Identification And Analysis Of Quinoa MADS-box Gene Family And Study On Agrobacterium-mediated Root Transformation

Chenopodium quinoa is an annual dicotyledonous plant with high nutritional value and strong resistance to stress,so more and more biologists begin to study it.In 2017,the reference genome of quinoa was announced,which alternately set off an upsurge in quinoa research and also promoted the breeding and cultivation of quinoa.But the deficiency of agronomic symptoms such as sterility caused by high temperature in the flowering period of quinoa restricts the popularization and cultivation of quinoa.Therefore,exploring the mechanism of sterility caused by high temperature in the flowering period of quinoa can provide guidance for the breeding and popularization of quinoa.Until now,the quinoa transformation system is immature,and the application of the CRISPR/Cas9 gene editing system in quinoa has not yet been seen,which has caused a great degree of restriction on the study of quinoa’s gene function.To this end,this research has carried out three parts of work: Firstly,the transcriptome of quinoa inflorescence after high temperature stress was sequenced to explore the pathway of quinoa responding to high temperature from the transcriptional level,and to explore the reason of quinoa sterility caused by high temperature at flowering stage combined with the changes of phenotype and transcription factor expression level;Secondly,in view of the decrease in the number of quinoa inflorescences under high temperature stress,and the functional characteristics of plant MADS-box gene family involved in regulating the development and flowering process of plant flower organs,this study identified the members of quinoa gene family by bioinformatics,and systematically analyzed their protein physicochemical properties,phylogenetic relationships,expression patterns,especially under high temperature stress.Finally,candidate target gene was identified based on gene family analysis results and transcriptome data.Gene editing vector targeting CqMADS33 was constructed.Agrobacterium rhizogenes were injected into the stem of quinoa to induce the production of transgenic hairy roots for root transformation.The main findings are as follows:(1)Quinoa variety Dave was used as experimental material,and the samples were treated at high temperature for 1 day,6 days and 11 days when they grew to flowering stage.Transcriptome analysis revealed a large number of differentially expressed genes,quinoa response to high temperature stress can affect carbon metabolism,starch and sucrose metabolism,phenylpropane metabolism and other pathways.At the same time,46 transcription factor families were annotated in differential expression genes,including MADS-box gene families related to flower development and flowering process,which was consistent with the phenotypic changes of quinoa inflorescence after high temperature treatment.(2)Identification and analysis of quinoa MADS-box gene familyA total of 103 MADS-box genes were identified in quinoa using bioinformatics methods,of which 39 were type I MADS-box genes and 64 were type II MADS-box genes.The physicochemical properties,chromosomal localization,phylogeny,gene structure and protein motif,cis-acting elements,gene collinearity and expression patterns of these 103 genes were analyzed.It was found that tandem repeat and fragment repeat were the main driving forces for the expansion of MADS-box gene family in quinoa,and these are also responses to high temperature stress.CqMADS33 is a homologous gene of AGL24 and may also have the function of regulating flowering in plants.Therefore,the research target is placed on the CqMADS33 gene.(3)Construction of CqMADS33 gene editing vector and preliminary study of quinoa root transformationCqMADS33 was selected as the target gene,and pK7WGF2::hCas9 was used as the background vector.The At U6 promoter-driven sg RNA expression cassette was constructed on the vector,named pK7WGF2::hCas9-CqMADS33.The availability of the vector was verified by sequencing and transient expression.The hairy roots were induced by direct injection of A4 Agrobacterium rhizogenes into the stem of quinoa.The detection of hairy roots verified the occurrence of transformation events,which provided a way for the quinoa transformation and gene function validation.The target genes of CqMADS33 were explored through DAP-seq analysis,and the expansin in the target point provided a direction for further experimental exploration.