Effect Of Low Temperature Stress And Cold Hardening On Physiology Character Of Strawberry And Molecular Cloning Of Class Ⅱ Chitinase Gene

Strawberry production is the biggest planting area at present. The temperature is warmin south, the overwintering safeguard of strawberry is not severe and the low temperaturereversion phenomena occurred so that strawberry would suffer little low temperature injurycontingently during planting and great loss was peoduced. But the research of lowtemperature effects and cold hardening enhancing strawberry cold-resistance was seldomreported. This study investigate the effect of 0℃low temperature stress on antioxidantmetabolism of Fragaria ananassa cv. Toyonaka and cv. Akihime Meanwhile, the effects of0℃, 4℃and 10℃cold hardening and dehardening on physiology and biochemistrychange of Fragaria ananassa cv. Toyonaka leaves were researched. The aim of thisresearch was in an attempt to clarify the physiology and biochemistry mechanism of thecold resistance formation about strawberry.Otherwise, it was reported that the chitinase gene would be induced during coldhardening which had dural function and played an imporment role in improving plant coldresistance. So, our experiment also isolated chitinase gene from straeberry cv. Toyonakaand cv. Akihime leaves by PCR in order to research the role of chitinase gene in enhancingthe strawberry cold resistance from the molecular level during cold hardening further. Theresults are showed as follows:(1) Low temperature stress increased the contents of O₂^-, H₂O₂, PMP and MDA anddecreased the contents of Chl and Car significantly which was relative with the damage ofbiomembrane.(2) Low temperature stress activated the antioxidant defense system of strawberry.The contents of soluble sugar and protein increased quickly, and the activities ofsuperoxide (SOD), catlase (CAT), ascorbate peroxidase (APX), monodyhydroascorbate reductase (MDHAR), dehydroascorbate (DHAR) and glutathione reductase (GR) and theratio of AsA/DHA increased except the activities of DHAR and GR, the ratio of AsA/DHAand the contents of AsA decreased in leaves of cv. Akihime. Every antioxidative enzymeand antioxidants lessened the oxidative damage induced by low temperature stresstogether.(3) The activities of antixoidative enzymes increased quickly was the response ofstrawberry protection itself to adapt environment at the initial stages of stress.(4) APX was very sensitive to environment that had strong oxidation ability andAsA was regenerated by mainly by MDHAR.(5) Under low temperature stress, cv. Toyonaka has stronger antioxidant ability thanthat of cv. Akihime,(6) Cold hardening damaged the membrane a little, however, this kind of damagewas reversible which damage degree was lessened after dehardening.(7) Cold hardening increased the contents of soluble sugar, soluble protein, prolineand AsA, meanwhile, the activities of SOD, CAT, POD, APX, DHAR, MDHAR and GRalso increased which strengthened the stability of membrane and enhanced thecold-resistance. The antixoidative defense system had still strong ability to eliminate ROSafter 2d dehardening.(8) The ratio of AsA/DHA increased during cold harening which strengthened theabilty of alleviating H₂O₂.(9) Two genes were isolated from Strawberry cv. Toyonaka and cv. Akihime leavesby PCR. Sequencing revealed that the length of the cloned fragments were 1575bp.Structural analysis showed that the two genes were 988 bp in length and had 2 extron and 1intron. The homology of nucleotide and deduced amino acid are 99.05% and 98.56%respectively. Although the two genes and another strawberry cv. Chandler Chi2-1geneshare a homology of the nucleotide and deduced amino acid sequences of 99% and 98%respectively, the homology of deduced amino acid between strawberry and other species'schi2 gene were about 50%.