Transcriptome Analysis Of The Dehydration Response Of Caragana Intermedia Kuang Et H.C.Fu And The Expression Profiling Of Some Secondary Metabolism Related Genes

Caragana intermedia Kuang et H.C.Fu, also known as Caragana, belongs to perennial shrubs of Caragana Fabr. It is a well-adapted plant to fix sand and conserve the soil and water in arid steppe or desert steppe zone with high ecological and economic value. The remarkable capabilities to tolerate drought make it a good candidates to investigate drough-tolerance mechanisms and to identify differentially expressed drough-response genes. However, the genetic information of this species is very limited. In this study, four sequencing libraries prepared from the control （without dehydration₀h） and dehydration-treated samples （dehydration₁hr, dehydration₃hr, and dehydration₁2hr） were sequenced by Illumina to obtain the transcriptome information of C.intermedia, and changes in the transcriptome level in response to drough stress were analyzed. In addition, the simple sequence repeat （SSR） of the expressed sequence in the transcriptome were predicted and validated. The content of total flavonoids in secondary metabolism was also determined primarily. The main results of this research are as follows:1. By the de novo assembled, Clean Reads of the control, dehydation-1hr,3hr and 12hr were assembled into 32,560,395,33,669,219,24,681,821 and 23,805,919 Unigenes. After removal of the redundant sequences,362,633 All-Unigenes left eventually. Among them,64,750 Unigenes were subjected to 25 clusters of the COG library. Totally 200,422（55.27%） Unigenes showed significant BLAST hits on the Nr Library （the protein database）. And 130,951 Unigenes were categorized into 9,104 Gene Ontology （GO） Terms. The physiological process of the annotated genes mainly involved in the oxidation reaction, transmembrane transport, metabolism, transcription regulation and amino acid hydrolysis. The cell compartments of the genes concentrated in the nuclei, chloroplast, plasmodesmata and other parts. The physiological function carried out mainly belongs to hydrolysis, RNA binding, activity of serine/threonine kinase.2. Among the 269,857 Unigenes, the number of up-regulated genes was more than that of the down-regulated genes. With the dehydration time prolonged, the differentially expressed genes increased. The 856 up-regulated DEGs and the 439 down-regulated DEGs were enriched inl 09 and 66 GO terms beween the samples of 1hr and 0hr. The serine/threonine kinases were enriched, the signaling pathway mediated by receptor-like proteins and enzyme-coupled receptor proteins were also down-regulated, which suggested that protein kinases might be associated with early response signal transduction of C. intermedia under dehydration stress.The 538 up-regulated DEGs and the 341 down-regulated DEGs were enriched into 82 and 46 GO terms respectively beween the samples of 3hr and 1hr. The 1,299 up-regulated DEGs and the 1,902 down-regulated DEGs were enriched in 15 and 216 GO terms respectively beween the samples of 12hr and 3hr, which indicated that abscisic acid mediated response was involved in the late response under dehydration stress. There are 766 up-regulated Co-DEGs and 505 down-regulated Co-DEGs. Among them,367 up-regulated Co-DEGs and 160 down-regulated Co-DEGs were enriched in 57 and 20 GO terms. Ethylene mediated and DNA-binding region:AP2/ERF related pathways were significantly enriched in the up-regulated Co-DEGs, which may be an important mechanism of C. intermedia to tolerate drought. qRT-PCR confirmed that the most of 10 out of the 12 randomly selected DEGs showed similar expression pattern compared with the trascriptome data.3. Totally 305 SSR motifs representing of 45,706 SSRs were predicted from the 362,633 Unigenes, with a frequency was 10.38%. The average length of the SSR was 115.37bp. PCR experiments showed that 79 out of the 150 primer pairs could be amplified with expected PCR products.4. qRT-PCR verified some genes related to the secondary metabolism pathways. Orthogonal experimental design for the ultrasonic extraction of rutin in C. intermedia was carried out, the optimal treatment was 60 min,60℃, with 80% ethanol, and the ratio of liquid to solid is 1:10. The content of rutin in leaves was much higher than that in stems, in addition, the rutin content in the tissues collected at the late stages was significantly higher than that collected at the the early stage. With the prolonged drought treatment, the rutin content increased gradully.