Research On Cloning And Anti-reverse Function Of CgWRKYs Gene In Cymbidium Goeringii

Cymbidium goeringii is one of the top ten famous flowers in China.It has a peculiar shape and elegant color,which is of great ornamental value.However,C.goeringii is susceptible to the adverse effects of abiotic stress during its growth and development.The molecular mechanism of C.goeringii to cope with abiotic stress has not been elucidated.WRKY protein is a large family of transcription factors and plays an important role in response to biological stress,abiotic stress and plant growth and development.So far,little is known about the C.goeringii WRKY gene family.Therefore,in this study,C.goeringii’songmei’was used as the material to obtain 6CgWRKY genes named CgWRKY2,CgWRKY3,CgWRKY21,CgWRKY24,CgWRKY53 and CgWRKY57 using RT-PCR technology for bioinformatics analysis.Real-time fluorescence quantitative PCR was used to investigate the expression patterns of 6 genes in different tissues and under different stress treatments.Finally,transgenic technology was used to transfer 6 genes into arabidopsis for functional analysis.The results were as follows:（1）Cloning of CgWRKY geneUsing Chunlan’Songmei’leaves as experimental materials,Chunlan RNA was extracted,c DNA was obtained by reverse transcription,and PCR amplification was carried out to isolate six CgWRKY genes.Among them,the protein encoded by the CgWRKY2,CgWRKY3,and CgWRKY24 genes contains two WRKY domains and one zinc finger structure（C₂H₂）,belonging to Group I of the WRKY gene family.The CgWRKY2 gene CDS is 2031 bp in length and encodes 676 amino acids with a protein molecular weight of 74.05 k D and a theoretical isoelectric point of 5.85.The CgWRKY3 gene CDS has a full length of 1491 bp and encodes 496amino acids with a molecular weight of 53.90 k D.The point is 7.37;the CgWRKY24 gene CDS has a full length of 1050 bp and can encode 349 amino acids with a protein molecular weight of38.90 k D and a theoretical isoelectric point of 8.77.The protein encoded by the CgWRKY21 gene contains a WRKY domain and a C₂H₂ zinc finger structure.Phylogenetic tree analysis indicated that the gene belongs to the IId subgroup of the WRKY gene family Group II.The CgWRKY21gene CDS is 1026 bp in length and encodes 341 amino acids with a protein molecular weight of37.63 k D and a theoretical isoelectric point of 10.00.The protein encoded by the CgWRKY53 and CgWRKY57 genes contains one WRKY domain and one zinc finger structure（C₂HC）,belonging to Group III of the WRKY gene family.The CgWRKY53 gene CDS has a full length of 1080 bp,encoding 359 amino acids,a protein molecular weight of 39.95 k D,and a theoretical isoelectric point of 6.15.The CgWRKY57 gene CDS has a full length of 912 bp and encodes 303 amino acids with a protein molecular weight of 33.67 k D.The theoretical isoelectric point is 5.23.（2）Expression pattern analysis of C.goeringii tissuesCgWRKY2 gene had the highest expression in leaves,while CgWRKY57 gene had the highest expression in roots.CgWRKY3,CgWRKY21,CgWRKY24 and CgWRKY53 were most expressed in flowers,among which CgWRKY3 and CgWRKY53 were most expressed in the labellum,CgWRKY21 was most expressed in petals,and CgWRKY24 was most expressed in sepals.（3）Expression patterns under different stressesCgWRKY2 gene was induced to express under low temperature and waterlogging stress,and responded to high temperature and ABA stress.CgWRKY3 gene was up-regulated and inhibited under low temperature stress,and responded to waterlogging and ABA stress.CgWRKY21 gene was up-regulated by low temperature stress,and inhibited by high temperature and ABA stress in response to waterlogging stress.CgWRKY24 gene was strongly induced by low temperature stress and responded to high temperature stress,waterlogging stress and ABA stress.CgWRKY53gene was induced to express under low temperature stress,and rapidly responded to high temperature stress,waterlogging stress and ABA stress,and its expression pattern was similar under these two stresses.CgWRKY57 gene was induced by low temperature and inhibited by high temperature in response to waterlogging and ABA stress.（4）Functional analysisComparing the morphological characteristics of overexpressing plants and wild-type Arabidopsis plants under normal growth conditions,it was found that overexpressing CgWRKY2gene plants were slow-growing,dwarfed,accompanied by pre-senescence phenotypes such as leaves and yellow;overexpression There was no significant difference in the morphology of CgWRKY3 gene plants;there was no significant difference in the morphology of CgWRKY21gene transgenic plants;the overexpressed CgWRKY24 gene plants were partially dwarfed;the overexpressed CgWRKY53 gene plants were dwarfed,the leaves were shrunk,and the hair was deformed;the CgWRKY57 gene was significantly dwarfed..The transgenic plants and wild-type Arabidopsis thaliana were subjected to low temperature stress and their phenotypes were observed.Compared with WT,the leaves of CgWRKY2 gene showed yellowing and no wilting.The leaves of CgWRKY3,CgWRKY53 and CgWRKY57 plants were dried and the plants were partially dead.The leaves of CgWRKY21 and CgWRKY24 were partially yellow,and some leaves were still green.Therefore,overexpression of CgWRKY2 gene may enhance the cold tolerance of Arabidopsis,while overexpression of CgWRKY3,CgWRKY21,CgWRKY24,CgWRKY53,CgWRKY57 genes may reduce the cold tolerance of the plants.The expression levels of cold stress and ABA response-related genes in transgenic plants under low temperature stress were analyzed.The results showed that overexpression of CgWRKY2 gene enhanced the expression of cold stress-related genes and inhibited the expression of ABA-related genes.Overexpression of CgWRKY3,CgWRKY21,CgWRKY53,CgWRKY57 The gene reduced the expression of cold stress and ABA-related genes;overexpression of CgWRKY24 gene inhibited the expression of At COR47,At RD29A and ABA responsive genes,and promoted the expression of At COR15A gene.It is speculated that CgWRKY gene may regulate the response of plants to cold stress by regulating the expression of At COR47,At COR15A and At RD29A genes downstream of cold signaling pathway,and may regulate cold signaling pathway through ABA signaling pathway.