Analysis Of Physiological Changes And Diferential Gene Expression Of Cercidiphyllum Japonicum During Drought Stress

Drought is one of the major abiotic stresses that adversely affect plant growth and yield worldwide. Lots of genes are thought to be involved in responses to abiotic stress.However,only a few genes have been known precisely until recently and moreover most of the studied genes originated from a few annual herbaceous plants like Arabidopsis,tobacco and rice.Information on drought-resistant genes of perennial trees is scare,even though drought stress constitutes a major challenge to forestry development in the future.To understand the molecular mechanism of water stress and the role of differential gene expression in drought tolerance of the trees,using Cercidiphyllum japonicum as material,we studied its transcriptome during drought stress by suppression subtractive hybridization (SSH) method.Based physiological and biochemical change of the seeds and seedlings,we analyzed the mechanism of drought tolerance of C.japonicum.At the same time,we used qRT-PCR to verify eight differentially expressed genes in the SSH data,and to confirm the SSH technique is accurate and reproducible.Main results as follows:(â…°) The morphological,physiological and biochemical indexes associated with seed germination and seedling growth of C.japonicum were measured under seven different polyethylene glycol 6000 (PEG 6000) concentrations from 95 to 345 g·L^-1.The results showed that the suitable PEG concentration was range from 140 to 301 g·L^-1.In this range,the membrane-repair capacity and the drought resistance of seed were improved,accompanied with the changes of the following indicators. The contents of three osmoregulation substances(soluble protein,sugar and proline) and the activities of three protective enzymes(superoxide dismutase(SOD),peroxidase(POD) and catalase (CAT)) were increased,while,in contrast,the content of a final product of lipid peroxidation, malondialdehyde(MDA) was decreased.In addition,seed germination and seedling growth were inhibited in higher PEG concentrations under water stress.With the increment of PEG concentrations,the contents of soluble sugar,proline and MDA,and the activities of two enzymes (SOD and CAT),were continuously elevated,in contrast,the enzyme activities of POD was strongly decreased.In particular,the content of soluble protein was increased first and then decreased during this process.These results also indicated that the seed germination and seedling growth were inhibited under the high PEG concentration(≥95 g·L^-1).A tentatively conclusion is that C. japonicum seed is sensitive to water stress.(â…±) The two years old seedlings of C.japonicum were treated with PEG 6000 at different concentration under water stress,which were used to study the physiological effects of drought stress on leaves.The results showed that the content of chlorophyll in leaves were strongly decreased with the increase of the concentration of PEG.The content of the soluble protein exhibited dismiliar change trend in different water stress,which increased under the low PEG concentration(50～150 g·L^-1),and increased first and then decreased under the high PEG concentration(200 g·L^-1). Accmpoany with the increase of water stress,we found the contents of soluble sugar,proline and MDA and the activity of POD were increased,in contrast,the activity of CAT was decreased. Stringly,the activity of SOD was firstly increased and then decreased.These findings suggest that the C.japonicum could adjust the contents of osmotic substance for the adaption of the water stress, and improve the activity of protective enzymes to enhance the ability of antioxygenic.(â…²) Using mixed cDNAs prepared from drought treated seedling(SB₄) and unstressed seedling (SB₀).A forward and reversal subtractive cDNA libraries were constructed,from which 768 recombinant clones were picked amd amplified.Through differential screening of the subtractive cDNA library by dot blot,three hundred and nine positive colonies were picked and sequenced,and Two hundred and forty one effective sequences were obtained.BlastX and BlastN alignment results revealed that 199 fragments showed significant similarity to the function-known sequences in NCBI database,and 109 fragments were similar to function-unknown genes.(â…³) All the sequences in the forward and reversal subtractive cDNA libraries were subjected to Gene Ontology(GO) annotation for understanding their roles in cell component,molecular function and biological process.According to their physiological function,they can be subdivided into 13 categories among them,103 genes were drought stress-related genes,and their function including signal transduction and gene expression regulation,osmotic substances synthesis,defense,transport facilitation and ions balancing,active oxygen elimination,cell rescue and defensive protein synthesis, photosynthesis repair and protection system and many other physiological and biochemical pathway.(â…´) We get many drought resistant related gene fragments including trehalose-6-phosphate phosphatase,S-adenosylmethionine decarboxylase,G protein,calmodulin,calcium-binding EF hand family protein,ADP ribosylation factor,bZip transcription factor,serine/threonine phosphatase protein,lipid transfer protein,H⁺-transporting two-sector ATPase,aquaporin,ABC transporter protein,catalase,ascorbate peroxidase,thioredoxin,metallothionein-like protein,glutathione S-transferase,Rubisco,oxygen-evolving enhancer protein,chlorophyll a/b binding protein, germin-like protein,expansin,late embryogenesis abundant protein,jasmonate-induced protein, cytochrome P450,selenium binding protein,and many other crucial genetic information.(â…µ) qRT-PCR was used to verify eight differentially expressed genes in the SSH data.The results of qRT-PCR were consistent with the SSH.The SSH technique used in this study is accurate and reproducible.