Study On The Effect Of Environmental Temperature On Rhythmic Expression Of Abiotic Stress Resistance Genes In Tibetan Hulless Barley

As an endogenous time controlling system,the circadian clock can help living organisms to predict the diurnal changes of the environmental factors which caused by the rotation of the earth within 24 hours,which also actuates the living organisms to make appropriate and timely responses to the daily changes of environmental factors in advance.In plant genomes,the transcription and translation process of most stress-resistant genes are finely modulated by their endogenous circadian clocks.In the plants grown under their regular growth conditions,the intracellular circadian clock will regulate the expression of most stress-responding genes to exhibit rhythmic profiles.Correspondingly,when the occasional or slight changes of environmental factors occurred within the normal physiological range,the circadian clock system in plants will maintain the stable operation of the circadian clock itself,as well as the stability of the primary metabolisms in plant cell by the compensational mechanism of plant endogenous time keeping systems.The above mentioned process have prevented the plants to make an over reaction to the unremarkable environment changes with unnecessary energy costs.While the environment changes have raised beyond the physiological range of plants growth,or have caused the occurring of abiotic stresses,the circadian clock genes will not only significantly alter their regular expression manner,but also produce different alternative spliced transcripts of clock genes.Eventually,the changes in the expression pattern of clock genes responding to the environmental stresses will also trigger the stress-responding reactions in the clock controlled downstream metabolic pathways,and initiate the stress-responding signaling pathways in plants.Tibetan hulless barley(Hordeum vulgare L.var.nudum hook.f.)also known as "hulless barley",is the most important food crop cultivated by the residents of the Qinghai-Tibet Plateau.The habitat of plant is considered to be the origin of the most modern barley species and their cultivars.Meanwhile the hulless barley is the only Hordeum crops that have really adapted to the extreme climate of the Qinghai-Tibet Plateau which is also known as the “roof of the world”.Having been cultivated for thousands of years by the local residents in the Qinghai-Tibet Plateau,hulless barley was thought to have evolved strong endogenous stress resistance systems and have accumulated some unique stress-resistant genes in its genome during the process of adapting to the extremely environment in Qinghai-Tibet Plateau such as the low temperature,lack of atmospheric oxygen,and intense ultraviolet radiation.Owing to its inherent physiological and genetic features,hulless barley was used as an excellent model plant for the researches on the abiotic stress tolerance of gramineae plants.For a reason that the high efficiency regeneration system and transgenic method for hulless barley has not been established,the traditional forward genetic strategies on the basis of genesis followed by the gene identification via transgenic and gene knockout method,can hardly be used in the molecular researches performed in hulless barley.However,in recent years,with the quickly developing and widely applicating of the next generation sequencing technique,the genome of hulless barley have been fully sequenced,and the transcriptome sequencing tasks of this plant under some identical growth conditions were also implemented by RNA seq methods.All of these above mentioned evidences have provided a solid basis for the further studies on the molecular mechanism of stress-resistance in hulless barley by molecular genetic methods.The main purpose of this dissertation is to detect and analyze the expression patterns of the endogenous circadian clock genes and some key resistance genes in hulless barley under various environment temperatures.The effect of low temperature on the rhythmic expression of circadian clock genes and the stress-resistant genes were also discussed,which has established an experimental basis for the future studies on the molecular mechanism by which,the circadian clock system has used to regulate the expression of down-stream stress-resistance genes.The main contents of this paper are listed as following:1.Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barleyAccording to the relevant reports,10 so-called house-keeping genes,including actin,e2,α-tubulin,β-tubulin,gapdh,hsp90,ef-1α,samdc,pkaba1 and pgk,were selected as candidates for the following expression stability tests.Subsequently,the q RT-PCR method was used to determine the relative expression of all these candidate genes under various stressed conditions.Then,3 commonly used software packages including ge Norm,Norm Finder,and Best Keeper,were used to analyze the expression stability of each candidate reference gene by relative expression data obtained by q RT-PCR amplifications.The results showed that under salt stressed conditions,hsp90 and tubα were the most stable reference genes.While under drought and cold stressed conditions,the most stable reference genes were act,e2 and tubα,ef-1α respectively.Finally,the feasibility of the optimized reference genes determined by software,were verified by using a circadian clock gene-cca1 as target gene for calibrating.The results indicated that the optimal reference genes obtained from ge Norm,Norm Finder and Best Keeper,were perfectly suitable for the expression analyzing of target genes by q RT-PCR in hulless barley.2.The selection of circadian clock genes and stress resistance genes and analysis of the expression patterns of these chosen target genes in Tibetan hulless barleyAccording to the reported circadian clock genes(cca1,toc1,lux,elf3,gi,co1,ft1 etc.)from different plants species such as Arabidopsis,rice,wheat and barley,the ortholog genes in hulless barley were collected from nucleic acid database of barley in NCBI.By the same strategy,the sequences of stress-responding genes to abiotic stress(such as cbf4,nhx1,nhx2,nhx3,cbl2,cbl4,cbl6,cbl8,p5 cs,p5cr,cat1,cat2,cmo1,m6 pr,sod1 and lea3 etc.)were also identified online.The m RNA sequence or EST sequence from c DNA library of the all the target genes were used for the expression analysis in following experiments.For the amplifying of these abovementioned genes,the software of Primer Premier5.0 and the program of Primerselect in software DNASTAR were employed to design the primers suitable for subsequent q RT-PCR experiments.The efficiency of the primers were verified by semi-quantitative RT-PCR.The results of this study revealed that in the hulless barley,all the circadian clock genes(such as cca1,toc1,lux,elf3,gi,co1,co2 and ft1)and some of the resistance genes(such as cbf4,cbl2,cbl4,cbl8,p5 cs,p5cr,cat2,nhx1,cmo1,m6 pr,sod1 and lea3 etc.)showed obvious circadian rhythmic expression profiles under 25°C.Most of these genes still maitained their expression pattern under 15°C.However,when the environmental temperature decrease to 5°C,most of these genes lost or damped their rhythmic expression patterns in varying degrees.Among all the tested genes,the rhythmic expression profiles of all circadian clock genes were completely depressed by a sudden shock of low temperature pulse,and their peak relative expression levels were significantly inhibited by the chilling environment.The expression pattern and abundance of most of the resistance genes are also affected by the sudden low temperature shock.Interestingly,the rhythmic expression profiles of cbl4 gene seems to be unaffected by the same sudden chilling environment in a short period of time,though the prolonged cold stress will also suppress the rhythmic expression of cbl4 gene.3.Cloning of the Hvcbl4 in Tibetan hulless barley and its induced expression in E.ColiThe results of previous experiments in this present study have indicated that short-term chilling environment does not affect the rhythmic expression of Hvcbl4 in hulless barley.Therefore,we hypothesize that the maitaining of Hvcbl4 rhythmical exprssion under short-term chilling condition is conducted by an unknown time cues which are not generated from the well-studied cca1/lhy-toc1 feedback loop.In order to perform further studies on the new function of Hvcbl4 and its modulating mechanism,we cloned the Hvcbl4 and successfully constructed it into a prokaryotic expression vector designated as p MAL-c2X-Hvcbl4.Then a ready to use prokaryotic expression system was constructed by transferring the p MAL-c2X-Hvcbl4 plasmid into the host E.Coli TB1.Finally,SDS-PAGE electrophoresis showed that the Hvcbl4 gene was successfully expressed in E.coli by the fusion protein MBP-Hv CBL4.For perspective,the purified MBP-Hv CBL4 fusion protein will be used to produce the specific antibody for Hv CBL4 protein,which will establish a solid experimental basis for function identification of Hv CBL4 protein in the further researches.