你的位置:水利论文 >> 论文 >> 福建省 >> 厦门大学 >> 详细内容 在线投稿

对虾白斑杆状病毒全基因组转录图谱分析及功能基因研究 【作者】兰永胜 【导师】

热度94票 浏览68次 【共3条评论】【我要评论 时间:2003年7月01日 16:58
水利论文S@*r'HK

对虾白斑杆状病毒全基因组转录图谱分析及功能基因研究

iyc8[h4Ffy0水利论文.fG~G] y/}M+K


%w,} VtOB)i S0作者兰永胜导师水利论文/E9o [)U boAbW*OW'G
【作者基本信息】厦门大学,,2003年,博士

0N]3U9wFLQ_?0水利论文ad^c8d,L,[:CFb3x,W

水利论文(MUe:Ax&v3k]J
【中文摘要】 对虾白斑杆状病毒(White spot bacilliform virus WSBV),或称白斑综合症病毒(White spot syndrome virus WSSV),是对虾养殖业最主要的病原。是一种具有囊膜的、无包涵体的、类杆状双链环状DNA病毒。分子生物学特征显示,WSBV不属于杆状病毒,分类待定。感染病毒的对虾在3-7天时间内死亡率可达90%-100%。WSBV具有广泛的宿主,包括许多甲壳类动物,并感染宿主大部分组织,对病毒的防治研究带来困难。近年来,感染范围已经波及到世界范围。对生态环境也存在潜在的威胁。WSBV全基因组序列为305,107bp,对WSBV进行全面并具有针对性的研究是解决病害的关键。基于WSBV以上的特点,我们以DNA microarray技术开展全基因组的转录图谱分析,试图研究WSBV全基因组的转录表达规律,并从中选择重要的功能基因进行针对性研究,为病害的防治提供理论依据。 本文首先采用竞争性定量PCR技术,对WSBV人工感染对虾后2-9小时内,病毒感染和增殖规律进行了研究。研究发现在病毒感染4小时以前,病毒含量呈下降趋势,4小时后,病毒含量开始呈现上升趋...更多势。同时我们观察了在4.6克至11.6克范围内,感染对虾的存活时间与对虾体重不存在线性相关。同时也对感染对虾致死后病毒累积量进行观察,发现4hr后病毒呈现逐渐累积增加的过程,至死亡时达到大于1011病毒粒子/毫克组织。研究确定感染后6小时以前作为早期时相,与感染晚期(濒死)的转录图谱进行比较研究。 本文以同位素标记的Microarray技术对WSBV的转录图谱进行研究分析。共设计259对引物,合成的DNA片段长度平均为578bp,设计的基因组DNA序列覆盖了绝大多数可预测的开放读框(ORF)。设立阴性对照、感染后2小时、6小时、濒死样品4个实验组别。对样品总RNA进行标记和杂交。实验结果表明绝大多数预测的ORF在感染对虾体内进行表达。有51个基因在感染早期(2-6hr)能够检测到转录产物。其中Wsv238基因在感染早期即有高丰度表达,并且在病毒生命活动中高丰度表达。分析认为Wsv238基因是病毒生命活动中的重要功能基因。 本文还报告了WSBV基因组大片段的天然缺失研究。不同对虾病毒株缺失片段大小不一,分别为4.6,4.8和8.1kbp。在缺失的序列两端发现富含AT的核苷酸序列,揭示出序列的缺失可能包括一些基因重组的过程。缺失的基因片段包摘要含5个开放读框,分别为wsv4sg,wsv492,wsv493,wsv495和wsv497。初步实验结果表明,基因片段缺失仅导致病毒的毒力减弱,并不影响其感染活性。这提示着病毒的基因缺失可能与病毒的毒力相关。 本文选择了缺失区基因WSV492、WSV493及极早期基因WSV238进行了表达和功能研究。其中wsv492基因能在原核载体pGEX一4T-1中全长可溶性表达,其重组蛋白(wP228)与随机 DNA发生非特异结合反应说明可能涉及基因调控。wsv493(wP136)为全长包涵体表达,由于未能获得可溶的重组蛋白,未能进行功能研究。 生物信息学分析结果显示WSV238所编码蛋白WP486在N端有明显的核定位信号,并富含甘氨酸。WP486因N端的甘氨酸簇在原核细胞中未能全长表达,但成功表达了WP486C端376个氨基酸的片段,制备了抗一WP486特异性抗体,对WP486蛋白进行了M几stern blot分析,发现表达产物的分子量约为32kD,小于理论预测51kD的分子量,说明可能病毒特殊的蛋白剪切和修饰机制。另外免疫电镜定位分析表明WP486定位在对虾细胞核内,认为该蛋白在病毒生活史中可能起重要的调控功能。  还原水利论文\v!nb2}&}s$l

S6dh5JB,S uz*h'^0【英文摘要】 Shrimp white spot bacilliform virus (WSBV), also called white spot syndrome virus (WSSV), is the heaviest pathogen to shrimp culture. It is an enveloped, non-occluded, bacilliform and circular dsDNA virus. WSBV is unclassified for the lack of molecular information and considered at a specific taxonomic position. Infection of penaeid shrimp by WSBV can result in mortality up to 90 to 100% within 3 to 7 days. Prevention and inhibition of this virus can be difficult due to its extremely virulence and a wide host range including most of aquatic crustacean and targets various tissues. The virus has spread into all over the world. It is also a threat to entironment. The complete genome of WSBV is 305,107bp. The pertinent study for import genes may be the most important for WSBV study. To obtain overall information of gene transcription, we studied the transcriptional profile of WSBV with DNA microarray. Some important gene would be further studied.The viral proliferation was studied at 2-7hr...更多 postinfection by competitive quantitative PCR. After infection, the quantity of WSBV was declined before 4 hrs and increased gradually thereafter. The accumulation of WSBV was more than 10n/mg tissue in dead infected shrimps. There was no correlation between survival time and weight of prawns ranging from 4.6g to 11.6g. As indicated in the proliferation study, 6 hr postinfection was putatively selected as early stage of infection and moribund as late stage in subsequent microarray assay.For the filter microarray (membrane) assay, 259 primers sets were synthesized to amplify WSBV DNA fragments by PCR. Most of putative open reading frames (ORFs) of WSBV could be detected by these amplicons. RNA extracted from negative control, samples of 2hr postinfection, 6hr postinfection and moribund stage was analyzed respectively. Most of putative ORFs are transcriped. Fifty-one of ORFs were found at early stage. For high frequency of expression at early stage, gene of Wsv238 gene selected for further study.This report described a spontaneous deletion in the WSBV genome. The size of the deleted fragment was approximately 4.6, 4.8 and 8.1 kb in different species. The deletion was later confirmed to be associated with the virulence of WSBV. There arefive ORFs (WSBV489, WSBV492, WSBV493, WSBV495 and WSBV497) in the deleted fragment. They might be responsible for the virulence but not for its infectivity and finally leading to the attenuation of the virus.WP228 (Wsv492) was expressed in E.coli vector as soluble fusion protein. The recombinant protein was shown nonspecific binding with DNA that mean relating to the gene regulation function. WP136 (Wsv493) was expressed in same vector but the recombinant protein was insoluble and the function research could not be processed.There are nucleus localization signals (NLS) and rich of glycines in the sequence of WP486 protein analyzed by biological informatics. The fragment of WP486 (376Aa at c-terminal) was expressed and the anti-WP486 antibody was raised for localization of WP486 with immune electron microscopy and western blot analysis. Natural protein of WP486 is shown about 32kD. The WP486 was determined localizing in the nucleus of infected shrimp cell in localization study. It was assumed as an important regulatory protein of WSBV.  还原水利论文$c Q1? D0U5^9yNp

水利论文1Y \U0g2J/sA

【中文关键词】 对虾白斑杆状病毒; Microarray; 蛋白表达与纯化; 免疫电镜技术; 蛋白质-DNA结合水利论文iX1AK:`Q sx7n
【英文关键词】 White Spot Bacilliform Virus (WSBV); Microarray; Expression and purification of protein; Immune electron microscopy; Protein-DNA binding interaction
` tM CM \\C*B`I1^4p0水利论文-s TL1oc/m

VOD4O-vQ3h G0 

1PWp1i V$~ ?0
TAG: 对虾 基因 图谱 兰永胜
顶:11 踩:13
【已经有71人表态】
13票
极差
10票
很差
9票
较差
6票
稍差
10票
稍好
7票
较好
8票
很好
8票
极好
下一篇:九龙江上游五川流域农业非点源污染研究【作者】张玉珍 【导师】洪华生
上一篇:南美白对虾类Ig的定性、功能和免疫分子进化的研究【作者】章跃陵 【导师】彭宣宪
水利论文安徽省电信网友 [mlia0t9c] ip: 117.67.*.*
2016-09-08 02:57:20
It can not only help you promote your brand but can also destroy yo <a href="http://www.bearsjerseyslockroom.com/martellus-bennett-bears-jersey/">Authentic Martellus Bennett Jersey</a> ur brand image completely. However which residence improvement jobs in fact “pay  <a href="http://www.bearsjerseyslockroom.com/pat-odonnell-bears-jersey/">Pat O'Donnell Jersey</a> for themselves” or maximize the appeal of your residence the most? DirectBuy,Authentic Martellus Bennett Jersey, the leading members-only showroom and  <a href="http://www.bearsjerseyslockroom.com/will-montgomery-bears-jersey/">Will Montgomery Bears Jersey</a> r
水利论文Unknown网友 [osdj44l3] ip: 183.160.*.*
2017-04-25 11:34:24
And to some extent, like in baseball with a baseball bat, the weight  <a href="http://www.wholesalenfljerseysonline.us.com/">Wholesale NFL Jerseys</a> of a ball used can depend upon the skill level of the bowler.If you do not w <a href="http://www.wholesalenfljerseysonline.us.com/">Cheap NFL Jerseys</a> ant to create videos yourself, get others to do it for you. "I don't know what's going to happen.tumblr. Your doctor will probably schedule follow-up visi <a href="http://www.wholesalenfljerseysonline.us.com/">Wholesale Jerseys</a> t
水利论文安徽省电信网友 [mlia0t9c] ip: 117.68.*.*
2017-04-25 12:39:55
On some occasions,botas ugg baratas espa?a, individuals will enter  <a href="http://www.comprarbotasrebajas.es/">botas ugg baratas espa?a</a> into a relationship just for a good ti <a href="http://www.comprarbotasrebajas.es/botas-ugg-bailey-button-baratas/ugg-bailey-button-mini.html">botas ugg mini bailey button</a> me with no intention of something more serious and in this situation it is not truly defined as something. Final thoughts, to research keyword effectively in PPC, you must foll <a href="http://www.comprarbotasrebajas.es/">botas ugg rebajas</a> ow the above keywor
查看全部回复【已有3位网友发表了看法】

广告投放

广告投放