Transcriptome Analysis Of Freezing Tolerance Mechanism For Tibetan Saussurea Laniceps Callus

Cold damage is one of the serious natural disasters in agriculture and forestry and it is important to reveal the molecular mechanism of plant cold resistance formation.Alpine plants growing in the Qinghai-Tibet Plateau have a very harsh living environment.Long-term natural selection and adaptive evolution have formed a special molecular mechanism for resisting freezing stress to complete their life history.In this study,the callus of Saussurea laniceps,an alpine plant growing at an altitude of about 3200-5280 meters in Tibet,was used as experimental material to analyze the molecular basis of cold acclimation for improving the freezing tolerance by transcriptome sequencing.The main results are as follows:1.Low temperature acclimation improved the freezing resistance and ABA level of Saussurea laniceps.After cold acclimation,the semi-lethal temperature(LT50)of callus of Saussurea laniceps decreased greatly.The LT50 decreased from-3.5? to-10.6? after 9 days of acclimation,indicating that cold acclimation significantly improved the freezing tolerance of Saussurea laniceps callus.The content of ABA in callus was increased by cold acclimation.On the 3rd day,the content of ABA in cold-allimated callus was 3.87 times than that before cold acclimation.Although it decreased after 3 days,it remained at 2.79 times that that before cold acclimation until 12 days.2.Saussurea cryogenic transcriptome database.The callus of Saussurea laniceps which was acclimated at low temperature for 0 d(CK),6 d(A)and 9 d(B)were used as materials.Illumian Seq sequencing platform was used for sequencing.The cryogenic transcriptome database of Saussurea laniceps was assembled by de novo.A total of 88,862 Unigenes were assembled and spliced.After comparing and screening at three different stages,12362 differentially expressed genes were obtained from CK vs A and 14253 differentially expressed genes were obtained from CK vs B.Among them,6597 differentially expressed genes were up-regulated and 5765 down-regulated in CK vs A,while 7695 were up-regulated and 6558 were down-regulated in CK vs B.3.Cryogenic transcriptome database analysis.GO enrichment of differentially expressed genes revealed that 155 terms were associated with low temperature,such as low temperature response,oxidative stress response and plant hormones(ABA,jasmonic acid,ethylene,salicylic acid,etc.).KEGG significant enrichment analysis showed that plant hormone signal transduction,phenylalanine biosynthesis,ribosome,fatty acid metabolism,unsaturated fatty acid biosynthesis and other pathways were significantly enriched in response to cold in Saussurea laniceps.According to the results of differential gene enrichment,differentially expressed genes were screened from plant hormone signaling(39),ROS process(12),Ca2+signaling(19),protein kinase(9),lipid metabolism(29),carbohydrate metabolism(27),secondary metabolism(17),and heat maps were drawn according to their expression level.Ten differentially expressed genes were selected for qRT-PCR as database validation analysis.The results were basically consistent with the results of RNA-seq sequencing,indicating the reliability of transcriptome data.4.Cold-related transcriptional factor analysis.Based on transcriptome sequencing(RNA-Seq)data and TFDB database,the transcription factors in transcriptome database were screened and identified using NR,NT,Swiss-Prot and PFAM databases and NCBI websites.AP2-EREBP(6),WRKY(7),BHLH(5),MYB(8)and NAC(6)were identified from 43 transcription factor families.Subsequently,the conservative domains,physicochemical properties and phylogenetic evolution of different members of the family were predicted and analyzed by bioinformatics.In all,by analyzing the cryogenic transcriptome database of Saussurea laniceps,we screened out the high expression genes related to freezing resistance of Saussurea laniceps.The results of this study can preliminarily reveal the molecular mechanism of the freezing tolerance of Saussurea laniceps,and provide stress tolerant gene basic information for breeding new trees,ornamental plants and medical plants by genetic engineering.