Cloning And Expression Analysis Of CaWRKY8 Gene In Pepper

As one of the largest families of transcription factors in plants,the WRKY gene family is widely involved in many biological processes such as plant growth and development,material metabolism,senescence and stress resistance.In the previous stage,71 WRKY transcription factors were identified based on the whole genome sequencing database of pepper,and the first line of cDNA was used as template to clone the CaWRKY8 gene of pepper and bioinformatics analysis.The expression of CaWRKY8 gene in pepper under different hormones and stress treatments was studied,and the defense response of the gene may be analyzed.The main findings are as follows:1.Cloning and bioinformatics analysis of Ca WRKY8 geneThe open reading frame（ORF）of this gene is 1647 bp in length and encodes 548 amino acid residues.The 7 amino acids between 214～220 and 387～393 constitute the WRKYGQK conserved domain,at 234～264 and 407～438.There is a sequence of typical C2H2 zinc finger structures,belonging to Group I in the WRKY classification.Blastn analysis showed that the gene and NM₀01325015.1（E:0.0）,DQ402421.1（E:0.0）,NM₀01287907.1（E:0.0）,AB192884.2（E:0.0）,XM₀15224023.1（E:0.0）and NM₀01319981.1（E:0.0）are 99%,99%,91%,90%,89%and 89%,respectively.NCBI-BLASTp online comparison found that CaWRKY8 has the highest homology with capsicum hypothetical protein T459₁7066（PHT79014.1）,reaching 100%,with pepper WRKY25（PHU14775.1）,potato WRKY（NP₀01274836.1）,tomato prediction WRKY26（XP₀15079509.1）,tobacco prediction WRKY33（XP₀16492389.1）,kiwi WRKY（PSS09530.1）,grape prediction WRKY24（XP₀02272040.1）,soybean WRKY39（NP₀01348302.1）,cocoa tree WRKY（EOY34631.1）,cork The homology of cork oak WRKY24（XP₀23888322.1）was 99%,89%,89%,80%,67%,66%,66%,65%and 63%,respectively.The analysis of phylogenetic tree analysis software MEGA6.06 showed that the cloned CaWRKY8 gene had higher homology with pepper WRKY25,and it also had higher homology with potato WRKY,tomato prediction WRKY26,tobacco prediction WRKY33 and kiwi WRKY.2.Effect of exogenous hormone treatment on the expression of CaWRKY8 geneThe expression of CaWRKY8 gene in pepper was induced by exogenous substances SA,MeJA and ABA.The results showed that the relative expression of Ca WRKY8 gene in pepper was very similar after treatment with three different hormones.Under the three hormone treatments,the relative expression levels peaked at 12 h,which were 8.53 times,7.87 times and 6.52 times of the control group,respectively.3.Effects of stress treatment on the expression of CaWRKY8 geneAbiotic stresses such as high temperature,drought and salt damage can induce the expression of CaWRKY8.Among the three abiotic stresses,the up-regulation of CaWRKY8 gene was most significant after drought stress treatment;it was significantly up-regulated under different treatment time,and reached the peak at 121 h after treatment for 3 h,which was 121.10 times of the control group.The CaWRKY8 gene of pepper has high expression level under drought stress treatment.The relative expression of CaWRKY8 gene in pepper seedlings was significantly upregulated after inoculation with Phytophthora capsici,and the overall trend of inverted U-shaped was observed.The relative expression reached a peak at 12 h,which was 6.81 times that of the control group.Therefore,high temperature,drought,salt damage,Phytophthora and other stress can induce the expression of CaWRKY8.We predict that the CaWRKY8 gene is very similar to the regulation of signal transduction after treatment with phytohormone.