Palli S R., Ladd T R., Ricci A R., Primavera M, Mungrue I N., Pang A S.D., Retnakaran A
Great Lakes Forestry Centre, Canadian Forest Service, P.O. Box 490, Sault Ste. Marie, P6A 5M7, Ontario, Canada
J Insect Physiol. 1998 May;44(5-6):509-524. doi: 10.1016/s0022-1910(97)00123-6.
Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.
云杉芽虫幼虫会产生大量与滞育相关的两种蛋白质（云杉芽虫滞育相关蛋白1和2，CfDAP1和CfDAP2）。这些蛋白质在孵化后不久出现，丰度增加，在一龄幼虫4天时达到最高水平，并在滞育结束后3天一直保持在高水平。这两种蛋白质被纯化至同质，并获得了它们的NH2末端序列。基于这些NH2末端序列设计的寡核苷酸引物用于RT-PCR，以分离编码这些蛋白质的cDNA片段。然后将这些PCR片段用作探针，以分离包含完整编码区的cDNA。编码这些蛋白质的2.5kb mRNA在孵化后24小时开始出现，在一龄和二龄幼虫中检测到大量此类mRNA，直到二龄幼虫进入滞育。在准备进入滞育的二龄幼虫、处于滞育期的幼虫以及滞育结束的幼虫中检测到低水平的这些mRNA。在蜕皮至三龄后的第1天和第2天也检测到低水平的CfDAP1 mRNA。然而，在四龄和五龄幼虫阶段未检测到CfDAP1和CfDAP2 mRNA。五龄幼虫蜕皮至六龄后24小时，这些mRNA再次出现，并在蜕皮后60小时增加到最高水平。mRNA水平在蜕皮至六龄后156小时（化蛹前36 - 48小时）一直保持高水平，之后再次消失。免疫细胞化学分析表明，CfDAP1蛋白存在于二龄和六龄幼虫脂肪体中，但不存在于五龄幼虫脂肪体中。因此，在云杉芽虫幼虫进入滞育前首次表达相同的两个基因，在化蛹前第二次表达。
