采用实时荧光定量PCR技术对刺参高温胁迫抑制性消减cDNA文库中2个上调表达差异基因hsp70和l(2)efl,2个下调表达差异基因pcsk9和myp,在不同温度、不同胁迫时间以及不同组织中的应激表达特性进行了分析.在15-30℃范围内,温度越高hsp70和l(2)efl基因相对表达量越多；而pcsk9、myp基因随着温度升高其表达量下降.在30℃高温胁迫0-3h时间内,hsp70表达量的变化最为显著,表现为先升后降再升;l(2)efl在1h后开始明显的上调表达,在2.5h之后呈下降趋势;pcsk9和myp基因表现为表达下调,表达量最小值分别在3h和2h.常温下hsp70、l(2)efl和pcsk9均在体壁中的表达量最高,在呼吸树中表达量最少,myp在消化道中表达量最高,在纵肌中表达量最少；高温胁迫后hsp70、l(2)efl均在呼吸树中上调表达量变化最显著；pcsk9和myP分别在呼吸树和纵肌中下调表达量变化最显著.这4种基因通过表达产物量的变化或作为功能蛋白直接参与代谢调节,或作为调节蛋白调控胁迫功能蛋白的表达及活性来提高刺参对高温胁迫的耐受能力.%To analyze gene expression in response to environmental temperature changes in Apostichopus japonicus, two up-regulated genes hsp70 and l(2)efl, and two down-regulated genes myp and pcsk9 in suppression subtractive cDNA library of heat stress, were picked out to investigate under different stress time, different temperature and different tissue using real-time quantitative PCR. At 15-30 ℃, the higher the temperature, the more relative expression of hsp 70 and l(2)efl were. The genes of myp and pcsk9 showed reduced expression under the same conditions. During 1-3h of heat stress of 30℃, hsp70 changed significantly, representing a fluctuate pattern. L(2)efl significantly increased after lh, and reached the maximum at 2.5h, then followed by a downward trend. The expression of myp and pcsk9 reached minimum at 3h and 2h, respectively. Under room temperature, hsp70, l(2)efl and pcsk9 had the highest expression in the body wall and the least in the respiratory tree, while the highest expression and least expression of myp were in the digestive tract and longitudinal muscle, respectively. After high temperature stress, hsp70 and l(2)efl changed significantly in the respiratory tree, as pcsk9 and myp varied most obviously in breathing trees and longitudinal muscle. Via variation of expression, the protein products of the four genes involved in the regulation of metabolism as function proteins, or control the expression and the activity of the stress proteins as regulatory proteins, in order to improve the heat stress tolerance of A. japonicus.
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