Salt Tolerance Gene Expression Profile Analysis And BADH Gene Transformation Of Lisianthus (Eustoma Grandiflorum)

Eustoma grandiflorum,a popular fresh cut flower,and a Gentianaceae herbaceous plant,is tolerant to salt to some degree.It is an important research material with economic value.To analyse its' molecular mechanism of salt tolerance,to isolate its' salt tolerant genes,and to enhance its' salt tolerant ability,three programs were done.1.Selection of salt tolerant genes from Eustoma grandiflorumTo get the salt tolerant genes of it,a suppression subtractive library was constructed, useing the leaves of plants watered with 400 mmol/L NaCl solution as tester and with pure water as driver.After sequencing and blasting,658 non-repeat sequences were gotton.These 658 sequences were classified by gene ontology(GO),and 123 of them got annotation,which are 19%of the total sequences.They were sorted to 14 classes in Biological Process classification,including mitotic spindle checkpoint,response to oxidative stress,protein sumoylation,transportation,electron transportation,etc.;21 classes in Molecular Function classification,including catalase activity,catalytic activity,epoxide hydrolase activity, monooxygenase activity,glutathione dehydrogenase(ascorbate) activity etc.;and 10 classes in Cell Component classification,including mitochondrion,chloroplast,peroxisome,glyoxysome, nucleus etc..2.Gene expression analysis of salt stressed Eustoma grandiflorumTo analyse the gene expression profile of salt stressed Eustoma grandiflorum,microarrays were manufactured with that library.The mRNA from leaves of the plants watered with 600 mmol/L NaCl solution and pure water were first reverse transcripted,then labeled with Cy3 and Cy5 seperatelly,finally hybridized with microarrays.The differently expressed genes were got by limma software package.We got 14 up-regulated genes,16 down-regulated genes,5 of them with known functions,including ubiquitin-like protein SUMO,peptide transport protein, cytochrome P450,thioredoxin,and dehydroascorbate reductase.3.Transformation of Eustoma grandiflorum with BADH geneTo enhance its' salt tolerant ability,Eustoma grandiflorum was transformed with BADH gene.The full length of Arabidopsis Arabidopsis BADH CDS was first cloned and verified by sequencing,then constructed into plant expression vector pH2GW7.The expression vector was transformed into Agrobacterium LBA4404.The resistance plants were got via agrobacterium-mediated transformation,and identified by PCR and southern blotting.The results indicated that the BADH gene has been inserted into the genome of Eustoma grandiflorum.