Cloning,Identification And Expression Pattern Of Three Fructan Metabolism Related Genes In Salt Tolerant Barley (Hordeum Vulgare L.)

Fructan exohydrolase(FEH)and sucrose invertase(INV)play an important role in plant growth and resistance to adversity stress.So far,the research on the fructan exohydrolase gene has been reported in wheat and ryegrass plants while sucrose invertase is also reported in wheat.But those enzymes have not been fully studied in barley.Therefore,this study took"Yanmai 3"as the experimental object,and cloned three genes related to fructan metabolism from barley for the first time,and named them Hv6-FEH I,Hv1-FEH I and Hv INV I.In this paper,all three genes were transferred into the Pichia pastoris X-33 expression system for protein expression,and the recombinant protein was analyzed for substrate specificity,optimal temperature and optimal p H,which further clarified the function of three genes.At the same time,under simulated salt stress(150 m M Na Cl solution)and drought stress(15%PEG6000 solution),the expression of these three genes in roots,sheaths and leaves was determined by real-time fluorescent quantitative PCR.We also studied the changes of 1-FEH and 6-FEH enzyme activities in barley rhizome and leaves and sugar content in tissues,so as to further explore the regulation mode of barley FEHs under abiotic stress.The main contributions of this work can be summarized as follows:1.According to the FEH gene sequences of other species that have been reported,we used BLASTEN and compared with barley database,and selected the sequences with higher similarity,and spliced with the software(Seq Man)to obtain three reference sequences.Then,according to these reference sequences,we designed primers in NCBI and cloned three ORFs,Hv6-FEH I,Hv1-FEH I and Hv INV I.The homology of amino acid sequence between Hv6-FEH I and Pp6-FEHI was 68.5%,and the homology of amino acid sequence with Ta6-FEH was 69.1%.The homology between Hv1-FEH I and Ta6-FEH was 88.8%,and the homology between Hv INV I and Ta6&1-FEH was 92.8%.Among them,both Hv6-FEH I and Hv1-FEH I contained the conserved domain NDPNG,FRDP,WECPD of the FEH family,while the conserved domains of Hv INV I are NDPCG,FRDP and WECLD.2.Three genes were transferred into Pichia pastoris X33 for protein expression individually,and analyzed its enzymatic properties after a series of purification.The substrate specificity analysis showed that Hv6-FEH I only specifically hydrolyzedβ(2,6)glycosidic bond linked levan.Hv1-FEH I could hydrolyzeβ(2,1)linked1-kestose,Nystose and 1~F-fructofuranosylnystose,and had certain hydrolysis ability to sucrose while Hv INV I could only hydrolyze sucrose.At the same time,the optimum temperature of Hv6-FEH I and Hv1-FEH I was 35℃,and the optimum p H was 5.5,which was similar to that of FEHs in grass and ryegrass.3.The quantitative expression of three genes in roots,sheaths and leaves of barley was studied under the simulated drought stress and salt stress.The results showed that the transcriptional level of Hv6-FEH I in roots was more than 7 times higher than control after the fifth day of treatment under the simulated drought stress.While under the salt treatment,the expression level of Hv6-FEH I was slightly inhibited in roots,sheaths and leaves.Compared with the salt stress,the expression of the three genes was more significant under drought stress and the expression of Hv1-FEH I in leaves reached more than 70 times.4.After 5 days of drought and salt treatment,the changes of enzyme activity and sugar content of 1-FEH and 6-FEH were measured in the rhizomes and leaves of barley.The results showed that the enzyme activities of 6-FEH and 1-FEH were increased in varying degrees,but the sucrose content was increased significantly in roots,sheaths and leaves under the abiotic stress.However,compared with the control,the sucrose content was increased by 2 times under the drought stress,and it was increased by 3 times in roots,sheaths and leaves under the salt stress.But the fructose content was relatively low under the drought and salt stress.The preliminary exploration may be due to the inhibition of the activity of FEH by sucrose accumulation,which leads to the decrease of the content of fructose.In addition,this article also made a preliminary discussion on the inconsistency between FEH activity and fructose content.In conclusion,three genes related to the metabolism of fructan were cloned related to the metabolism of fructan were cloned and identified in barley.