Molecular Cloning And Expression Analysis Of PtDHAR And PtFer From Puccinellia Tenuiflora

Puccinellia tenuiflora (Turcz.) Scribn. is a kind of grazing which grows in the soil that is excessive alkalization. Puccinellia tenuiflora plants are tolerant to salt stress. It can live in circumstance of pH10 depending on its salt-resistant ability. It is theoretically and practically important to study saline-alkaline resistance of Puccinellia tenuiflora for the purpose of developing the antisaline-alkaline stress capacity of ornamental and crop. Saline-alkaline stress can induce oxidative stress in terms of early research. Increasing the capability of oxidative stress tolerance could help plants to resist Saline-alkaline stress. DHAR and Fer are the important genes in the antioxidant system and the Expressed Sequence Tag (ESTs) were gained in terms of early research. In this study, the full-length of the two genes were gained and the expression levels were analyzed through Northern blotting. Sequence analyses, phylogeny reconstruction were performed. All of above can provide important information regarding the salt-resistant mechanism of Puccinellia tenuiflora. The overall results have been summarized as followings:1. The RACE (Rapid Amplification of cDNA Ends) cDNA library was constructed, using the Puccinellia tenuiflora plants which were from QingGang county in HeiLongjiang province. The Gene-specific primers (GSP) of DHAR and Fer were designed in terms of their ESTs. Full-length cDNA of the two genes (were named as PtDHAR and PtFer respectively ) were cloned by using RACE methods.2. Sequence analyses revealed that the full lengths of PtDHAR and PtFer were 987bp and 751bp respectively. The deduced amino acid sequences were 213 and 111 amino acids in length. The deduced amino acid sequences of PtDHAR contained two conserved domains: GST-N-family and GST-C-DHAR. In addition, the phylogeny reconstruction analyses revealed that the two sequences both had a high degree of identity in the predicted protein sequence with which from Oryza sativa and Triticum aestivum etc.3. Eight months old Puccinellia tenuiflora plants had been sampled after 200 mmol/L Na₂CO₃ stress treatments for Oh, 2h, 6h, 12h, 24h, and then, total RNA were extracted from the overground part and roots respectively. Northern blotting analysis showed that the expression levels of PtDHAR under stresses were all significantly higher than that with no stress in both overground part and roots. The expression levels of PtFer under stresses were much higher than that with no stress in overground part. In addition, the Thinflower Alkaligrass had been sampled with 0 mmol/L,50 mmol/L,100 mmol/L,150 mmol/L and 200 mmol/L Na₂CO₃ stress treatments for 24h, and extracted total RNA respectively. Northern blotting analysis showed that the expression levels of the two genes under stresses were all significantly higher than that with no stress. There was no remarkable expression levels difference among concentration grads of PtDHAR, while the expression levels of PtFer after 200mM Na₂CO₃ was the most. Both the cDNA microarray result and Northern blotting analysis indicated that PtDHAR and PtFer genes are related to saline-alkaline stress. This study provided preliminary experiment basis for further study of PtDHAR and PtFer gene functions.4. Over expression vector in plant was constructed by fusing PtDHAR gene sequence with promoter CaMV35 in plant expression vector pBI121, and through electric shocks method, the recombinant plasmid pBI121-DHAR was transformed into the Agrobacterium tumefaciens strain LBA4404, and then was successfully transformed into tobacco by Agrobacterium tumefaciens-mediated method.