Transcriptome Sequencing Of Larix Olgensis Henry Induced By Nitric Oxide And Functional Characterization Of LoERF017 Gene

Larix olgensis Henry is an important coniferous species found in plantation forests in northeastern China. Its wood products are high pressure intensity, corrosion resistance and high aesthetic value.We performed the transcriptome sequencing of L. olgensis seedlings to nitric oxide using Illumina sequencing and de novo transcriptome assembly. A significant number of genes associated with the both abiotic and biotic stress differentially expressed. We have cloned the LoERF017 gene which was significantly increased and analyzed the gene expression at transcription level in different tissues of L. olgensis treated with different stress. In addition, the gene was transformed into Arabidopsis thaliana for functional verification. Specific results are as follows:(1) Transcriptome sequencing of L. olgensis seedlings to nitric oxide were performed using Illumina sequencing technology. It generated 9.09 G high-quality data,51176 unigene sequences, with an average length of 834nt and N50 length of 1365nt. About 40206 unigenes were annotated. The sequencing results indicated that NO treatment induced a large number of differentially expressed genes, among which,2671 were up-regulated, and 3674 were down-regulated. An analysis of metabolic pathways found that the expression of genes involved in plant biotic and abiotic stresses differentially expressed, such as ERF、LEA、NAC、 WRKY33、PR1 and so on.(2) By rapid amplification of cDNA ends (RACE) cloning technology, the gene ERF017 of L. olgensis has been cloned. The open reading frame length of the gene is 624bp, which encoded the 207 amino acids. The gene contains a AP2/EREBP domains, which belongs to AP2/EREBP family DREB subfamily. The recombinant LoERF017-GFP fused gene was transformed into onion epidermal cells by particle gun for subcellular location analysis. The results showed that the gene is located in nucleus and cytoplasm of cell.(3) We analyzed the expression patterns of LoERF017 under abiotic stress. The seedling of L. olgensis were treated with NaCl, Mannitol, ABA and SNP for 0 h,3 h,6 h,12 h,24 h respectively and the roots, stems and leaves were collected for qRT-PCR. We found that the LoERF017 gene expression had been changed under various abiotic stress conditions. The transcript levels of the LoERF017 gene in leaves markedly increased in response to NO. Moreover, root expression also increased nearly five-fold under the PEG Stress.(4) We successfully constructed the plant expression vector of pBI121-LoERF017 was transformed into Arabidopsis thaliana via floral dip protocols. The analysis of the survival rate and development of transgenic plants under mannitol revealed that overexpression of LoERF017 in Arabidopsis displayed enhanced tolerance to drought and salt. To further study of its drought resistance, we did the histochemical stain analysis (NBT and Evans blue) and physiological index analysis (MDA, SOD, POD) between the transgenic Arabidopsis and control. The T3 transgenic Arabidopsis overexpressing LoERF017 showed considerable stress tolerance under drought stress, by comparing with wild type. This suggests that overexpression of LoERF017 gene may directly or indirectly induced the antioxidant enzyme systems to enhance drought resistance capacity in plant.