Screening Of Proteins Interacting With Phytophthora Sojae Elicitins In Tobacco

Soybean Phytophthora root rot caused by Phytophthora sojae Kaufmann et Gerdemann, is one of the most devastating diseases in soybean production of the world. In the long run, the most economic and effective measures to control this disease is developing resistant cultivars. However, the resistance of cultivars is easily loss due to the high genetic variability of isolates. Mining durable resistance genes has become a key problem in disease resistant breeding. Unlike the gene-for-gene resistance governed by single resistance (R) genes, the non-host resistance is the most effective and lasting resistance mechanism in plant. Thus, identifying the key genes involved in the response of nonhost resistance to P. sojae may be helpful to using genetic engineering to create lasting and broad-spectrum resistant materials. The main purpose of this study is to find genes involved in signal recognition and resistance response of non-host Arabidopsis and Nicotiana to P. sojae, and give new evidence to explain the molecular mechanism of non-host resistance to P. sojae. The main results were as follows:1. To search the receptor of Phytophthora sojae elicitin protein, a bait vector of P. sojae elicitin gene was constructed for using in yeast two-hybrid system, and then effect of the vector on the yeast cell growth and the self-activating function were tested. The target segment in TOPO vector containing P. sojae elicitin gene was amplified by PCR and was cloned into the bait vector PGBKT7. The recombinant plasmids were transferred into yeast strain AH109. The testing results showed that constructed vector containing elicitin protein was no activating function to reporter gene in AH109 and was no poisoning to the yeast. This bait vector constructed was the basis to find receptor proteins which were interacted with P. sojae elicitins from plant cDNA library.2. A dscDNA was Synthesized with common tobacco K326 leaves by using cDNA Library Construction Kit.Total RNA and LD-PCR products were screened by Electrophoresis.The result indicate that the quality and integrality of total RNA was good and obtain cDNAs were from 0.5-5kb.3. Proteins interacted with the ELICITIN-like and ELICITIN were screened from Nicotiana by indicate that yeast two-hybrid system. To ELICITIN-like PPSE7 and ELICITIN SOJB, 11 proteins and 14 proteins from Nicotiana were screened. The results indicated that both PPSE7 and SOJB have the same binding protein in Nicotiana, suggested that PPSE7 and SOJB may share some similar biological function. In Nicotiana, ubiquitin-conjugating protein and peptidase were found binding with SOJB but not PPSE7. The functions of them were involved in protein degradation. Both SOJB and PPSE7 can bind the transmembrane transport protein, SOJB with copper transporter protein, and PPSE7 with water channel protein. The results suggested that the target of SOJB and PPSE7 may be located in cytoplast.5. For further functional analysis on the candidate genes, gateway compatible RNAi expression vector containing 8 target genes were constructed.