| Medicago sativa cv.Pianguan is a local fine variety in Shanxi Province,drought is one of the main factors limiting its growth.It is of great significance to mine drought resistance related genes and apply them to genetic improvement of Medicago sativa cv.Pianguan for the re creation of excellent local germplasm.The miR397-5p related to drought was screened from Medicago sativa cv.Pianguan by high-throughput sequencing and successfully transferred into tobacco at early stage of our research group.In this experiment,20% PEG-6000 was used to stress wild type and overexpression miR397-5p tobacco,the physiological characteristics of two kinds of tobacco were tested at 0,12,24,36,48,60 and 72 h of stress,and the metabolic pathways of lignin and proline were studied at 0,24,48 and 72 h of stress,to explore the drought resistance function of miR397-5p gene,an efficient regeneration system of Medicago sativa cv.Pianguan was established,obtaining Medicago sativa cv.Pianguan overexpressing miR397-5p gene,which provided theoretical basis and methodology basis for further revealing drought resistance effect and mechanism of Medicago sativa cv.Pianguan controlled by miR397-5p,the main results are as follows.1.The soluble protein content and soluble sugar content of overexpression miR397-5p tobacco were higher than that of wild type tobacco,and the degree of water deficit was lower than that of wild type tobacco.miR397-5p gene can enhance water holding capacity of plants under drought stress by increasing osmotic adjustment substances in plants.At the same time,the content of MDA of overexpression miR397-5p tobacco was lower than that of wild type tobacco,and the activities of CAT,SOD and POD were higher than those of wild type tobacco,overexpression miR397-5p tobacco has a stronger protective system to scavenge reactive oxygen species.2.Except that the lignin content of overexpression miR397-5p tobacco was significantly lower than that of wild type(P<0.05)after 72 h stress,the lignin content of overexpression miR397-5p tobacco was significantly lower than that of wild type at other drought time points(P<0.001).The relative expression of genes related to lignin synthesis pathway was determined,except for PAL,F5 H,CCo AOMT and PAO genes,the relative expression levels of overexpression miR397-5p tobacco were lower than that of wild type tobacco at each drought time point,at the same time,the relative expression levels of PAL,F5 H and CCo AOMT in overexpression miR397-5p tobacco decreased significantly under drought stress for 48 h,miR397-5p gene may reduce lignin content by regulating the expression of lignin synthesis related genes.3.The proline content of overexpression miR397-5p tobacco was significantly higher than that of wild type tobacco under 24 h drought stress(P<0.05),the proline content of overexpression miR397-5p tobacco was significantly higher than that of wild type tobacco under 36 h stress(P<0.01).By measured the activities of the key enzymes of proline metabolism,we can get the followed results,at all stress time points,δ-OAT and P5 CS activities of overexpression miR397-5p tobacco were higher than those of wild type tobacco,while Pro DH activity was lower than that of wild type tobacco,the relative expression ofδ-OAT gene in overexpression miR397-5p tobacco was significantly higher than that in wild type tobacco at 0 h and 24 h of drought stress,and the relative expression of P5 CS gene in overexpression miR397-5p tobacco was significantly higher than that in wild type tobacco at 72 h of drought stress(P<0.05).In the early stage of drought stress,miR397-5p gene was involved in the regulation of ornithine-proline pathway,and changed to the regulation of glutamate-proline pathway with the extension of drought stress time.4.The work build a complete and optimized regeneration system of Medicago sativa cv.Pianguan,which was disinfected with 0.1% Hg Cl2 for 6 min and then inoculated on 1/4MS medium,It were the best germination culture conditions,MS + 2 mg·L-1 2,4-D + 0.4 mg·L-1 KT was the best medium for callus induction,UM + 1.6 mg·L-1 KT was the best medium for callus differentiation,hypocotyls were better than cotyledons in callus induction rate and differentiation rate.In addition,the overexpression vector PEG100-pre-miR397-5p was successfully constructed and introduced into Medicago sativa cv.Pianguan by Agrobacterium mediated genetic transformation,and the miR397-5p transgenic plants were obtained. |
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