| Parashorea chinensis is the evergreen tall tree in the Parashorea Kurz Dipterocarpaceae,and the distribution of it is narrow,only in southern China,southeastern and southwest of Guangxi have a natural distribution,is a rare and endangered species in China.Ex situ conservation is one of the effective ways to preserve germplasm resources of precious tree species,and it also helps to expand its range of cultivation.But temperature is an important environmental factor affecting ex situ conservation.In this study,the 1-year-old Parashorea chinensis seedling was selected as the experimental material,and the low temperature treatment experiment of 0? low temperature induction of Od,1d,2d and reverting to 25 ? recovery treatment were carried out to study the growth status,physiological and biochemical changes of the seedlings in the four stages of,and the proteomic technique was used to analyze the expression of differential proteins in leaves at 0? low temperature induction of 2d,the main conclusions are as follows:1.With the induction of low temperature,the whole plant leaves of the seedlings were sagging,and the young leaves were withered at 2d.After re-rising to 25 ?,the young leaves were still curled and even dried,while the functional ripe leaves began to stretch.2.The total chlorophyll a,b and chlorophyll content in the leaves of Parashorea chinensis seedlings were higher than that of the control treatments at the low temperature of 0? and re-rising to 25 ?.And their contents changes were generally increased first and then decreased,and reached the maximum at 2d.The ratio of chlorophyll a/b was lower than that of the control group at 0?induction of Od,1d and 2d,and the ratio was greater than that of the control group when the temperature returned to 25 ?.The total content of carotenoids was lower than that of the control group at 0?induction of Od and 1d,while it increased gradually and even was higher than that of the control group at 0? induction of 2d and at the temperature returned to 25?.The content of MDA,free proline,soluble sugar and soluble protein and relative conductivity of leaves gradually increased during induction at 0?,reached the peak at 2d,and their contents were reduced when the temperature returned to 25?,but in the whole process the values of the above indicators were higher than their control.The activities of SOD,POD,CAT and APX were stronger than those of the control group,and the changes of enzyme activity were increased first and then decreased,and reached the peak at 2d,but the change of PPO activity was opposite.During the induction of low temperature at 0?,the counts of IAA and ZR had no significant difference.When the temperature rose to 25 ?,the content of both increased significantly.However,with the prolongation of stress time,the content of GA3 and ABA increased significantly,and the increase of both was the largest at 2d.When the temperature rose,the increase of these two hormones had a very significant decline.In conclusion,at 0? induction for 2d,the leaves of Parashorea chinensis seedlings sag were the highest,and the young leaves were even withered and curled,and the contents of chlorophyll,free proline,soluble sugar,soluble protein,GA3 and ABA were the highest,and the activities of SOD,POD,CAT and APX were the strongest,after the temperature rise,the value of the above indicators reduced significantly,and functional maturity of the leaves began to resume stretching,which indicated that Parashorea chinensis had a certain resistance to transient hypothermia stress.3.The total protein of leaves of Parashorea chinensis seedlings was extracted by modified TCA-acetone precipitation and phenol-methanol/ammonium acetate extraction.The total protein content extracted by method TCA-acetone precipitation was 4.3 times higher than that of modified phenol-methanol/ammonium acetate precipitate,and the electrophoretic bands were more.Therefore,the modified total TCA-acetone precipitation method was used to extract the total protein from the leaves of the leaves to analyze the differential protein expression.4.After induction at 0?for 2 days,the total protein was extracted by modified TCA-acetone precipitation method.The extracted protein was analyzed by LC-MS/MS combined with TMT quantitative labeling technique,and then the SEQUEST software was used to search the Uniprot database.The results showed that a total of 481 proteins were identified,among which 96 proteins showing differences,5 2 were up-regulated and 44 were down-regulated.5.The bioinformatics analysis of 96 differential proteins was carried out,?1?GO comments:the differential proteins were involved in four biological processes,which were cell processes,precursor metabolites and energy,photosynthesis and nucleotide metabolism.The cell components of the differential proteins were three,which were protein complexes,thylakoids and light systems,respectively.And the molecular function of the differential protein was only two:nucleoside binding and chlorophyll binding;?2?KEGG-Pathway biological pathways analysis:the differential proteins were annotated into sevn pathways,which were photosynthesis,oxidative phosphorylation,metabolic pathway,splice,RNA degradation,phagocytosis and ribosomes.The differential proteins involved in photosynthesis and metabolism were all the photosystem ?family proteins,the light system II subunit Q-2 and F1 ATP enzyme y subunits,and the expression of these proteins was up-regulated.The FlATPase y subunit was also involved in oxidative phosphorylation Pathway.The differential protein involved in splicing and RNA degradation pathways was small nuclear ribonucleoprotein family proteins and it was also upregulated.The differential proteins involved in phagosome and ribosome pathway were respectively ?-6 tubulin isoforms 1 and 60S acidic ribosomal protein P2B,both of which were down-regulated;?3?STRING protein interaction analysis:it was found that there was only an interaction between light system II subunit QA and light system II subunit Q-2 which were related to light and electron transport relationship.Combining with GO and Pathway analysis showed that the low temperature stress allowed the photosynthesis of Parashorea chinensis to be most affected,these three analyzes are related to photosynthesis-related proteins.In summary,Parashorea chinensis through differences in expression of different proteins in a variety of biological pathway to regulate a variety of physiological and biochemical mechanisms and then to deal with short-term low-temperature environment.This study provided a scientific basis for further clarifying the cold tolerance mechanism of Parashorea chinensis. |
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