Walnut JrWRKY4 And JrWRKY21 Transcription Factor Functions In Response To Infection By Colletotrichum Gloeosporioides

Walnut?Juglans regia?is an important nut tree species.With the rapid increase of cultivated area,serious occurrence of walnut anthracnose caused by Colletotrichum gloeosporioides.It causes fruit gangrene to fall off,resulting in a significant loss of walnut yield and quality.It can harm the shoots and leaves,resulting in debilitating or even death of trees.It is a catastrophic disease in the development of the walnut industry.In this experiment,the JrWRKY4 and JrWRKY21 of walnut were cloned from‘Ruijia'cultivars and their functions were studied,the main results are as follows:1.The WRKY of walnut were downloaded from PlantTFDB 4 database and 71 walnut WRKY transcription factors were screened.The isoelectric point and molecular weight of walnut WRKY were predicted by Ex PASy Proteomics Server.The phylogenetic analysis of walnut WRKY transcription factors was performed by MEGA 5.0 software.The WRKY transcription factors of walnuts were classified into 3 group??,?,??reference the grouping of Arabidopsis thaliana.Class I contains 13 WRKY,each transcription factor contains 2 WRKY conserved domains.Class II contains 49 WRKY,and Class II can be subdivided into IIa,IIb,IIc,IId,and IIe.IIa has 5 WRKY,IIb has 8 WRKY,IIc has 16WRKY,IId has 12 WRKY,IIe has 8 WRKY.Class?has 9 WRKY transcription factors.2.The JrWRKY4 and JrWRKY21 of walnut were cloned from the disease-resistant variety'Ruijia'.The open reading frame of JrWRKY4 was 1554 bp and encodes 517 amino acids.It contains two conserved WRKY domains and has a zinc finger structure of C₂H₂type.It has 71.16%homology with Prunus avium,belonging to the first class of the WRKY family.The open reading frame of JrWRKY21 was 1035 bp and encodes 344 amino acids.It contains a WRKY conserved domain with a zinc finger structure of C₂H₂ type.It has82.02%homology with Prunus avium,and has a close genetic relationship.It belongs to the IId of the WRKY family.Subcategories.3.Real-time PCR was used to detect the expression of JrWRKY4 and JrWRKY21 in different tissues of walnut.We found that JrWRKY4 and JrWRKY21 are expressed in different tissues,but there were significant differences between tissues.The expression of JrWRKY4 and JrWRKY21 were highest in mature leaves.4.The walnut leaves were infected by C.gloeosporioides and the expression changes of JrWRKY4 and JrWRKY21 were detected at 0,3,6,12,24,48 and 72 hours after infection.We found that the expression trends of JrWRKY4 and JrWRKY21 were consistent,and reached the highest expression at 6 h.The relative expression level of JrWRKY4 reached 28times at 6 h and JrWRKY21 reached 63 times.The expression level then began to decline and was lower than the contrast.This suggests that the JrWRKY4 and JrWRKY21 may be involved in the early response to walnut anthracnose stress.5.The plant fusion expression vector was constructed and the leaves of Nicotiana benthamiana were transiently expressed by Agrobacterium-mediated method.we find that both JrWRKY4 and JrWRKY21 proteins were localized in the nucleus in two-photon laser confocal microscope.6.The JrWRKY4 and JrWRKY21 of walnut were overexpressed in Arabidopsis thaliana and the transgenic A.thaliana was identified for resistance to anthracnose.We found that compared with wild-type A.thaliana,overexpression of JrWRKY4 and JrWRKY21 A.thaliana,incidence was late,and the symptoms were mild at the same time after infection by C.gloeosporioides.The resistance to anthracnose of the transgenic plants was significantly increased.