Cloning And Characteristics Of Transcriptional Factors BeMYB140 And BeMYB680 And Their Response To Abiotic Stresses

The current study mainly comprises of two different parts.The part I highlight the cloning of MYB family related transcriptional factors;while,part ? elaborate the effect of salinity on Phyllostachys edulis plants.Part I elaborates;the MYB family is very well known for its important role in the plants against abiotic stresses and also in plants improvement both physiologically and anatomically.From MYB family related Transcriptional factors(TFs)were thoughtfully selected;which were having role in abiotic stresses.Overall Cloning from Bdamboo is a tough task and very slight success has been accomplished because of its incomplete genome sequence.However,we have successfully cloned two MYB family related TFs and were designed as "BeMYB680"and "BeMYB140".The selection of TFs from bamboo plants is carried because its genome offers the best for future improvement of transgenic plants.Bamboo is fast-growing species and also provide very good podium because of its adoptable genomics to harsh climatic conditions.China is rich in bamboo resources,especially in Sichuan,Yunnan and Fujian provinces covering larger areas Bamboo is an important and renewable timber resource,which has a fast growth rate,high productivity,versatility and high economic value with 70%or more contents of high quality fibers.Bamboo is good raw materials for paper-making industry.Bambusa emeiensis is one of the most important species of bamboo;which is native to Yunnan Plateau and Sichuan province.This specie has several uses i.e.from household weaving to agricultural tools,and for ornamental and soil-protection purposes.Bambusa emeiensis has a substantial ability to grow in a diverse range of habitats and successfully survive in harsh environmental conditions.The major goal of the current research is the identification,cloning and characterization of selected TFs which make Bambusa emeiensis unique and transformation of these TFs into the wheat and tobacco for the development of strong cultivarThe successfully cloned TFS i.e.BeMYB140 and BeMYB680 were analyzed through bioinformatics analysis,Yeast one-hybrid(HIY),subcellular localization and expression analysis.Latterly,the overexpression vector was constructed and transformed into the wheat genotype"DN7742"and tobacco through particle bombardment and agrobacterium.The current research is a good baseline for the understanding of the different roles played by the cloned TFS in the improvement of plants especially the robust cultivars of wheat.The outcomes of our research are illustrated as under;1.While adopting the bioinformatics and the available database of the selected specie the TFS were identified.The Two TFs were cloned from the transcriptome database of the Barmbusa emeiensis and Phyllostachys edulis.The isolated TFs were named as "BeMYB140"(NCBI GenBank accession number MG763924)and "BeMYB680"NCBI(GenBank with accession number MG763924)respectively2.Through bioinformatics analysis of "BeMYB140"and "BeMYB680"it is analyzed that BeMYB140 has an open reading frame(ORF)of 723 bp and encodes a protein of 240 armino acids with a predicted relative molecular weight of 27.34346 kDa.Whereas BeMYB680 have 1146 bp open reading frame(ORF)that encodes a protein of 381 amino acids with predicted molecular mass 40.97185 kDa3.The multiple proteins Sequence alignment defines that BeMYB140 and BeMYB680 protein sequence contains a typical MYB domain structure.Through phylogenetic analysis it is revealed that BeMYB140 showed high homology with HvMYBl,ZmMYB38 and AtMYB4 Whereas BeMYB680 showed high homology with AtMYB46,AtMYB61,AtMYB86;HvMYB33 and AtMYB264.The YIH screening results showed that the "BeMYB140 and"BeMYB680"are the transcriptional activators.The blue staining showed the protein-protein traction;while the yeast EGY48 with empty vectorpEG202 has no positive staining5.Subcellular localization assay revealed that "BeMYB14" and "BeMYB680"are localized in the cell nucleus.The cells of onion were used and the GFP was analyzed through fluorescent microscope.The DAPI staining was also used for the confirmation of cellular nucleus.6.The results regarding expression profiling of BeMYB140 showed that in response to the all abiotic stressors(ABA,PEG,NaCl,H2O2,and Na2SO4)BeMYB140 was expressively up regulated.In some cases,like ABA and Na2SO4 down regulation was followed by up-regulation.7.BeMYB680 expression profiling toward different abiotic stresses was up-regulated when treated with ABA,PEG,NaCl,and H2O2;while down-regulated was seen with Na2SO48.The overexpression vector of BeMYB140 was constructed and transformed into tobacco through agrobacterium.The successfully transformation of survived plants were verified through the semi-quantitative PCR.The results showed that the BeMYB140 is successfully transformed into tobacco plants9.The overexpression vector of BeMYB680 was transformed into the wheat through particle bombardment.The successfully transformation of survived plants were verified through the semi-quantitative PCR.The results showed that the BeMYB680 is successfully integrated into the 5 transgenic linesThe part II elaborates the effect of salinity on the Phyllostachys edulis plants growth,development and metabolism.Salinity halts metabolic processes which can be gauged through physiological,enzymatic and genetic analysis.Phyllostachys edulis is used as a plant source against different kinds of salts i.e.KCl,NaCl and Na2SO4 with different concentrations as limiting factors.The physiological parameters always have great variations in response to salinity.The biomass has also been decreased greatly by NaCl followed by KCl and Na2SO4.The 600 mM NaClthrough time interval cause the death of all plants.Few plants were dead because of higher concentration of KC1;while Na2SO4 didn't cause the plants death and majority of the plants survived.The photosynthesis Fv/Fm is always significantly damaged and halted by different kinds of salts such as NaCl,KCl and Na2SO4 In current research the NaCl was having the greatest impact followed by KCl and Na2SO4 The Fv/Fm lower value for NaCl 600 mM was zero,KCl 600 mM was 0.16 and Na2SO4 300 mM was 0.33.These results through physiological parameters showed that the NaCl was the most devastating salt for Phyllostachys edulis.The enzymatic activity i.e.SOD,POD,CAT,MDA and proline were analyzed.The results for SOD showed that in response to KCl the activity was increased significantly to all concentration while NaCl and Na2SO4 have no significant impact on the SOD activity.The results of POD and CAT showed that the Na2SO4 havehigher the activity to maximum level;while KCl and NaCl have little positive effect on POD and CAT activity.The Na2SO4 increase the POD and CAT activity with the passage of time with increase in concentration.Through MDA results it is showed that all the salts have increased the MDA activity.The KCL have the maximum positive effect with 600 mM concentration.The NaCl and Na2SO4 have non-significant change with the time interval and increase in concentration.Through proline results it is concluded that the KCl and NaCl increase proline contents.Especially the KCl has the maximum effect on proline activity.Na2SO4 have nearly no effect on proline contents.This showed that the Na2SO4 have induced low level of stress to Phyllostachys edulis plants.The four genes which are BeSNAC1,BeWRKY2,BeMYB140 and BeMYB680 were analyzed and through bioinformatics analysis it was confirmed the sequences with similarities.The expression analysis showed that the BeSNACl and BeVWRKY2 were expressed in all kinds of salts stresses.There are no significant effects of salts concentrations on BeSNACl and BeWRKY2 expression level.The maximum fold change was observed in KCL 350 mM followed by lower concentration of KCl,Na2SO4 and NaCl.The BeMYB140 was significantly expressed in response to KCl and Na2SO4 but to NaCl there is no increase in expression level.The higher concentrations of KCI and Na2S04 have increased the expression level of BeMYB140.This results showed that The BeMYB140 have positive role against KCl and Na2SO4.The BeMYB680 expression level is increased to lower level in response to different kind of salts.The expression level is irregular and it is trigger in small patches.The higher concentrations also have positive effect on the expression level.the results of Na,K and Ca minerals deposition showed that the NaCl salt does not promote the deposition of Na in plants leaves;while Na2SO4 enhance the Na deposition level.Both are sodium salts but they showed different results.The KCl have nearly no effect on Na deposition in comparison to control.KCl salts increase the deposition of K;while NaCl and Na22SO4 have no significant effect on K level in plants leaves.The KCl,NaCl and Na2SO4 does not have any significant impact on Ca deposition in leaves except for KCl 600 mM and NaCl 600 mM.Over all NaCl have the worse effect on Phyllostachys edulis plants followed by KCl and Na2SO4.