| Liriodendron chinense Sarg.belongs to genus Liriodendron of family magnoliaceae,which is endemic to China.It naturally occurs in mountain forests with altitude of about 1000meters in southern China.In the process of introduction of the species to other places,tolerance to cold temperature in northern China has been a problem for a long time,making it hard to grow L.chinense extensively.Up to date,very few studies on molecular mechanism of cold resistance L.chinense resources have been reoported.In this study,high-throughput sequencing technology was used to establish L.chinense transcriptome database and screen differentially expressed genes related to cold resitance under different conditions,in order to provide foundational information for studying L.chinense on gene level.Meanwhile,experiment on measuring physiological parameters relating to cold resistence was made to obtain a comprehensive evaluation at both physiology level and genetic level.The aim is to select genetic resources of high qulity for conservation and production,and also provide theoretical basis for molecular genetic breeding.Main results are as follows:1.Cold-resistant physiology indicator evaluation was carried to identify Liriodendron chinense provenance with high cold-resistant ability.Cold-resistant physiological indicators of different provenances showed obvious seasonal variation,and had the same trend in the four provenances.Specifically,malondialdehyde content,soluble protein content,SOD activity,soluble sugar content,proline content of leaf bud in winter were higher than in autumn,relative electric conductivity and water content in winter were lower than in autumn.According to the measurement data of physiological indicator,subordinate function value of four provenance were calculated as the comprehensive evaluation value of cold resistance,and the value of Yunnan,Zhejiang,Guizhou,Dabie mountain was 0.834,0.286,0.021,0.488 respectively.According to the result of comprehensive evaluation on four provenances,the cold-resistant ability was sorted as:Anhui>Zhejiang>Guizhou>Yunnan.Correlation analysis showed that relative electric conductivity and water content had significantly negative correlation with the subordinate function value,while proline content and the subordinate function value has significant positive correlation.Therefore,these indicators can be used as suitable evaluation indicatores.2.Leaf bud transcriptome library of Liriodendron chinense was established.The total number of clean reads through sequencing is 89 832 89,the total base number is 11.4Gbp,GC content was 46.42%.The percentage of one fragment with 20 base in length is96.05%,and the GC value was 46.75%,which indicated that the transcriptome sequencing could be used for subsequent analysis.162092 non redundant Unigenes were obtained,with the sequence information of 88714395 bp,and the fragment size ranged from 201-16808bp,with N50 fragment 719bp in length and N90 fragment 242bp in length.In the transcriptome Unigene functional annotation,1.69%of the total 162092 Unigenes were annotated successfully in each database,28.92%of the Unigenes were annotated successfully at least in one database.The high annotation ratio facilitated the subsequent gene function analysis to obtain the purpose gene study and low temperature response mechanism of Liriodendron chinense on gene level.Differentially expressed genes were screened from two transcriptome libraries,with the conditions qvalue<0.005 and|log2?foldchange?|>1.Yunnan and Anhui transcriptome database were compared?YUN vs AN?,and 611 significant differentially expressed genes were selected,of which 351 were up regulated expressed genes,260 were down regulated expressed.Results on GO enrichment analysis of differentially expressed genes showed,Unigenes were divided into biological processes and molecular features,with 27 small classes.Among them,Biological processes had 512 differentially expressed genes,with 13categories.Molecular function has 570 differentially expressed genes,with 14 categories.KEGG enrichment analysis was also used to screen the differentially expressed genes.Result showed that,there were 422 differentially expressed genes were annotated in 102 pathways of KEGG.In the most 20 enrichment metabolic pathways of differentially expressed genes,Fatty acid degradation and Arginine and proline metabolisminvolved in plant cold resistance changes of physiological and metabolic activity,which may be associated with the low temperature adaptability of plants.3.Cold-resistant related differentially expressed genes were selected by digital gene expression profiling technology21 samples were analysised through digital gene expessed profile technology.More than 0.5 G of clean bases were obtained of each sample,and more than 90%clean bases of each sample were compared successfully on the transcriptome sequencing sequence.Seven groups were made to analysis gene expression in different condition.Result showed that,a large number of differentially expressed genes were sellceted under the condition of different provenances,season,and low temperature stress treatment.The differentially expressed genes were analysied in the GO database and KEGG database function enrichment.A batch of cold-resistant related differentially expressed genes were annotated in the metabolic pathways,which are mainly divided into three types:one is cold-regulated signal transduction,including Plant hormone signaling,Calcium signaling pathways and MAPK signal transduction pathways.Second is photosynthesis,including Photosynthetic antenna and photosynthesis.The third is cold-resistant physiology metabolism,including Fatty acid metabolism,Biosynthesis of unsaturated fatty acid,Arginine and proline metabolism,Peroxidase,Starch and sucrose metabolism,and Beta-amylase metabolism,etc.At the same time,a large number of differentially expressed genes related to cold-response had been selected.Combining the predecessors'research on cold-resistant gene transcription factor,and according to gene function annotation information,26 differentially expressed Unigenes were selected.Accurate information about the candidate genes were obtained through Blastx on GenBank.Difference on cold-resistant physiology indicator between provenances probably related to gene expressing differentially in some cold-related physiological metabolic pathways.4.Cold-response genes were verified by fluorescence quantitative experimentsReal-time fluorescent quantitative PCR detections were used to verify the expression level of the selected 26 genes.Optimal reaction system was established.18S rRNA was used as reference gene.2-??Ct method was used to calculate relative expression of the genes.Correlation value data of the seven groups ranged between 0.79-0.91,which showed gene expression level obtained from RT-PCR kept consistency with transcriptome sequencing.The result indicated that the transcriptome sequencing results are accurate and reliable. |
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