Identification Of Mirabilis Laccase Genetic Mir-LAC Transformation To Arabidopsis Thaliana

With the development of society and the advancement of science and technology,the use of fossil energy is also increasing day by day.The exploitation of fossil energy has caused a large amount of soil crude oil pollution.Crude oil contaminated soil contains a large number of carcinogenic teratogenic components,which have a great impact on people's health and income.Some experiments have shown that Mirabilis laccase plays a role in the degradation of crude oil pollutants in the soil of Mirabilis jalapa.In this experiment,the jasmine laccase gene was transferred into the model plant Arabidopsis by means of transgenic method,and the Arabidopsis thaliana plants were identified by PCR amplification and Southern blot,and the transgenic plants were identified by crude oil.The mustard was subjected to stress to identify physiologically,in order to obtain the laccase gene material,and to provide materials for studying the role of the laccase gene in petroleum degradation.The research results obtained in this study are as follows:1.The high-yellow jasmine laccase gene screened by high-throughput sequencing of transcriptome was cloned by RT-PCR.The results of bioinformatics analysis indicated that the open reading frame of jasmine laccase gene(ORF,Open Reading Frame)is 1 719 bp in length and encodes 572 amino acids with a protein molecular mass of 63.34 k D and an isoelectric point of 7.97.The homologous sequence alignment and phylogenetic tree analysis found that the protein encoded by the gene was more conservative and did not differ greatly with evolution.At the same time,the results of subcellular localization indicated that the jasmine laccase was present in the organelles of the nucleus,endoplasmic reticulum,Golgi apparatus and the like,and in the organelles of protein synthesis and vacuoles,mitochondria and other cells.The eukaryotic expression vector p BI-Mir LAC of the jasmine laccase gene was extracted and transferred into GV3101 Agrobacterium.PCR and sequencing results showed that the eukaryotic expression vector was successfully transferred into Agrobacterium.2.The eukaryotic expression vector p BI-Mir LAC of the jasmine laccase gene was extracted and transferred into GV3101 Agrobacterium.PCR and sequencing results showed that the eukaryotic expression vector was successfully transferred into Agrobacterium.3.Transfer the Mirabilis laccase gene into the wild-type Arabidopsis genome using the pollen tube pathway method.The T0-T2 generation seeds were tested for resistance plate.The wild type Arabidopsis genomic DNA was used as the negative control,the deionized water was used as the blank control,and the p BI-Mir LAC plasmid was used as the positive control to identify the total DNA of the transgenic Arabidopsis thaliana.Six strains of T2 transgenic Arabidopsis carrying the Mirabilis laccase Mir LAC.4.Southern blot analysis of transgenic Arabidopsis thaliana.The results showed that 4 of the 6 plants identified above had positive signals,indicating that the target gene fragment had been inserted into the Arabidopsis genome.It can be used as a material for subsequent research.5.Physiologically identified T3 transgenic Arabidopsis thaliana.Physiological indicators such as laccase activity,relative conductivity,superoxide dismutase(SOD)activity and malondialdehyde content of transgenic Arabidopsis thaliana were determined under crude oil stress.The results showed that the laccase activity of transgenic Arabidopsis was different in 4 strains,and no laccase activity was detected in wild type Arabidopsis.Under the conditions of crude oil stress treatment,the relative conductivity,SOD activity and malondialdehyde content of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana increased with the prolongation of oil stress time,while transgenic Arabidopsis thaliana was relatively wild-type.The rate of rise of mustard is slower.The SOD activity of wild-type Arabidopsis thaliana increased and then decreased with the increase of crude oil pollutant concentration in the same stress time.The SOD activity of transgenic Arabidopsis thaliana increased continuously,indicating that transgenic Arabidopsis thaliana had crude oil pollutants.The resistance is stronger.The malondialdehyde content of transgenic and non-transgenic Arabidopsis thaliana increased during the same stress period,but the malondialdehyde content of transgenic Arabidopsis was always lower than that of non-transgenic.