| Apple(Malus domestica Borkh.)is a perennial woody plant of the genus Malus of the Rosaceae family.It is one of the main fruit trees in the world.It is also one of the most important fruit tree species in China.Apples will encounter various biological and abiotic stresses during the growth and development process.Among them,drought stress and salt stress will cause severe oxidative stress to the plant,thereby inhibiting plant growth and reducing fruit quality.Abiotic stress-related genes play a vital role in responding to plant stresses such as drought and salt damage.Studies have shown that melatonin in plants is not only a growth regulator,but also a powerful antioxidant,so it plays an important role in plants against stress.Therefore,this study constructed the plant expression vector of the melatonin synthase gene MzASMT1 in Malus zumi Mats to study the function of this gene in plant stress resistance by transferring into K326 tobacco and ’Gala’ apple.The main results were as follows:1.Successfully constructed the plant expression vector pCAMBIA2300-35s-GUS-CaMVterm-MzASMT1 of the melatonin synthase gene MzASMT1.Using the Agrobacterium-mediated genetic transformation method,the expression vector containing the MzASMT1 gene was transferred into model plant tobacco and stained by GUS.Three positive lines were obtained by genomic PCR identification and screening.The results of qRT-PCR showed that compared with wild type,the expression of MzASMT1 gene in transgenic tobacco increased significantly,and the expression of MzASMT1 gene in different transgenic lines was significantly different.The melatonin content of transgenic lines and wild type was determined by enzyme-linked immunosorbent assay.The results showed that the melatonin content of transgenic lines was significantly higher than that of wild type,which was consistent with the gene expression results.2.After a 30-day drought treatment of MzASMT1 transgenic tobacco and wild-type tobacco,a comparative analysis of phenotype,physiological and biochemical indicators,and drought related gene expression showed that after 30 days of drought treatment,the transgenic plants and wild-type plant growth is more robust,leaf wilting and root damage are less severe.The relative water content and chlorophyll content of leaves of transgenic plants are significantly higher than that of wild type.At the same time,transgenic plants also significantly increased proline content and reduced MDA.The amount of accumulation indicates that the transgenic plants have a lesser degree of oxidative damage to the cell membrane system under drought stress.Further research found that the transgenic plants increased the activity of antioxidant enzymes(POD,SOD,CAT)and the expression levels of related genes NtPOD,NtSOD,NtCAT.The expression of drought-tolerance related genes showed that the gene expression of NtERD10C,NtERD10D,NtLEA5,NtP5CS and NtDREB3 in transgenic plants increased.The above results indicate that the MzASMT1 transgenic tobacco increased its drought tolerance by increasing the relative water content,chlorophyll content,proline content,antioxidant enzyme activity,drought-tolerance related gene expression and reducing the accumulation of MDA.3.After transgenic MzASMT1 tobacco and wild-type tobacco were treated with 200 mM NaCl for 14 days,the phenotype,physiological and biochemical indicators and salt-stress related gene expression were compared and analyzed.The results showed that:transgenic plants compared with wild-type plants,the growth is more robust,the wilting of the bottom leaves and the damage to the root system are lighter.The relative water content of the roots,stems and leaves of the transgenic plants is 1.6 times,1.5 times,1.2 times of the wild type,chlorophyll content and chlorophyll fluorescence parameters(Fv/Fm)were significantly higher than the wild type,the proline content increased by about 2.3 times on average,and the MDA content was reduced to about 75%of the wild type.At the same time,the transgenic plants significantly increased the antioxidant enzyme(POD,SOD,CAT)activity,related The expression levels of genes NtPOD,NtSOD,and NtCAT also increased significantly.Chemical tissue staining of leaves after salt stress treatment with DAB and NBT showed that the transgenic plants were lighter in color.The quantitative determination of H2O2 and O2-content showed that:the transgenic plants H2O2 And O2content is significantly lower than that of wild type.Related gene test results show that:NtERD10C,NtERD10D,NtLEA5,NtP5CS and NtDREB3 in transgenic lines increased gene expression.The above results show that:MzASMT1 transgenic tobacco can increase the relative water content of roots,stems and leaves,chlorophyll content,chlorophyll fluorescence parameters,proline content,antioxidant enzyme activity,salt-tolerance related gene expression and reduce MDA,H2O2 and O2-accumulate the amount to improve its salt tolerance.4.Using the Agrobacterium-mediated genetic transformation method,the expression vector containing the MzASMT1 gene and vacant vector were transferred into ’Gala’ apples respectively.Through GUS staining and genomic PCR identification,three MzASMT1 gene-transmitted Gala lines and vacant vector Gala lines.The results of qRT-PCR showed that the transgenic plants significantly increased the expression of MzASMT1 gene compared with wild-type and transgenic vector plants,and the expression of MzASMT1 gene was significantly different among different transgenic lines.The melatonin content of transgenic plants and wild type and vacant vector plants was determined by enzyme-linked immunosorbent assay.The results showed that the melatonin content of transgenic plants was significantly higher than that of wild type and vacant vector plants.In addition,drought stress and salt stress treatment were carried out on transgenic plants and wild-type and vacant vector plants,and the expression levels of stress-related genes were also measured.The results showed that Gala transgenic MzASMT1 gene up-regulated antioxidant enzyme related genes(MdSOD,MdCAT),ABA synthesis and signal transduction genes(MdNCED3,MdAREB2,MdSnRK2.4),proline synthase gene(MdP5CDH)and stress response Genes(MdRD22,MdDREB2A,MdRD29A)expression abundance,improve their drought and salt tolerance. |
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