| Bermudagrass(Cynodon dactylon)and Seashore paspalum(Paspalum vaginatum)are two major perennial warm-season turfgrass.Bermudagrass is wildly utilized for turfgrass in tropical and subtropical regions south of the Yangtze River in China,because of fast-growing and strong tolerance of drought and heat.Case it’s poor salinity tolerance,it’s difficult to grow in the soil salinization areas.Exploring the genes associated with salinity tolerance in bermudagrass,will carry a great implication for improving it’s salinity-tolerance.Seashore paspalum is a halophytic grass species and mainly used in salinity-affected areas,but the molecular mechanisms regulating salinity tolerance is unknown.Exlploring the genes related with salinity tolerance will have a great potential for futher understanding of the molecular mechanisms involved in regulating salinity tolerance,and providing excellent candidate genes for the breeding of salinity-tolerance in turfgrass.We explored and cloned 11 zinc finger protein genes form the EST database of bermudagrass,two gene of CCCH family CdZFP3,CdZFP7 were as the research object.We analyzed the.sequences and the expression patterns of CdZFP3 and CdZFP7 under abiotic stresses,detected the salinity tolerance of the transgenic Arabidopsis.Besides,we constructed a full length cDNA library,and exlplored the genes related with salinity tolerance of seashore paspalum by yeast expression library screening system.The results of the study were showed as following:(1)By sequence analysis we found that,the CdZFP3 gene full-length cDNA consists of 1727bp,comprising a 1326bp ORF,encoding 442amino acids,and it was most intimate with OsC3H63_Os11g28270 in Oryza brachyantha.The CdZFP7 gene full-length cDNA consists of 1206bp,comprising a 963bp ORF,encoding 321 amino acids,and it was most intimate with OsC3H25_Os03g61110 in Oryza brachyantha.CdZFP3 was induced by salinity stress and ABA;CdZFP7 was induced by salinity stress,ABA and low temperature.(2)The CdZFP3 and CdZFP7 genes were inserted into the plant expression vector,and transformed into wild type Arabidopsis thaliana through floral-dip method.Five transgenic plants of CdZFP3 and CdZFP7 gene were obtained separately via glufosinate ammonium screening.Salinity-tolerant testing of transgenic plants found that the seed germination rate,the content of soluble sugar and proline,the photosynthetic characteristics of transgenic Arabidopsis increased,and the electrolyte leakage and the ROS content of transgenic Arabidopsis decresased compared with the wild-type Arabidopsis under salinity stress.This suggested that the CCCH family genes,CdZFP3 and CdZFP7 regulating the salinity tolerance of Arabidopsis.(3)Under salinity stress,CdZFP3 increased the expression of AtSOS1、AtP5CS、AtSOD、AtPOD in transgenic Arabidopsis,and CdZFP7 increased the expression of AtNHX1、AtAKT1、AtP5CS、AtAPX1、AtSOD、AtPOD in transgenic Arabidopsis.This suggested that CdZFP3 and CdZFP7 improved the salinity-tolerance of Arabidopsis by regulating the antioxidant systems and the osmotic adjustmentment system.(4)Based on the CAP capture and Gateway-compatible vector system,a high-quality cDNA entry library and yeast expression library of seashore paspalum were constructed.The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes,using the yeast transform screening system.Reconstructing the ORF sequence of each screened gene into the yeast expression vector and retransforming to yeast cells confirmed that 18 salinity-tolerance genes improved the salinity-tolerance of yeast.(5)Functional analysis indicated that 18 salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism(psaK,Cha/b and Elip),antioxidant systems(CS,AKR and APX),protein modification(14-3-3,LRR),iron transport(IRT,YS),vesicle traffic(VAP33),and phospholipid biosynthesis(CCT).qRT-PCR analysis confirmed that most of the 18 salinity-tolerance genes were up-regulated at the transcript level in response to salinity stress in seashore paspalum. |
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