| Hevea brasiliensis is the main source of natural rubber. The juvenile clone of Hevea brasiliensis has great potential a pplication as a new type of planting material, with fast growth speed, high volume production of rubber, and the advantages of strong resistance. Now the research on morphology, physiology and cultivation of Hevea brasiliensis juvenile clone had made considerable progress, and the volume production of rubber reached a certain level. But there is no sufficient evidence of molecular biology to directly prove its high-yielding mechanism. The molecular biology research of rubber is still backward, thus limited the production applications and large-scale promotion of Hevea brasiliensis juvenile clone.So it has important theoretical and practical significance to do some work on the biological function of differentially expressed genes in rubber latex of old and juvenile clone of Hevea brasiliensis. A differentially expressed fragment of latex in old and juvenile clone of Hevea brasiliensis was obtained through the suppression subtractive hybridization by our reseach group.It encodes a thioredoxin-like amino acid analyzed by BlastX. Because thioredoxin is a class of multi-functional acidic protein existed in vivo and can regulate the activity of some key enzymes during the photosynthesis process in plant. The study of thioredoxin in rubber tree is shortage, and its function is not clear, therefore, by using the information about the acquired sequence (HbSSH), the thioredoxin was cloned by the technology of RT-PCR and RACE, named HbTRX. Sequence analysis revealed that the ORF of HbTRX encodes 123 amino acid. The deduced amino acid sequences of HbTRX showed high identities (79%,78%,77%,77%,72%,69%) to those of thioredoxin from Ricinus communis, Populus trichocarpa, Vitis vinifera, Codonopsis lanceolata, Arabidopsis thaliana and Pseudotsuga macrocarpa. Bioinformatics shows that HbTRX was a member of TRX-family, with a similar CXXC domain.Semi-quantitative RT-PCR analysis indicates that the HbTRX is most highly expressed in callus, and more highly expressed in latex than in flower, in enbryos, in leaf and bud. And it is highly expressed in old clone of every type of rubber tree. The transcription of HbTRX was not induced in the latex by jasmonic acid and ethylene. In order to further study the function of HbTRX, construct HbTRX prokaryotic expression vector pET-30a-HbTRX, specific protein band was detected by SDS-PAGE in the cell homogenate by induction with IPTG in the E.coli cells harboring pET-30a-HbTRX produced recombinant proteins with a total predicted molecular mass of 21.296kD. The recombinant proteins is a form of soluble protein. Ultrasonic broked cells with recombinant proteins. The fact was that recombinant protein was a soluble protein in E. coli (BL21) by SDS-PAGE.In order to understanding the features and functionality of HbTRX protein, the study separated recombinant proteins by Ni2+ chelate affinity chromatography, and constructed two reaction system with the presence of DTT. The first reaction system is composed of 25μg/ml recombinant protein and bovine insulin, the second is composed of 50μg/ml recombinant protein and bovine insulin. The results showed that, the two groups displayed that the light absorption value was gradually increased under in the 650nm light waves. The light absorption value of the second reaction was higher than the first reaction, and both of reaction groups went into plateau 90min later. This shows that, the recombinant protein in E. coli, can reduce bovine insulin disulfides bridges, with weak redox activity.
|
PDF Full Text Request