Some Results Of The Research On Plant Antifreeze And Disease-Resistance

This postdoctoral research work mainly focuses on two aspects: 1)isolation and identification of antifreeze gene in Ammopiptanthusmongolicus; 2) isolation and cloning of disease resistant gene in potato.A. mongolicus is a specific evergreen leguminous plant of Chinawhich lives in the temperate zone. One antifreeze protein which has highantifreeze activity was identified during my doctoral work period(thermal hysteresis acitivity is 0.9℃at 20 mg/mL). The purpose of thiswork is to amplify cold-induced cDNA fragment from the leaves of A.mongolicus by the 5' specific primer designed according to the sequenceof the 20 N-terminal amino acids of the antifreeze protein, and the3'primer—Oligo(dT)₁₆. A cold inducible cDNA fragment was isolatedand cloned, named am1. Squence analysis of am1 shows that it is 68ï¼…homologous to tobacco chloroplast F₀-ATPase subunitâ… andâ…¢. Southernblotting confirms that aml fragment derived from the genomic DNA ofA. mongolicus, but it is not sure whether am1 gene has double copies ornot. The result of Northern blotting shows that am1 transcripts appearedin 1 hour's cold treatment, and the transcription is constant within 5d'scold treatment. Furthermore, a cDNA library was constructed and prepareto scan for the full length of this cDNA squence.By the conservative character of R gene, PCR primers weredesigned according to the squence of NBS region. The genomic DNA ofdifferent potato varieties (Pseudomonas solanacearum resistant var.898006, P. solanacearum sensitive var. Zhongshu No.3, Trident, JinshuNo.1, Chunshu No.3, Wishchip etc.) were prepared and used as thetemplates of PCR in order to find some differences between the twovarieties, but haven't got any definite reults yet. Similarly, the cDNA ofresistant variety 898006 before and after inoculating with P.solanacearum was amplified and obtained two cDNA fragments. Thecloning and identification of these two fragments were under hand.Cloning P. solanacearum related gene from potato may be carried outthrough the following ways: 1) abolish the effect of inhibitors in potatoextraction, and establish high performance PCR system of potato; 2) usemore pair of PCR primers; 3) compare suitable P. solanacearum resistantand sensitive variety by AFLP technique.