| Magnoliaceae are valuable landscape trees mainly distributed in the southeast and southwest of China and gradually reduced to the northeast and northwest of China.It has the characteristics of cold resistant species are not evergreen and the evergreen species are not resistant to cold.Freezing injury is the main limiting factor for Magnoliaceae plants to be introduced to the northern high latitude area.It was gradually limited the application of Magnoliaceae in the northern cities of China.Therefore,screening of cold resistant evergreen Magnoliaceae plants and study physiological and molecular mechanisms under low temperature will provide excellent reserve gene and theoretical basis for further systematic research on cold-related gene and molecular breeding of Magnoliaceae.It will has a great significance to introduce evergreen Magnoliaceae plants to higher latitudes and to improve the landscape in winter in the northern of China.By means of data analysis,specimen identification,field investigation and resource collection the resources and distribution patterns of Magnoliaceae in China were fully understood,and the resources and geographical distribution of Magnoliaceae in Hunan province were further studied.In the main cultivation areas of Magnoliaceae in China,cold resistant evergreen Magnoliaceae species of grafted scion and seedlings were collected from the northern margin of the geographical distribution and the upper limit of altitude to establish the Germplasm Repository of Magnoliaceae.The leaf anatomical structure of 6 varieties of Magnoliaceae was observed by paraffin section and fluorescence microscopy to clarify the relationship between leaf structure and cold resistance.The physiochemical indexes were measured of each varietie and the cold resistence evaluated by means of average membership function method.In order to further analyze the cold resistant mechanism of Magnoliaceae plants,the 2 years old cutting seedlings of Parakmeria Lotungensis were selected as materials.Physiological indexes and anatomical structure of leaves in different time at 0? low temperature were observed while transcriptome sequencing was performed on Oh,2h,6h and 24h under low temperature stress of 0?.RNA sequencing quality were evaluated and DEGs were functionally annotated and bioinformatics analyzed.Key transcription factors and signal responsive genes of p.lotungensis under low temperature stress were screened out and identified and analyzed by qRT-PCR technology,and the gene expression regulation and mechanism of response to low temperature stress were explored.The main results are as follows:1.The results showed that Hunan is one of the modern distribution center of Magnliaceae plants in our country.There are 6 genera and 43 species,including varieties,of the family Magnoliaceae in Hunan Provinces.These plants are distributed in 47 counties and the distribution centers are in the south,southwest and northwest of Hunan province.Their vertical distribution center occurs between 500?1500m above sea level,and its horizontal center limits about 28°?26°N and 108°47'?111°E.The similarity coefficient of Magnoliaceae species in Hunan compared with Guangdong and Guangxi are 58.46%and 55.42%,respectively,followed by Guizhou,Sichuan and Jiangxi and the lowest with Yunnan.2.Two hundred and two germplasm resources of Magnoliaceae plants were collected,and the germplasm bank of Magnoliaceae was established.Six kinds of high cold resistance evergreen Magnoliaceae varieties of Parakmeria lotungensis,Michelia.martinii'Tinny',M.platypetala,M.wilsonii,M.martinii'Tiny'?× M.doltsopa? and M.maudiae var.Rubicunda were selected as the further research materials.3.From leaf paraffin section and fluorescence imaging microscopic observation of six varieties of evergreen Magnoliaceae,we found that the leaf had 1-3 layers of palisade tissue cells and the differences among the indexes of nine anatomical structures were extremely significant.The thickness of palisade tissue,the ratio between palisade tissue and spongy tissue,and the thickness of the vein were the key factors affecting cold resistance.4.Analysis on physiochemical indexes of six varieties of Magnoliaceae showed that the LT50 ranged from-22.06 ? to-10.64?.During low temperature,the indexes of soluble protein(SP),proline(Pro),superoxide(SOD)and peroxidase(POD)showed the trend of increasing first and then decreasing,while those of soluble sugar(SS)and malonaldehyde(MDA)kept accumulating.Relative electrical conductivity(REC),MDA,SP,SS and Pro can be used as the key indicators for the evaluation of the cold resistance of the 6 Magnoliaceae species.Cold resistance capabilities from strong to weak were P.lotungensis>M.martinii'Tiny'>M.platypetala>M.martinii 'Tiny'? xM.doltsopa?>M.wilsonii>M.maudiaevar.Rubicunda5.The cDNA libraries of P.lotungensis on Oh,2h,6h and 24h under 0? were constructed.Approximately 238.067 x106 high quality clean reads were generated,these clean reads were trimmed and assembled into 273,252 nonredundant putative transcripts with a mean length of 590.52 bp and E90N50 length of 1144 bp respectively.The error rate of base<1%and total BUSCO was 100%,transcriptome sequencing data were reliable,reproducible and high integrity..Of the unigenes,52,599(26.57%)had the best BLAST matches in four databases.A total of 70624(25.85%),25189(9.22%),49622(18.16%),9079(3.32%),5819(2.13%),6139(2.25%)and 69070(25.28%)unigenes had the most significant BLAST matches with known proteins in the Uniprot,Pfam,GO,KEGG,Pathway,eggNOG,and Nr databases respectively.6.According to the threshold of qvalue<0.05 and |log2FoldChange|>1,the differential expressed gene(DEGs)were screened from Unigenes produced by transcriptome analysis of P.lotungensis.We found that 10753,18364 and 19231 DEGs in sOh vs s2h,sOh vs s6h and sOh vs s24h respectively.Among them,5174,9222 and 8956 genes were up-regulated,and 5581,9144 and 10277 genes were down-regulated.3373 and 2146 up-regulated DEGs and 1843 and 3433 down regulaed DEGs were found in s2h vs s6h and s2h vs s24.1280 up-regulated DEGs and 1958 down-regulated DEGs were found in s6h vs s24h respectively.Based on the functional annotation through Basic Local Alignment Search Tool(BLAST)with public protein database,there were 3544,2369 and 2173 entries of Unigenes from Leucine rich repeat family(LRR),ATPase family and protein kinase family annotated in Pfam domain respectively.GO annotation showed that 47,705,34,675 and 67,917 entries of Unigenes involved in biosynthetic process,cellular component and molecular function and these Unigenes were significantly enriched in photosynthesis,response to stress,response to oxidative stress,glutamate metabolic process,hydrolase activity,lipid binding,protein kinase,oxidoreductase activity,transferase activity and cell wall organization.Most of the annotations in KEGG pathway were metabolic pathways,with 6299 entries(41.85%)of Unigenes.Among them,1401 entries of Unigenes were related to carbohydrate metabolism and significantly enriched in starch and sucrose metabolism pathway.777 entries of Unigenes were related to amino acid metabolism and significantly enriched in arginine and proline metabolism,isoquinoline alkaloid biosynthe,alanine,aspartate and glutamate metabolism pathways.681 entries of Unigenes were related to lipid metabolism and significantly enriched in fatty acid elongatio pathway.1337 entries of Unigene were involved in the process of environmental information processing,the most involved part was signal transduction pathway with 1241 entries of Unigenes,these Unigenes were significantly enriched in MAPK signaling pathway and Phospholipase D signaling pathway.7.Based on physiochemical and transcriptomic data,the key protein kinases,transcription factors(TFs)and responsive genes related to cold response of P.lotungensis were preliminarily excavated and the functions and regulation mechanism in response to low temperature stress were clarified.The cold signal is first perceived through cold-induced membrane rigidification(LRR_TPK,FAD3,LEA5,FAO)and transduced further via Ca2+,MAPK,PLD,cMAP and ABA signaling transduction pathway and protein kinases(MAPK,PPP3R,ATPase,CDPK28)resulting in the activation of downstream transcription factors(WRKY33,Myb4,APL,bZIP,NAC).The action of the TFs,trigger a cascade of downstream cold related genes,including ROS scavenging enzymes(Cu ZnSODCAT,POD,LOX2S,GR),starch synthesis and glycometabolism related genes(Beta-amylase,GTs,GPA1,RCA),proline metabolism related genes(PRODH,HRGP)and other secondary metabolism related genes(CytP450,SHSP,ACBP,LTP2),whose activations further modulates cellular metabolic homeostasis and improved tolerance to cold stress of P.lotungensis.8.Sixteen DEGs were selected and performed for real-time fluorescence quantitative(qRT-PCR)to validate the RNA-seq data.qRT-PCR analysis results showed that these sixteen DEGs had four expression modes with the extension of treatment time,in which relative expressions of RAC?SHSP?PLCPs and FAO genes with the expression mode of decreasing at 2h first and then increasing;those of ?-amylase,LRR_RLK,ACBP,CTs,LOX2S,LEA5,WRKY33 and[Cu_Zn]SOD genes appeared up-regulated expression mode;while those of FAD3,Myb4,d LTP2 genes with increasing before 6h treatment and then decreasing expression mode;ATPase gene with the expression mode of increasing at 2h and then decreasing.The results showed that the relative expression(??CT)of these 16 DEGs by qRT-PCR was basically consistent with the dynamic change trend of the relative expression(FPKM)in RNA-seq,which further verified the reliability of the data obtained by RNA-seq of P.lotungensis... |
PDF Full Text Request