Preliminary Studies On Curcin Genes And Their Promoters In Jatropha Curcas L.

Four putative full-length curcin genes, termed curA2, cur A3, curB2 and curB3 respectively, were isolated by the polymerase chain reaction (PCR) amplification according to a full-length curcin gene sequence data and revealed to encode the type-1 Ribosome-Inactivating Proteins (RIPs). The sequence alignment and analysis of the four putative curcin genes and the other two reported curcin genes indicates that the curcin gene-family in the Jatropha curcas genome can be divided into two subfamilies. The identity of the gene sequences in the two subfamilies is 86.5%, and the identity of the gene sequences in each subfamily is 91.5% or 99.6%. One subfamily's expression might be induced by stresses, such as fungi, low temperature and drought. The other subfamily might express in the endosperm of physic nuts. Genomic Southern blot suggests there are three or more curcin gene copies in the genome of Jatropha curcas.Five DNA promoters of curcin genes, termed cpa1, cpa2, cpa3, cpa4, cpa5, in the Jatropha curcas genome, were amplified by PCR amplification. Sequence comparison shows that the fragments are different in some parts from the existed sequence data and some cis regulatory elements are concerned in these parts. Analysis of these promoters' sequence data suggests these promoters root in threecurcin genes. Subsequently, the five promoters were inserted into pBI121 vectorlarge fragments generated from digesting pB1121 by BamH â…  and Hindâ…¢ to construct the expression vectors of the promoters.The results of tobacco leaves transient expression assay and histochemical GUS activity staining show cpal and cpa2 can promote GUS activity after 60hr co-culture, but the other promoters, cpa3, cpa4, cpa5, are not able to promote GUS activity in this period. In the leaves of the transgenic tobacco regenerated plantlets, all the promoters cannot promote GUS activity. This results suggest cpal and cpa2 can be induced by some signals, but cpa3. cpa4, cpa5 may act differently.This research is an important foundation to study the expression and regulation mechanism and molecular evolution of curcin gene-family.