was the highest.(2)Cloning and functional analysis of different U6 promoters in quinoaAccording to the published quinoa reference genome information,five quinoa U6 snDNAs were screened by homology alignment method.Based on their upstream sequences,primers were designed and cloned and constructed into the promoter region of pSCZ3 to optimize Good protoplast transformation conditions were used to transform the five vectors,and the transformation efficiency was observed under a fluorescence microscope.The results showed that the five quinoa U6 promoters contained conserved upstream cis-acting elements,of which the CqU6-3P promoter had the highest transcriptional activity and could be used for sgRNA transcription in the quinoa CRISPR/ Cas9 gene editing system.(3)Construction of CqASMT12 gene CRISPR / Cas9 targeted editing vectorIn order to determine the ASMT target gene,the sequence containing ASMT domain in the whole quinoa genome was analyzed by homology alignment method.The results showed that the quinoa contains 49 ASMT family gene members,the protein sequence length is distributed between 224-639 amino acids,and there are 0-6 introns.The gene family may be enlarged due to tandem replication and.RNA-seq results showed that the four genes CqASMT6,CqASMT12,CqASMT20 and CqASMT25 were constitutively expressed in different tissues of quinoa.CqASMT12 was selected as the target gene,pCAMBIA3301 was used as the background vector,the original 35 S promoter was replaced with the pYAO promoter,and the sgRNA expression cassette driven by theendogenous CqU6-3P promoter of quinoa was constructed to a specific location and named pCAMBIA3301-pYAO-Cas9.Agarose gel electrophoresis and sequencing results showed that pCAMBIA3301-pYAO-Cas9 was successfully constructed.