| Tobacco Bacterial Wilt is a soil dissemination disease caused by Ralstonia solanacearum,which lead to the occurrence of tobacco wilt disease.Ralstonia solanacearum can overwintering in soil or in diseased bodies.The germs will still cause harm to tobacco production.Bacterial wilt is a bacterial vascular bundle disease,traditional methods of disease prevention could comprehensively prevent and control the outbreak of bacterial wilt,but it will consume a lot of human and material resources,in the meantime chemical control is often accompanied with environmental pollution and resistance to pathogenic bacteria,in addition,there is almost no effective treatment once bacterial wilt outbreak with a large-scale.Therefore,the development of tobacco germplasm resources with tolerance to bacterial wilt and stable inheritance has practical application value.Cross-breeding is a common breeding method,but it lacks clear antigens in tobacco bacterial wilt control,while in mutagenesis breeding methods,the direction of induced mutations is hard to grasp,and mutant tobacco plants which have effectively tolerant to bacterial wilt are difficult to obtain.By means of transgenic breeding,beneficial genes of interest can be quickly integrated into the tobacco genome,so that tobacco obtains high resistance to bacterial wilt.Not only that,the host of the bacterial wilt infestation is very extensive.It contains important economic crops,vegetables and food.The diseases will also cause serious ecnomic and production losses of their hosts.Through the study of tolerance to bacterial wilt in transgenic tobacco,the ability of the candidate genes heterologously expressed in plants to inhibit R.solanacearum was identified,and the development of transgenic resistance to bacterial wilt materials such as other important cash crops could also be guided.The staphylococcal antibacterial peptide deathin Thanatin is the smallest insect antibacterial peptide with a β-sheet structure,and has only 21 amino acid residues.It is an ideal candidate for transgenic peptides due to its small molecular weight,wide antibacterial spectrum and high efficiency.Studies have shown that transfer the antibacterial peptide CecB gene into tomato,transgenic lines obtained resistance to bacterial wilt;obtained rice blast resistance and Arabidopsis leaves with increased resistance to fungi through the rice and Arabidopsis heterologous expression of Thanatin.The development of these resistant transgenic materials provides a theoretical basis for the study of plant disease resistance.In this study,the Thanatin gene with the secretory peptide(SP)coding sequence was controlled by the CaMV 35 S promoter.The transgenic tobacco plants with integrated Thanatin were transformed with Agrobacterium tumefaciens-mediated transformation,and the expression level of Thanatin gene in T1 plants was analyzed.Tobacco plants were inoculated with Ralstonia solanacearum,the index of tobacco disease,changes in leaf chlorophyll content,and tissue culture of roots and stems were analyzed.The effect of exogenous Thanatin on tolerance to bacterial wilt in transgenic plants was investigated.Provide theoretical basis for breeding and research of transgenic resistance to bacterial wilt.The mainly results are as follows:1.The binary expression vector pVCT2365 containing the 2x35S::spThanatin gene was constructed by linking the spThantin gene which is coding sequence of tabaco PR-1 gene secreting singnal peptides Thanatin(synthesized by company)to the CaMV 35 S promoter..2.Transgenic Tobacco T1 generation plants were obtained and the expression level of Thanatin gene was clarified The tobacco leaf were transformed with LBA4404(pVCT2365)by Agrobacterium-mediated method and screened in tobacco differentiation medium containing kanamycin(Kan)to obtain9 strains of plant-cultured seedlings with Kan resistance.Then,the thanatin gene in the tissue culture seedlings was detected by PCR,and the positive plants transfecting the thanatin gene were screened for L4,L5,L7,L8,and L9.The positive individuals in the T1 population of each strain were screened by PCR,and the leaves of three positive plants in each strain were randomly cut,RNA was extracted from the mixed samples,and single-stranded cDNA was synthesized by reverse transcription;detection and analysis of expression of Thanatin Gene by SYBR GREEN qRT-PCR.The expression level of the T1 generation strain L5 was taken as the reference number 1.The T1 generation strains L4,L7,L8,and L9 were 3,3.2,and the T1 generation strain L5,respectively.25.6,4.5 times relative expression level.3.Overexpression of Thanatin Gene Increases Resistance to Bacterial Wilt in Transgenic Tobacco Inoculation of Ralstonia solanacearum in L4,L5,and L8 transgenic Tobacco Lines and WT Wild Type Tobacco.In the early stage of tobacco bacterial wilt disease,the wilting of plant leaves is reversible and does not accompany changes in chlorophyll content.As the progress of the disease,the leaf of the plant become yellow-brown and withered.The diseased part of the leaf blade starts from the petiole and spreads from the leaf vein to the surrounding area;After inoculation,the L8 strain and the WT wild tobacco rhizome-associated tissue were isolated and sterilized after surface disinfection.Red colonies grew when the longitudinal section was coated on the TTC medium,indicating that the green-stem successfully infectsthe plants and proliferation in plants.The L4,L5 strains and WT tobacco had similar disease index values.The L8 strain was significantly lower than the other three strains during the treatment period;From the analysis of disease index trending,compared with the other three treatment strains,the L8 strain can inhibit the reproduction of pathogens in the early stage of R.solanacearum vaccination,delay the onset of bacterial wilt,and prolong the survival time of tobacco,indicating that the relatively higher expression of thanatin gene increases the tolerance of tobacco plant bacterial wilt,shows that the heterologous expression of the thanatin gene enhanced the tolerance of transgenic tobacco to bacterial wilt. |
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