Construction Of CDNA Library Of Puccinellia Teniflora Roots Grown In Saline-Alkali Land And Cloning And Expression Analysis Of PtLIR1Gene

Puccinellia tenuiflora, a halophyte with high capacity of salt tolerance, is an excellenttarget for isolation of salt tolerant genes and study on salt tolerance mechanism in plants.The cDNA library of Puccinellia tenuiflora roots grown in saline-alkali land wasconstructed, recombinant clones with the insert fragment larger than500bp were randomlyselected and sequenced, preliminary bioinformatic analysis was performed. PtLIR1genewas cloned from Puccinellia tenuiflora roots by RT-PCR method and plant expressionvector of pPtLIR1-GFP35S was constructed. Content of water soluble sugar and sucrose inPuccinellia tenuiflora roots under Na₂CO₃stress was determined. Expressioncharacteristics of PtLIR1and PtSS in Puccinellia tenuiflora under Na₂CO₃stress wereanalyzed by semi-quantitative RT-PCR. The association between expression of PtLIR1andPtSS and sucrose content was also analyzed. The main results are as follows:1. The cDNA library of Puccinellia tenuiflora roots grown in saline-alkali land wasconstructed. After amplification, the titer of cDNA library was1.747×10⁹cfu/mL, thepercentage of recombinant phages were70.83%. Total25positive clones randomlyselected from the cDNA libray were sequenced. Blastx analysis showed that8ESTs werehomologous to the nucleic acid or protein sequences with definite annotation in database,4ESTs had no homology with the sequences in the database,8ESTs was predicted protein orunknown protein. Several ESTs associated with salt tolerance of plants were screened suchas AdoMet synthase gene and Ubiquitin-protein ligase gene.2. PtLIR1gene was isolated from Puccinellia tenuiflora roots by RT-PCR method, andligated to pMD19-T Simple Vector. Positive recombinant plasmid of pT-PtLIR1wasobtained by colony PCR and enzyme digestion. Sequencing and blastN analysis showedthat sequence of PtLIR1gene fragment had99%homology with the known sequence in thedatabase.3. The expression characteristics of PtLIR1gene in Puccinellia tenuiflora roots underNa₂CO₃stress were as follows: （1） PtLIR1gene was expressed in Puccinellia tenuiflora roots and leaves under bothsalt stressed and non-stressed conditions.（2） The changing trend of PtLIR1expression in Puccinellia tenuiflora roots both underhigh and low concentration of Na₂CO₃stress are opposite to non-stressed conditions.PtLIR1gene displayed transient high expression level in response to low concentration ofNa₂CO₃stress, while showed continuous high expression level at certain stage in responseto high concentration of Na₂CO₃stress. PtLIR1paricipates in response of Puccinelliatenuiflora roots to Na₂CO₃stress and the expression characteristics of PtLIR1are closelyassociated with Na₂CO₃concentration.4. The plant expression vector of pPtLIR1-GFP-35S was constructed with codingregion cDNA fragment of PtLIR1without stop codon, which layed an important foundationfor further analysis of PtLIR1localization in Puccinellia tenuiflora.5. The expression characteristics of PtSS gene in Puccinellia tenuiflora roots underNa₂CO₃stress were as follows:（1） PtSS gene expressed in Puccinellia tenuiflora roots under non-stressed conditonand the expression level was relatively stable, which showed that PtSS functions in plantnormal growth. PtSS paricipates in response of Puccinellia tenuiflora roots to Na₂CO₃stress.（2） The expression of PtSS gene was suppressed to certain extent under Na₂CO₃stressin comparison to non-stressed condition. The expression level of PtSS gene in Puccinelliatenuiflora roots changed mildly under low concentraion of Na₂CO₃stress, while changedacutely under high concentraion of Na₂CO₃stress.6. PtLIR1and PtSS gene co-regulated the accumulaiton of sucrose in Puccinelliatenuiflora roots under Na₂CO₃stress.（1） In Na₂CO₃stressed Puccinellia tenuiflora roots, the expression level of PtLIR1wasclosely associated with sucrose content, which indicates that PtLIR1play an important rolein regulation of sucrose accumulation, while the expression level of PtLIR1was regulatedby water soluble sugar.（2） Both under non-stressed and low concentraion of Na₂CO₃stressed conditions, thechanging trends of PtLIR1and PtSS expression level in Puccinellia tenuiflora roots were opposite at the early stage, while coincident at late stage. The changing trends of PtLIR1and PtSS expression level were highly coincident in Puccinellia tenuiflora roots under highconcentration of Na₂CO₃stress. The consistency of PtLIR1and PtSS expression inPuccinellia tenuiflora roots was higher under high concentration of Na₂CO₃stress than lowconcentration of Na₂CO₃stress.