Analysis Of The Responsiveness Of Flowering Chinese Cabbage BcAMT1;3 And BcAMT1;4 Promoter To NH₄⁺

Flowering Chinese Cabbage is a variant of Chinese Cabbage of Cruciferae canola,and nitrogen is one of the main elements needed for the growth and develop mentof Flowering C hinese Cabbage.nitrogen transport protein mainly includes ammoniumtransporters and nitrate transporters,according to the absorption kinetics,ammonium transporters and nitrate transporters can be divided into high affinity and low affinity.BcAMT1;3 and BcAMT1;4 gene encode high-affinity ammonium transporters of Flowering C hinese Cabbage,earlier stage laboratory research found,under the condition of N itrogen deficiency BcAMT1;3 and BcAMT1;4 gene expressioncan be significantly induced.BcAMT1;3 mainly expressed in the root,and is responsible for the uptake of NH₄⁺ in root from the environment,BcAMT1;4 mainly expressed in leaves,and in charge of the metabolites of NH₄⁺assimilation,it is benefit to adapt to N itrogen deficiency,in addition,NH₄⁺is a kind of nutrients,is also a signal.Ammonium transporter encoded by BcAMT1;3 and BcAMT1;4 gene can response N H4+ signal,different ratio of ammonium nitrate have different way of response,from the Angle of the promoter,This research studied the response to N itrogen deficiency?NH₄⁺ signal?200 um/L??different ratio of ammonium nitrate of BcAMT1;3 promoter and BcAMT1;4 promoter.Explore the promoter cis element and the rule response to nitrogendeficiency?NH₄⁺ signal?200 um/L??different ratio of ammonium nitrate of BcAMT1;3 promoter and BcAMT1;4 promoter.This study has the following three main content:Cloned BcAMT1;3 and BcAMT1;4 gene promoters by the Genome Walking.Bc AMT 1;3 and BcAMT1;4 gene promoters' Total length are 2149 bp and 2086 bp respectively,the sequencing results analysis showed: BcAMT1;3 promoter sequence is highly conserved,The DNA sequences obtained from Flowering C hinese Cabbage BcAMT1;3 promoter and brassica napus Bn AMT1;3 promoter sequences on chromosome 5 are highly homologous sequences,homology is as high as 99%,Flowering C hinese Cabbage BcAMT1;4 gene promoter and Brassica napus Bn AMT1;4 promoter sequences contain homologous sequences onchromosome 3 and chromosome 1,homology is lower compared with BcAMT1;3,different species,the same gene promoter sequences exist differences.analyzes the promoterpotentiAlcis element Through Plant Care online software,BcAMT1;3 and BcAMT1;4 promoters contain common enhanced transcriptional regulatory elements,such as TATA box,CAAT-box,And 5'UTR Py-rich strech of improving the level of transcription.Promoters also Contain a lot of light signal response element and day and night cycle element;In addition,contain hormone signaling pathways including salicylic acid,auxin,ethylene and gibberellin,methyl jasmonic acid response element,etc.;and Induced disease resistance,stress response components including the high and low temperature stress,anaerobic induction,induced defense response element;And meristematic tissue specific expression of related components,development momentum effect element.To explore the BcAMT1;3 and BcAMT1;4 gene promoter in response to low Ncondition order element position,Lay the foundation for using yeast single miscellaneous technical appraisal in the next step that can adjust BcAMT1;3 and BcAMT1;4 gene expression of transcription factor,so as to improve the absorption of nitrogen efficiency.This experiment will respectively 5' lack of different length of four segments of the BcAMT1;3 and Bc AMT 1;4 promoter,and then cloning to GUS reporter gene upstream of binary expression vector Pcambia1391,to build BcAMT1;3Pro::GUS and BcAMT1;4Pro: : GUS fusion gene expression vector.Using the method of agrobacterium mediated infection Flowering Chinese Cabbage seeds of dewing bud,get the two groups Transgene Flowering C hinese Cabbageseedling of transienting expression usion gene and then use the nitrogen deficiencyof 1/8 hoagland nutrient solution treated for two days,the transient expression of the two groups of fusiongene Flowering C hinese Cabbage seedling are analyzed by GUS histochemical staining method,t The results found that eight carry BcAMT1;3Pro609::GUS? BcAMT1;3Pro1009::GUS?BcAMT1;3Pro1429::GUS?BcAMT1;3Pro2149::GUS?BcAMT1;4 Pro555::GUS?BcAMT1;4Pro1186::GUS?BcAMT1;4Pro1406::GUS?BcAMT1;4Pro2086::GUS fusion gene transgenic Flowering C hinese Cabbage seedling,can start the GUS gene expression,rin the root andleaf can be dyed dark blue espectively in the histochemical staining.lacking of promoter length to 609 bpof BcAMT1;3Pro: :GUS,GUS staining is still deep blue,indicate that the BcAMT1;3 and BcAMT1;4 promote cis element response to nitrogen deficiency signal role mainly in gene transcription start site ATG upstream within 609 bp,more exact position,still need to be further shortened to explore.NH₄⁺ as a kind of nutrients,but also can be used as a signal,adjusting BcAMT1;3 and BcAMT1;4 gene expression,also can adjust the happening of the lateral root;Using t he method of agrobacterium mediated infection Flowering C hinese Cabbage seeds of dewing bud,get the two groups Transgene Flowering C hinese Cabbage seedling of transienting expression usion gene,to explore the low concentration of NH₄⁺?200 um/l?,Flowering Chinese Cabbage BcAMT1;3 and BcAMT1;4 promoter cis element position of response to NH₄⁺ signal,By GUS histochemical staining method,this study found that the Bc AMT 1;3 promote cis element response to nitrogen NH₄⁺ signal role mainly in gene transcription start site ATG upstream within 609 bp,more exact position,still need to be further shortened to explore;BcAMT1;4promote cis element response to NH₄⁺deficiency signal role mainly in promoter 1186 bp to 555 bp.With different ratio of ammonium nitrate?0:10 captured the 0,5,50:50,100:0?1/4 hoagland nutrient solution processing the transient expression of the two groups of fusion gene Flowering Chinese Cabbage seedling,then using histochemical staining method,exploring the Flowering C hinese Cabbage BcAMT1;3 and BcAMT1;4 promoter activity under different ratio of ammonium nitrate;and indirectly reflect.Flowering C hinese Cabbage BcAMT1;3 and BcAMT1;4 promoterresponse rules under different ammonium nitratea,under ammonium nitratea?0:10 captured the 0,5,50:50?,GUS histochemical staining,the BcAMT1;3 and BcAMT1;4 promoter activity are strong,Flowering Chinese Cabbage seedling can be dyed blue,and the difference is smal between the treatments,under the treatment of ammonium nitrate ratio?100-0?,promoter activity are suppressed on a degree,the histochemical staining becomes shallow blue.In conclusion,BcAMT1;3 and BcAMT1;4 promoter by GenomeWalking acquisitioncan response tonitrogen deficiency signal and NH₄⁺ signal,BcAMT1;3 and BcAMT1;4 promoter response of N and NH₄⁺ signal promoter sequence in gene transcription start site ATG upst-ream within 609 bp and 1186 bp to 555 bp;Under different ratio of ammonium nitrate,BcAMT1;3 and BcAMT1;4 promoter has a certain response law,promoter activitys are strong in lower concentration of NH₄⁺ treatment,are weak in high concentration of NH₄⁺ treatment,promoter activitys areinhibited;BcAMT1;3 promoter activitys in root specific expression,BcAMT1;4 promoter activitys in leaf specific expression,bear the different functions.