Abstract：Cysteine proteinases (CP), an essential component of proteolysis are widely implicated in physiological processes in plants, e.g. plant growth and development, biotic and abiotic stresses. The full-length cDNAs of 2 CPs were cloned from the latex of para rubber tree(Hevea brasiliensis), named HbCP2 (1 245 bp) (GenBank accession No. KF771829) and HbCP3 (1 268 bp) (GenBank accession No. KF771830), and predicted to encode proteins of 41 and 40 kD, respectively. The genomic DNA sequences of HbCP2 (1 919 bp) and HbCP3 (1 634 bp) shared the same genomic structure of 3 introns and 4 exons. The 2 Hevea CPs were classified into the papain C1 family based on the existence of a typical papain C1 domain and phylogeny analysis. Homology analysis showed that HbCP2 was highly homologous to a CP homolog of Ricinus communis (86.86%), whereas HbCP3 homologous to a CP homolog of Jatropha curcas (84.74%). Real-time PCR analysis revealed that HbCP2 and HbCP3 showed different tissue expression patterns as assayed among Hevea tissues (leaf, bud, female flower, male flower, seed, young bark, mature bark and latex). HbCP2 had the highest expression in male flowers, followed in mature and young barks, and the least in buds. In comparison, HbCP3 showed predominant expression in latex, followed in leaves, and much less expression in the 6 other tissues with the least in buds and mature barks. Expression of HbCP2 and HbCP3 in the latex were regulated to different levels by the treatments of tapping, wounding, ethrel, GA and JA, not affected by the treatments of 2, 4-D, ABA and SA. In virgin rubber trees, with the process of tapping cuts, the expressions of HbCP2 and HbCP3 showed a similar manner of first down-regulation and then gradual recovery. HbCP3 expression decreased with the time of wounding treatment, whereas HbCP2 expression showed a fluctuation with increased wounding treatment. Hormones treatment showed overall low regulation on HbCP2 and HbCP3 expressions, only GA treatment led to a more than 2-fold expression change in gene HbCP2 of the 6 hormones examined. In addition, the expression of HbCP3 in Hevea leaves was apparently regulated by Corynespora inocualtion. In comparison, HbCP2 expressed stably at 3 d after Corynespora infection, increased significantly at 5 d, which most possibly resulted from leaf senescence. These results suggested that HbCP2 might play a role in stress responses and tissue senescence regulation, whereas HbCP3 might be involved in the regulation of latex regeneration and disease response in Hevea tree. The study provides useful information for research on cysteine protease gene family which regulates latex regeneration and stress response of rubber tree.
