Evaluation Of Reference Genes For Normalization Of Quantitative RT-PCR In Soybean Under Abiotic Stresses Such As Aluminum Toxicity And Functional Analysis Of A Soybean Aluminum Tolerant Candidate Gene GmZFP3

Soybean is one of the major crop of oil and food.In Southern China,the primary factor that limits the production of soybean is the acidic soils,where soluble aluminuminhibits the growth and development of soybean.Therefore,identification and functional study of candidate genes for aluminum tolerance is critical to breed new cultivars that are more tolerant to aluminum toxicity,which would increase the yield of soybean in acidic soil.Gene expression pattern is essential to study gene function.Due to its high efficiency,convenience and accuricy,qRT-PCR technology is used widely in many fields.The stability of reference genes is one of the most important factor affecting the accuricy of qRT-PCR.Therefore,we first evaluated the stablilities of 10 reference genes under stresses including aluminum toxcity in this study.In addition,we cloned and characterized a gene encoding a C2H2 type of zinc finger protein,based on the previous study on the transcriptome of soybean under aluminum toxcity.The expression pattern and functional of this gene was investigated.The main results are as follows:1.Under the treatments of 25 μmol·L-1 AICl3.100 μmol·L-1 CdCl2 and 42 ℃ heat shock,the stabilities of ten candidate reference genes are estimated with geNorm,NormFinder,BestKeeper and Delta Ct approach.RefFinder gives a comprehensive ranking as the following:(1)Under 25 μmol·L-1 AICl3 treatment,the ranking forthe stabilities of ten reference genes in the roots of soybean Kefeng No.1 is TUA>AC11>Fbox>UKN2>TUB>60S>ElF-1α>ACT2/7>ABC>CYP.TUA is the most stable reference gene under aluminum toxcity treatment by geNorm,BestKeeper and Delta Ct approach,while CYP is the least stable one by geNorm,BestKeeper,andNormFinder.(2)As for the 100 μmol·L-1 CdCl2 condition,60S,UKN2 and ACT2/7 are the top three most stable reference genes in both roots and leaves,and Fbox and TUB should be avoided.Fbox and UKN2 is the most stable reference in roots and leaves,respectively.(3)Uner 42℃ heat shock,UKN2,ACT2/7 and 60S are identified as the most stable reference genes in both roots and leaves.ACT217 is also reported as a stable reference geneuner cold stress,indicating its insensitivity to temprature change.Meanwhile,CYP and TUB are not recomanded for 42 ℃ heat shock.TUA and UKN2 has the most stable expression in roots and leaves,respectively.2.Based on a previous study on the transcriptome of soybean under aluminum toxcity,a gene encoding a zinc finger protein was cloned and designated as GmZFP3(Glycine max Zinc Finger Protein 3).The results on GmZFP3 are as follows:(1)GmZFP3 is located on Chromosome 13 in soybean.Its Opening Reading Frame is 549 bp in length,encoding 182 amino acids.The molecular weight of GmZFP3 protein is 20441.7Da,and its predicted isoelectric point is 9.60.(2)GmZFP3 protein was localized in the nucleus of arabidopsis protoplasts cells.(3)The expression patterns of GmZFP3 under abiotic stresses(AICl3,PEGQ NaCl and ABA)were investigated by qRT-PCR.The results showed that GmZFP3 was expressed in both roots and leaves,with higher relative expression in roots than leaves.GmZFP3 is up-regulated by 25 μmol·L-1 AlC13,and the relative expression is more than 1600 fold higher than control at 24 h in roots.GmZFP3 has the highest relative expression at 3-6 h when treated with 20%PEG in roots,and 6h,24-48 h in leaves.The relative expression of GmZFP3 is more than 500 fold higher than control at 24 h under 200 mmol·L-1 NaCl treatment in roots,and showed two peaks at 6 h and 48 h in leaves.When plants were treated by 100 μmol L-1 ABA,the relative expression of GmZFP3 peaked at 24 h in roots and 6 h in leaves.Because of its strong responses to AIC13 and NaCl stresses,GmZFP3 may participate in the pathways of soybean tolerance to aluminum toxcity and salinity.(4)We transferred GmZFP3 into arabidopsis by floral dipping method.The germination rates of wild type,homozygous T3 GmZFP3 overexpressing lines,GmZFP3-1 and GmZFP3-2,were recorded after 3 days under 0,50,100 and 150 mmol·L-1NaCl treatments.These three lines showed no dififerences under 0 and 50 mmol L"1 NaCl.GmZFP3-1 showed significantly higher germination rate than the wild type and GmZFP3-2 under 100 mmol·L-1.Under 150 mmol L-1 NaCl stress,GmZFP3-l and GmZFP3-2 showed significant higher germination rates than wild type,indicating that GmZFP3 may play a important role in plant tolerance to salt stress.No significant difference in the relative root growth was observed between the wild type and homozygous T3 GmZFP3 overexpressing lines under 15 μmol·L-1 AICl3.The role of GmZFP3 in plant tolerance to aluminum toxicity need to be further studied.