Mechanisms Of Tea Seeds Sensitive To Dehydration Treatment And Genome-wide Identification And Expression Analyses Of The LEA Protein Gene Family

Tea seeds are the offsprings of tea plant generated by natural propagation and own completely genetic information.Acting as the storage material,tea seeds have several excellent characteristics such as the convenience of carrying,transportation,storage and so on.However,it is very difficult for seed storage and preservation for a long time due to its sensitiveness to the desiccation and low temperature.Presently,relative researches mainly focused on the cellular,physiological and biochemical levels,while scarce studies focused on the underlying molecular mechanisms.Therefore,the key genes associated with desiccation-sensitivity were explored using high throughout RNA-Seq technology with the materials of seed embryos of tea plant variety 'Echa 1' at three different stages of desiccation.Meanwhile,genome-wide identification and analysis of LEA genes family was performed,and CsLEA genes involved in the growth and development especially seed development,abiotic stresses including low and high temperature,drought and ABA,and desiccation of mature seeds were explored in this study.The main results were as following.1.The content,germination ratio and index of mature seeds from tea plant showed a gradual decline,and relative conductivity presented a gradual enhancement with the increase of desiccation time.Tea seeds lost completely the vitality at 18 h of desiccation.The results indicated that tea seeds were super sensitive to desiccation,which belonged to the classical recalcitrant seeds.2.In this study,100628270 clean reads were obtained by transcriptomic sequencing,and then 91925 Unigenes were generated by de novo assembling with an average length of 854 bp and an N50 length of 1480 bp,of which 58,472?63.6%?were extensively annotated.By the analysis of expression profiling,we found that 525 differentially expressed genes were first upregulated and then downregulated,482 were first downregulated and then upregulated,5875 were continuous upregulated and 8929 were continuous downregulated.In conclusion,the number of upregulated expression genes were obviously larger than that of downregulated expression.Go enrichment analysis indicated that differentially expressed genes involved in carbohydrate metabolism,amino acid metabolism,coenzymes metabolism and so on were remarkable accumulated.Pathway analysis showed that the pathway of arginine and proline metabolisms,fatty acid biosynthesis and carotenoid biosynthesis were significantly enriched.3.By deep analysis,we found a large number of genes,which have been reported to function in dehydration process,including the genes related to ABA biosynthesis and signal transduction,transcription factor,antioxidant enzyme,LEA protein,and proline metabolism were upregulated expression,which might be the reason why recalcitrant seeds lacked the tolerance to desiccation.At the same time,qRT-PCR analysis of 12 selected genes from ditlferentially expressed candidate genes were carried out in the study.The results were in good accordance with transcriptomic data with 92.2%similarity,indicating that transcriptomic data was high reliable.4.48 CsLEA genes were identified in tea genomes and transcriptome databases by the use of HMM model.They were grouped into 7 subgroups including LEA₁?2??LEA₂?32??LEA₃?4??LEA₄?2??LEA₅?1??DHN?4?and SMP?3?based on the conserved structural domain and phylogenetic evolution.Bioinformatic analysis indicated that the molecular weight of CsLEA proteins varied from 7.55 to 49,47 kDa andthe theoretical isoelectric point varied from 4.72 to 10.16,and most of CsLEA proteins were hydrophillic proteins,which were mainly targeted into the cytoplasm,the nucleus and the chloroplast etc.Conserved motif analysis showed that high conservation of the motifs was found in the same subgroup,however the significant difference was observed in different subgroups.5.qRT-PCR results showed that CsLEA39 were only expressed in the seeds,and the others?47 CsLEAs?were constitutive expressed in detected tissues including the roots,the stems,the leaves,the flowers and the seeds.Among 47 CsLEAs,24,14,3 and 2 CsLEAs represented the highest expressions in the roots,the seeds,the flowers,the stems and the leaves,respectively.Furthermore,23,44,33 and 28 CsLEAs responsed to the low temperature,high temperature,drought and ABA,respectively.Notably,the abundances of 11 CsLEAs were all changed in exposure to low temperature,high temperature,drought and ABA.6.Moreover,47 CsLEAs showed a changed expression during the seed growth and development,of which 39 were upregulated at the stage of maturation drying in the seeds.14 CsLEAs were found to be changed in response to desiccation,of which 8 were down-regulated with desiccation.7.Collectively,the results suggested that downregulation of 4 CsLEAs including CsLEA11,CsLEA32,CsLEA36 and CsLEA45 might be attributed to desiccation sensitivity of recalcitrant seeds in tea plant.