In previous studies, mutation of Lys296 or Glu113 in opsin has been shown to result in constitutive activation of the protein--that is, these mutants can activate the G protein transducin in the absence of chromophore and in the absence of light. These and other data have led to the suggestion that a salt bridge between Lys296 and Glu113 helps to constrain opsin to an inactive conformation. It is shown here that of 12 different amino acids substituted at position 296, all, except Arg and the wild-type Lys, are constitutively active at neutral pH, lending further support to this suggestion. However, activation of opsin appears also to be influenced significantly by the size of amino acid side chain at position 296. Thus, there are multiple effects of the mutations. Wild-type opsin is also shown to be weakly active at pH 6.1. Five other charged amino acids in the membrane-embedded region of the protein (Asp83, Glu122, Glu134, Arg135, and Glu201) were mutated to see if they affect constitutive activity. Of these amino acids, only mutation of Glu134 results in an increase in the activity of opsin. Changing Glu134 to Gln increases the activity of opsin, while changing Glu134 to Asp inhibits activity. These results suggest that a negative charge on Glu134 is important in stabilizing the inactive state of opsin. Glu134 is highly conserved in all visual pigments and most of the other G protein-linked receptors.

Abstract-This

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Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (18S, 28S, EF, UBA, UBQ, TIF, NTB, TUA, RPS, and TIF5A) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S, TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.