Construction Of A CDNA Library For The Wild Watermelon PI296341 And Cloning ClWRKY1 Transcription Factors Gene

Fusarium wilt of watermelon (Citrullus lanatus Thunb.) caused by Fusarium oxysporum f. sp.niveum is the most important soil borne pathogen limiting watermelon production throughout the world. Fusarium from the soil can infect roots. Management strategies to avoid Fusarium wilt epidemics rely on grafting and rotating crop. However, these two methods are time- and labor-intensive. Therefore, the bio-engineering to improve crop resistance to Fusarium wilt is the most cost-effective methods. The objectives of the present study are to construct a full-length cDNA library of watermelon PI296341 root tissues induced by Fusarium wilt pathogen with SMART method,and to identify and clone the WRKY transcription factor.The results are as follows:1.A full-length enriched cDNA library of watermelon PI296341 were constructed, which the titter of primary cDNA library was 2.21×106 pfu/mL containing 1.11×106 independent clones and library amplified was 2.0×1010 pfu/mL .The insert fragment size ranged from 0.5 kb to 2.0 kb.2. The cDNA fragments of WRKY transcription factors were obtained by RT-PCR. Using RACE technique the whole WRKY gene ClWRKY1 was cloned. The nucleotide sequence of ClWRKY1 cDNA was 1,981 bp, which contained an open reading frame (ORF) of 1,185 bp encoding a putative polypeptide of 394 amino acids. The molecular mass of the predicted protein was 43.77 kDa and the isoelectric point was calculated to be 7.17.It's accession in GenBank is HM0308793. The bioinformatic analysis showed that ClWRKY1 protein without signal peptide and transmembrane domain, which localized in the nucleus. The putative functions were predicted, e.g. it may have transcription, transcription regulation, signal transducer, stress response and immune response activity.4. The amino acid sequence of ClWRKY1 protein contained two conserved WRKY domains with Zin-finger motif C-X4-5-C-X22-23-H-X-H, belonging to the groupⅠof WRKY protein5. Homology comparison and cluster results showed that the ClWRKY1 gene and several kinds of grapes were grouped together.