| Nelumbo nucifera is the only aquatic flower among the top ten traditional flowers in China,which has high ornamental value,economic value and ecological value.However,in terms of the current environmental form,the area of saline-alkali land is still increasing,and the growth and development of lotus are limited.As a unique and large number of transcription factors in plants,NAC transcription factors play an important role in plant growth and resistance to external environmental stress,and are important candidate genes for improving plant stress resistance.In this study,the NnNAC35 transcription factor gene of lotus was cloned from the salt-tolerant variety ’Huangshuai’,and its expression pattern and function were preliminarily discussed,which provided a theoretical basis for further elucidating the regulation mechanism of NnNAC35 gene in lotus response to saline-alkali stress.The main results are as follows :(1)NnNAC35 was cloned from the leaves of Nelumbo nucifera.The full length of the gene sequence was 894 bp,encoding 297 amino acids.It had a typical NAC domain and belonged to the NAC transcription factor superfamily.The molecular weight of the protein was 34.47 k Da.There was no transmembrane structure and signal peptide,and it was an unstable hydrophilic protein.The content of random coil structure was the highest,accounting for 56.23%.Multiple sequence alignment showed that it had the highest homology with At NAC036,and phylogenetic analysis showed that it had the closest genetic relationship with Dl NAC6 gene.(2)The expression pattern of NnNAC35 was analyzed by q RT-PCR.The results showed that NnNAC35 was expressed in roots,petioles,leaves and flowers of lotus under normal growth,and the expression level was higher in leaves and roots,with tissue expression specificity.Saline-alkali stress treatment could significantly induce the expression of NnNAC35 gene in petiole.(3)The plant expression vector p C2300-NnNAC35-GFP was constructed,and the tobacco leaves were infected by Agrobacterium-mediated method to observe its subcellular localization.It was found that the gene had a fluorescence signal in the nucleus of tobacco leaf cells,indicating that the gene was a nuclear localization protein.The p GBKT7-NnNAC35 and its segmented expression vector were constructed and verified by yeast self-activation activity.The full-length and C-terminus of NnNAC35 have transcriptional self-activation activity,while the N-terminus does not have activation activity.NnNAC35 is a transcriptional activator.(4)The overexpression and gene silencing lotus were constructed by transient transformation method,and the function of NnNAC35 gene was preliminarily verified by gradient mixed saline-alkali stress treatment.Under mixed saline-alkali stress,the leaves of overexpressing lotus showed obvious yellowing,the total chlorophyll content was significantly lower than that of wild type,the malondialdehyde(MDA)content was significantly higher than that of wild type,the superoxide dismutase(SOD)activity and soluble protein content increased at low concentration,and decreased significantly at high concentration.The photochemical efficiency of gene silencing lotus decreased,the chlorophyll content was significantly higher than that of wild type,and the MDA content under high concentration treatment was significantly lower than that of wild type.It shows that overexpression of NnNAC35 gene can affect the response of transgenic lotus to mixed saline-alkali from many aspects such as photochemical efficiency,chlorophyll content and antioxidant enzyme activity.The mixed saline-alkali stress tolerance of the gene silencing plants was slightly better than that of the overexpression genotype lotus,but similar to that of the wild-type lotus.In contrast,overexpression of NnNAC35 gene can increase the sensitivity of lotus to saline-alkali treatment and limit its growth and development under saline-alkali stress.(5)The transgenic Arabidopsis thaliana was obtained by inflorescence infection method,and four positive strains with different degrees of overexpression of NnNAC35 gene were selected for functional analysis and verification under saline-alkali stress.Under single salt and alkali stress,the seed germination rate of overexpression lines was lower than that of WT,and the inhibition of root elongation was significantly higher than that of WT,but the number of lateral roots of transgenic Arabidopsis thaliana increased significantly,indicating that NnNAC35 may be involved in regulating the growth of lateral roots of Arabidopsis thaliana under stress to make it have a certain degree of stress tolerance in seedling stage.Under mixed saline-alkali stress,compared with wild-type Arabidopsis,the active oxygen scavenging ability and osmotic adjustment ability of overexpressed plants showed different trends due to the different degrees of overexpression of the lines,and the expression of stress-related genes was significantly down-regulated,indicating that overexpressed Arabidopsis showed different degrees of saline-alkali stress sensitivity.In general,the saline-alkaline tolerance of transgenic Arabidopsis may be negatively correlated with the degree of overexpression of NnNAC35 gene. |
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