Analysis Of Resistance Genes In The Wintersweet Flowers' ESTs, Chitinase Gene Cloning And Its Expression In E.coli

Chimonanthus praecox (L.) Link(Calycanthaceae) is a deciduous shrub and an endemic plant lived in China. It is famous for its landscape usage and medicinal value. Chimonanthus praecox (L.) Link could bloom at low temperature. Because of this, we believed that there must be some kinds of antifreeze materials in wintersweet flower. We constructed the wintersweet flower's cDNA library and analyzed some expressed sequence tags by bioinfomatics tools to screen biocontrol genes. Through this method, we could get the message of antifreeze mechanism in wintersweet flower.A cDNA library was constructed with SMARTTM cDNA Library Construction Kit using the RNA from the wintersweet flowers of the initiating bloom period .Then, we sequenced and analyzed some expressed sequence tags. The result of the analysis is that the primitive titer of the library was 1.16×105cfu/ml, the recombination efficiency of the cDNA library was 96.43%. We removed the part of vector and found the effective lengths of the insertion elements were between 600 bp and 800 bp. After randomly selected 1000 clones was sequenced and analyzed, we got 955 high-quality sequences,including 202 contigs and 745 singlets. After blast alignments on internet, 706 ESTs were genes of known function, 126 ESTs were genes of unknown function, and 123 ESTs were genes of putative function.Of all genes, genes of known and putative function accounted for 86.81%. Classified all genes of known and putative, we found that there were 54 antifreeze genes in resistance genes, including lipid transfer protein, late embryogenesis abundant protein, stearoyl acyl carrier protein desaturase, glyceraldehyde-3-phosphate dehydrogenase and so on. The analysis of those proteins were in favor of helping understand the antifreeze mechanisms. Chitinase is a spieces of induced glucosidase, which widespreadly resides in animal, plant and microorganism. Chitinases are enzymes that catalyze the hydrolysis of the beta-1, 4-N-acetyl-D-glucosamine linkages in chitin polymers. In usual, it is a low level of chitinase in most plants, but it is a high level of chitinase when bacteriumand virus aggressed the plant. Some reports illuminated the mechanisms of biocontrol, but there have been little test about its antifreeze mechanisms. We blasted the ESTs and found an EST which encoded the ClassⅠb chitinase in cell outside. We extracted the apoplastic protein from the wintersweet flower,and found that the chitinase was existed in cell outside. Some reports indicated that the chitinase was antifreezed, we hoped to cloned the gene and expressed in E.coli, then authenticated its function. It could be an important theoretical significance and economic value to get the message of chitinase's function.