Transcriptome Sequencing Of Ammopiptanth Us Mongolicus And Cloning Of AmDREB1F Gene

Ammopiptanthus mongolicus has very strong cold and drought tolerances and is a rare plant species for studying both cold and drought tolerance mechanisms and the related stress-tolerant genes. However, only limited genomic data are currently available for this plant. At present study, we performed the transcriptome sequencing and de novo assembly of cold-and drought-stressed A. mongolicus using Illumina technology. We also cloned the AmDREB1F gene from A mongolicus. The main results are as follows:(1) The sequencing output generated4.26gigabases of high-quality data,47.29million bases of clean reads and86058unigene sequences, with an average length of756bp and N50length of1279bp.(2) Twelve unigene sequences were succesfully amplified using RT-PCR method, indicating high-quality assembly of our data.(3) By similarity search against protein databases, a total of51014unigenes matched to known gene models, most of which were functionally classified into44GO terms,25COG classes and125KEGG pathways. About35044unigenes were not annotated, and some of them may represent specific genes in A. mongolicus.(4) A total of2533unigenes were annotated to encode transcription factors and can be assigned to57gene families.(5) By searching total unigenes against the TIGR Plant Transcript Assemblies database, A. mongolicus was found to be most similar to Glycine max at transcriptome level.(6) A DREB gene induced strongly by low temperature and drought stress was identified from A. mongolicus using the RNA-Seq technology. Both the complete coding region cDNA and gDNA fragments of AmDREB1F gene were cloned by PCR method, which all contain726base pairs and therefore no introns. The predicted protein of this gene is composed of241amino acid residues, contains a conserved AP2domain and a nuclear localization signal. The protein is most likely to be localized in nucleus.(7)The expression of AmDREB1F was obviously induced by low temperature but weakly induced by drought and high salinity.The complete coding region cDNA fragment of AmDREB1F was successfully constructed into plant expression vector pCAMBIA3301, then introduced AmDREB1F into Arabidopsis by Agrobacterium-mediated transformation and the35T1generation transgenic plants were obtained. The work laid a foundation for further functional analysis of AmDREB1F.