| NAC transcription factor family is a kind of plant-specific transcription factor family,which is composed of NAM(no apical meristem),ATAF1/2(Arabidopsis transcription activation factor)and CUC2(cup-shaped cotyledon).It plays an important role in regulating plant growth and development and responding to abiotic stress.It is widely involved in plant growth and development,signal transduction and regulation under stress.In this study,an unknown functional protein OsNAC071 was screened and cloned by analyzing the data of FST gene expression profile chip.Through bioinformatics analysis,overexpression,RNAi and other binary vectors,the corresponding transgenic lines were obtained by Agrobacterium-mediated genetic transformation.Combined with subcellular localization,RT-PCR detection,GUS staining,phenotypic analysis and other aspects of its temporal and spatial expression patterns and functions,the main results are as follows:1.A NAC transcription factor family gene OsNAC071 and its promoter were successfully cloned.Bioinformatics analysis showed that the gene was located on chromosome 11.The ORF(Open reading frame)was 990 bp in length,encoding 329 amino acids.The molecular weight of the protein was 35.5 k D.It was a hydrophobic unstable protein,without signal peptide,no transmembrane structure,and contained a typical NAC domain.By constructing a phylogenetic tree,it was found that it was closely related to Ob NAC071 protein and had the same motif distribution,all of which were unknown functional proteins.Through protein interaction prediction,it was found that it interacted with multiple pollen fertility and seedling growth regulatory proteins.Promoter element analysis showed that the gene promoter contained a large number of hormone response,light regulation,stress induction and tissue-specific elements.2.Five fusion expression vectors,p OsNAC071 ∷ GUS and p OsNAC071 ∷OsNAC071:EGFP,were successfully constructed for functional study.p Ca MV35S×2∷ OsNAC071:EGFP for overexpression and subcellular localization;the RNAi interference vectors p Ca MV35S×2 ∷ OsNAC071:RNAi1 and p Ca MV35S×2 ∷OsNAC071: RNAi3 of different interference fragments were obtained,and the corresponding transgenic plants were obtained by Agrobacterium-mediated genetic transformation.3.Subcellular localization showed that OsNAC071 gene was located in the nucleus;RT-PCR detection showed that the expression of OsNAC071 was different in different parts of each stage.The expression of OsNAC071 was stronger in panicles and meiotic stems at each stage,followed by young panicle differentiation stage and meiotic leaves and sheaths,and roots at young panicle differentiation stage.GUS staining results showed that OsNAC071 was widely expressed in cell division and vigorous growth sites,especially in embryos during germination,root elongation and lateral roots of root hairs,internodes during meiosis,ovaries during meiosis and developing seeds.GUS staining and expression database analysis showed that abiotic stress drought,high salt and low temperature could induce the expression of OsNAC071.4.After inhibiting the expression of OsNAC071,the plant height,panicle length,total grain number per panicle,pollen fertility and seed setting rate of the two RNAi interference lines RNAi1 and RNAi3 decreased significantly,but did not affect the normal pollen germination and pollen tube elongation.5.The transgenic plants were subjected to high salt and drought treatment experiments.The results showed that overexpression of OsNAC071 enhanced the salt tolerance and drought resistance of rice,indicating that OsNAC071 gene could respond to a variety of abiotic stress and positively regulate the tolerance of rice to abiotic stress.6.By observing the root phenotype and paraffin section staining of each transgenic plant,it was found that there were differences in root robustness,lateral root development and lignin content in secondary xylem,fiber and thick wall tissues of each transgenic plant.The roots of overexpression plants were stronger,the number of lateral roots and roots were larger,and the content of lignin was higher,indicating that OsNAC071 would affect the growth and development of roots and the content of lignin,which in turn affected the drought resistance of rice.This study preliminarily explored the biological function and expression pattern of OsNAC071 gene in rice,which provided a theoretical basis for further exploring the function of NAC gene family in plant growth and development,laid a foundation for revealing the regulatory network of NAC protein,and opened up new space for molecular breeding of crop resistance to abiotic stress. |
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