Cloning Of Aquilegia Vulgaris Pectin Lyase Gene AvPll17 And Validation Of Its Salt Resistance Function Based On Transcriptome

Aquilegia vulgaris is a genus of Aquilegia in Ranunculaceae family,which has beautiful foliage,unique flower shape,rich flower color,certain salt tolerance,ornamental value and economic value characteristics.Currently,the impact of salt stress on land is increasing year by year and there is an urgent need for a large amount of salt-tolerant plant material.Therefore,it is important to investigate the molecular response of Aquilegia vulgaris to salt stress and to screen for salt-tolerance-related genes in Aquilegia vulgaris for breeding and application promotion.In this study,we analyzed the molecular response to salt stress and screened Av PLL17,a key gene that responds to salt stress,cloned and constructed a plant expression vector for transformation into tobacco,and preliminarily verified the Av PLL17 gene function based on the transcriptome sequencing data of the Aquilegia vulgaris under salt stress.The main findings are as follows:1 The transcriptome was sequenced from leaves and roots of Aquilegia vulgaris seedlings treated with 200 m M Na Cl at 0 h,12 h,24 h and 48 h.A total of 24 libraries were constructed;28088 Unigenes were obtained;16,479 differential genes were screened;GO enrichment indicates that the cell wall may play an important role in response to salt stress;KEGG enrichment revealed that the Pentose and glucuronate interconversions pathway associated with the primary cell wall is significantly enriched in the roots,which is the main focus of GO and KEGG analyses;the pathway mainly includes PME,PLL and PG,and PLL differential expression ploidy and number of differential genes are higher than PME and PG,so the focus is on PLL genes.2 17 Av PLL genes were identified,all containing the conserved domain of Pec＿lyase＿C;the number of motifs contained in Av PLL proteins ranged from 6 to 10;a phylogenetic evolutionary tree was constructed with At PLL,and a total of 43 PLL genes were divided into eight subfamilies;PLL expression patterns were similar under the same subfamily,and most PLL genes changed in expression under salt stress,and showed a certain change pattern with increasing stress time;Av PLL17 is a differentially expressed gene,and it has high homology and similar expression trend with At PLL26 gene in Arabidopsis in response to salt stress,both down-regulated expression,so Av PLL17 was identified as the target gene.3 Av PLL17 was successfully cloned by PCR using Aquilegia vulgaris c DNA as template;Av PLL17-PBI121-GUS plant expression vector was constructed,and positive tobacco plants were obtained by genetic transformation of tobacco,through resistance screening and c DNA identification of resistant plants.4 Anatomical analysis of WT and Av PLL17-OE seedlings showed that the cell walls of Av PLL17-OE plants became thinner and the pore size of ducts in the bast became smaller and less numerous.After treatment with 300 m M Na Cl,the SOD and POD activities of Av PLL17-OE plants were significantly lower than those of WT plants,and the MDA content of Av PLL17-OE plants was significantly higher than that of WT plants.According to the analysis of the above results: overexpression of Av PLL17 plants with thinner cell walls resulted in increased sensitivity of plants to salt stress;Av PLL17 regulates salt tolerance of plants through expression changes in the process of salt resistance in plants.