Cloning And Function Study Of Cryptogein Gene From Phytophthora Cryptogea

Elicitins purified from culture filtrate of Phytophthora spp. are a class of small proteinaceous elicitors and act as a trigger in the plant-Phytophthora interactions. Cryptogein (Crypt) from .P cryptogea elicits the hypersensitive response (HR) and defense reactions in many plants at concentration of nanomolar level, including the transcriptional activation of defense-related genes and the synthesis of antimicrobial phytoalexins. Genetic engineering of the proteinaceous elicitor in plants may provide a clue of obtaining crops a wide spectrum of disease resistance. In this study, Crypt was manipulated to express in an inducible system and in a "low toxic" form by mutation of a key amino acid involved in HR. Transgenic tobacco plants were obtained by the method of Agrobacterium-mediated transformation. Some of the transgenic plants showed a wide spectrum disease resistance and enhanced salt tolerance. Results were summarized as follows:1 Cloning of Crypt and CryK13V genes Crypt gene was obtained by PCR amplification of genomic DNA of P. cryptogea withpfu enzyme, using a pair of primers designed according to the published sequences. The amplified fragment was subcloned into a T-vector and verified by DNA sequencing. Since the lysine residual at position 13 of Crypt plays a critical role in induction of cell death, overexpression of Crypt gene in tobacco may cause severe damage to the plants. Therefore, the lysine at position 13 was mutated to valine (K13V) through PCR site-directed mutagenesis. The fragment (CryKlBV) obtained was confirmed by enzyme digestion and sequencing.2 Construction of the plant expression vector Both Crypt and CryK13V genes were fused to a signal sequence of an extracellular pathogenesis-related (PR) Ib protein of tobacco, then the chimeric genes were cloned into a CHF3 vector under the control of the promoter of rice phenylalanine ammonia-lyase (PAL) gene and 35S promoter of cauliflower mosaic virus (CaMV35S), respectively.3 Tobacco transformation and obtaining of transgenic plants The twobinary vectors harbouring Crypt gene (CHF3-PAL-PRs-Crypt) and CryK13V gene (CHF3-35S-PRs-CryK13V) were transformed into tobacco by Agrobacterium-mediatedimethod via leaf disc, and 55 kanamycin-resistant plants were obtained. Most of the transgenic plants grew normally and set seeds. Firstly, the transgenic plants were detected by PCR amplification with a pair of specific primers. One band of the expected size (about SOObp) was obtained from 51 kanamycin-resistant tobacco plants, whereas no such band was amplified from the control plant. Further, twelve of the PCR positive transgenic lines were selected randomly for Southern blot analysis. The blot result indicated the integration of the transfer gene into tobacco genome with low copy numbers (1~3).Expression of Crypt and CryK13V genes in the transgenic plants was detected by Northern blot analyses. Only faint hybridization bands were observed by using [ a -32P]-dCTP labeled Crypt cDNA as probes even at low stringency wash condition. Alternatively, RT-PCR was used to determine the accumulation of mRNA of Crypt and CryKlSV genes in the transgenics. The band of expected size was amplified from total mRNA of transgenic plants by RT-PCR, and no such a band was observed in the non-transformants. These results indicated the exogenous Crypt and CryKlBV genes have been transcribed but at low level in the transformants.4 Enhanced resistance to pathogens in transgenic plants The transgenic plants Were challenged by black shank fungi Phytophthora parasitica var nicotianae (Ppn), brown spot fungi Alternaria alternata, and wild fire bacterium Pseudomonas syringae pv tabaci, respectively. Among the tested transgenic plants, 54.9% exhibited highly resistance to all the three pathogens and 80% showed enhanced disease resistance level compared with control.5 Salt tolerance test of transgenic plants The transgenic plantlets could grow normally with salt treatment (in 1/2 MS medium containing 200 mM NaCl) but a l...