【作者】 张占会；

【导师】 张其中； 林小涛；

【作者基本信息】 暨南大学 ， 水生生物学， 2010， 博士

【摘要】 热休克蛋白70 (heat shock protein 70, HSP70)是热休克蛋白中的重要家族,具有“分子伴侣”的生物学功能。现有研究表明海洋双壳贝类HSP70s基因对海洋环境污染物有广泛反应性,是被环境科学家看好的海洋环境污染潜在早期综合预警分子,但其对污染物的反应灵敏度和持续时间却未知,急待阐明,以便正确评价其作为海洋环境污染早期综合预警分子的可行性和应用范围。围绕这一核心,首先采用简并PCR、RACE和基因组步移法(Genome Walker),克隆了海洋环境污染指示种近江牡蛎(Crassostrea hongkongensis Lam and Morton,2003)(又称香港巨牡蛎,但国内多数人已习惯“近江牡蛎”这一名称,所以,仍沿用旧中文名)的三种HSP70s (HSC70, HSP69, HSP68)cDNA和包括5′调控区序列的基因全序列,以及HSP69一异构基因和HSC70一异构基因,在此基础上,研究了HSC70基因对多种污染物的反应灵敏度和持续时间以及反应规律。具体结果和结论如下：近江牡蛎HSC70基因含有6个内含子,其中最长内含子(322bp)位于5′端非编码区,其余5个内含子(长度依次为：119bp、203bp、104bp、110bp和215bp)在编码区以内。同时,利用基因组步移法获得HSC70基因5′端上游序列1231bp,序列包含有1个CAAT BOX、1个启动子核心序列(Core promoter region)和6个热休克元件(heat shock element, HSE),其中还包含有数个Poly C结构。HSC70异构基因2789bp片段,共包含6个内含子,长度分别为174、119、203、104、112和215bp,其中后5个内含子与HSC70基因序列中相应位置内含子序列几乎相同,只是第一个内含子不同。两者外显子碱基序列相似度99.48%,而推导的氨基酸序列完全相同。近江牡蛎HSP69基因的全长cDNA序列为2251bp,开放阅读框(Openreading frame, ORF)为1911bp,编码633个氨基酸。HSP69与其它物种的HSP69高度同源。HSP69基因不包含内含子。共得到5′端上游基因序列1933bp,包含启动子核心序列、1个GC BOX、和6个HSE。同时从基因组中克隆得到一HSP69异构基因,序列全长2623bp,包含一ORF长1911bp,编码633个氨基酸组成的蛋白质。两基因ORF序列相似度为96.48%,氨基酸相似度为98.42%。异构基因5′端上游序列506bp,包含有1个启动子核心序列和3个热休克元件(HSE)。实验证实该异构基因在近江牡蛎热休克时也可被诱导表达。近江牡蛎HSP68基因序列和5′端上游序列长度共2794bp,包含一ORF1848bp,可编码615个氨基酸(GenBank Accession No. GU586491),推导氨基酸序列中含有HSP70家族三个标签序列,其3′-侧翼区具有AATAAA加尾信号,126bp 5′上游序列序列中包含启动子核心序列和1个HSE序列。在非胁迫状态下HSC70、HSP69和HSP68三种基因表达具有组织特异性,在闭壳肌中三种基因表达水平均最高,外套膜中最低。其中HSC70的基础表达水平要远高于另外两种基因。热休克处理(从24℃至37℃)1h后,三种基因表达量均在3-6h出现显著增高,之后逐渐恢复至对照水平,HSC70基因显著高表达维持时间最长至24h,HSP69基因表达最长至12h,而HSP68只在3h左右高表达,各基因最高表达水平在不同组织间有差异。用Cu2+(100μg／l)、孔雀石绿(0.5μg／l)和甲醛(1μg／l)处理近江牡蛎,不同大小个体的牡蛎组织器官中HSC70的表达水平显著不同。在预实验的基础上,使用一系列浓度的不同污染物对近江牡蛎处理不同时间后,检测其外套膜、鳃和消化腺中HSC70基因的表达水平。结果显示近江牡蛎HSC70基因对孔雀石绿、Cu2+、Zn2+、甲醛和溴氰菊酯反应灵敏,最敏感组织或器官的临界诱导浓度分别在消化腺：0.001-0.01μg／l、消化腺：0.1-1μg／l、鳃：0.1-1μg／l、鳃：0.0001-0.001μg／l,以及鳃和消化腺：0.05-0.1μg／l。当处理浓度大于或等于污染物临界浓度范围上限时,近江牡蛎HSC70基因表达显著高于对照,反应的总体规律是随处理时间的延长,表达水平先上升,后下降,并逐步恢复到对照组水平,甚至出现表达显著低于对照水平。随着污染物浓度增加,HSC70的表达水平逐步升高到一峰值,当浓度增加到一定值后,继续增加,表达水平又逐步下降,同时,表达峰值出现的时间点在逐步提前。这些规律在使用对近江牡蛎毒性较小的孔雀石绿和两种重金属Cu2+和Zn2+处理时,表现最明显,但是在用对牡蛎毒性强的甲醛和溴氰菊酯处理时,HSC70表达只在一很窄的浓度范围内出现短时间显著高于对照表达,当浓度超过这个范围,表达会被抑制,且浓度越高,出现抑制的时间越早。HSC70基因显著高于对照表达的维持时间长度,随污染物浓度升高,维持时间先变长,浓度继续升高,又缩短。在鳃中HSC70显著高表达的时间较外套膜和消化腺中长,一般可以维持数天至十几天。用高于临界浓度范围上限孔雀石绿和敌百虫同时联合处理或敌百虫先单独处理10天,再与孔雀石绿联合处理,或者使用低于临界浓度范围下限的孔雀石绿和敌百虫联合处理近江牡蛎时,诱导鳃HSC70基因高表达的联合效应类型均属于协同作用,即两污染物的联合诱导效应明显高于两者单独诱导效应。但联合诱导HSC70高表达出现的时间与两污染物单独诱导出现的时间并不同步。上述结果表明近江牡蛎HSC70基因对污染物反应灵敏,且反应信号能维持一段时间,可作为海洋环境污染早期综合预警分子。不同大小个体的近江牡蛎HSC70基因表达往往存在显著差异,所以,在研究和实际海洋环境污染预警中,应尽可能选择个体大小相同或接近的个体为材料,而近江牡蛎鳃是适合的指示器官,消化腺次之。更多还原

【Abstract】 at shock proteins 70kDa (HSP70s) were the most important and highly con(?)d proteins among members of the Heat shock protein family, and mainly function;as molecular chaperone which play an important role in protein folding.sent researches showed that marine bivalves HSP70s could be induced by a wid(?) ange of marine environmental pollutants. The bivalve HSP70s are ideal candi(?)es for comprehensive biomarkers of early marine environmental pollution. However, the sensitivity of HSP70s expression response to pollutants and the times of sig(?)icantly higher expression duration are unknown, which are important for correcdy evaluate feasibility and application range of HSP70s as early comp(?)ensive biomarkers of marine environmental pollution. In order to solve the problems three HSP70s (HSC70, HSP69 and HSP68) of Crassostrea hong(?) gensis (Lam and Morton,2003) were cloned with RT-PCR, RACE and geno(?) walker methods. Then, the 5’-flanking regions of them were also cloned by mean(?) of genome walker method. At the same time, we get one isogene of HSP69 and (?) HSC70, respectively. On this basis, the sensitivity of HSC70 gene expression and t naintenance times of significantly higher expression duration response to pollu(?)s are studied.Through PCR amplification in C. hongkongensis genome DNA, six introns were found in the HSC70 gene. And the longest intron (322bp) was located in the 5’unt islated region (UTR) and the others (length:119bp,203bp、104bp、110bp and 21(?)bp) in the open reading frame (ORF) region. A 1231 bp 5’-flanking region of HSC70 gene was obtained using genome walker method, including a putative core moter region and transcription elements including 6 heat shock elements (HSF(?)one CAAT box and a few poly C region. At the same time, one isogene of HSC fragment was cloned. There are 2789bp sequences including 6 putative intro with differences only in the first intron of both the gene and isogene of HSC70 Sequence analysis revealed that the identity of the exon nucleotide seque(?)between the gene and its isogene we obtained in the HSC70 is 99.48%. and th(?) of the partially deduced amino acid sequence is 100%.terms of the features of the HSP69 in C. hongkongensis, it was found that the f(?)ength HSP69 cDNA of 2251 bp contained an open reading frame (ORF) of 1911bp with the 5’UTR of 130 bp and the 3’UTR of 218 bp. The deduced 633 amino acid sequence showed the highest identity with HSP69 of other mollusks. No intron was found in the HSP69 gene. The 5’-flanking region sequence of the HSP69 gene contained a putative core promoter region and transcription elements including 6 heat shock element (HSE) and GC box. At the same time, one isogene of HSP69 fragment was cloned, obtaining 1933bp sequence including one ORF of 1911bp that encoded 633 amino acids. Sequence analysis revealed the identities of ORF nucleotide sequence and amino acid sequence between the gene and isogene of HSP69 were 98.42% and 96.48%, respectively. A 506 bp 5’upstream sequence of the HSP69 isogene contained a putative core promoter region and 3 heat shock elements (HSE). The results indicated that HSP69 isogene expression could be induced by heat.A 2794bp sequence of C. hongkongensis HSP68 gene was obtained using genome walker method, including a ORF of 1848bp that encoded 615 amino acids. Three classical HSP70s signature motifs were detected in the deduced amino acid sequence. A ployadenylation signal was found on the 744bp of 3’end, and a putative core promoter region and a heat shock element (HSE) in 126bp 5’ upstream sequences.The investigation about tissues distribution of HSC70, HSP69 and HSP68 with real-time fluorescence quantitative RT-PCR indicated that HSC70, HSP69 and HSP68 transcriptional expression levels were detectible in digestive gland, muscle, mantle, gill and heart from un-stressed C. hongkongensis. Tissue-specific variation was observed in all three HSP70s:relatively high expression in muscle and lowest in mantle. A similar trend occurred that significantly increased at 3-6 hours and then dropped and returned to the control level after heat shock(24℃to 37℃, 1h). Significantly higher expression of the genes could maintain about 24 hours for HSC70, about 12 hours for HSP69 and just at 3 hours for HSP68. The highest gene expression levels of three HSP70s varied in different organs.Our results showed that expression levels were different among various tissues or organs of small and large individuals after malachite green, formaldehyde and Cu2+challenge.The mRNA transcription levels of HSC70 were analyzed with real-time fluorescence quantitative RT-PCR technique in different organs of C. hongkongensis responding to a series of concentrations for Cu2+, Zn2+, malachite green, formaldehyde and deltamethrin. The results showed that the threshold induction concentration of HSC70 expression was 0.1-1μg/l in the digestive gland for Cu2+,0.1-1μg/l in the gill for Zn2+,0.001-0.01μg/1 in the digestive gland for malachite green,0.0001-0.001μg/l in the gill for formaldehyde and 0.05-0.1μg/l in the gill and digestive gland for deltamethrin, respectively. All these proved that the HSC70 gene was sensitive to pollutants.HSC70 expressions were significantly induced at different time after pollutants exposure when treated concentration was greater than or equal to the upper limit of the threshold induction concentration range. The overall response rule was as follows:with the treatment time elongation, the expression level of HSC70 gene increased at first, end then decreased, gradually returned to control levels, even became significantly lower than the control; with increastment of pollutant concentration, HSC70 expression levels also raised gradually to a maximum value, and then the expression level progressively decreased, meanwhile, the peak expression time point gradually went ahead. The rules described above always occurred prominently when the less toxics, malachite green and two heavy metals (Cu2+and Zn2+), treated C. hongkongensis. However, HSC70 expression levels were significantly higher than control group only in a narrow range of concentrations of formaldehyde and deltamethrin, and the HSC70 expression will be inhibited when the treated concentration exceeds the scope. At the same time, the higher the concentration treated, the earlier inhibition occurred.The expression time of the oyster HSC70 at a significantly higher level than the control was different after treatment with different concentrations of various pollutants. The higher the treated concentration in a range of pollutants, the longer the HSC70 expression time, but when the concentration was over some upper limit of range, the expression time became shorter. In general, the significantly higher expression time maintained for several days, up to more than ten days. HSC70 expression time in gill was longer than that in the mantle and digestive gland.When the oysters were exposed to either the mixture of malachite green and trichlorfon (no matter the two pollutants was at over or blow the threshold induction concentrations) for 35 days or trichlorfon for 10 days at first, then malachite green for another 25 days, the two pollutants induced HSC70 mRNA expression in a synergistic effect in the oyster gill. On the basis of the above-mentioned results, the HSC70 gene of C. hongkongensis was sensitive to pollutants, and the significantly higher expression time maintained for several days, up to more than ten days. Therefore, it is feasible that the HSC70 gene of C. hongkongensis was used as early comprehensive warning biomarker of marine pollution. Due to the significant difference of HSC70 expression between small and large individuals, the same or similar size oysters should be used for research and practical application on early warning of marine environment pollution. At the same time, the gill of C. hongkongensis was the most appropriate organs for indicator, and digestive gland could also be used.更多还原