Analyses Of Transcriptome Of Cotton (Gossypium Hirsutum) In Response To Drought Stress And The Role Of DRPP1 Gene In Cotton Drought Resistance

Drought is one of the most important abiotic stresses that affect the survival,growth and distribution of plants.The reduction of crop production due to lack of water accounts for more than half of the crop yield reduction caused by all environmental stress factors.Cotton is one of the most important economic crops in the world,but drought and little rain occurred quite frequently in Xinjiang and the Yangtze River Basin especially in Hubei,Hunan.Jiangxi,Jiangsu,Anhui and other provinces during summer and autumn,causing the deficiency of water supply,wilting of cotton leaves,retardation of cotton growth and development and increased shedding of cotton bolls,which seriously influenced the yield and quality of cotton.Therefore,it is extremely important to create the cotton with resistance to stabilize the development of cotton industry.The reversible protein phosphorylation plays a very important role in various signal transduction pathways and regulates majority of biological processes in eukaryotic cells.Protein kinases?PK?and phosphatases?PPs?are essential for maintaining the phosphorylation state of cells under normal and unfavorable conditions.It has been found that protein phosphatases play important roles in regulating cellular metabolism and 2C-type protein phosphatases play important roles in cell cycles,stress signal transduction,transcription,translation and post translation modification.In plants,in addition to class A PP2Cs,the other categories of PP2Cs also play important roles in plant development and stress resistance.As a kind of protein phosphatases family of plants,PP2Cs have special structure characteristics and physicochemical properties,and they are involved in a variety of signaling pathways.The diversity of 2C-type protein phosphatases family in plants suggests that they have diverse signal transduction mechanisms in different organs and tissues.However,it is a long-term and arduous task to understand the functions and the mechanisms of PP2C clearly.Furthermore,the research of the direct substrate of PP2Cs in plants resistant stress is not clear.And the functions of PP2Cs in cotton were reported very little,so it has important theoretical significance and application value to study the function of PP2C protein in cotton.In this paper,we made a comparative transcriptional analysis of the leaves of two different drought resistant cotton varieties under drought stress by RNA-seq technology,and we found that many genes were differentially expressed under drought stress.Among these up-regulated genes,a gene encoding a PP2C protein was identified and was named GhDRPPl?Drought Related PP2C1?.The function and mechanism of GhDRPP1 in cotton drought response process were studied by the method of excess expression and gene silencing.The main results are as follows:1.Comparative transcriptional analysis of drought resistant variety Jinmian 13 and drought sensitive variety Lumian 6 under drought stress.We made a comparative transcriptional analysis of the leaves of two different drought resistant cotton varieties?drought-resistant cultivar Jinmian13 and drought sensitive cultivar Lumian 6?under drought stress by RNA-seq technology.The statistical results showed that the percentage of differentially expressed genes was between 13.38%and 18.75%.Compared with the control sample,the number of differentially expressed genes of Jinmian13 was gradually increased with the drought stress time increasing.The result of functional clustering of differentially expressed genes showed that a large part of differentially expressed genes were involved in the biological pathways of metabolism,transportation,defense response,protein modification,hormone signal response and so on,which indicated that the drought stress may affect cotton normal growth and metabolic processes.We analyzed the gene expression profiling in Jinmian13 samples and found that there were 229 continuously downregulated genes and 627 continuously up-regulated genes,which implied that a lot of genes were expressed temporally and specifically in plant to respond to drought treatment.The genes related to cell wall synthesis,response to abiotic stress and abscisic acid stumulus,synthesis and metabolism of carbohydrates and so on were continuously up-regulated,while the genes related to ethylene signaling pathway were continuously down-regulated.These results suggested that Jinmian 13 may resistant to drought by activating the expression of genes related to ABA signaling pathway and other metabolic pathways.Furthermore,there were about ten thousands genes differentially expressed beween Jinmian 13 and Lumian 6 after 6 days drought treatment.Compared with Lumian 6,the genes involved in response to abiotic stimulus,defense response to bacterium,response to abscisic acid stimulus and gluconeogenesis were up-regulated after drought treatment for 6 days.However,the genes involved in ethylene response,jasmonic acid biosynthetic and metabolic process were down-regulated in Jimian13.These results implied that these signaling pathways may be involved in response to drought stress and tolerance of drought stress in cotton.2.Characterization and expression analysis of GhDRPPl gene.Through analyzing the data of RNA-seq mentioned above,a gene of PP2C family was isolated and identified from cotton tissues,and it was named GhDRPPl?The gene number is CotAD₁8479?.The transcript of GhDRPPl could be detected in every cotton tissue,and the expression of this gene in cotton leaves and roots was strongly induced by ABA,mannitol and NaCl.During the period of drought treatment,the expression of GhDRPP1 was significantly up-regulated in cotton leaves of Jinmian13 and Lumian6,and the expression level of GhDRPPl gene in Jinmian13 was higher than that of Lumian6 after 3 days and 5 days drought treatment.These results indicated that GhDRPP1 was induced by drought stress and that GhDRPP1 may be involved in the response process of drought stress in cotton.3.GhDRPPl encodes a PP2C protein phosphatase.The results of GhDRPPl protein alignments and phylogenetic analysis revealed that this protein may be a PP2C protein phosphatase,and the subcellular localization result showed that this protein was localized in the nucleus.The fusion expression vector of MBP-GhDRPPl was constructed,and the activity of GhDRPPl protein phosphatase was detected in vitro.This result showed that the protein had the activity of PP2C protein phosphatase.4.The Overexpression of GhDRPPl resulted in Arabidopsis thaliana sensitivity to drought.The GhDRPP1 excessive expression vector was constructed and transformed into Arabidopsis thaliana,and transgenic plants were obtained.The seed germination rate and green seedling rate of transgenic Arabidopsis under ABA and mannitol treatment were analyzed and the results showed that overexpression of GhDRPPl resulted in Arabidopsis thaliana insensitivity to ABA and sensitivity to mannitol.After 20 days drought treatment,transgenic Arabidopsis became welting,whereas the wild type grew well.After 23 days drought treatment,the plants were recovered with water,and the survival rate of the plants was statistically analyzed after recovered water for 2 days.The results showed that the survival rate of the transgenic plants was significantly lower than that of the wild type.These results suggested that the overexpression of GhDRPPl enhanced the sensitivity of Arabidopsis thaliana to drought.The expression levels of ABA and stress response related genes in GhDRPP1 transgenic plants under ABA and mannitol treatment were analyzed.The expression levels of RD22,COR15,NHX1 and CBF3 genes in transgenic plants were significantly lower than those in wild type after ABA and mannitol treatments.These results suggested that GhDRPP1 overexpression transgenic Arabidopsis sensitivity to drought may be caused by the down regulation of ABA and stress response related genes expression in plants.5.GhDRPPl plays an important role in the process of cotton response to drought.The overexpression and gene silencing?RNA interference?vectors of GhDRPPl were constructed and transformed into cotton respectively.The phenotypes of T1 and T2 transgenic cotton were analyzed respectively after harvest,and the results showed that GhDRPP1 overexpression transgenic lines were more sensitive to drought than wild type?WT?,while GhDRPPIRNAi transgenic lines were more tolerant to drought than WT.The results of physiological parameters showed that the levels of malondialdehyde?MDA?and H2O2 in GhDRPPl overexpression transgenic plants were higher than those in WT and the levels of malondialdehyde?MDA?and H2O2 in GhDRPP1 RNAi transgenic plants were lower than those in WT,which suggested that the extent of cellular damage of GhDRPP1 overexpression ransgenic plants was more serious.In contrast,the activities of protective enzymes whose functions were clearance of ROS?e.g.SOD,POD,etc.?in GhDRPP1RNAi transgenic plants were higher than those in WT,while the activities of protective enzyme in GhDRPPl transgenic overexpression plants were lower than those in WT,and similar results were obtained for the determination of proline content,which implied that the improvement of drought tolerance of GhDRPPIRNAi transgenic plants may be due to the enhancement of these ROS scavenging enzymes activities and the amount of proline.In addition,the expression levels of stress response genes?e.g.,GXH-PX8,GST8,RD22,KIN10,etc.?in GhDRPP1RNAi transgenic plants were higher than those in WT and lower than those in GhDRPP1 overexpression transgenic plants.These results indicated that GhDRPP1RNAi transgenic plants could resist drought stress by enhancing the expression of genes related to stress response.6.Screening and interaction analysis of GhDRPPl interacting proteins.In order to study the mechanism and molecular function of GhDRPPl in drought resistance of cotton,the yeast two hybrid cDNA library of cotton leaves under drought stress was constructed,andthe GhDRPP1 protein was used as a bait protein to screen the library,trying to find the interaction proteins of GhDRPPl.Among the 23 candidate proteins screened from the library,there were two interaction proteins interested us,and their functions together with GlDRPP1 in the drought resistance were to be further studied.In addition,yeast two hybrid experiments showed that interactions between GhDRPPl and GhPYL proteins were selectively.