Screening And Identificaiton Of Transcirpiton Factors Interacted With Fungus-inducible Core Element In BjC-P

Chitinase is a group of pathogenesis–related proteins. It can hydrolyze chitin, a major constituent offungal cell wall, and enhance the disease resistance of plants. BjCHI1, a gene identified in Brassicajuncea, encodes an unusual chitinase with two nearly identical chitin-binding domains. The expressionof BjCHI1was highly induced by Methyl jasmonate, wounding, caterpillar feeding and fungal infection,suggesting that the promoter of BjCHI1(BjC-P) might contain corresponding stress-inducible elements.Previous studies revealed that BjC-P was strong induced by fungi and the W–box–like–4(GTAGTGACTCAT) is the core element responsive to fungal infection. The fragment (409to371)coupled to the W-box-like-4element was needed for full magnitude of the fungal induction. In thisstudy, we used a wild type BjC-P fragment(desiganted Bait1) containing the W–box–like–4element andits mutatant Bait1–m (derived from Bait1by mutation of the W–box–like–4) to screen thetranscriptional factors from the Brassica juncea cDNA library by the yeast one–hybrid system. Thescreened transcriptional factors were analyzed by bioinformatics method. Furthermore, we intensivelystudied Bj26, one of the3transcription factors that specifically bind to the W–box–like–4element. Theresults are as follows:1. Eleven proteins interacting with Bait1were screened out from the Brassica juncea cDNA library.Among theem,3proteins, named Bj13, Bj26and Bj39, specifically interacted with Bait1, but not withBait1–m.2. Bioinformatics analysis showed that Bj13and Bj39are MYB-type transcription factors and Bj26isa C2H2-type transcription factor. The Bj13contains297amino acids (aa) with a molecular weight of32.6KD, an isoelectric point of6.29and two different conserved MYB domains. Bj39contains220aawith a molecular weight of24.1KD, an isoelectric point of5.88and one conserved MYB domain. Bj26contains244aa with a molecular weight of26.6KD, an isoelectric point of9.2and two typicalzinc–finger domains and two conserved QALGGH amino acid sequences.3. Alignments of Bj26CDS sequences with the C2H2–type proteins-encoding genes fromArabidopsis thaliana (At) and Oryza sativa (Os) and phylogenetic analysis (MEGA6) showed that Bj26shares higher similarity with those of Arabidopsis thaliana (At);4. Subcellular localization showed that Bj26locates in the nuclear;5. Transient gene expression in Nicotiana benthamiana leaves showed that Bj26can bind to the coreelement W–box–like–4and activate the function of BjC–P;6. qRT–PCR analysis showed that the Bj26expression obviously increased upon induction of thefungal elicitor (Hexa–N–Acetyl–Chitohexaose).The above results suggested that Bj26encodes a new C2H2–type zinc finger protein which canactivate the activity of BjC–P by binding to the fungus–inducible core element W–box–like–4and further mediate the process of plant response to fungal pathogen.