【作者】 张元；

【导师】 陈昌生；

【作者基本信息】 集美大学 ， 水产养殖， 2011， 硕士

【摘要】 本研究以耐高温型坛紫菜Z-61F4、Z-26F4纯系叶状体作为实验材料，以野生型坛紫菜叶状体作为对照，分别从生长发育特征、生理生化指标以及基因差异表达等方面对坛紫菜叶状体在高温胁迫下的响应机制进行了初步研究。结果表明：1、生长发育特征方面：两个耐高温型坛紫菜品系叶状体经高温（30℃、26℃）胁迫10d后，藻体颜色分别由正常温度（21℃）时的红褐色与褐红色逐渐变浅，但将其转入正常温度下培养能够基本恢复正常生长，而野生型坛紫菜叶状体经30℃高温胁迫2d后即出现细胞质外溢、逐渐坏死的现象，转至正常温度下培养不能恢复正常生长；2、在品质指标方面：本研究测定了2个耐高温型F4与1个野生型坛紫菜叶状体在30℃、26℃、21℃三个温度下分别处理0d、2d、4d、6d、8d、10d后的5个品质指标（藻胆蛋白、叶绿素a、粗蛋白、总氨基酸及游离氨基酸），3个品系在21℃下培养10d后各指标变化不显著（P>0.05），30℃、26℃下多数指标动态变化，胁迫10d后呈现显著或极显著降低（P<0.05或P<0.01），耐高温型品系降低幅度较野生型小；3、在生理生化指标方面：测定了2个耐高温型与1个野生型坛紫菜叶状体在30℃、26℃、21℃三个温度下分别处理0d、2d、4d、6d、8d、10d后的7个生理生化指标[自由基含量、过氧化氢（H₂O₂）含量、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、过氧化物酶（POD）活性、过氧化氢酶（CAT）活性及游离脯氨酸（Pro）含量]，根据各指标在胁迫周期内的动态变化，推测耐高温型坛紫菜叶状体应答高温胁迫的生理生化过程为：高温胁迫→活性氧含量上升→细胞膜氧化受损，产生大量膜脂过氧化物→细胞感受到过量的活性氧信号→抗氧化系统和渗透压调节系统共同响应，清除活性氧→自由基含量下降；同时比较了两种坛紫菜对高温胁迫的生理生化响应过程，发现耐高温型坛紫菜在高温胁迫下能同时启动抗氧化系统和渗透压调节系统，而野生型坛紫菜则只能启动渗透压调节系统，且两种坛紫菜抗氧化系统的本底含量也存在着显著差异（P<0.05）；4、利用引物退火控制技术（ACP），对耐高温型坛紫菜Z-61F₄叶状体胁迫6d后与正常温度培养下的cDNA进行扩增筛选得到多个基因差异表达片段，并对其中6个（GW-1、GW-2、GW-3、GW-4、GW-5、GW-6）片段进行测序分析，片段大小分别为387bp、512bp、702bp、360bp、228bp与132bp，同源性比对发现这些基因与信号转导、蛋白代谢等相关。对前5条基因进行实时荧光定量PCR分析显示，在0d与6d间的表达量均存在显著或极显著差异（P<0.05或P<0.01）。其中GW-3为全长基因，编码核糖体蛋白S7，GenBank数据库登录号JF719273。预测该基因编码195个氨基酸，理论等电点为9.99，不稳定系数为37.43，脂肪系数为88.00。残基构成螺旋（H）、片层（E）和环状（L）占总氨基酸比例分别为38.46%、21.54%与40.00%。更多还原

【Abstract】 This response characteristics of gametophytic blades of two high temperature-tolerancelines (Z-61, Z-26) and a wild-type line of Porphyra haitanensis under high temperature stresswere studied from growth characteristics, quality parameters, physiological indexes and thedifferential gene expression:1．Growth characteristics: Under two high temperatures(30℃,26℃) stress, the blade colourof Z-61and Z-26was faded gradually, but they could restore to normal growth state whenremoved to culture under normal temperature for some days. However, the cytoplasm ofwild-type line leaked out and necrosis gradually after cultured under30℃for two days, andfather, they could not restore to normal growth state when removed to culture under normaltemperature.2．Quality parameters:5quality parameters (included the contents of phycobiliprotein,Chlorophyll a, crude protein, total amino acids and free amino acids) were detemined. Theresults showed that all the5quality parameters of Z-61, Z-26and wildtype line were nosignificantly changes under21℃, but significantly or highly significantly declined(P<0.05orP<0.01) under26℃and30℃. The declined amplitude of Z-61and Z-26is smaller than thewild-type line.3．Physiological indexes: several physiological indexes (included the contents of freeradical, hydrogen peroxide (H2O2), malondialdehyde (MDA), proline (Pro) and the activity ofsuperoxide dismutase (SOD) and peroxidase (POD), and catalase(CAT)) were determined. Theresults showed that all these indexes were presented as dynamic changes on the stress days. Sobased on these data, we concluded the physiological process of the gametophytic blades of Z-61and Z-26response to high temperature stress was: high temperature stress→levels of reactiveoxygen species(ROS) increased→cell membranes were damaged by oxidation and MDA levelsincreased→cells received signals of excessive ROS→antioxidation and osmoregulation systemsbegan to clean out excessive ROS→free radical levels declined. By comparing the responsesmechanism of high temperature-tolerance lines and wild-type lines to high temperature stress, weknowed that the high temperature-tolerance lines can initiate both antioxidation andosmoregulation systems to resist to stress, but the wild-type line only can initiate osmoregulationsystem.4. Differential gene expression: The ACP technology was used to investigate the genes ofZ-61which differential expressed under high temperature stress for0days and6days,6gene fragments which senquence length each was387bp(GW-1),512bp(GW-2),702bp(GW-3),360bp(GW-4),228bp(GW-5) and132bp(GW-6) were gained. Furthermore, relative expressivelevel of gene GW-1, GW-2, GW-3, GW-4and GW-5on the blades of Z-61which cultured underhigh temperature stress for0,2,4,6,8,10days were also determined by qPCR, respectively, theresults showed that the expressive level of each gene was significantly changed on the differentstress days. Protiens which encoded by these genes were relative with signal transduction,protein supersession and others by analyzing with blast-x.5. A full length cDNA sequence of702bp(GW-3) was obtained and was assigned to a newNCBI accession number JF719273. It encoded a protein which included195amino acids,theoretical isoelectric points was9.99, instability index was37.43, coefficient of fat was88.00,the proportions of amino acids which was constructed helix, pleated sheet and loop were38.46%,21.54%and40.00%, respectively.更多还原