乙酰胆碱受体在神经突触传导过程中具有重要作用，也是氯化胆碱类杀虫剂的作用靶标。采用RACE技术，成功地从棉蚜中克隆了3个nAChR亚基，其中2个为α亚型，1个为β亚型，分别命名为Agα1 、Agα2和A gβ1。通过锚定mRNA 的5'mG结构，5'RACE 结果表明Agβ1有三个不同的剪接变体，具有不同长度的5′UTR区，表明Agβ1亚基具有多重的转录起始位点。其中，最短的剪接变体A gβ1C 在蛋白编码区域也存在选择性剪接，位于D环区域的186 bp碱基缺失。3'RACE 实验结果表明，Agα1亚基虽然具有ploy (A) 和加尾信号AATAAA等完整的mRNA基因结构，但缺失了终止子和乙酰胆碱受体α亚基保守的第4个跨膜区，文中对此做了进一步分析。分子进化树的分析表明，昆虫乙酰胆碱受体亚基应当被划分为三个不同的亚类群αⅠ,αⅡandβ。本文的研究揭示了昆虫乙酰胆碱受体亚基复杂的基因结构。

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33, 680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDSPAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25℃. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations. Arch.

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5′-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09: A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5′-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.

The systematic analysis of very short alternative splicing (VSAS) in the human genome showed that VSAS might contribute more to protein-function diversity than expected. More than 65% of VSAS fragments have different secondary structures from flanking regions. They tend to have a non-loop structure and have an important influence on protein functions, as shown by the predicted 3D structure of human IL-4d2. The observed VSAS events can be classified into two groups depending on whether they insert new structure domains in the proteins, and they might be of different evolutionary status.

Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.