Physiological Responses And Transcriptome Analyses Of Oxyria Sinensis ?Polygonaceae? To Cu²⁺ Stress

Screening plant species that are tolerant to or enrich heavy metals could be important ecological restoration of mining areas.Oxyria sinensis is a common plant in copper-zinc mining area and is considered as the indicator plant of copper mine.Furthermroe,O.sinensis can reproduce clonally via roots in drought environments.Therefore,O.sinensis can occupy the harsh habitats through clonal growth,suggesting that this species it is a good plant in ecological restoration of mining areas.However,it still remains unclear how O.sinensis responds to heavy metals physiologically,and survival of O.sinensis in mining areas via tolerance to and or enrichment of heavy metals still needs to be clarified.In this thesis,plants of O.sinensis from mining?Dongchuan,Yunnan?and non-mining?Lijiang,Yunnan?areas were subjected to different contents of Cu²⁺stress?0,50,150,300 and 500mg/L?for different time?one and two months?,with an aim to find the physiological responses of this species to heavy metal.In addition,transcriptome sequencing was performed to analyze the genetic depressions of plants subjected to different treatments.The main results are in the following:?1?The content of superoxide dismutase?SOD?in the leaves was significantly higher than that in the roots.The content of peroxidase?POD?in the leaves after one month treatment was significantly higher than that after two months,and the POD contents in the roots and stems were significantly higher than that in the leaves.Ascorbate peroxide?APX?in stem was significantly lower than that in root and leaf.However,no significant difference was found in catalase?CAT?among sex morphs,treating times,sources of material,Cu²⁺contents and plant parts.?2?The contents of protein in roots and stems was significantly higher than that in leaves,but no significant difference was found in MDA among sex morphs,treating times,sources of material and Cu²⁺contents.In addition,the contents of free proline,chlorophyll and Cu²⁺in the plants treated for one month were significantly higher than those in the plants treated for two months,and the content of chlorophyll in the plants from Dongchuan were significantly higher than those from Lijiang.The content of Cu²⁺in the plants subjected to Cu²⁺stress was significantly higher than that in the control group.?3?43.55Gb Clean Data were acquired after transcriptome sequencing of six types of treatments,with an average of 6.76Gb for each sample.There were 170952contigs,with a total length of 248,903,417 nt and an average of 1,456 nt.N50 of contigs was 2,026 nt.After clustering,60,095 unignes were acquired,with a total length of 843,173 nt and an average of 1,112 nt.N50 of unigenes was 1,755 nt.All unigenes were blasted to each database?COG,GO,KEGG,KOG,Swiss-port,eggNOG and Nr?,and a total of 38,860 unigenes were annotated,and The number of annotated unigenes in COG,GO,KEGG,KOG,Swiss-port,eggNOG and Nr were11,413?29.3%?,20,394?52.5%?,14,677?37.8%?,24,225?62.3%?,23,626?60.8%?,36,764?94.6%?and 34,306?88.3%?,respectively.?4?Compared with cultured control plants,3,610 genes with differential expression were detected in plants collected from Lijiang and subjected to Cu²⁺stress of 150mg/L for one month,of which 3174 genes were up-regulated and 436 genes were down-regulated.The annotations showed that these genes were mainly enriched in ribosome,oxidative phosphorylation,endoplasmic reticulum protein processing and RNA transport pathway.When plants were subjected to Cu²⁺stress of 150mg/L for two months,2,178 genes with differential expression were detected,of which1,284 genes were up-regulated and 894 genes were down-regulated.These genes were mainly concentrated in phenylpropanoid biosynthesis,horny biosynthesis,alkaloid and wax biosynthesis,?-linolenic acid metabolism,starch and sucrose metabolism,and plant hormone single signal transduction pathway.Furthermore,in comparison with plants that were just collected in Lijiang,3,972 differentially expressed genes were detected in the plants subjected to Cu²⁺stress of 150mg/L for one month,of which 3,442 genes were up-regulated and 530 genes were down-regulated.These differentially expressed genes were mainly enriched in ribosomes and oxidative phosphorylation pathways.In contrast,a total of 2,823 differentially expressed genes were detected in the plants subjected to Cu²⁺stress of 150mg/L for two months,of which 1,744 genes were up-regulated and 1,079 genes were down-regulated.These differentially expressed genes were mainly enriched in photosynthetic antenna protein,?-linolenic acid metabolism and phenylpropanol biosynthesis.Cysteine and methionine metabolism and photosynthesis pathways.?5?Further analysis showed that,in those genes related with Cu²⁺transports and tolerance to Cu²⁺stress,the expression level of oxidase genes were down-regulated,but the expression of metallothionein genes,iron-zinc regulatory protein ZIP and ATP binding cassette protein ABC family were up-regulated.Small molecular heat shock protein were also found in the regulation process of O.sinensis in response to Cu²⁺stress,but the expression of these genes did not change consistently.Conslusively,the results of this study showed that plants of O.sinensis subjected to Cu²⁺stress are not damaged obviously,indicating that this species are tolerant to Cu²⁺stress.Furthermore,the tolerance to Cu²⁺of plants from mining areas is stronger than that from non-mining area,in which both SOD and POD in antioxidant system play an important role,indicating that special genotypes of O.sinensis might exist to adapt to the soils in the mining areas.Transcriptome sequencing showed that ribosomal and oxidative phosphorylation pathways of O.sinensis have contributed to the tolerance to short-term Cu²⁺stress,but phenylpropanol biosynthesis and?-linolenic acid metabolism pathways might have contributed to long-term Cu²⁺stress.The gene expressions of antioxidant system related genes,flavonoids biosynthesis,fatty acid elongation and phenylpropanoid biosynthesis were up-regulated,suggesting that O.sinensis is tolerant to Cu²⁺stress to a certain extent.