| As a necessary trace element for plants,copper is widely used in pesticides and becomes a common pollutant in the environment.Excessive copper environment has toxic effects on plants,and plants have formed a variety of defense mechanisms in response to stress.WRKY transcription factors play an important role in the transcriptional regulatory response related to plant defense.Nelumbo nucifera has high ornamental value and can purify water.A large number of studies have shown that Nelumbo nucifera can resist metal pollution.The effects of different concentrations of copper on the growth and physiology of Nelumbo nucifera were studied and transcriptome sequencing was carried out.In this study,lotus cultivar ’ Yanyangtian ’ was used as the experimental material,and the transcriptome sequencing results of our research group were analyzed.The cloned gene NnWRKY22 was screened,and its transcriptional regulation function was studied.The main research results are as follows:(1)Nn WRKY22 gene was cloned.The full length of the gene was 573 bp,encoding 190 amino acids,containing a WRKY domain.The isoelectric point was 9.03,and there were no transmembrane domain and signal peptide structures.It was predicted that the subcellular localization was in the nucleus.The sequence expression analysis showed high homology with Corchorus olitorius L.,Durio zibethinus,Macleaya cordata,Theobroma cacao and Vitis vinifera.(2)The expression levels of NnWRKY22 were different in different tissues of lotus.The highest expression was in roots,followed by leaves and stems,and the lowest expression was in flowers,indicating that the gene mainly played a role in the roots of lotus.The expression level of Nn WRKY22 gene under copper stress was significantly higher than that under normal conditions.The difference increased with the increase of copper sulfate concentration,and the relative expression level reached the maximum after 7 d of stress treatment and began to decrease.(3)Subcellular localization to the nucleus is consistent with prediction.The inflorescence infection method was used to transform the model plant Arabidopsis thaliana,and 16 transgenic Arabidopsis thaliana strains were obtained.On the MS solid medium containing kanamycin resistance(kana),the transgenic Arabidopsis thaliana grew normally,and the wild-type Arabidopsis thaliana seedlings did not germinate or yellow and the roots did not grow.The OE-7,OE-8 and OE-15 transgenic lines were further selected for the next experiment.(4)The root length of transgenic and wild-type Arabidopsis seedlings was significantly different under copper stress or salt stress,and Nn WRKY22 gene played a certain regulatory role in abiotic stress.The chlorophyll fluorescence Fv/Fm and Fv/Fo of wild-type Arabidopsis thaliana were significantly lower than those of transgenic Arabidopsis thaliana under abiotic stress.The chlorophyll content measurement results showed that the chlorophyll a content of wild-type Arabidopsis thaliana was significantly lower than that of NnWRKY22 transgenic Arabidopsis thaliana,and the decrease of chlorophyll a was significantly higher than that of chlorophyll b,indicating that copper stress affected chlorophyll a content and reduced photosynthetic efficiency.At the same time,the activities of superoxide dismutase(SOD)and peroxidase(POD)of NnWRKY22 transgenic Arabidopsis thaliana were significantly higher than those of wild-type under abiotic stress.This indicated that NnWRKY22 gene could participate in increasing chlorophyll a synthesis and antioxidant enzyme activity under abiotic stress,thereby reducing the damage of stress on growth.Under copper stress,the relative expression of copper transporter COPT6 in transgenic Arabidopsis was significantly higher than that in wild-type Arabidopsis,indicating that NnWRKY22 gene may be involved in regulating copper transport pathway to increase copper resistance. |
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