[Objective] To investigate the effects of mitochondrial unfolded-protein response (UPRmt) on the aggregation toxicity of Aβ protein in Alzheimer's disease (AD).[Methods] By cloning the mitochondrial outer membrane tomm-22,inner membrane E04A4.5 and atfs-1 genes of Caenorhabditis elegans (C.elegans) and constructing the L4440 interference vectors,HT115 competent cells were transformed to prepare tomm-22,E04A4.5 and atfs-1 RNAi bacteria.The effects of tomm-22 and E04A4.5 RNAi on the process of paralysis were investigated through transgenic AD disease models CL4176 and CL2006.The life span of wild type N2 C.elegans was observed after RNAi of tomm-22 and E04A4.5.The regulatory role of ATFS-1 signaling by atfs-1 RNAi in inhibition of Aβ protein aggregation was detected.The dynamic changes of UPRmt in transgenic SJ4100 nematode and the autophagy level in transgenic DA2123 nematodes were analyzed by tomm-22 and E04A4.5 RNAi.[Results] We successfully established the UPRmt model by cloning mitochondrial tomm-22 and E04A4.5 of C.elegans and further constructing RNAi bacteria,and showed that they can suppress aggregation toxicity of Amyloid-β (Aβ) protein in AD model CL4176,and slow down paralysis process.The life span of wild type N2 was significantly shortened after feeding with the tomm-22 and E04A4.5 RNAi bacteria.At the same time,the progressive paralysis AD model CL2006 shows a delayed paralysis in the early stage of life cycle but get acceleration in the late.These results illustrate that the UPRmt can alleviate the mitochondrial stress and improve the function of mitochondria at least in the short term.The atfs-1 RNAi confirmed that delayed paralysis process of AD model CL4176 is not directly related to the ATFS-1 signal.However,tomm-22 and E04A4.5 RNAi can gradually increase the UPRmt response and induce the expression level of autophagy-related molecules LGG-1,suggesting that tomm-22 and E04A4.5 RNAi may play a role in delaying the AD disease process by enhancing the activity of autophagy in C.elegans.[Conclusions] The study found that the UPRmt can inhibit the accumulation of A β protein by coordinating the signal transduction between mitochondria and nucleus,and can help to restore mitochondria and even intracellular protein homeostasis for protecting the normal physiological function of cells,and also provides new targets for prevention and treatment of neurodegenerative diseases such as AD.

OBJECTIVETo study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers, providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood (Artemisia annua).

METHODEST-SSR detection was performed using Perl program MISA. Gene Ontology (GO) annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category. Primer 3 software was used to design 18 pairs primers, amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining.

RESULTBy searching with Active Perl, totally 2 110 SSRs were detected, accounting for 8.6%. The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%, respectively. The most common repeat motifs of trinucleotide were ACC/GGT, accounting for 9.8%. Three hundred and twelve SSR-ESTs were annotated using GO terms. The suitable PCR system of 15 pairs primers was established, and revealed microsatellite polymorphism in 36 individuals.

CONCLUSIONThere are a variety of motifs at EST-SSR locus in sweet wormwood, and more effective amplification and polymorphism in 18 pairs detected primers. Therefore, EST resource is an effective and feasible approach to develop SSR markers, and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.

Artemisia annua ; genetics ; Expressed Sequence Tags ; Microsatellite Repeats ; Polymorphism, Genetic