Comprehensive Evaluation Of Drought Resistance And Transcriptome Analysis Under Drought Stress In Camellia Oleifera

As a kind of drought resistance plant,the serious water shortage in hilly lands limit Camellia oleifera growth.Drought is regarded as the major threat to this plant.In this study,morphologic changes and physiological responses of the seedlings of four C.oleifera cultivars ‘Cenruan No.2',‘Cenruan No.3',‘Guiwu No.1' and ‘Guiwu No.4' were studied.Then,8 libraries of ‘Cenruan No.3'(drought-resistant)and ‘Guiwu No.4'(drought-sensitive)were sequenced using the Illumina Hiseq 2500 platform.With the help of the new generation of sequencing technology,we can understand the molecular mechanisms of drought tolerance at the transcriptome level.The results show that:(1)Morphological indexes,physiological changes,photosynthetic physiological indexes and fluorescence parameters of four C.oleifera cultivars were observed.The experiment set 10% PEG,20% PEG and 30% PEG stress concentration,and drought stress duration 0 h,12 h,24 h,36 h and normal nutrient solution 12 h stress time.Finally,with the help of subordinate function values analysis,a comprehensive evaluation on drought resistance of four different C.oleifera cultivars were successfully gained.The results indicated that the ‘Cenruan No.2' and ‘Cenruan No.3' are drought-resistant cultivars,nevertheless,‘Guiwu No.1' and ‘Guiwu No.4' are drought-sensitive cultivars.The order of drought resistance of four C.oleifera cultivars was ‘Cenruan No.3' > ‘Cenruan No.2' > ‘Guiwu No.1' > ‘Guiwu No.4'.(2)Using the Illumina Hiseq 2500 platform,8 libraries of ‘Cenruan No.3' 0 h,12 h,24 h,36 h and ‘Guiwu No.4' 0 h,12 h,24 h,36 h were sequenced.In total,we obtained 76,585 Unigenes,among them,52,531 Unigenes were functionally annotated.46,171 Unigenes were enriched belong to 338 KEGG pathways.Based on the functional annotation,a total of 46,440 coding sequences(CDSs)were extracted from Unigene sequences and translated into peptide sequences.Finally,the expression levels of the 20 Unigenes tested of q RT-PCR were consistent with the results of RNA-seq.(3)A large amount of differential expressed genes(DEGs)were received from RNA-seq,some key genes related to drought torlerance were contained.Based on the metabolic pathways enrichment analysis,we identified 8,836 DEGs which were enriched in 39 metabolic pathways,including the drought torlerance pathways such as map00500(Starch and sucrose metabolism),map00941(Flavonoid biosynthesis)and map00750(Vitamin B6 metabolism).A total of 1,209 Unigenes were identified to be involved in transcription,which belong to 63 transcription factors families,such as C2H2 family,b HLH family,C3 H family,WRKY family,AP2/ERF family,b ZIP family.Most of the transcription factors were proven to increase the drought resistance of plants.In total,2,299 protein kinases were successfully annotated,including functional enzymes which can improve the drought resistance of plants.(4)The expression level of POD,CAT and SOD activity related genes were obtained from the 8 libraries,we found that POD activity related genes may play a major role in the drought resistance of C.oleifera.Drought stress causes damage in ‘Cenruan No.4',lots of LOX related genes were expressed after 24-hour drought stress.The Unigenes of PP2 C,Sn RK2 and NCED were obtained in the research which are important to ABA signal transduction.The PYCR2 and P5 CS related genes were obtained in the 8 libraries,they were the key genes about proline biosynthesis.Betaine-aldehyde dehydrogenase related genes(BADH)and sucrose synthase related genes(SUS)were recorded,all of these genes play an important role in the osmotic adjustment of C.oleifera.In our study,28 Unigenes were involved in the biosynthesis of sesquiterpenoid and triterpenoid,including mixed amyrin synthase genes and squalene epoxidase genes.60 Unigenes were verified to flavonoid biosynthesis.The differential expressed genes of metabolic systems of vitamin B6 in two C.oleifera cultivars may enhance the drought resistance of ‘Cenruan No.3'.(5)Antioxidant enzyme related genes and osmotic substance related genes were validated in ‘Cenruan No.3' under drought stress.The expression level of CL4548Contig1(POD1),CL216Contig1(POD12),contig?11343(CAT3)and contig?18823(CAT)showed a continuous upward trend with stress time,the same trend of the activity of POD and CAT.But the expression level of contig?91532(Cu Zn SOD)and contig?73481(Mn SOD)which related to SOD activity continued decline.The expression level of contig?29559(PYCR2)and CL10313Contig1(P5CR),CL65Contig1(SUS2),contig?2255(SUS2)with the same trend of the content of prolina and soluable sugar.The expression level of CL4202Contig1(BADH)increased with stress time.All these genes might be the key genes of C.oleifera to resistance drought stress.In this research,drought resistance of four main C.oleifera cultivars in south China were comprehensive evaluated.Large quantities of drought tolerant genes were annotated at the transcriptome level for the first time.These works would supply the new breeding resources and favorable drought tolerant genes for the breeding of resistant C.oleifera.