Cloning And Functional Verification Of Stress-tolerance Related Gene Tamyb01 And Tapp2c01 In Common Wheat (Triticum Aestivum L.)

Wheat(Triticum aestivum L.)has important significance to food security and economic development as an important food crop.In this study,winter wheat J841 used as experimental material,selecting two stress-toleranc related genes Tapp2c01 and Tamyb01 from high-throughput sequencing results,cloned and systematically analyzed the expression characteristics of two candidate genes were during abiotic stress,VIGS technique and expression vector constructed were applied to primary function verification.The main resecrch results are as following:1.Tapp2c01 gene was cloned,which contain a 960 bp coding box;the ORF encoding a 320 amino acids protein,which molecular weight is 34.7 KD,PI is 8.41;The DNA sequence contains four exons and three introns.Conserved domain analysis showed that the 82-317 amino acids of Tapp2c01 had a PP2 C domain belonging to the PP2 C family.PP2 C homology comparison indicated that Tapp2c01,Aegilops tauschii and Triticum Urartu have a near relationship,followed by Oryza sativa Indica;Manihot esculenta and Ananas comosus PP2 C protein affinity were relatively far away.2.Real-time PCR(q RT-PCR)Result showed that,the expression patterns of Tapp2c01 gene was positively regulated under drought stress(20%PEG6000)in different organizational structures.The relative expression of Tapp2c01 gene increased first and followed by decrease in the root tissue,drought stress treatment for 6 hours,and the amount of expression increase rapidly.With the prolongation of stress treatment time the expression level of Tapp2c01 gene decreased gradually.In the shoot tip tissue,the expression of Tapp2c01 gene was the same as that of root tissue under drought stress.In the leaf tissue,the relative expression of Tapp2c01 gene relative expression fluctuates relatively large under drought stress.In the process of stress treatment,the expression of Tapp2c01 gene in wheat showed three turning points were 6h,24 h,72h,the maximum appeared in the treatment 72 h.The expression patterns of Tapp2c01 gene was positively regulated under abiotic stress.3.Tamyb01 gene was cloned,which contain a 522 bp coding box;The ORF encoding a 174 amino acids protein,which molecular weight is 19.7 KD,PI is 7.61;The DNA sequence contains two exons and one intron.Conserved domain analysis showed that the 4-129 amino acids of Tamyb01 had a MYB domain belonging to the MYB family.MYB homology comparison indicated that Tamyb01 and Triticum Urartu have a near relationship,followed by Aegilops tauschii;Zea and Sorghum bicolor MYB protein affinity were relatively far away.4.Real-time PCR(q RT-PCR)technique result showed that,the expression of Tamyb01 gene showed a decreasing trend in roots、shoots and leaves of wheat seedlings under salt stress(Na Cl,100μmol·L-1).In the root tissue,the relative expression of Tamyb01 gene reached peak at 3h after stress treatment,with the prolongation of salt stress,the expression was relatively stable.in the shoot tip tissue,with the prolongation of salt stress,the expression level of Tamyb01 gene decreased gradually.In the leaf tissue,the relative expression of Tamyb01 gene same as the control increased first and followed by decrease,same as the control,but under salt stress the relative expression of Tamyb01 gene was lower than that of control.5.Winter wheat Jing 841 was used as the experimental materials to establish technology system of VIGS gene function verification,by VIGS technology.The results indicated that: After 16-day virus inoculation,the plants of control grew normally,but the plants of BSMV-?:00 showed weak photo bleaching phenomenon,and BSMV-?:Ta PDS 、 BSMV-?:Tamyb01 、 BSMV-?:Tapp2c01showed obvious bleaching symptom or banded white sports.Transcription level test results showed: the expression quantity of wheat plants inoculated with BSMV-?:Ta PDS 、BSMV-?:Tamyb01、BSMV-?:Tapp2c01accounted for 56 %,36% and 42% of that of negative control respectively,indicating that the expression of three genes had been significantly inhibited.The chlorophyll content of wheat plants with BSMV virus was significantly lower than that of BSMV-?:00 and the content of MDA were significantly higher than BSMV-?:00.6.Overexpression vector p CAMBIA1304-Tapp2c01 was constructed,through transfixion Arabidopsis by tidbits infection method,selecting stable expression Arabidopsis thaliana plant.Under drought stress,p CAMBIA1304-Tapp2c01 Arabidopsis compared with p CAMBIA1304 Arabidopsis and wild-type Arabidopsis,all with death phenomenon,the survival rate were 40%,8% and 3% respectively,after watered for a week,the survival rate were 78%,10% and 5% respectively.The results suggest that the Tapp2c01 gene has a positive regulation in enhancing the drought resistance of Arabidopsis thaliana.