Cloning And Functional Analysis Of A U-box Ubiquitin Ligase Gene TaPUB15 From Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is one of the three major grain crops in China and has a vital role in Chinese food security.As the global climate changing,abiotic stress such as drought,salinity,extreme temperature seriously restricts the wheat production in China.Finding and utilizating the stress-tolerant genes in wheat and cultivating new cultivars having resistance against abiotic stress became one of the most effective way to improve wheat yield to meet the growing demand for food.Ubiquitin ligase E3 plays an important role in participating in response to abiotic stress.In the present research,wheat cultivar Hanxuan 10 was used for the study of the relationship between TaPUB15(Plant U-box 15)and the abiotic stress tolerance.We cloned a ubiquitin ligase gene TaPUB15.The present research exhibited the expression patterns under abiotic stress conditions and the phenotype of transgenic Arabidopsis and rice in salt stress.All the results showed that TaPUB15 improved salt stress tolerance in Arabidopsis and rice.The major results are exhibited as follows:1.A ubiquitin ligase E3 gene TaPUB15 was cloned from wheat.The full length of c DNA contains 2496 base pairs and encodes a protein containing 831 amino acids with a molecular mass of 89.80 k D.TaPUB15 contains a conserved U-box domain and a ARM repeat motif and belongs to U-box E3 ubiquitin ligase.Subcellular localization indicated that TaPUB15 was located in the cytoplasm and nucleus.TaPUB15-A,TaPUB15-B and TaPUB15-D keep highly conserved in the modern cultivars of wheat.2.TaPUB15 expressed in multiple tissues and was more abundant in roots than in other tissues.It was induced by salt,abscisic acid,low temperature,and polyethylene glycol(PEG).The expression of TaPUB15 under Na Cl,ABA and low temperature stresses increased steadily and reached a peak and then decreased.Transcription of TaPUB15 under PEG stress peaked at 1 h,and then maintained a level slightly higher than that at the initial time.3.The TaPUB15-D transgenic rice had stronger slat tolerance and more developed root systems.In normal conditions,the transgenic rice at seedling and heading stages developed more crown roots and longer root system than the wild type(WT).Transgenic rice was more tolerant to high salt than WT,which was supported by improved total leaf chlorophyll contents,cell membrane stability(CMS),and osmotic potential in both hydroponic and soil culture conditions.Salt-stress related genes such as Os SOS1,Os NHX1,Os NHX2,Os P5CS1,Os AKT1 and Os HKT1;1 were up-regulated in transgenic rice under salt conditions.After salt treatment,the Na~+ content in transgenic rice was lower than that in the WT,whereas the K~+ content has no significant difference between the transgenic rice and the WT.All these results showed that TaPUB15 participated in regulating the expression of salt-stress related genes,and then regulated Na~+ and K~+ homeostasis to maintain a low Na~+/K~+ ratio to enhance the salt tolerance in the transgenic rice.4.The TaPUB15-D enhanced salt tolerance at the germination and seedling stages in transgenic Arabidopsis.Under the condition of salt treatment,germination of seeds of transgenic lines was less sensitive to Na Cl than WT.It was evident that transgenic seedlings stayed green whereas WT were bleached.A physiological index assay indicated no significant difference in CMS under normal conditions,whereas the CMS of transgenic lines was significantly higher than that of WT under salt-stressed conditions.Salt-stress related genes such as At SOS1,At NHX1,At P5CS1,At CBF3,At RD29 A and At KIN1 were up-regulated in transgenic Arabidopsis under salt-stressed conditions.The efflux rates of Na~+ in the transgenics were much higher than that in WT.However,for K~+ transgenic seedlings showed lower K~+ efflux rates than that in WT.All these data suggested that overexpression of TaPUB15-D in Arabidopsis regulated the expression patterns of salt-stress related genes and finally maintained a low Na~+/K~+ ratio to enhance tolerance to salt.5.TaPUB15-D had no autoactivation.By screening the c DNA library of yeast,six candidate proteins interacted with TaPUB15-D were identified.All the above results revealed that TaPUB15 can not only involve in plant root systems development,but also regulate the expression patterns of a variety of salt-stress related genes,suggested that TaPUB15 play a key role in regulating the homeostasis of Na~+ and K~+ to improve plant salt tolerance.This research provided a potential gene resource for breeding salt tolerant wheat.