Isolation And Characteristic Of Salt-tolerance Related Genes From Aspergillus Glaucus CCHA

A halotolerance strain Aspergillus glaucus CCHA was isolated from the surface of wildvegetation around saltern, the salinity range was0-31%. To isolate salt-tolerance genes fromAspergillus glaucus CCHA, the genome DNA was sequenced. Salt-tolerance genes were alsoselected from cDNA library. These new genes could be used to improve plant salt tolerancethrough genetic engineering.The genome of Aspergillus glaucus CCHA was sequenced through a whole-genomeshotgun approach. The draft genome sequence consists of313sequence contigs longer than1kilobase, ordered orientated within105scaffolds. The total length of all sequence contigs is26megabases (Mb), the total length of the scaffolds, including estimated sizes of the gaps, is26Mb. Within the A. glaucus CCHA genome,10066unigenes were predicted with proteinproducts of longer than100amino acids.A cDNA library of A. glaucus CCHA under salt stress was constructed according toimproved SMART cDNA library contruction protocol. Total RNA were extracted from A.glaucus under170g·L NaCl stress. The size of cDNA library in E. coli DH5α wasapproximately4.8×106pfu·mL-1. One hundred thirty clones were sequenced; the resultsshowed that the sizes of inserted fragments were about500-1500bp. The sequences wereannotated by aligning with the deposited ones in diverse protein databases including NationalCenter for Biotechnology Information (NCBI) nonredundant protein (Nr) database, NCBInon-redundant nucleotide sequence (Nt) database. The analysis indicated that among the100unigenes,82(82%) had significant matches in the Nr database. The annotated genes werefunctionally involved in signal transduction, osmolyte synthesis transport, regulation oftranscription.The size of the library in E. coli BL21Star (DE3) was approximately1.6×104pfu·mL-1.Total60transformants that can grow on LB plate with0.7M NaCl were sequenced, the sizes of the inserts were ranging from300-1,500bp. Five of them (CCHA-2142, CCHA-2114,CCHA-2227, CCHA-2229, CCHA-2247) can also grow at1M NaCl, which were selected forfurther analyses. CCHA-2247showed97%identity to Ribosomal L27e protein, CCHA-2229was a member of putative plasma membrane low affinity zinc ion transporter, CCHA-2142belong to DUF3431superfamily. All of the five transformants showed better growth comparedto the control stain with empty vector under1M NaCl stress. Although the folds changed weredifferent from each other, all the five genes showed up-regulation in response to salt stress in A.glaucus under170g·L-1NaCl.The cDNA sequences of CCHA-2142and CCHA-2114were digested with Sfi I, and thenconstructed into pBI121G. Then those were transformated into Arabidopsis thaliana ecotypeColumbia plants by floral dip. The results showed, under salt stress, the levels of SOD intransformed plants were higher than that in control; this means that CCHA-2142andCCHA-2114overexpression may enhance the tolerance of transgenic plants throughinfluencing the levels of active oxygen scavengers to fit the salt stress. The reduction inchlorophyll contents was much more dramatic than that in transgenic plants. MDA content thedegree of membrane damage of the transgenic plants were both significantly lower than that incontrol. This may indicate that the overexpression of CCHA-2142and CCHA-2114help tocombat the inhibition of chlorophyll synthesis caused by salt stress. CCHA-2142andCCHA-2114gene may play important role in protecting the cell membrane. These data furtherconfirmed highlighted the functions of CCHA-2142and CCHA-2114in enhancing plants salttolerance.