【作者】 谢彦海；

【导师】 文春根；

【作者基本信息】 南昌大学 ， 动物学， 2011， 博士

【摘要】 本研究采用RACE技术从褶纹冠蚌中克隆到HSP70和HSP90基因的cDAN序列,以及从褶纹冠蚌基因组中克隆出HSP70和HSP90基因组序列。HSP70基因cDNA序列全长为2664bp(基因登录号：ADM64336),其中5’UTR有364 bp；3’UTR有383 bp,在N末端存在4个RNA不稳定模体ATTTA和26bp的poly (A); HSP70基因的开放阅读框为1917bp,编码638个氨基酸组成的蛋白质,该蛋白理论等电点为5.61,分子量为70.3kD。根据HSP70基因cDNA序列推导氨基酸序列发现,氨基酸序列含有HSP70家族的三个标签序列,含有4个糖基化位点,以及保守的EE (V/M) D多肽结构。HSP70氨基酸包括一个由380个氨基酸组成ATP酶结构域,一个由159个氨基酸组成的底物肽结合结构域,以及一个由82个氨基酸组成的C端结构域。HSP70基因组全长发现,HSP70基因的ORF内不存在内含子。HSP90基因cDNA序列全长为2674bp(基因登录号：ADN87332),其中5’UTR有83 bp；3’UTR有410 bp。该序列包括1个28bp的poly (A),1个AATAAA加尾信号以及1个RNA不稳定模体ATTTA; HSP90基因的ORF为2181bp,编码726个氨基酸组成的蛋白质,该蛋白理论等电点为5.05,分子量为83.47kD。根据HSP90基因cDNA序列推导氨基酸序列发现,氨基酸序列含有HSP90家族的5个标签序列,3个糖基化位点,以及C末端存在保守的MEEVD多肽结构。同时,推测该序列含有由155个氨基酸组成ATP酶结构域。HSP90基因组序列全长9739bp；其中在ORF中有8个外显子,7个内含子。利用半定量PCR和荧光定量PCR研究了热处理、嗜水气单胞菌、重金属刺激下,HSP70 mRNA和HSP90 mRAN的表达量。热休克刺激后,蚌5种组织的HSP70 mRNA表达量均显著高于对照组,其中血液和鳃内的表达量变化最显著,其次是外套膜和肝胰腺,而肌肉内的表达量变化不明显。嗜水气单胞菌刺激6h后,仅血液和外套膜组织中HSP70 mRAN的表达量显著增加,而其它组织增加不明显,12h后各组织的表达都达到峰值,其中血液、鳃和肌肉内的表达量增加最为显著,随后逐渐下降,48h表达水平恢复到正常状态。不同温度刺激下,蚌血细胞中HSP70 mRNA的表达变化显著,其中35℃时表达量是5℃时表达量的99042倍,而15℃,25℃时HSP70 mRNA的表达量仅为5℃时的16.1倍和13.4倍。在重金属(Cd2+)刺激下,HSP70 mRNA的表达量都随时间的增加而升高,表达峰值出现在15d,而20d时表达量急剧下降,接近正常表达水平。在重金属(Cr6+)刺激下,5d时,HSP70 mRNA的表达量增加不显著,随刺激时间的增加,表达量逐渐的升高,15d时表达量都达到最大值,随后表达量开始逐渐下降,20d时表达量下降到正常状态水平。热休克刺激后,蚌5种组织HSP90 mRNA表达量明显的增加,其中在血液、肌肉和肝胰腺内的表达量增加最大,而鳃和外套膜的表达量相对小。嗜水气单胞菌刺激后,HSP90mRNA的表达量明显地增加,但5种组织间的相对表达量没有明显的差异。不同时间段的刺激中,发现随刺激时间的增加,HSP90 mRNA的表达量也增加,到12h时表达量达到最大值,之后表达量趋于稳定,随后表达量开始下降,48h后表达量恢复到刺激前的水平.不同温度刺激下,HSP90 mRNA的表达量随温度的升高而增加,35℃时,蚌处于热休克状态,此时HSP90 mRNA的表达量是5℃时表达量的130倍。不同浓度Cd2+刺激蚌后,蚌内HSP90 mRNA的表达具有不同的变化趋势,但都表现为正调节作用。当Cd2+的浓度为50μg/L和100μg/L时,表达峰值分别出现在15d和20d,随后表达量开始下降,而Cd2+浓度为200μg/L时,峰值出现在5d,随后表达量趋于高位稳定。重金属Cr6+刺激后,随着刺激时间的增加,HSP90mRNA的表达量随之增加。浓度1μg/L和10μg/L时,表达峰值出现在15d,而Cr6+浓度为100μg/L时,HSP90表达峰值均出现在10d,随后表达量逐渐下降,20d时恢复正常水平。为了进一步探讨HSP70和HSP90的功能,通过双酶切连接,构建了pET30-HSP70和pET30-HSP90原核表达系统,成功表达了两种蛋白。SDS-PAGE分析发现HSP70和HSP90在IPTG的诱导下,随诱导时间的增加,蛋白表达量逐渐增加,8h时表达量最大。利用纯化的重组蛋白制备多克隆抗体。用Western-blotting分别检测褶纹冠蚌血、鳃、肌肉、肝胰腺和外套膜五种组织中HSP70和HSP90的蛋白表达量。结果显示,热休克(35℃)后,五个组织中HSP70的表达量相对于室温(20℃)时的表达量显著增加,其中血、鳃、外套膜和肝胰腺组织的表达量高,而肌肉的表达量相对较少。热休克(35℃)后,五个组织中HSP90的表达相对于室温(20℃)时的表达量增加明显,其中血液的表达量最大,其次是肝胰腺和肌肉,而鳃和外套膜的表达量增加相对较少。褶纹冠蚌和背角无齿蚌血细胞形态极其相似,都可分为大颗粒细胞、小颗粒细胞、透明细胞和淋巴样细胞四种。两种蚌不同亚型之间所占的比例有所差别,其中小颗粒细胞为数量最多的细胞类型,分别占血细胞总数比例分别为(56.6±1.41)%和(59.28±0.89)%；透明细胞也是数量较多的细胞类型之一,占血细胞总数比例分别为(37.8±2.11)%和(27.74±0.56)%；大颗粒细胞数量较少,所占比例分别为(4.2±0.23)%和(4.28±0.35)%。淋巴样细胞比例分别为(2.4±0.12)%和(8.70±0.23)%。更多还原

【Abstract】 The HSP70 and HSP90 cDNA cloned from fresh water mussle, Cristaria plicata, using rapid amplification of cDNA ends, and the sequences of HSP70 and HSP90 genome were cloned from genomic DNA of C. plicata.The full-length cDNA sequence of HSP70 was 2664bp (GenBank accession number:ADM64336), consisting of a 5’-terminal untranslated region (UTR) of 364 bp, a 3’-terminal UTR of 383 bp with a 26bp poly (A) tail and four ATTTA (RNA instability motifs), and the open reading frame of 1917bp. The HSP70 cDNA encoded a polypeptide of 638 amino acids with a predicted molecular mass of 70.3 kDa, and a theoretical isoelectric point of 5.61. The deduced amino acid sequence of HSP70 cDNA includes three HSP70 family signatures. There were 4 glycosylation sites and a highly conserved, the cytoplasmic HSP70 carboxyl terminal region of EE (V/M) D (residues 635-638). The amino acid sequence of HSP70 was included of an ATPase composed of 382 amino acids, the substrate peptide binding domain composed of 159 amino acids, and the C-terminus domain composed of 82 amino acids. There was no intron in ORF of HSP70 cDNA.The full-length cDNA sequence of HSP90 was 2674bp (GenBank accession number:ADN87332), consisting of a 5’-UTR of 83 bp, a 3’-terminal UTR of 410 bp with a canonical polyadenylation signal sequence AATAAA, a 28bp poly (A) tail and a ATTTA. The open reading frame of HSP90 was 2181 bp, which encoded a polypeptide of 726 amino acids with a predicted molecular mass of 83.47 kDa and a theoretical isoelectric point of 5.05. The deduced amino acid sequence of HSP90 cDNA includes five HSP70 family signatures. There were 3 glycosylation sites and a highly conserved, the cytoplasmic HSP90 carboxyl terminal region of MEEVD. The HSP90 cDNA was deduced that an ATPase domain was composed of 155 amino acids. The full length sequence of HSP90 genome was 9739 bp, with 7 introns and 8 extrons in ORF of HSP90.The temporal expression of HSP70 mRNA and HSP90 mRAN were measured by semi-quantiative RT-PCR and fluorescent real-time quantitative RT-PCR after thermal treatment, bacterial challenge and heavy metal stress.After thermal treatment, the HSP70 mRNA relative express value in five tissues from C. plicata were significantly increased compared with the control group, and the HSP70 levels of blood and gills were the most significant, then were the mantle and hepatopancreas, the least were muscles. The pathogenic bacteria Aeromonas hydrophila challenged, the relative express value were increased obviously in only blood and mantles at 6h, but were not obvious in other tissues. The HSP70 levels reached the peak at 12h, then decreased gradually, and recovered the normal level after 48h.Fluorescent real-time quantitative RT-PCR showed the HSP70 levels in blood were increased significantly at different temperature, the maximum levels at 35℃were the 99042-fold higher than that at 5℃, however, the levels at 15℃and 25℃were 16.1- and 13.4-fold respectively higher than that at 5℃. At heavy metal (Cd2+) stressed, levels of HSP70 mRNA were increased with temperature ascended compared with control group, the expression peak was at 15d, however, the levels were rapidly dropped, were got close to the normal level at 20d. At heavy metal (Cr6+) stressed, HSP70 mRNA levels were not obviously increased at 5d, however, with the stressed time lengthened, the express value rose rapidly. The maximum levels were at 15d, followed the level was descended gradually, the expression of HSP70 recover the control level at 20d.The HSP90 levels in five tissues from C. plicata were significantly increased compared with the control group after thermal treatment. The relatively higher expression was in blood, muscles and hepatopancreas, while mantles and gills were lower level. At A. hydrophila challenged, the relative express value was not significant difference among five tissues. At different time points, the HSP90 level reached the peak at 12 h, followed maintained a relatively high level, then decreased gradually, and recovered the normal level after 48h.HSP90 expression level increased with the temperature lengthened at different temperature treatment, the maximum level were at 35℃,130-fold higher than that at 5℃. HSP90 levels showed different change tendency at different heavy metal Cd2+ concentration stressed, but all showed up-regulated. When the Cd2+ concentrations were 50μg/L and 100μg/L, the peak of HSP90 mRNA expression appeared at 15d and 20d, respectively, followed the level were descended gradually, however, when the Cd2+ concentrations were 200μg/L, the peak appeared at 5d, followed maintained a relatively high level compared with the control group. At heavy metal (Cr6+) stressed, levels of HSP90 mRNA were increased with stress temperature lengthened. When the Cr6+ concentrations were 1μg/L and 50μg/L, the peak of HSP90 mRNA expression appeared at 15d, while the Cr6+ concentration was 100μg/L, the peak appeared at 10d, followed the level were descended gradually, then recovered the normal level at 20d.In order to further inquire into the functions of HSP70 and HSP90, pET30-HSP70 and pET30-HSP90 prokaryotic expression system were constructed, and successfully expressed these proteins. SDS-PAGE analysis showed HSP70 and HSP90 expression levels were increased with induced time to add, by IPTG induction, the maximum level was at 8h.Polyclonal antibodies were prepared with rabbit and mouses using purified recombination proteins. Western-blotting was used to detect the expression level of HSP70 and HSP90 in bloods, gills, muscles, hepatopancreas and mantles from C. plicata. The results showed that expression levels of HSP70 were increased rapidly in five tissues at heat shock temperature (35℃) compared with at room temperature (20℃), relatively higher expression was detected in blood, gills, hepatopancreas and mantles were high, while muscles were relatively low. The expression level of HSP90 were also increased obviously in these tissues at heat shock temperature (35℃) compared with 20℃group, and the maximum was in bloods, then were muscles and hepatopancreas, the least were the mantle andgills.The morphology of C. plicata and Anodonta woodiana was exceedingly similar, which could be divided into four subpopulation, large granulocytes, small granulocytes, hyalinocytes and lymphoid hemocytes. The ratio of different subpopulations in two mussles was obviously difference, small granulocytes were the most subpopulation in two mussles, (56.6±1.41)% and (59.28±0.89)% in the hemocyte population, respectively. Hyalinocytes were also the main types, (37.8±2.11)% and (27.74±0.56)%, respectively. Large granulocytes were the little percentage in hemocyte population, (4.2±0.23)% and (4.28±0.35)%. The ratio of lymphoid hemocytes was(2.4±0.12)% and(8.70±0.23)%,respectively.更多还原