| Drought is one of the most important environmental factors affecting crop yield and quality.Arid and semi-arid areas account for about 50% of the land area in our country.Moreover,in the wet areas alsohave been suffered seasonal and cyclical droughts.To solve the drought problem,cultivating new varieties with drought resistance and tolerance is one of the important means.These require us to understand the response and regulation mechanisms of crop under drought stress.Roots play an important role in absorption of water,nutrients and signal transmission of water stress,on other hand,root tissues in direct contact with dry soil are severely affected by drought.So it is important to understand how the root system responds to and deal with drought stress.Watermelon(Citrullus lanatus)is an important cucurbit crop planted widely throughout the world and watermelon cultivation area accounts for about 7% of the world's vegetable cultivation area.A large amount of water is required during watermelon life cycle,but frequent drought has seriously affected the quality,production of watermelon,even the entire watermelon industry.M08 is a cultivated inbred watermelon accession and exhibits relatively high resistance to water deficit.The root tissues were used to m RNA and mi RNA sequencing.The information underlies the osmotic stress response and adaptation mechanism and genetic improvement of drought resistance in watermelon.The activity of proline related synthesis enzymes and the expression pattern of relative genes were investigated.The long distance transport of proline was clarified,and the Cl P5 CS was transformed to Arabidopsis for characterizing function.The main results of the study are as follows:(1)The inbred watermelon accession “M08”was treated with 20% PEG6000.Root samples were harvested at 6 h after PEG treatment and untreated samples were used as controls.Transcriptome analyses were carried out by Illumina RNA sequencing.After removing adaptors and low quality sequences,a total of 32.99 Gb clean and high quality data were obtained,and over 40 million(M)pair-end reads of 125 bp in length were obtained from each library.In total,5246 differentially expressed genes(DEGs)were identified between PEG-treated and untreated watermelon root samples.Among these 5246 genes,2753 were up-regulated and 2493 were down-regulated under water deficit stress.A total of 5246 differentially expressed genes were identified.Gene ontology enrichment andbiochemical pathway analyses of these 5246 genes showed that osmotic stress affected osmotic adjustment,signal transduction,hormone responses,cell division,cell cycle and ribosome,and “M08” may repress root growth to adapt osmotic stress.A total of 825 DEGs were assigned to 108 different biochemical pathways.The top five pathways comprising the most DEGs were ribosome,plant hormone signal transduction,plant-pathogen interaction,purine metabolism and starch and sucrose metabolism.Of which,ribosome pathway was significantly changed.(2)Illumina Hiseq 2500 platform was used to sequence the mi RNA of PEG treated watermelon root samples.Bioinformatics and degradome were applied to predict the target genes of mi RNA.14,662,351?14,263,084?15,767,557?16,743,057?16,615,676?13,354,601 and 27,618,848 clean reads were got form S01?S02?S03?S04?S05 and S06 libraries,respectively.Clean data in every libraries is larger than 13.35 M and the Q30 >91%.1060 mi RNAs were predicted in six samples,and the length is mainly 24 nt.456 target genes were predicted for 174 mi RNAs among 1060 mi RNAs.While,241 targets genes were annotated among 456 target genes.There were 42 differently expressed mi RNA.Furthermore,20 DEGs were up regulated and 22 DEGs were down regulated.14 target genes were annotated among 75 target genes of differently expressed mi RNA.In degradome library,66 target gene degradation sites and 66 target genes were detected.28 target genes were obtained by the intersection of target genes predicted by mi RNA sequencing and degradome sequencing,and the 28 target genes involved in SPL,HD-Zip transfactors and signal transmission of plant hormone.(3)Under the PEG stress,the content of proline in stems and leaves increased gradually during the initial 12 h.In the roots,the proline content were gradually increased and then decreased at 3 h.The activity of proline related synthesis enzymes and the relative expression of the coding genes showed that the synthesis of proline was mainly via glu pathway.Among the two P5 CS genes,Cla006553 was predominant and Cla017928 was inhibited.The changes of P5 CS activity were similar to expression trend of Cla006553.OAT enzyme activity changed slightly,and relative expression was inhibited in the roots,stems and leaves.The watermlon aboveground and underground were cut off andtreated by drought stress.The results showed that leaves are the main place of proline synthesis,moreover,the proline synthesized by leaves could be transported to roots under PEG stress.The activity of Cla006553 promoter in leaves was the highest,and the activity was low in roots.While the promoter of Cla017928 had lower activity in roots,stems and leaves under PEG stress.These further showed that Cla006553 mainly encodes P5 CS enzyme and proline synthesis is in leaves under PEG stress.(4)The CDS of Cla006553 and Cla017928 were cloned.The CDS of Cla006553 was2166 bp and the ORF encoded a protein of 720 amino acid residues,while Cla017928 was2145 bp and encoded 714 amino acid residues.The nucleotide and amino identity of Cla006553 and Cla017928 was 74.19% and 77.12%,respectively.The protein of Cla006553 and Cla017928 contain the putative ATP-binding site,Leu rich regions,Glu-5-kinase domain,GSA-DH domain,NADPH-binding site and Pro feedback inhibition amino acid residue.Overexpression of Cla006553 and Cla017928 genes in transgenic Arabidopsis resulted in elevated proline content and enhanced ability of drought and salt tolerance. |
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