Effects Of Cold Stress On The Physiological Characteristics And Related Gene Expression Of Phalaenopsis

The changes in chlorophyll fluorescence and photosynthetic parameters of six Phalaenopsis cultivars the Big Chili,the Zhengshi Rosy Dawn,the Fuller’s Sunset,the Fire Phoenix,the Wedding promenade and the Queen Beeer ’Red Sky’were studied during low temperature in this research,And select the cold-resistant vatiety the Big Chili and the not cold-resistant variety the Fuller’s Sunset.And researched the chlorophyll,osmotic adjustment,H2O2 and MDA contents,and the activity of PEPC and Rubisco and several kinds of antioxidase in the leaves of the the Big Chili and the Fuller’s Sunset.The results showed that during the treatment period,the chlorophyll fluorescence parameter Fo was increased gradually and the other were all reduced,and by the end of the treatment,the Fv/Fm of the Big Chili was highest;The chlorophyll content of the Big Chili was higher than that of the Fuller’s Sunset.Pn,Gs and Tr are decreased gradually,while the Ci of the Queen Beeer Red Sky and the Fuller’s Sunset tended to increase in the later period of low temperature treatment.The activity of PEPC increased gradually to stability and the value of the Big Chil’s was significantly higher than that of the Fuller’s Sunset.The increase of Rubisco activity in Big Chili was greater than that in Fuller’s Sunset.The contents of soluble sugar and soluble protein were increased firstly and then decreased,and the accumulation of the Big Chili was more than that of the Fuller’s Sunset.The change trends of proline content were not consistent.The activity of SOD was gradually increased,and the activity of POD,CAT and APX showed a trend of increased at first and then decreased.The activity of each antioxidase of the Big Chili was increased faster than that of the Fuller’s Sunset.The content of H2O2 and MDA in Fuller’s Sunset was increased significantly greater than that in Big Chili.The results showed that the activity of PEPC,Rubisco and SOD of the Big Chili were significantly higher than that of the Fuller’s Sunset,which may be an important reason for its stronger resistance to cold.The results provide a theoretical basis for the breeding of cold resistant varieties and resistance molecular mechanism research of Phalaenopsis.The results of space-time expression analysis of photosynthetic enzyme genes in Phalaenopsis under low temperature showed that the expression of RCAα,RCAβ and PEPC in leaves and roots of two varieties of Phalaenopsis had the same trend,the expression of RCAa and RCAβ in leaves and roots of Big Chili and Fuller’s Sunset had a decreasing trend,while the expression of PEPC in leaves and roots of two varieties had an increasing trend.AP2/ERF family is a kind of plant-specific transcription factors which includes AP2,ERF,DREB and RAV subfamilies and plays an important role in gene regulation.In this study,four AP2/ERF family genes named PhAP2/ERFl-4 were cloned from the leaves of the cold-resistant variety Big chili of Phalaenopsis by RT-PCR.Sequence analysis showed that the full length of PhAP2/ERFl was 594 bp,containing 588 bp open reading frame and coding 195 amino acids;the full length of PhAP2/ERF2 was 106 9 bp,containing 100 2bp open reading frame and coding 333 amino acids;the full length of PhAP2/ERF3 was 127 7 bp,containing 111 9 bp open reading frame and coding 372 amino acids,and the full length of PhAP2/ERF4 was 896 bp,containing 813 bp open reading frame,encoding 270 amino acids.PhAP2/ERF1、PhAP2/ERF3 and PhAP2/ERF4 contained an AP2 domain and PhAP2/ERF2 contained an AP2 domain and a B3 domain.The phylogenetic tree analysis showed that PhAP2/ERF1-4 had the closest relationship with members of AP2/ERF family in Orchidaceae.PhAP2/ERF1 belonged to the A1 group of DREB subfamily of AP2/ERF family,PhAP2/ERF2 belonged to RAV subfamily,PhAP2/ERF3 belonged to the B4 group of ERF subfamily,and PhAP2/ERF4 belonged to the A2 group of DREB subfamily.Real-time fluorescence quantitative PCR(qRT-PCR)was used to investigate the expression levels of PhAP2/ERF1-4 in the leaves of Big chili and Fuller’s Sunset under low temperature acclimation and]ow temperature stress.The results showed that the expression pattern of PhAP2/ERF1-4 in the leaves of Big chili was consistent:they were induced at the stage of low temperature acclimation and the expression level reached the highest at the early stage of low temperature stress and reduced slightly at the middle and late stage of stress.Whereas the expression of PhAP2/ERF1-4 genes in leaves of Fuller’s Sunset was significantly different:the expression of PhAP2/ERF1 and PhAP2/ERF2 were not induced or only increased slightly during the whole process of low temperature treatment.The response time of PhAP2/ERF3 in leaves of Fuller’s Sunset noticeably lagged behind that of Big chili under low temperature,and the expression level reached the highest at the late stage of stress.There was little difference of PhAP2/ERF4 between Fuller’s Sunset and Big chili.The difference in the expression of AP2/ERFs in the leaves of cold-resistant variety Big chili and unresistant variety Fuller’s Sunset suggests that the AP2/ERF gene family of Phalaenopsis plays an important role in the regulation of cold stress response of Phalaenopsis.