Cloning And Expression Analysis On Of Cysteine Protease Gene HbCP1 From Hevea Brasiliensis

Natural rubber, which is a plant secondary metabolism, has been well recognized as an important raw industrial material due to its unique properties that can not be substituted by any alternative synthetic ones. The rubber tree (Hevea brasiliensis) has been the only commercial source of natural rubber mainly because of the abundance of rubber in the tree,its quality, and the ease of collection. The morphology,physiology,or cultivation of Hevea brasiliensis are well understood, the yield is also very large, but compared with other crops, the research about Hevea brasiliensis molecule biology is lagged correspondingly.Natural rubber synthesis depends on laticifer system. Latex, which is the cytoplasm of laticifer cell, contains a number of proteins and enzymes .The roles of those latex proteins and enzymes are as yet poorly understood. Cysteine protease is one of the important protease families found in plants, which is generally involved in seed germination, seedling development, stress response, organ differentiation and senescence, or programmed cell death(PCD) et al. It has important academic meaning and applied value to study potential function of cysteine protease in Hevea brasiliensis. In this reach, we cloned a cysteine protease gene, analyzed its expression and did some forecast about its protein function.A pair of degenerate primers was designed according to the conserved regions which encode the cysteine proteases activesites in cysteine proteases genes. Using the method of PCR and RACE, cloned a cysteine proteases gene from latex named cysteine proteases1(HbCP1) (GenBank,DQ642886).The HbCP1 has a 1597 bp full length cDNA sequences with an ORF of 1374 bp, which encode 457 amino acid residues with a total predicted molecular 50.63KD, a theoretical pI 5.37. The deduced amino acid sequences of HbCP1showed high identities of 85%,76%,74%,70% and 70% to those of the cysteine proteases from Manihot escu lenta, Phaseolu svulgaris, Platycodon grandiflor s, Arabidopsis thaliana, Zea mays.Biology informatics analysis indicated that HbCP1 has a N-terminal signal peptide composed of 16 amino acid residues and three domains Peptidase C1A, GRAN and RTX. Peptidase C1A contains cysteine proteases activesites: THIOL_PROTEASE_CYS(147-158) Eukaryotic thiol (cysteine) proteases cysteine activesite, THIOL_PROTEASE_HIS (287-297) Eukaryotic thiol (cysteine) proteases histidine activesite) and THIOL_ PROTEASE_ASN(304-323) Eukaryotic thiol (cysteine) proteases asparagine activesite. GRAN is a granular protein, whish is concerned with wounding restoration. RTX is domain of bacterial toxins, which has not been found in cysteine proteases in plant. HbCP1 was a no-transmembrane stable hydropathic protein, predicted that it located in the vacuole and maybe had the function of signal- transducer and stress-response.Southern analyses of genomic DNA suggest that there are low copies of HbCP1 gene in rubber trees.The expression of HbCP1 gene was determined using RT-PCR analysis. The HbCP1 mRNA levels are high in latex, and little in young leaf and bark. Ethylene and wounding could induce HbCP1 gene expression, whereas jasmonic acid had little effect. There was no obvious difference in HbCP1 gene expression between in various organizations of healthy tree and in TPD tree.The results of pilot study indicated that HbCP1 was concerned with ethylene signal transmission, could respond to wounding stress, it also maybe had defense function in the latex.Four procaryotic expression vectors pET-30a-HbCP1, PGEX6P-1-HbCP1, pET-30a-EHbCP1 and PGEX6P-1-EHbCP1 were constituted and were expressed in E. coli. The result revealed that the HbCP1 has physiological toxicity to the host, which blocked the expression of itself. Moreover, the reason of HbCP1 had physiological toxicity was RTX domain.