Verification Of Drought Tolerance Function Of CiMYB4 Gene Of Chrysanthemum Indicum Var.Aromaticum And Construction Of RNAi Vector

Chrysanthemum indicum var.Aromaticum is a newly discovered wild plant with excellent traits in recent years.Transcription factors of MYB are widely present and distributed in plants,and have important regulatory effects on stress resistance of plants.In the early stage of the laboratory,the CiMYB4 gene was excavated,analyzed and cloned based on the existing transcriptome data of Chrysanthemum indicum var.Aromaticum and the CiMYB4 gene was successfully transferred into tobacco and Dendrathema indicum.In order to explore the regulatory role of CiMYB4 gene in plant drought resistance,this study focused on the CiMYB4 transgenic tobacco and CiMYB4 gene Dendrathema indicum obtained in the laboratory earlier.Observe and measure the changes of physiological indexes and photosynthetic indexes of the plants under each drought line under natural drought stress;At the same time,through the"enzymatic digestion-ligation" method,a CiMYB4 gene inhibitory expression vector was constructed,and the Agrobacterium-mediated method was used to transfer into the Dendrathema indicum.The preliminary study of the function of the CiMYB4 gene was conducted for the later RNAi research and further study of the function of the CiMYB4 gene Laying the foundation.The main findings are as follows:1.Wild-type tobacco(WT),transgenic vector tobacco(EV),and CiMYB4 transgenic tobacco(S1,S2,S3)were simultaneously subjected to natural drought stress and rewater treatment.Observation of the phenotype of each line,physiological indicators such as SOD,POD,CAT,MDA,Proline and net photosynthetic rate(Pn),stomatal conductance(Gs),transpiration rate(Tr),intercellular CO2 concentration(Ci)photosynthesis Index measurement.we get:Compared with WT and EV tobacco lines,CiMYB4 transgenic tobacco plants wither and wither at a slower rate,with higher rehydration survival rates;better protection of membrane systems and ability to regulate moisture in the plants;under severe drought,CiMYB4 transgenic tobacco can still perform important life activities.Comprehensively showed that the transgenic tobacco has stronger drought resistance and higher growth advantages than wild-type tobacco;CiMYB4 gene has enhanced the drought resistance of tobacco to a certain extent.2.Subject wild-type Dendrathema indicum(WT),transgenic vector Dendrathema indicum(EV),and CiMYB4 transgenic Dendrathema indicum strains(A1,A2)to the same degree of natural drought stress and rehydration treatment.By observing the phenotype of each strain,measuring the physiological indexes of SOD,POD,CAT,MDA,Proline,and measuring net photosynthetic rate(Pn),stomatal conductance(Gs),transpiration rate(Tr),intercellular CO2 concentration(Ci).we get:Compared with the WT and EV Dendrathema indicum strains,the transgenic CiMYB4 gene Dendrathema indicum plants have a slower wilting and yellowing process,a higher rehydration survival rate,and a stronger regulation ability;under severe drought,they can maintain a certain vitality.The results showed that the transgenic CiMYB4 gene had higher growth advantages and stronger drought resistance than the WT and EV Dendrathema indicum strains.The CiMYB4 gene enhanced the drought resistance of the Dendrathema indicum to a certain extent.3.Based on the sequence of CiMYB4 gene of Chrysanthemum indicum var.aromaticum,a 425bp length target fragment was selected,and Xhol,KpnI/Xbal I,Hind III restriction sites were added at both ends to construct a cloning vector pGEM-CiMYB4RNAi-T.The cloning vector was double-digested to obtain forward target fragments and reverse target fragments,respectively.Recombinant CiMYB4(+)/pHANNIBAL/CiMYB4(-)recombinant vector was constructed by double digestion with Xhol,KpnI/Xbal I,and Hind III.SpeI and SacI were used to double digest and construct pRNAi-CiMYB4 plant expression vector.The pRNAi-CiMYB4 plant expression vector was introduced into Agrobacterium EHA105 by electrotransformation,and stored in a-80? refrigerator for future use.4.Spectinomycin 10mg/L is the optimal critical concentration for leaf differentiation screening during the genetic transformation of the pRNAi-CiMYB4 recombinant plasmid Dendrathema indicum.Spectinomycin 12 mg/L is the optimal critical concentration for the selection of resistant seedlings for rooting during the genetic transformation of the pRNAi-CiMYB4 recombinant plasmid Dendrathema indicum.200mg/L is the optimal bacteriostatic concentration of cephalosporin.The Agrobacterium-mediated method was used to transfer the pRNAi-CiMYB4 recombinant plasmid into the Dendrathema indicum plant,and the relative expression of CiMYB4 gene in the resistant plant was tested by resistant rooting screening,PCR detection of the resistant plant's cDNA and qRT-PCR,One strain of transfection of pRNAi-CiMYB4 recombinant plasmid positive Dendrathema indicum has been successfully obtained.