Molecular Cloning Of Cinnamomin Gene Family And Study Of Antifungal Protein From Guilder Rose

This dissertation includes two parts: 1. Molecular cloning of cinnamomin gene family and study of their expression patterns Cinnamomin, which has three isoforms, is a type II ribosome-inactivating protein (RIP) purified from the mature seeds of camphor tree (Cinnamomum camphora). In a previous study, an incomplete cDNA that encoded the A- and B-chain of Cinnamomin but lacked signal peptide sequence was cloned. In the present paper, its full-length cDNA was obtained by 5.RACE. Subsequently, PCR amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products revealed three cinnamomin genes with >98.0% sequence identity. One of them corresponded to the published cDNA and was designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and cinnamomin III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III manifested that these two genes were functional. The three genes have no intron. Three Cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. Northern blot analysis demonstrated that the cinnamomin genes only expressed in cotyledons of C. camphora seeds and the acmes of expression emerged at 75-90 DAF when seeds were close to maturity. Based on the primary structure of 3 cinnamomin, the stereo structures of A- and B-chain were modeled by technique of bioinformatics. Finally, it is proposed that the three cinnamomin genes may encode three isoforms of Cinnamomin. The physiological function of Cinnamomin in C. camphora seeds is briefly discussed. 2. Purification and characterization of an ethylene-induced antifungal protein from leaves of guilder rose Phytopathogenic fungi infect crops during most periods of plant development and cause great loss to agriculture. In contrary, plants produce many types of antifungal proteins and peptides to defend the invasion of phytopathogenic fungi. These natural antifungal agents are constitutively expressed or induced in different organs and tissues of higher plants, and are regulated by normal developmental processes, ethylene and other plant hormones. Further study on the structure, function and regulation of these proteins will be greatly helpful for crop defense against fungi. In this paper, we report the purification and characterization of an antifungal protein from leaves of Hydrangea macrophylla. This ethylene-induced, chitin-binding protein (designated HM30) was purified to apparent homogeneity by chitin affinity chromatography followed by FPLC on a Superose 12 column. The molecular mass of HM30 was 30010.0 Da determined by mass spectrometry and its is...