Identification And Functional Verification Of OSC Gene Family In Quinoa(Chenopodium Quinoa Willd.)

Quinoa(Chenopodium quinoa Willd.)Native to high altitudes,it has good stress resistance,can grow in harsh environments such as saline-alkali land,and has extremely high nutritional value.Its seed coat contains saponins,which will make quinoa seeds bitter and affect the taste,after collecting the seeds,the use of water washing or mechanical methods to remove saponins,which not only increases the cost,but also loses part of the nutrients,so it is of great significance to study the molecular genetic mechanism of quinoa seed saponin synthesis,use biotechnology to carry out quality improvement,and reduce the amount of quinoa seed saponin synthesis at the molecular level.The cyclization reaction catalyzed by 2,3-squalene oxide cyclase(OSC)is a key step in triterpene saponin synthesis.With the release of the whole genome of quinoa and the accumulation of transcriptome research data related to different tissues,organs and growth and development of quinoa,bioinformatics technology can be used to quickly identify and analyze the members of the CqOSC gene family in the quinoa genome,and analyze their expression,regulation and function.In this study,three parts of the CqOSC family were studied: first,bioinformatics methods and transcriptome(RNA-seq)database data were used to identify the members of the quinoa CqOSC gene family,and a series of predictive analyses were carried out on its gene and protein structure,phylogenetic evolution,expression pattern and cis-acting elements;Secondly,RT-q PCR was used to quantify the expression of CqOSCs in leaves and flowers during the growth cycle of quinoa,and the changes of CqOSCs gene expression,antioxidant enzyme,soluble protein content and chlorophyll content with Me JA concentration were analyzed under different concentrations of Me JA.Finally,an overexpression vector containing CqOSC gene with p SCZ 3 as the background vector was constructed,and the function of CqOSCs gene was studied.The main findings are as follows:(1)Family identification and analysis of CqOSC geneA total of 15 members of the CqOSC gene family were identified in the whole genome of quinoa.In a series of predictive analyses of gene and protein structure,phylogenetic evolution,expression pattern and cis-acting elements of CqOSC gene family members,it was found that:(1)There are similarities between CqOSCs members,both genes and protein structures,and they all contain two highly conserved domains,DCTAE and QW,and have more protein homologous sequences.Evolutionary development analysis showed that the CqOSC gene was obtained from continuous hybridization,chromosome doubling,tandem replication and fragment replication of OSC genes from the two ancestral species,and these15 CqOSC members evolved in three directions,resulting in three subpopulations.(2)In the promoter sequence of CqOSCs gene,there are rich types of cis-acting elements upstream,all of which contain photoresponse elements and more hormone-response elements,but the overall difference is large,and the different upstream elements of CqOSCs genes give different expression and regulation to different CqOSCs gene family members;(3)Expression pattern analysis showed that there were great differences in expression pattern analysis between 15 CqOSC genes.The two genes,CqOSC9 and CqOSC10,showed relatively continuous and stable expression in various tissues of quinoa.For example,CqOSC6 is mainly involved in the pre-and mid-growth stage of quinoa,both CqOSC1 and CqOSC8 are mainly expressed in apical meristem and inflorescences,and CqOSC7 and CqOSC12 are mainly expressed in flowers and seeds.(2)Quinoa CqOSC gene expression verification and effects of Me JA hormone treatment on gene expression of CqOSCs and physiological and biochemical effectsAccording to the biographical analysis of the CqOSC gene family,several paired genes in the three evolutionary branches were selected and the expression of CqOSCs genes in the leaves and flowers of quinoa throughout the growth cycle was analyzed using RT-q PCR technology.At the same time,different concentrations of Me JA were applied externally to observe its effects on gene expression of CqOSCs and physiological and biochemical effects.The results showed that:(1)CqOSC9 and CqOSC10 maintained high expression during the whole growth process of quinoa,and the expression of CqOSC7 and CqOSC12 was extremely high during flowering.Although CqOSC2/CqOSC6 and CqOSC8/CqOSC14 branched in the same branch during phylogenetic evolution,gene expression showed different phenomena.CqOSC2 gene expression is low in both leaves and flowers,while CqOSC6 is expressed in both leaves and flowers.The expression level of CqOSC8 in leaves has always been relatively stable,but the gene expression in flowers is significantly upregulated,and the upregulation factor can reach nearly 12 times,while the gene expression of CqOSC14 in the same branch,whether in leaves or flowers,is relatively high in the early stage,and the expression in the later stage is very low.(2)Using the comparative analysis of three quinoa ecotypes with different sources,Faro,NL-6 and DAVE,the results showed that the relationship between the saponin content of the three quinoa varieties was as follows,Faro >NL-6 > DAVE。(3)External application of different concentrations of Me JA found that the expression of CqOSCs gene and hormone concentration showed the phenomenon of "low promotion,high inhibition",combined with bioinformatics analysis results of the promoter’s "contained Me JA response element",the results showed that the expression of CqOSCs gene was related to the number of Me JA response elements in its upstream promoter,and the more Me JA response elements,the more obvious the concentration change.(4)At the same time,physiological and biochemical experimental data showed that the content of antioxidant enzymes,soluble protein and chlorophyll in quinoa increased with the increase of concentration,which indicated that external application of Me JA would improve the stress resistance and physiological activity of quinoa.(3)Construction and functional verification of CqOSC overexpression vectorsAccording to the results of bioinformation analysis and RT-q PCR,CqOSC6,CqOSC9 and CqOSC12 were selected as target genes,and an overexpression vector with p SCZ 3 as the background was constructed,and Arabidopsis thaliana was transformed using Agrobacterium GV3101,and the gene function was preliminarily verified by Basta’s screening of homozygous positive transgenic Arabidopsis thaliana,and the results showed that:(1)CqOSCs are important coding genes in the saponin anabolic pathway,the content of saponins in transgenic Arabidopsis thaliana seeds overexpressing CqOSC6,CqOSC9 and CqOSC12 was significantly higher than that in wild-type Arabidopsis,and the content of saponins was CqOSC12 > CqOSC6 > CqOSC9 > wild-type Arabidopsis thaliana.(2)Overexpressed transgenic Arabidopsis thaliana seeds germinated later than wild-type Arabidopsis thaliana seeds,and the higher the saponin content,the later the seed germination.