Salt-tolerance Gene Mining And Functional Verification Of Casuarina Equisetifolia

Casuarina equisetifolia Forst.is a typical coastal shelter forest species.It is one of the a few salt-tolerant tree species distributed in the subtropical areas.The availability of sequencing data of its genome the characters of short vegetative period and abundance seed production make it an ideal tree for salt-tolerance research.In this study,salt stress treatment and transcriptome sequencing were performed with C.equisetifolia treated with 0 mM,100 mM and 200 mM NaCl.The CeCIGR1 was identified as a candidate gene responsive to salt tolerance.The expression of CeCIGR1 was confirmed by real-time PCR.Overexpression of CeCIGR1 in Populus trichocarpa with Agrobacterium tumefaciens-mediated transformation exhibited enhanced salt tolerance.The main result are listed as below:1..57850 Unigenes were obtained by RNA-Seq.The shortest Unigene was 201 nt and the longest was 13035 nt.A total of 55.48% of Unigenes were annotated in the eggNOG database,which is the highest one of six databases((Nr?eggNOG?GO?KEGG?Pfam?Swiss-Prot).In addition,the highest proportion of GO functional classification is cell components,accounting for 33%,and metabolic pathways was the most pathways in KEGG,accounting for 32.8%.According to the comparison of protein sequence similarity in the Nr database,it was found that 46.63% of the protein sequence is homologous to the Juglans regia,so it is the closest species related to Casuarina.The GO functional classification of differentially expressed genes showed that the differentially expressed genes of stem of Casuarina under salt stress were mainly enriched in Carbohydrate Metabolism,Hormone response and transmembrane transport,and the most of the differentially expressed genes in root were related to Ribosome.KEGG enrichment analysis showed that the KEGG pathway of differentially expressed genes in the steam is mainly related to Carbohydrate Metabolism,including Starch and sucrose metabolism,Galactose metabolism and ECT.The most enriched KEGG pathway in the root is also Ribosome-related,as well as Nitrogen metabolism,Pantothenate and CoA biosynthesis,Oxidative phosphorylation etc.2.Ten differentially expressed genes were selected from the transcriptome data.Five genes(CeATHB7,CeIKU2,CeNCS2,CePM19 L,CeCIGR1)were successfully cloned and constructed into overexpression vectors and CeNCS2,CePM19 L and CeIKU2 were transmembrane.Transport-related,CeATHB7 is a transcription factor that specifically binds to DNA,and CeCIGR1 responds to gibberellin.The transgenic technology was used to transform the Populus trichocarpa stem explants,and finally the transgenic lines of CeCIGR1 were identified.3.The quantitative analysis of CeCIGR1 by real-time PCR showed that the relative expression level of CeCIGR1 gene was decreased in control,and the relative expression level in 100 mM and 200 mM salt-treated was increased in a short time and then decreased,indicating that CeCIGR1 responds to salt stress.4.The physiological indicators of Populus trichocarpa overexpressed the CeCIGR1 gene under salt stress were measured.According to the fast fluorescence parameters of chlorophyll,the leaves of overexpressing plants were less damaged after salt treatment for 24 h compared with wild type;Compared the wild-type and over-express of POD and CAT and MDA under salt stress,the decrease of POD and CAT activity and the increase of MDA content in leaves of over-express plants were less than wild type under salt stress.It indicates that the transgenic plants are more tolerant to high salt environments.