Abstract:

【Objective】The research purpose is to figure out the expression patterns of defense genes, screen candidate genes related to resistance to Fusarium wilt, and further clarify the resistance mechanism. 【Method】A normalized cDNA enriched in full-length sequences was constructed from roots of banana cultivar Nongke No. 1 inoculated with FOC race 4 using DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′ end of the RNA transcript) technique. Furthermore, the expressions of defense genes were considered with multiplex semi-quantitative PCR. 【Result】The liter of original cDNA library was 3.1×106 cfu•mL-1, and amplified library was 2.8×108 cfu•mL-1. Then, 421 unigenes containing 42% unknown genes from the sequenced results of 580 clones selected randomly were obtained after the removal of low-quality sequences. The average size of cDNA inserts ranged from 750 to 3 000 bp with a recombination rate of 100%. The rate of full-length gene was 68%. Some protein genes associated with defense were obtained on the basis of cDNA library. By RT-PCR analysis, the expression levels of phenylalanine ammonialyase (PAL), catalase (CAT), β-1,3-glucanase (PR-2) and pectin acetylesterase (PAE) in non-inoculated condition were very low. However, seven defense genes were obviously increased after inoculated by FOC race 4, and peroxidase (POX), endochitinase (PR-3), PAE and PAL reached the peak in the sixth day. Though the expression patterns of seven defense genes were similar with the controls, the intensities were much less than the inoculations. 【Conclusion】 These results indicated that the normalized full-length cDNA library was established successfully, which can be used for isolating required gene in the light of the rate of recombinant and integrity. Meanwhile, seven defense genes were identified, which played a key role in resistance to Fusarium wilt in some extent.

Key words: banana, Fusarium wilt, cDNA library, defense genes, expression