Cloning And Functional Identification Of A GRAS Protein SpSCL1from Sesuvium Protulacastrum

The GRAS proteins are a plant-specific protein family playing critical and diverse roles in plant growth and development, ranging from GA and light signal transduction to root and axillary shoot meristem formation. To study the molecular mechanism of the salinity tolerance of halophyte Sesuvium portulacastrum, a differentially expressed cDNA subtractive library of Sesuvium portulacastrum treated with seawater and fresh water was constructed and from which an EST fragment homology to GRAS gene was screened. In this study, the full-length cDNA sequence of the GRAS gene was obtained by applying RACE technique, which was2170bp long with an open reading frame of1638bp encoding545amino acids and was named SpSCL1. Amino acid sequence alignment showed that it might belong to the AtPAT1Subfamily of GRAS family. A1254bp of promoter sequence of SpSCLl was obtained by genome walking. The sequence was then analysed by the soft PLACE and the result showed that it contained a typical eukaryotic core promoter region (-60bp--10bp) and several promoter elements such as TATA-box and CAAT-box. Also, many cis-regulatory elements related to salt, dehydration stresses were identified in this sequence suggesting that SpSCL1gene may play important roles in salt, and drought tolerance responses.The expression profiles of SpSCL1were analysed by semi-quantitative RT-PCR technology. The result showed that SpSCLl ubiquitously expressed in roots, stems, leaves with a higher level. With0.5M NaCl treatment, the expression of SpSCL1increased gradually and reached the peak after2h and then falled and maintained a relatively high level.when treated with20%PEG8000, the expression had no obvious changes untill a sharp increase observed after4h, followed by decreasing to the normal level.These results suggested that SpSCLl could be induced by salt and drought stresses and was involved in the responses of salt and drought tolerance.To further reveal the function of SpSCL1, we constructed a yeast expression vector and transformed it into Pichia pastor is to study the salt tolerance of yeast. The results showed that the growth rate of the yeast expressing SpSCL1was significantly fast than that of the control yeast transfected with an empty vector in the BMMY medium within1M NaCl.This phenomenon was particularly evident in1.5M NaCl medium. In the plate experiment, when coated with same concentration of cells in the1M and1.5M NaCl YPD plate, the transgenic yeast growed more clones than the control. All these results proved that SpSCLl had endowed the yeast with the ability of salt tolerance.Further, we constructed a transgenic tobacco lines to investigate the physiological functions of SpSCL1.The results showed that the transgenic tobacco plants, respectively, after the treatment of0.2M NaCl,20%PEG8000,growth significantly better than the control group.Under the stress of salt and physiological drought,the transgenic tobacco plant can basically grow normally,while the control group tobacco plants have shown significant wilting, yellow leaves, slow growth and other symptoms.This means SpSCL1gene increase the abiliy of salt tolerance, drought tolerance, overexpression of SpSCLl gene enhanced salt tolerance, drought tolerance of the tobacco plant.Transgenic yeast and transgenic tobacco trial initially confirmed the SpSCLl genes involve in the salt tolerance, drought tolerance physiological processes in Sesuvium porrulacastrum. Transgenic technology using SpSCL1as target genes can improve salt-tolerant and drought-tolerant in plant.