Universal, Rapid, and Visual Detection Methods for Phytoplasmas Associated with Coconut Lethal Yellowing Diseases Targeting 16S rRNA Gene Sequences.

Coconut lethal yellowing (LY) diseases caused by phytoplasmas are devastating diseases for coconut cultivation and seriously threaten the coconut industry around world. The phytoplasmas associated with the LY diseases belonged to six 16Sr groups containing 16SrI, 16SrIV, 16SrXI, 16SrXIV, 16SrXXII, and 16SrXXXII with comparatively higher variable levels. Conserved regions of the 16S rRNA genes of LY phytoplasmas belonging to the six 16Sr groups were obtained in the study. Based on the conserved region sequences of 16S rRNA genes, two sets of LAMP primers, Co-4 and Co-6, were designed and screened, and the rapid and visual detection methods universal for different groups LY phytoplasmas were established. The entire detection reactions of the universal detection methods could be completed with only 30 to 40 min of constant temperature amplification at 64°C, and the detection results were judged by the color changes of the reaction systems, which are convenient and quick. For the six groups of phytoplasmas, the estimated minimum detection limit range of the universal detection primers Co-4 and Co-6 were identical: 4.8 × 101 to 4.8 × 107 copies per 200 µl. The universal detection methods for the LY phytoplasmas established in the study are of great significance for the rapid diagnosis and identification and the efficient monitoring and early warning as well as the port inspection and quarantine of the LY phytoplasmas and their related diseases.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

Recent advances on the GAP promoter derived expression system of Pichia pastoris.

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.