Cloning And Functional Analysis Of Mono Domain TaFKBPs Genes In Wheat (Triticum Aestivum L.)

Wheat production is often suffers from abiotic stresses like high temperature,drought and soil salinity and etc.It is effective ways to cultivate wheat stress-resistant varieties for ensuring the safety of wheat's production.The breeding based on conventional methods is blindness.However,molecular breeding has some advantages such as high targeted,short cycle and so on.Crop molecular breeding premise is to search key genes involved in stress-response.Previous studies have found that FK506-binding protein family?FKBPs?are one kind of molecular chaperone and assist the target protein to fold correctly or remove damaged target proteins.So FKBPs can improve cell resistance.FKBPs genes family is divided into mono domain and multi-domain.Mono domain genes contain only one FK506 binding domain?FKBd?.At present,the study of wheat FKBPs is rare,and that of mono FKBPs genes is rarer.In this study,we have analysed expression patterns in stress of four mono domain TaFKBPs genes?TaFKBP19-2AS,TaFKBP16-2-6AL,TaFKBP16-3a and TaFKBP16-3b-7AS?by real-time fluorescent quantitative PCR?qRT-PCR?.Then three heat response genes were cloned and amino acid sequence analysis.TaFKBP19-2AS gene has also done subcellular localization,the promoter cloned,transgenic Arabidopsis function verification etc.,to complete wheat mono domain TaFKBPs genes preliminary molecular properties and functional verification.The main reserch results are as follows:?1?By real-time quantitative PCR?qRT-PCR?assay,we found that wheat mono domain TaFKBP19-2AS,TaFKBP16-2-6AL,TaFKBP16-3a and TaFKBP16-3b-7AS genes all were induced by high temperature.TaFKBP19-2AS gene is the most sensitive on heat stress and after 15 minutes it's expression was reached highest expression and was 5.5 times as untreated's.However,four geneswere down-regulated-expression by drought.Such as after 12 hours TaFKBP16-3a gene's expression is 0.02 times as untreated's.?2?The results of tissue-specific analysis showed that TaFKBP19-2AS,TaFKBP16-2-6AL and TaFKBP16-3a had expression in root,stem,leaf,spike,lemma and palea.The highest expressions in all tissues was leaf.?3?TaFKBP19-2AS,TaFKBP16-3a and TaFKBP16-3b-7AS were cloned and were 744 bp,778bp,843 bp,respectively and had 264 AA,236 AA and 247 AA,respectively.The protein sequences were prediction by SMART and found typical FKBd domains of FKBPs family.We drawed the Evolutionary tree by MEGA6.0and the they are closest relative with Brachypodium distachyon.?4?We constructed subcellular localization vector as16318 h GFP-TaFKBP19-2AS and inducted expression in wheat protoplast by PEG.TaFKBP19-2AS is located in the cytoplasm.?5?The promoter TaFKBP19-2AS was cloned and the sequence was 1769 bp.It was named PtaFKBP19.The promotor was predicted for cis-acting element and found that contained some stress elements,such as CCGTCC-box,ABRE?Abscisic acid response element?,ACE?ACGT-Containing Element?,HSE?Heat shock element?and LTR?Low temperature response element?etc.in Plant CARE website.We constructed transformed-gene vector p Cambia1391Z-PtaFKBP19 for plants and got transgenic lines.The T1 lines' s GUS staining result showed that young tissue had GUS staining signals,especially leaf vein and petiole in new leaf and root tip,but other plant tissues had not dyeing signal.It may explains that the promoter has really meristematic tissue specific expression.?6?We constructed transformed-gene vector p Cambia1302-TaFKBP19-2AS for plants and got Arabidopsis transgenic T₃ lines.After heat treatment,the survival rate of transgenic plants is higher than wild type.After PEG10 % stress treatment,OE lines whole biomass highly significantly bigger than wild type,this shows that TaFBKP19-2AS enhance the heat and drought resistance of transgenic lines.?7?By qRT-PCR assay,we found that TaFKBP19-2AS gene can effect the expression of related-stress genes in Arabidopsis.Such as At Hsp23.6,At Hsp22.0,At Hsf A2 genes etc.were higher significantly in transgenic plants than in WT.It is impossible that TaFKBP19-2AS improve the resistance of transgenic plants by regulation heat shock protein and heat stress transcription factors?Hsfs?.