Expression Pattern Of Three Drought-Induced Genes And Functional Analysis Of MaCDSP32 In Mulberry(Morus Alba)

Mulberry(Morus alba)is highly adaptable to environment and widely distributed all over the world,since they have developed roots,luxuriant foliage and vigorous vitality.In China,mulberry is selected as an ecological protection forest tree species to effectively prevent sand and soil,reduce surface runoff and maintain the original ecological soil structure.On the other hand,mulberry,due to its rapid growth and large abovementioned biomass,is used in a variety of natural ecological restoration systems,such as the remediation of soils contaminated by heavy metals,stony barren deserts and desertification areas.The indomitable survival ability of mulberry is due to its unique physiological characteristics,which has attracted much attention in the field of plant genetics in recent years,involving in the research on the exploration,development and utilization of the resistance gene resources in mulberry.In this study,three drought differentially expressed genes were cloned from mulberry leaves:(chloroplast drought-induced stress protein of 32 k Da,Ma CDSP32,IAA-amino acid hydrolase ILR1-like 5,Ma ILR1 and ascorbate peroxidase,Ma APX2)in mulberry.We have used bioinformatics analysis,expression characteristics analysis,transgenic plants stress tolerance analysis and transcriptome data analysis,mainly to explore the molecular functions of Ma CDSP32 in response to drought stress,and to pave the way for improving the environmental adaptability of plants by biotechnology engineering in the future and taking advantage of mulberry germplasm resources in actual production cornerstone.The main results of this study are as follows: 1.Gene cloning and subcellular localization.The encoding sequences length of Ma CDSP32,Ma ILR1 and Ma APX2 genes from mulberry were 909,1302 and 750 bp,encoding 303,434 and 250 amino acids,respectively.All the three proteins are predicted hydrophilic and stable.The protein encoded by Ma CDSP32 was located in the chloroplast stroma.Ma APX2 encodes a protease that is located in the cytoplasm,and mainly enriched near the membrane and nucleus;Ma ILR1 encodes a membrane protein,it's a secreted protein and is located on the cell membrane.2.The genes expression pattern under different conditions in mulberry.The Ma CDSP32 was mainly expressed in mature leaves of mulberry,and showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma CDSP32 reached its peak expression at 15:00 within one day,presenting a circadian rhythm expression pattern.The Ma CDSP32 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.The Ma ILR1 was mainly expressed in young leaves;it showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma ILR1 expression had no obvious circadian rhythm.The Ma ILR1 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.The Ma APX2 expression level was consistent in the aboveground tissues;it showed expression preference among different mulberry cultivars while has nothing to do with the plant ploidy.Ma APX2 reached its peak expression at 12:00 within one day,presenting a circadian rhythm expression pattern.The Ma ILR1 expression level changed with abiotic stress and exogenous hormones treatment,but they were different in leaves and roots.3.Study on gene function of Ma CDSP32 transiently transforming in mulberry.The Ma CDSP32 transiently overexpressed mulberry leaves showed increased water loss compared with the wild-type mulberry leaves during the natural desiccation.Besides,the gene expression of Ma MRSB,Ma P5 CS,Ma PRX,Makds A,Ma DREB and Ma MAPK,and also the accumulation of ROS were affected by overexpressed Ma CDSP32 under PEG treatment.What's more,the gene expression of Ma WRKY,Makds A,Ma DREB and Ma MAPK were affected by overexpressed Ma CDSP32 under Na Cl treatment.These results suggested that Ma CDSP32 could affect the expression level of stress-related genes and participate in plant abiotic stress response.4.Study on Ma CDSP32 gene function in transgenic tobacco.Compared with the wild-type tobacco,Ma CDSP32 overexpressed lines(OE-2 and OE-7)showed increased water loss of detached leaves during the natural desiccation process.Transgenic tobacco showed increased sensitivity to drought,the ROS accumulation and MDA content in leaves increased,the activity of various antioxidant enzymes and the contents of proline and soluble sugar decreased compared with the wild-type.However,the recovery ability of transgenic tobacco enhanced after post-drought rewater,the ROS accumulation and MDA content in leaves decreased,the activity of various antioxidant enzymes and the contents of proline and soluble sugar increased compared the wild-type.Moreover,expression levels of Nt CAT,Nt ADC2,Nt LEA5 and Nt MSRB in transgenic lines were also affected by Ma CDSP32 overexpressing under drought stress.Chlorophyll content increased in transgenic tobacco while decreased in wild-type under salt stress.Additional,Ma CDSP32 significantly increased the seed germination rate and seedling growth of transgenic tobacco under osmotic stress by increasing Nt MSRB expression and reducing ROS accumulation.These results suggested that Ma CDSP32 regulates plant stress tolerance mainly by participating in the redox pathway.5.Study on Ma CDSP32 gene function in transgenic Arabidopsis.Compared with the wild-type Arabidopsis plants and the At CDSP32 loss function mutant lines(mt-1 and mt-2),Ma CDSP32-overexpressing Arabidopsis lines(OE-3 and OE-9)showed increased severe wilting phenotype,increased ROS accumulation and MDA content,lower activities of SOD,POD and APX enzymes during drought process.However,the survival rate of OE Arabidopsis plants was the highest compared to that of wild-type and mutant lines after rewatering.The gene expressions of At SOD,At POD,At CAT,At PRX,At APX2,and At GR were affected by overexpressing of Ma CDSP32 or loss function of At CDSP32 under drought and salt treatments.The chlorophyll content of At CDSP32 mutant lines was reduced compared to the wild-type and OE lines under salt stress.The germination rate of OE Arabidopsis seeds significantly increased than the other two genotypes under osmotic stress.In addition,OE Arabidopsis lines showed early flowering and the gene expression of At SOC1 was significantly higher than the other two genotypes.These results indicated that Ma CDSP32 affects the plant's stress physiology and seed germination rate mainly through the redox pathway involved in the stress response,and Ma CDSP32 participated in plant flowering time regulation by affecting At SOC1 expression.6.Transcriptome analysis in transgenic tobacco of Ma CDSP32 under drought stress.The transcriptome data analysis of transgenic tobacco showed that the drought-differentially expressed genes of OE and wild-type lines were mainly in the physiological processes such as catalytic activity,nucleic acid binding transcription,transcription factor activity,photosynthetic membrane module,carbohydrate metabolism process and peptide chain enzyme regulatory factor,et al.;after rewatering,the differentially expressed genes of OE and wild-type lines were mainly in the physiological processes such as polymer biosynthesis,nitrogen biosynthesis,catalytic activity and hydrolase activityand,et al..Further analysis revealed that these differentially expressed genes were most up-regulated in the pathways of phenylpropanoid biosynthesis,photosynthesis-tentin,keratin,biosynthesis of cork resin and wax,carbon fixation of ribosomes and photosynthetic systems,while were most down-regulated in endoplasmic reticulum protein processing,galactose metabolism,sesquiterpene and triterpene biosynthesis,and fatty acid extension.In addition,the differentially expressed genes of OE lines involving in cysteine and methionine metabolism pathways,starch and sugar metabolism pathways,brassinosteroid metabolism pathways,plant hormone signaling metabolism pathways,and photosynthetic element carbon fixation,as well as antioxidant enzyme activity,proline anabolism,and sugar anabolism were down-regulated during drought stress and up-regulated after rewatering.These results suggested that these differentially expressed genes are likely the key sites for the interaction with Ma CDSP32 in modulating drought tolerance under drought stage and after rewatering.Taken together,the evidences show that the drought sustained expression gene Ma CDSP32 in mulberry enhances plant drought tolerance by participating in the ROS metabolic regulation proces,revealing the molecular mechanism that Ma CDSP32 involved in to improve plant post-drought recovery ability.This research screened genetic resources for the development and utilization of mulberry germplasm resources,and provided new ideas for improving plant environmental adaptability and viability with molecular biotechnology.