| The Bp TT2 gene is in the key position of the network regulation and is inextricably linked with several key family genes in salt stress regulation,and belongs to the pivotal gene.In this paper,we used Betula platyphylla as the experimental material,constructed the plant expression vector of Bp TT2 gene,optimized the genetic system of Betula platyphylla,introduced Bp TT2 gene into Betula platyphylla,and performed molecular assays and preliminary functional analysis of the screened transgenic plants for salt stress.The results of the study were as follows:(1)The Bp TT2 gene was cloned from the Betula platyphylla c DNA library.Through bioinformatics analysis,the full-length 789 bp,encoding 262 amino acids,has the specific domain SANT MYB DNA binding domain of the plant MYB transcription factor family,which belongs to R2R3-MYB family.Through multiple sequence alignment and phylogenetic tree construction of the Bp TT2 gene,it was found that it has 96.56% homology with Bl MYB3 gene.(2)The regeneration system of Betula platyphylla tissue culture was optimized.The Betula platyphylla leaves were selected as the experimental material,and the regenerated plants were formed after initial culture,healing tissue induction,differentiation,strong seedling culture and plant rooting,and the most suitable medium,hormone type and concentration were determined at each stage.When the hormone concentration was 0.2 mg/L NAA+1 mg/L 6-BA,the callus induction rate reached 96.6%;when the hormone concentration was 0.02 mg/L NAA+1.0 mg/L 6-BA,the differentiation rate was the highest,which could up to 99.5%;when the hormone concentration is 0.2 mg/L NAA,the rooting rate can reach up to 100%.(3)Betula platyphylla callus was used as test material,and transiently transformed with Agrobacterium tumefaciens GV3101,which was transiently transformed into the plant expression vector p BI121-Bp TT2-GFP.By analyzing the effects of different infection times,different bacterial concentrations and hormone ratios on the transformation efficiency,detecting the fluorescent expression signal of GFP green fluorescent protein within 72 hours,and studying the optimal conditions affecting its transient expression,and then improving the transient transformation of Betula platyphylla by Agrobacterium tumefaciens.The callus system lays a solid experimental foundation for the stable genetic transformation of the gene.(4)Stable transformation of Betula platyphylla leaves and stem segments using Agrobacterium tumefaciens,Betula platyphylla plants overexpressing the Bp TT2 gene were obtained,and a stable transformation system based on Betula platyphylla leaves and stem segments was established.On the DNA level and the RNA level,using specific primers and GFP primers,it was identified by PCR that the Bp TT2 gene had been successfully integrated into the Betula platyphylla genome.(5)Under normal growth conditions,there was no significant difference in the growth status of over-expressed plants and wild-type plants.Through salt stress experiments,it was found that the growth of over-expression plants and wild-type plants were severely inhibited,and the leaves turned yellow and fell off.The results of physiological indicators showed that the POD activity of the over-expressed plants was slightly higher than that of the wild type,indicating that the over-expressed plants could remove oxygen free radicals in the plant and reduce the damage suffered by the plants;the MDA content of the over-expressed plants was lower than that of the wild type,reducing It prevents the damage of cell membrane caused by salt stress,reduces cell damage and death,and improves the salt tolerance of over-expressed plants.In conclusion,it was indicated that the Bp TT2 gene may be involved in the growth and development of Betula platyphylla,protect the stability of the cell membrane,and improve the resistance of Betula platyphylla to salt stress. |
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