Expression Profile Analysis, Cloning And Functional Research Of MYB Genes From Fraxinus Velutina Torr.

Forestry plays a decisive role in the national economic development, maintaining ecologicalbalance and providing the high yield and good quality trees. However, high salt stress and otherabiotic stress factors severely restrict the growth and yield of trees. The salt tolerance traits ofplants are usually controlled by multiple genes, and their response genes involved in many sides,such as plant physiological metabolism, ion transport and material synthesis, etc.. Clarifying themolecular mechanism of plant salt tolerance has important theoretical guarding significance toenhance the salt resistance of tree species by genetic engineering. Fraxinus velutina Torr., themain greening tree species for saline-alkali land and urban, is one of the important species in theforest, also with strong resistance to salt, drought and other good features. Now the deepresearches in transcriptome and other molecular biology data under salt stress are mainly focus onthe model plants, but the studies of woody plants are very few.In this study, we analyzed the gene expression profile of Fraxinus velutina Torr. under differentperiods of salt stress through high-throughput sequencing technologies and selected a largenumber of genes related to salt induced. Besides these, expression differences of eight genesunder salt stress were verified by qRT-PCR. On this basis, we cloned and analyzed the functionsof two MYB transcription factor genes and their promoters, pointed out the possible regulationapproaches of the two genes involved in plant salt resistance. The results will provide insight intothe salt-tolerant molecular mechanism and lay a theoretical foundation for genetic improvementof Fraxinus velutina Torr.. The main results of this study were as follows:1．Gene expression profile analysis of Fraxinus velutina Torr. under salt stress(1) In this study, digital gene-expression (DGE) technologies were used to investigate theglobal gene expression profiles of Fraxinus velutina Torr. seedlings under salt stress. All the amounts of the data obtained from the six groups of Fraxinus velutina Torr. were around fifty-sixmillion reads after processing. By sequencing analysis,72,1345and1379differentially expressedgenes were detected, respectively. Among them,4differentially co-expressed genes were incommon,50,317and353were special expression genes in each library.(2) GO analysis indicated that, after0.5h NaCl stress, the significant increased GO termsmainly included oxidation stress response, adversity stress response, nitrate response, telomeraseactivity regulation and positive regulation of telomerase activity. And the number of GO termsincreased significantly after24h NaCl stress, which included dehydration drought response,stress response, response of stress stimulation and ABA signaling regulation, etc.. In addition,20pathways maybe related to salt stress were identified by KEEG analysis, which mainly includedplant hormone signal transduction, starch and sucrose metabolism, plant-pathogen interaction,phenylpropanoid biosynthesis pathway, apoptosis pathway, carotenoid biosynthetic pathway andpentose and glucuronate interconversions pathway, etc..(3) By gene expression profile analysis, a large number of salt stress response genes wereobtained and the main functions of these genes involved in osmotic regulation and ion balance,hormone signal transduction, reactive oxygen removal, transcription regulation and so on. Theresult showed that these genes are likely to participate in the salt-resistance process of Fraxinusvelutina Torr..(4) In order to test the accuracy of the sequencing results, eight differentially expressed geneswere selected to analyze the expression patterns using qRT-PCR. The results were consistent wellwith those obtained from expression profile sequencing.2. Cloning and expression analysis of MYB genes from Fraxinus velutina Torr.(1) Two cDNAs encoding MYB genes were cloned from Fraxinus velutina Torr. by RACEand designed as FvMYB1and FvMYB2. Sequence analysis of FvMYB1showed that it was1203bp long and encoded301amino acids, with a calculated molecular mass of33.38kDa, and thegene had no introns. FvMYB2encoding311amino acids was1201bp in full length, with a calculated molecular mass of35.00kDa, and the genomic DNA contained three exons and twointrons.(2) The expression patterns of FvMYB1and FvMYB2in different organs of Fraxinus velutinaTorr. were analyzed by semi-quantitative RT-PCR. The results showed that both of them werewidely distributed in all the tested tissues. It was also found that the expression level ofFvMYB1was highest in stem and lowest in root while FvMYB2gene expression showed noobvious differences in all the tested tissues. Further, the gene expression patterns of MYB genesunder abiotic stresses (salt, drought and ABA) were also analyzed. Results indicated that therewere some similarities and differences between them. For example, the highest amountexpression time of FvMYB1and FvMYB2was around12h or6h. However, FvMYB2couldresponse to all the stresses above while FvMYB1could not response to ABA. The resultssuggested that FvMYB2may play a more important role than FvMYB1in the process of plantresponse to abiotic stress.(3) The subcellular localization of FvMYB1and FvMYB2showed that both were mainlylocalized in nucleus and the results were consisted with the studies of other transcription factors.In addition, the transcriptional activities of FvMYB1and FvMYB2were analyzed using yeastone-hybrid system. Results indicated that both of them could activate the expression ofdownstream report genes and β-Galaetosidase activity assay showed that FvMYB2has highertranscriptional activity than FvMYB1.(4) The prokaryotic expression vectors of HIS-FvMYB1and HIS-FvMYB2were constructedand expressed in E.coli BL21. Further SDS-PAGE and western blot were carried out to test thesuccess expression of FvMYB1and FvMYB2fusion protein, respectively. This result would lay afoundation for preparing effective antibodies used for western blot of transgenic plants.(5) To further investigate the functions of FvMYB1and FvMYB2genes, twoplant over-expression vectors pROKII-35S-FvMYB1and pROKII-35S-FvMYB2wereconstructed and introduced into tobacco genome respectively by Agrobacterium-mediated transformation. The transgenic tobaccos after Kan selection were obtained and examined by PCRand Southern blot. In normal conditions, some stress-related genes, such as NtP5CS, NtLEA5andNtMnSOD, expressed more in the transgenic tobaccos than the wild plants. Resistance analysisshowed that the salt injury symptoms of strains over-expressing FvMYB1or FvMYB2gene werelighter than the wild strains, and the concentration of SOD, POD, CAT and free proline of thetransgenic plants were higher than control, and the concentration of MDA was reduced aftertreated with NaCl for24h. In addition, the chlorophyll content of FvMYB2transgenic strains wasincreased in comparison with the control. These results indicated the roles of FvMYB1andFvMYB2in salt stress regulation.3. FvMYB1and FvMYB2promoter cloning and analysis of Fraxinus velutina Torr.(1) For further identified the function of FvMYB1and FvMYB2, two promoters of l140bp and1600bp were cloned from Fraxinus velutina Torr. genomic DNA based on genomic walkingprocedure. By Plant Care software analysis of the DNA sequences showed that many basic motifswere found in the FvMYB1and FvMYB2promoters, besides, there were some light-response andstress-response motifs including GT1GMSCAM4, ABRE, MBS, TCA, HSE, LTR, etc..(2) To investigate the characterizations of the FvMYB1and FvMYB2promoters, differentpromoter fragments were used to replace the35s promoter of pCAMBIA3301to construct plantdeletion expression vectors. The results of GUS histochemical staining of transgenic plants withdifferent deletion vectors showed that three of FvMYB1promoter sequences could drive the GUSgene expression, which were242bp,523bp and792bp in length. The transgenic plants withFvMYB2promoters of858bp and1600bp length showed blue color after GUS staining. Besides,the GUS stainings of all of the four fragments after salt induced were raised. Therefore, thepromoters of FvMYB1and FvMYB2could be induced by salt stress to some extent.