Analysis Of Differentially Expressed Genes Of Haloxylon Ammodendron Adapting To Simulated Drought And Functional Identification Of HaASR

Desertification accounting for 27.2%of land in China with 2.612 million km²,drought and desertification pose adverse impact on plant growth and development.In the process of long-term evolution,plants have evolved several different types of stress resistance mechanism to adapt to specific habitats.Haloxylon ammodendron,a chenopodiaceae family shrub,possesing excellent tolerance to environmental factors,naturally distributed in desert region and is called the“desert guard”.While,the physiological and molecular mechanism underlying how H.ammodendron adapts to drought environment is still unclear.Investigating stress resistance mechanism and exploreing the stress tolerance genes from dominant species H.ammodendron are of great significance for genetic improvement of herbage and crop plants and ecological restoration.Therefore,transcriptome?RNA-seq?analysis were performed on H.ammodendron seedlings under simulated drought treatment?-0.75 MPa?to identify stress response pathways and drought-candidate genes.Moreover,ASR genes with higher expression upon simulated drought stress was cloned,and the function of ASR in Arabidopsis thaliana responding to abiotic stresses was analyzed.The main results are as follows:1.The final assembly which consisted of 87,109 unigenes with mean length of 680 bp were obtained through RNA-seq,and 13,486 SSRs were identified.3,353 DEGs were observed totally in shoot,and there were 811 and 1,220 genes up-regulated in 6 and 24 h,respectively;For root,4,564DEGs were observed totally,and there were 508 and 1,219 genes up-regulated in 6 and 24 h,respectively.Up-regulated genes were mainly involved in ethylene,jasmonic acid,salicylic acid stimulus response,flavonoid biosynthesis,oxidative and osmotic stress response pathway.2.In shoot,most of genes related to PPO,anthocyanin synthesis and GPX pathway were up-regulated,indicating PPO,anthocyanin and GPX pathway play vital roles in shoot of H.ammodendron adapting to oxidative stress.In root,most of genes related to SOD,AsA,anthocyanin synthesis and GPX pathway were up-regulated,indicating root of H.ammodendron posses strong antioxidant capacity to alleviate ROS damage at the earlier stage of drought stress.3.In shoot,most of genes related to ABA,ethylene and jasmonic acid were up-regulated,accumulating higher levels of ABA,ethylene and jasmonic acid.In root,most of genes encoding ACO and LOX,and genes encoding ACS and AOS were up-regulated,accumulatin higher levels of ethylene and jasmonic acid.20 up-regulated genes and 22 down-regulated genes related to photosynthesis for 6 h were identified.Most genes associated with chlorophyll synthesis,cytochrome B6/F complex and photosystem II were up-regulated,while genes associated with chlorophyll degradation were down-regulated,indicating H.ammodendron maintain photosynthetic efficiency by keeping chlorophyll stability and photosynthetic electron transfer at the earlier stage of drought stress.4.In shoot,almost all of genes encoding LEA,ASR,TPS,BADH,PEAMT and MDH were up-regulated,indicating shoot of H.ammodendron accumulate LEA,ASR,trehalose,betaine and mannitol to respond to osmotic stress.In root,most genes encoding LEA,ASR,P5CS and TPS were up-regulated,indicating LEA,P5CS,ASR and TPS play vital roles in shoot of H.ammodendron adapting to osmotic stress.5.HaASR1 with full-length of 382 bp,ORF of 333 bp,and HaASR2 with full-length of 479 bp,ORF of 324 bp were obtained,respectively.HaASR1 and HaASR2 possessing high hydrophilicity have an ABA/WDS domain.HaASR1 and HaASR2 were expressed most strongly in root,and significantly induced by salt and drought stress.HaASR1 and HaASR2 protein were localized in whole cell of tobacco subepidermis.A positive interaction of HaASR1 with HaPrxQ and HaBADH,and HaASR2 with HaNHX1 protein were observed throughyeast system and BiFC assay.Then the overexpression vectors of HaASR1 and HaASR2 were constructed and transformed into Arabidopsis,respectively.6.The expression levels of AtALDH10A9 and AtPrxQ in HaASR1over-expressed plant were significantly higher than WT.Upon ABA treatment,over-expressing HaASR1 promoted the germination and seedling growth,reducing ABA sensitivity.Upon NaCl treatment,over-expressing HaASR1 increased leaf proline and betaine accumulation and reduced osmotic potential,decreased plasma permeability,maintained water and chlorophyll content,photosynthetic capacity and plant growth,enhancing salt tolerance.Upon drought treatment,over-expressing HaASR1 reduced water loss rates,enhanced water-retaining ability,maintaining higher water content;reduced expression level of AtABA1 and AtAAO3,reducing the accumulation of ABA and delaying the response to drought;increased the transcript levels of AtPrxQ,AtCAT2,and AtAPX1 and reduced the accumulation of H₂O₂,protecting plasmalemma and chloroplast from oxidative damage,which maintaining photosynthetic capacity and the plant growth.7.The expression level of AtNHX2 in HaASR2 over-expressed plant was significantly higher than WT.Upon ABA treatment,over-expressing HaASR2 promoted the germination and seedling growth,reducing ABA sensitivity.Upon NaCl treatment,over-expressing HaASR2 increased proline accumulation,and reduced osmotic potential,maintaining water content;decreased plasma permeability;maintained chlorophyll content,photosynthetic capacity and the plant growth,enhancing salt tolerance.Upon drought treatment,over-expressing HaASR2 reduced water loss rates,and increased betaine and proline accumulation,maintaining water content;reduced expression level of AtABA1,reducing the accumulation of ABA and delaying the response to drought;increased the expression levels of AtCAT2 and AtAPX1 and reduced the accumulation of H₂O₂ and plasma permeability,which enhancing antioxidant capacity and maintaining photosynthetic capacity and the plant growth.The above results revealed that antioxidant?PPO,anthocyanin,GPX,SOD and AsA?,ethylene,jasmonic acid,ABA and organic osmolyte?LEA,ASR,trehalose,betaine,proline and mannitol?play vital roles in H.ammodendron responing to drought stress;HaASR1 and HaASR2 play an essential role in plants adapting to salt and drought stress.This research laid a certain foundation for invistigation of molecular mechanism of H.ammodendron adapting to adverse environment and identification of key functional genes related to drought and salt tolerance,and provide gene resources for genetic improvement of fine forage and crops.