| The lotus(Nelumbo nucifera Gaertn.)is one of China's ten traditional flowers and belongs to an aquatic plant of the genus Lonicera.Lotus is widely grown in China and has high economic value.However,with the prolongation of the planting time of lotus,various diseases will occur,among which the corruption of lotus is particularly serious.So far,there is no good chemicals to prevent lotus corruption.The disease has become an important obstacle to the cultivation and production of lotus flowers.There are few studies on the response of Lotus to spoilage and related molecular mechanisms.The regulatory mechanism is still unknown.In our previous production practice,it was found that flooding can enhance the resistance of lotus spoilage,and further through high-throughput sequencing analysis,histological data mining,physiological and molecular biological analysis,and genetics verification,initially revealing that NnWRKY41 responds to corruption in lotuses In the function.This study laid a foundation for exploring the molecular mechanism of lotus resistance.The main results are as follows:1.The WRKY gene may be involved in enhancing the disease resistance of lotus disease after submergence.In order to investigate the interesting phenomenon of enhanced of lotus after water flooding,we analyzed high-throughput sequencing techniques on the fully flooded Lotus.Under waterlogging stress,a total of 4002 genes were differentially expressed in lotus plants.Among them,1976 genes were up-regulated and 2026 were down-regulated.Functional annotation of these genes was performed by GO and KEGG enrichment analysis and they were found to be involved in redox,abiotic stress,cell metabolism and small molecule metabolism.Based on these data,previous studies and quantitative verification,we have constructed a flood response control network that involves several important approaches,including antioxidant systems,disease resistance,anti-anoxia and morphological adaptability.Further studies have confirmed that several WRKY genes associated with innate immunity are induced during flooding and can increase resistance to lotus spoilage diseases by regulating other resistance proteins.These results will provide useful information for the study of the molecular mechanism of the response of lotus to flooding stress.2.Whole genome identification and expression analysis of Lotus WRKY gene familyIn the above studies,it was found that multiple WRKY genes may play an important role in defending against diseases after flooding.Afterwards,we systematically identified and analyzed the WRKY gene family of lotus,including gene sequence,structure,conserved motif,evolution and Histoanalysis etc.to further screen candidate genes.We identified a total of 54 WRKY genes distributed on 14 large genome fragments from the entire genome of the lotus.Systematic tree analysis revealed that these genes can be divided into five subfamilies,including WRKY ?,?a,?b,?c,and ?.Gene structure analysis showed that the WRKYIIa subfamily contains the largest number of introns,suggesting that it may be the oldest one in the WRKY gene family.Most of the amino acid residues in the WRKY domain are highly conserved,and there are also certain variations,with dN/dS less than one.We found that 6 amino acid residues in the WRKY domain(875,878,879,881,912,and 914)may play a major role in the functional divergence of the WRKY family genes in the lotus flower.By pooling the data in the database,we revealed the expression patterns of various members of the WRKY gene family under different tissues,developmental stages,and stress conditions.In addition,we further analyzed high-throughput sequencing of WRKY gene expression under the infection of a spoilage pathogen and infection with water-flooded+ spoilage bacteria and found that the target gene NnWRKY41 was induced by infection with a spoilage pathogen and was associated with flooding-corruption disease.The expression level was further increased during treatment,revealing that NnWRKY41 may be a key factor mediating the enhancement of resistance to floral spoilage after flooding.3.Nn WRKY41 mediates the enhancement of resistance to submergence in waterfloodingIn order to verify that lotus flooding stress can enhance lotus disease resistance,we conducted flooding pretreatment analysis,The water-flooded pretreated 6-hour(Sub(uninfected)lotus leaf and non-flooded pretreated lotus leaf(Ck(no infection))were inoculated with Fusarium oxysporum.After 3 days,we observed that the lesion area(2 cm2)of Sub(infection)was significantly smaller than the lesion area(7 cm2)of Ck(infection).We screened and cloned the NnWRKY41 gene from the above studies and constructed a 35S::NnWRKY41 overexpression vector,which was transiently expressed in tobacco.Combined with the resistance phenotype and quantitative analysis of downstream disease-resistance genes,it was initially revealed that NnWRKY41 can be increased.Plants are resistant to disease and may play an important role in the prevention of disease susceptibility to spoilage after flooding. |
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