【作者】 李喜莲；

【导师】 刘小林；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2011， 博士

【摘要】 由于对虾养殖业在世界范围内迅速扩张,凡纳滨对虾正成为一个重要的经济物种。随着养殖技术的不断提高和抗病性的增强,通过遗传改良来提高虾的养殖速度已成为一个重要的技术。这有可能降低养成周期的长度,从而降低生产成本,缩短对虾养殖周期。但是目前有关对虾肌肉增长的相关基因,特别是调控基因,目前还不是很清楚。本研究以凡纳滨对虾雌虾极端体重个体腹部肌肉为实验材料,利用抑制消减杂交（SSH）技术构建极大体重个体和极小体重个体正反向消减cDNA文库、鉴定和克隆了差异表达的基因,并采用实时定量技术分析筛选出的体重调控相关基因的表达规律,利用生物信息学对其功能和结构进行预测,而且对筛选出的体重调控相关基因在肌肉组织的表达量和体重十个指标进行了相关性分析,同时构建原核表达载体,研究了RAS、P23和Troponin基因在大肠杆菌中的表达情况,为凡纳滨对虾的功能基因克隆和分子选育等研究奠定基础。本研究取得以下结果:1.利用SSH技术构建极大体重雌性个体和极小体重雌性个体腹部肌肉组织正反向消减cDNA文库:正库（以极大体重个体为试验组,以极小体重个体为驱动组,L-S）和反库（以极小体重个体为试验组,以极大体重个体为驱动组,S-L）消减cDNA文库,以β-actin为看家基因检测两个文库的消减效率分别为2¹⁰和2⁵,说明差异表达在极大体重个体和极小体重差异基因在文库中的富集效率为2¹⁰和2⁵。2.从S-L消减cDNA文库中挑出50个克隆,筛选出30个大小合适的阳性克隆进行测序。测序获得18个EST序列,其中14个为凡纳滨对虾已知基因,4个为凡纳滨对虾未知基因,但与其他物种同名基因同源性达95%以上。这18个基因分别是:延伸因子1α,NADH脱氢酶亚基（1、2）,细胞色素c氧化酶亚基（2B、Ⅱ、Ⅲ）,热休克蛋白90,高血糖激素,腺苷酸激酶,亚型原肌球蛋白,核糖体蛋白31,核糖体蛋白S24,肌钙蛋白C。从L-S消减cDNA文库中挑出150个克隆,筛选出100个大小合适的阳性克隆进行测序。测序获得49个EST序列,其中19个为凡纳滨对虾已知基因,19个为凡纳滨对虾未知基因,但与近缘物种具有较高的同源性。另有11个为线粒体序列,其差异基因主要包括:组织或细胞成分合成的cDNA序列为序列总数的37.21%;细胞生长或修复的cDNA序列,所占比例为13.93%;绑定蛋白、生物调节、催化活性相关的cDNA序列,占序列总数的4.65%、4.65%、4.65%。对S-L消减cDNA文库中的AK、CHH和Troponin基因片段进行实时定量分析,结果表明三个基因在大体重个体肌肉中的表达量低于在小体重个体肌肉中的表达量。挑选L-S消减cDNA文库中的CTSL、ER、FABP、P23、PK、PRDX、TM、RAS、VTG这9个基因片段进行实时定量分析,结果表明:这9个基因在极大个体肌肉中的表达量高于极小个体中的表达量。同时,实时定量分析结果表明:AK、CHH和Troponin基因在体重的调节中起下调作用,而CTSL、RAS、TM等其余9个基因在体重的调节中起上调作用。3.根据不同物种之间的基因编码区的保守性,设计引物并成功克隆了凡纳滨对虾RAS、P23和Troponin的基因,均包括完整的编码区序列,其GenBank登陆号分别为:RAS（ORF 564bp,GenBank No:JF806618）、P23（ORF 495bp,GenBank No:JF806619）和Troponin（ORF 480bp,GenBank No:JF806620）。本研究利用多个生物信息学的分析软件,对筛选出的差异表达的3个基因RAS、P23和Troponin进行氨基酸结构和潜在功能位点的预测,同时进行了蛋白质的二级和三级结构的预测和分析。结果发现RAS蛋白部分序列有强疏水性,且有强疏水结构域。P23蛋白也有部分序列有强疏水性,无跨膜螺旋结构,两种信号肽预测都显示P23含有信号肽,第12个氨基酸处存在信号肽的可能性是92%。Troponin蛋白无疏水性序列,在6-24氨基酸处有强跨膜螺旋结构,无信号肽区域。4.本实验对12个基因在凡纳滨对虾不同性别间表达量差异的分析中发现VTG基因不仅在雌虾肌肉中表达,在雄虾肌肉中也有表达,这一结果与前人研究不一致。对12个基因在五个组织（眼柄、心脏、肝胰腺、胃和消化道）的mRNA表达量研究中发现,AK在心脏和胃组织上的表达量最高,而CHH却在心脏和胃组织上的表达量最低,Troponin基因在肌肉上表达量最高,这些都与其功能有关。对9个基因与体重十个性状的相关性分析发现,AK、PK和TM这三个基因的相对表达量与其他基因都不相关,而只有VTG基因与体重呈负相关,且相关系数最高的两个基因为CHH和ER,这两个基因均为与对虾蜕皮有关（R²=0.795）。5.将RAS、P23和Troponin基因的编码区构建到pET32a （+）载体中进行原核表达,研究结果显示在大肠杆菌BL21中成功表达了RAS和Troponin蛋白;优化后的表达条件为30℃,1mmol/L IPTG诱导6h;而重组质粒pET32a （+）-P23在BL21（DE）中未能表达成功。更多还原

【Abstract】 Litopenaeus vannamei has become a significant economic species as the development of shrimp culture industry worldwide. Enhancement of growth rate by genetic improvement is an important technique due to the wide development of breeding technology and resistant isolates, which may decrease the length of grow-out cycles, resulting in the reduce of production cost and culture cycle of shrimp. Shrimp muscles are primarily present in the abdominal part of its body, but the genes related to muscle growth, especially regulatory genes is not well understood.In the present study, abdominal muscle samples of the female-shrimp between large female （LF; body weight>90 percentile of weight distribution curve） and small female （SF; body weight<10 percentile of weight distribution curve）were selected as the target tissue to construct forward and reverse subtracted cDNA libraries of abdominal muscle , aiming to identify and clone the differently expressed genes, and predict their structure and function by bioinformatics, and analyze their expression pattern by real-time reverse transcriptase-polymerase chain reaction, The expression levels of the selected body weight regulation genes in abdominal muscle were also analyzed. The main results were showed as follows:1. Suppression subtractive hybridization （SSH） is used to construct forward and reverse subtracted cDNA libraries of abdominal muscle of the female-shrimp between large female S-L（LF as tester, SF as driver） and small female S-L（SF as tester, LF as driver）. The subtraction efficiency was estimated by a housekeeping gene signated asβ-actin, and the results showed thatβ-actin was subtracted efficiently at 2¹⁰ and 2⁵ folds for SF and LF subtracted cDNA library respectively which demonstrated that differentially expressed genes were also enriched at the same folds.2. 50 clones were isolated from S-L subtracted cDNA library, and 30 clones were randomly selected from the library which different insert fragments were sequenced. The results showed that there exist 18 ESTs in S-L subtracted cDNA library,and 14 of them are known in L. vannamei, 4 are unknown in L. vannamei but have more than 95% homology with other species. These genes are categoried as: elongation factor 1-alpha （EF1A） , NADH dehydrogenase subunit 2 , NADH dehydrogenase subunit 1 , Cytochrome c oxidase subunitⅡ, Cytochrome c oxidase subunitⅢ, Cytochrome b , Heat shock protein 90 , crustacean hyperglycemic hormone B1, Arginine kinase, Tropomyosin slow isoform （sTm1）, Ribosomal protein 31 , Ribosomal protein S24 , Cytochrome c oxidase , subunit 2 B, troponin C;150 clones were isolated from L-S subtracted cDNA library, and 100 clones were randomly selected from the library which different insert fragments were sequenced. Searching GenBank by using these nucleotide sequences indicated that 49 cDNA fragments could not find their homologous sequences in the database. The results indicated that there exist 49 ESTs in L-S subtracted cDNA library,and 19 of them are known in L. vannamei, 19 are unknown in L. vannamei but could find their homologous sequences with other species in the database, and 11 of them is the sequence of mitochondrion, these differentially gene mainly include 5 groups: cDNA sequence that expressed with synthesis of tissue or cell components occupied 37.21% of total unigenes,Genes that expressed with growth or remediation of cell account for 13.935% of the total unigenes, Among the unigenes with molecular function classification,Binding protein, biological regulation binding activity and catalytic acitivity related proteins took up to 4.65%, 4.65% and 4.65%, respectively.3. We designed primers based on the high homology sequence in gene coding region among different species and existed sequence for L. vannamei, and successfully cloned the coding region of RAS、P23 and Troponin of L. vannamei for the first time, namely, GenBank:RAS（ORF 564bp ,GenBank No:JF806618）,P23（ORF 495bp ,GenBank No:JF806619）and Troponin（ORF 480bp, GenBank No:JF806620）,which laid a foundation for further functional research.The structure and potential function of RAS、P23 and Troponin,as well as the secondary structure and tertiary structure of the three genes, were analyzed by using bioinformatics software. The obtained results demonstrated that partial Sequence of the PAS gene had strong hydrophobicity with high structural domain. Partial Sequence of the P23 gene also had strong hydrophobicity but with no transmembrane helices structure. The signal peptide prediction in Troponin gene showed that there was a signal peptide with strong amino acid residues in the partial Sequence of the P23 gene, whose possibility was 90.2%. Hydrophobicity profile analysis and signal peptide prediction in Troponin gene demonstrated that there were strong transmembrane regions and no signal peptide with strong amino acid residues （6-24）, but with no structural domain.4.In the present study, analysis on 12 gene expression in Litopenaeus vannamei of different gender were performed and the results indicated that VTG gene expression was found in muscles of males sand females, which was inconsistent with the previous research. Study on mRNA expression of 12 genes in five tissues （eyestalk, heart, liver and pancreas, stomach and digestive tract） showed that high expression of gene AK was found in the heart and stomach where CHH gene expression was the lowest, While the highest expression of Troponin gene was found in the muscle, which were related to their functions. Correlation analysis on 9 genes and 10 characters of weight indicated that the relative expression of genes AK, PK, and TM was not associated with that of other genes, but only VTG gene was negatively correlated with body weight, and the highest correlation coefficient were observed in CHH and ER genes which are related with the shrimp molt （R²=0.795）.5. The coding region of the RAS,P23 and Troponin were constructed into pET32a （+） vector for proeukaryotic expression. The obtained results showed that RAS and Troponin were expressed successfully in BL21 E.coli and detected. The expression condition of RAS and Troponin recombinant protein was optimized at 30℃which was induced for 6 h by IPTG（1 mmol/L）.The recombinant protein pET32a （+）-P23 was not expressed in BL21 E.coli.更多还原