Study On The Drought Resistance Of GhDRPP1 Transgenic Cotton(Gossypium Hirsutum) And Its Physiological And Genetic Analyses

"Clothing,food,housing,and transportation' the four important things that relate to everyone's life,among which "clothing" is the first one,so its importance is evident.Clothes made of cotton fiber are hygroscopic and breathable;they are well received by many people.However,as an important economic crop,cotton is often subjected to various biological/biotic stresses such as diseases,drought,extreme weather,etc.,which severely affected the yield and fiber quality of cotton.Therefore,studying the molecular mechanism of cotton stress response in order to enhance the cotton resistance by means of transgenic technology is beneficial to cotton yield and fiber quality.In response to abiotic stresses,abscisic acid(ABA)acts as an important molecule in plants.In recent years,a relatively complete ABA signaling pathway has been studied in Arabidopsis,but the mechanism of ABA signaling in cotton has not been reported.Previously,a gene of PP2C family was isolated and identified from cotton,named GhDRPPl,which may plays an important role in the ABA signaling pathway and drought stress responses.Here we performed a phenotypic analysis and functional exploration of GhDRPP1 transgenic cotton progenies.1.Analysis of drought resistance and related physiological indexes of GhDRPPl overexpression(OE)and RNA interference(RNAi)transgenic cottonWe performed genetic and biochemical analyses on the GhDRPP1 overexpression and RNA interference transgenic cotton in T3 and T4 generations,in both normal and drought conditions.The results of phenotypes and physiological parameters showed that the overexpressed lines had a significantly drought-sensitive phenotype and the RNAi lines were more resistant to drought than the WT,and their phenotypes could be stably inherited.Consistent with thephenotypic observations,the levels of H2O2 and MDA in the overexpressed lines were significantly higher than that of the wild type under drought stress,reflecting the more serious damage caused by drought in OE lines;The low activity of enzymes such as SOD and POD in OE lines reflects their weak adaptability to drought stress.By contrast,H2O2 and MDA levels in the RNAi strain were lower than those in the wild type;the activities of SOD and POD were higher than the wild type.In order to fully understand the function of GhDRPPl,we compared the seed size and fiber length between GhDRPPl OE/RNAi transgenic cotton with wild-type cotton.The results showed that there are no significant difference between transgenic cotton and wild-type cotton.2.Analysis of the interaction between GhDRPPl and interacting proteinsIn our previous study,we used GhDRPP1 as bait protein to screen cDNA library of cotton leaves under drought stress conditions and screened some candidate interaction proteins.In order to search for proteins that may interact with GhDRPP1,the full-length cDNA of the five candidate genes Gh_D03G1306,Gh_A11G0835,Gh_A08G1057,Gh_A07G0741,and Gh_A12G2092 were isolated and cloned into the pGADT7 vector then used in a yeast two-hybrid assay with pGBKT7-GhDRPPl.The results showed that Gh_D03G1306 and Gh_A08G1057 could interact with GhDRPP1,but the other three proteins could not interact with GhDRPP1.GO clustering analysis results showed that Gh_D03G1306 encodes SNF1-related protein kinase regulatory subunit beta-3,which may be involved in sucrose response and cellular response to nitrogen levels,etc.Molecular function is related to protein binding;Gh_A08G1057 is a DNA methyltransferase,possibly participating in chromatin reorganization,DNA pairing,etc.3.Transcriptome Analysis and Validation of GhDRPP1 Transgenic Cotton Leaves under Drought ConditionTo investigate the molecular mechanisms of GhDRPPl involved in drought stress response,we performed a comparative RNA sequencing transcriptome analysis of the GhDRPP1OE,RNAi,and wild-type cotton after 5 days of drought.Compared with normal WT,OE,and RNAi lines,the differentially expressed genes of these three lines after drought treatment were:6805,6164,and 2417.It can be seen that under drought conditions,plants response with extensive regulation.Results showed that there were 11 differentially expressed genes in the GhDRPP1 OE transgenic line and 9 differentially expressed genes in the GhDRPP1 RNAi transgenic line compared to the wild type under normal conditions;Five days after drought,there were 1932 differentially expressed genes in GhDRPP1 OE transgenic cotton lines,of which 1072 were up-regulated and 860 were down-regulated.605 differentially expressed genes were found in GhDRPP1 RNAi transgenic lines,of which 389 were up-regulated and 216 were down-regulated.Under drought conditions,both GhDRPP1 OE and RNAi transgenic lines showed a large number of differentially expressed genes compared to the wild type,suggesting that GhDRPPl plays an important role in drought stress response.After annotating these differentially expressed genes,a co-expression analysis was performed with GhDRPP1,and qRT-PCR analysis was performed on some genes that were highly related to GhDRPP1,and some related proteins that might be regulated by GhDRPP1,such as the valine transport factor(PROT1),cysteine receptor-like kinase 25(RLK25),etc.Based on GO clustering analysis and KEGG analysis,it was speculated that GhDRPP1 mainly affects the abscisic acid and gibberellin signal transduction pathways,and inhibits a series of stress responses such as stomatal closure,then affects all hormonal signaling regulatory network,leading to GhDRPP1 OE transgenic cotton sensitive to drought.Compared with the wild type,the enhanced photosynthesis of RNAi strains may be one of the reasons for its enhanced drought resistance.A number of differentially expressed genes were selected for qRT-PCR analysis and the results showed that the above transcriptome data were basically true and reliable.Genes that may be regulated with ABA signaling pathway,marker genes that are under stress control,and genes that make up the photosynthetic system were expressed in heat maps in different strains.Based on the above results,we believe that GhDRPPl may play a negative regulatory role in response to drought stress in cotton,by participating in a variety of hormone signal transduction pathways such as abscisic acid and gibberellin signaling pathways,and affecting stomatal closure and photosynthesis.