| Strawberry(Fragaria × ananassa)is one of the most popular fresh fruits in the world.It tastes delicious and contains many nutritional elements such as vitamins.China is the largest producer of strawberries in the world,but it is very vulnerable to various diseases during strawberry cultivation.Gray mold is one of the three major diseases that harm the growth of strawberry.It can not only lead to reduced production of strawberry,but also cause large-scale decay of strawberry after harvest,which brings great loss to strawberry industry.At present,chemical control is one of the means to prevent gray mold.However,frequent use of fungicides makes the drug resistance of pathogen continuously enhanced,and the control effect is worse and worse.It is imperative to explore the key genes of strawberry resistance to gray mold and carry out molecular breeding for strawberry resistance.In this experiment,7different wild species and 28 different cultivated species of strawberry were inoculated with Botrytis cinerea.By observing the phenotype and calculating the disease index,a disease-resistant variety Fragaria pentaphylla was screened out.We inoculated B.cinerea at different time points,conducted transcriptome sequencing analysis,screened out the key gene WRKY33 resistance to B.cinerea,and further cloned FpWRKY33 cDNA sequence(GenBank accession number:OQ555767),the overexpression vector was constructed and transformed into Fragaria vesca ’Hawaii 4’ by Agrobacterium to study the disease resistance function of FpWRKY33.The results obtained are as follows:1.The sporulation suspension of B.cinerea was inoculated into 7wild strawberries and 28 cultivated strawberries,the disease index of different wild and cultivated strawberries were calculated.The wild strawberry Fragaria pentaphylla showed the highest resistance to gray mold.2.Used F.pentaphylla as experimental materials,inoculated with B.cinerea,and sampled at 0,12,24,48,72,and 120 h for transcriptome analysis.The results showed that 272 continuously up-regulated differentially expressed genes were found during B cinerea infection.Through GO enrichment and KEGG enrichment analysis,it was found that these 272 differentially expressed genes were mainly enriched in the processes of plant hormone signal transduction,MAPK signal pathway,and brassinosteroid biosynthesis,indicating that F.pentaphylla may resist the infection of B.cinerea through the hormone pathway and the synthesis of disease-resistant substances.The key resistance gene WRKY33 was identified by RNA-Seq technology from MAPK signaling pathway where differentially expressed genes were significantly enriched3.Cloned the cDNA sequence of FpWRKY33 from F.pentaphylla.The open reading frame length was 1554 bp,encoding 517 amino acid residues and containing two WRKY conserved domains.Constructed the overexpression vector,the overexpressed vector was transformed into forest strawberry ’Hawaii 4’ by Agrobacterium EHA105 by leaf disc method.4 positive transgenic strawberry ’Hawaii 4’ was identified by DNA and RNA levels.Wild type strawberries and transgenic strawberries were inoculated with B.cinerea,Phenotypic results showed that transgenic strawberry with FpWRKY33 enhanced resistance to gray mold.The RT-q PCR results showed that defense genes FvCOI1、FvJAZ1 and FvPDF1.2 were significantly up-regulated in transgenic strawberry compared with wild-type strawberry,suggesting that FpWRKY33 may induce up-regulated expression of these genes by activating JA pathways to improve resistance to gray mold. |
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