| Leaf senescence is the last stage of plant development.During leaf senescence,resources are degraded and transferred to new organs to maintain growth.According to delaying senescence,the growth period of plants can be extended.In the previous study,the acid phosphatase gene ZmAPRG was located in the target segment of 546 kb,in which 5 genes with functional annotation were included.Among them,it was found that the CDS sequences of ZmVQ52 gene in the two parents 082 and 107 were not different,while the promoters were different.Therefore,this study mainly focused on the ZmVQ52 gene,and used the CaMV 35 S promoter to drive the overexpression of ZmVQ52,so as to obtain transgenic Arabidopsis thaliana for subsequent studies.In this study,ZmVQ52 gene was cloned from maize inbred line B73 and its tissue specific expression was analyzed.Subcellular localization,interaction protein analysis,phenotype identification of transgenic Arabidopsis thaliana,hormone treatment,transcriptome sequencing and expression verification,it was found that ZmVQ52 gene plays an important role in the aging mechanism of maize.The main results of this study are as follows:(1)The full-length open reading frame(ORF)of ZmVQ52 gene is 576 bp,encoding 191 amino acids.After cloning,the CaMV-ZmVQ52 plant expression vector was constructed.The transgenic Arabidopsis thaliana strains were obtained by agrobacteria-mediated genetic transformation.The homozygous strains OE4 and OE5 of transgenic Arabidopsis thaliana were screened by hydomycin resistance and detected by PCR.(2)qRT-PCR was used to analyze the expression of ZmVQ52 gene in the roots,stems,leaves,ear and tasssel of maize inbred line B73 at the maturity stage.The results showed that ZmVQ52 was the most expressed in maize leaves and the least expressed in roots and stems.(3)The fusion protein expression vector of GFP-ZmVQ52 was constructed and co-located with the nuclear Marker plasmid by using the methods of maize protoplast extraction and instantaneous expression.It was found that the ZmVQ52-encoded protein was located in the nucleus and played its biological function as a nuclear protein.The interaction between ZmVQ52 and ZmWRKY20,ZmWRKY36,ZmWRKY50,ZmWRKY71 was found through Bimolecular fluorescence complementary experiment and the screening of the interaction proteins by the maize protoplast transformation method.(4)The overexpression of ZmVQ52 accelerated the leaf senescence of transgenic Arabidopsis thaliana,and it was found that the leaf yellowing was serious,the wilting aging was accelerated,and the chlorophyll content was reduced.The molecular mechanism of ZmVQ52 in transgenic Arabidopsis was analyzed by q RT-PCR in combination with the observed leaf senescence phenotype of ZmVQ52 transgenic Arabidopsis strains obtained from genetic transformation.Overexpression of ZmVQ52 promoted up-regulated expression of SAG12 and SAG13,up-regulated expression of WRKY53 and ORE1,and down-regulated expression of CCA1 and GLK2.(5)ZmVQ52 transgenic Arabidopsis thaliana treated with SA and JA showed rapid senescence of Arabidopsis thaliana leaves,the expression of the response genes PDF1.2a and PDF1.2b in JA pathway was up-regulated,and the expression of the response genes PR1 and PR2 in SA pathway was also significantly up-regulated,indicating that the overexpression of ZmVQ52 enhanced the sensitivity to JA and SA.ABA treatment showed that the taproot length of transgenic Arabidopsis thaliana increased relative to that of wild type plants,indicating that the overexpression of ZmVQ52 gene enhanced the tolerance to ABA.(6)Sequencing analysis of transgenic Arabidopsis thaliana line ZmVQ52-OE5 and WT transcriptome revealed that the differentially expressed genes were mainly concentrated in the circadian rhythm and photosynthetic pathway,suggesting that the gene may regulate leaf senescence through photosynthesis and circadian rhythm.Subsequently,gene expression was verified by qRT-PCR,indicating the reliability of transcriptome sequencing analysis. |
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