Preliminary Study On CoSQS Promoter And Its Interaction With CoWRKY Transcription Factors In Camellia Oleifera Abel.

Camellia oleifera is one of the non-wood forest trees with high economic and ecological benefits in southern China.It is widely cultivated from the Yangtze River valley to all parts of south China.The main product,tea oil,is a kind of high-quality vegetable edible oil,which is not only rich in unsaturated fatty acids,but also has a high content of biologically active substances such as squalene,tocopherols and sterols.It is known as the"Oriental Olive Oil".According to previous studies,in vegetable edible oil,the squalene content of tea oil is second only to olive oil.However,the molecular regulation mechanism of squalene-rich seeds in Camellia oleifera seeds is still unclear,and it is not convenient to use genetic modification to increase the content of squalene in Camellia oleifera Based on the previous research of our group,this study verified the biological function of the squalene synthase gene promoter from Camellia oleifera(pCoSQS).The CoWRKY transcription factors that interacted with pCoSQS were analyzed according to the Camellia oleifera seed transcriptome database,and preliminary verified by yeast single hybrid method.The main contents of this research are as follows:1 pCoSQS cloning and bioinformatics analysis.Using leaves as material from’Huashuo’ Camellia oleifera,genomic DNA was extracted and used as a template to amplify pCoSQS fragments.After cloning and sequencing,the results showed that the pCoSQS sequence is 624bp,mainly containing 18 TATA-boxes,9 CAAT-boxes,2 light-responsive elements(MRE and chs-CMAla),3 MYB binding sites,and a W-box domain(TGACG-motif).2 Study on the biological function of pCoSQS.Recombinant ligation was used to construct plant expression vectors pCAMBIA1304-pCoSQS and pCAMBIA1304-pCoSQS-CoSQS-NOS,etc.,then the vectors were successfully transferred into wild-type tobacco by Agrobacterium-mediated method.Determination results of squalene content in T2 generation seeds of positively transformed tobacco and wild-type tobacco seeds showed that the squalene content in wild-type tobacco,pCoSQS-only tobacco and pCoSQS-CoSQS-NOS-transformed tobacco T2 seeds were 1.948mg/kg,0.858mg/kg and 94.423mg/kg respectively.The results of ANOVA analysis showed that the squalene content in the T2 generation seeds of the pCoSQS-CoSQS-NOS group was significantly different with the others.When pCAMBIA1304-pCoSQS was transferred into wild-type tobacco,it was no function to promote the synthesis of squalene.But when pCoSQS is co-transformed with CoSQS,pCoSQS can perform the promoter function to regulate the successful expression of CoSQS and promote the synthesis of squalene.3 CoWRKY transcriptional gene analysis.A high-throughput transcriptome sequencing technology was used to construct a transcript database for the peak oil synthesis of Camellia oleifera seed kernel.The database contained 29.31Gb of readable data.After de-redundancy,a de-redundant database with a total length of about 192.41Mb was obtained.The transcript de-redundant data was functionally annotated,and 41 CoWRKY genes were selected through bioinformatics analysis.The results of cluster analysis showed that some Co WRKY genes may be related to the metabolism of terpenoid in Camellia oleifera,five CoWRKY genes may be related to triterpene metabolism,and the CoWRKY1 gene is the most relevant.Previous studies showed that the CoSQS gene and the squalene content of Camellia oleifera have a significant positive correlation.Correlation analysis of CoWRKY1 gene expression in different seed development stages and CoSQS gene expression in corresponding periods showed that CoWRKY1 and CoSQS gene expression were significantly positively correlated.So,it indicated that CoWRKY1 gene expression may also affect Camellia oleifera seed squalene synthesis.4 A preliminary study on the interaction between CoWRKY1 and pCoSQS.The CoWRKY1 and pCoSQS promoter were selected for interaction research by yeast single hybrid technology.The results showed that the-94bp to-327bp fragment of the pCoSQS promoter was intercepted as the bait fragment.The bait vector-transformed yeast could grow normally on the screening medium after being transferred to the hunting vector.The result indicated that there may be an interaction between CoWRKY1 and pCoSQS.