一九九八年博士研究生论文摘要
一九九八年博士研究生论文摘要
论文题目：分子进化工程用于抗草鱼出血病病毒的研究
博士研究生：王冰
专&& 业：微生物学
导&& 师：田波
&& 草鱼出血病病毒（GCHV873）属于呼肠孤病毒科新成员。采用基因工程方法构建随机噬菌体九肽表位库以及采用化学合成方法建立随机RNA库。（1）以完整的GCHV873颗粒作为受体,进行三轮亲和筛选,用ELISA法测定噬菌体与病毒颗粒结合能力,从肽库中的300个克隆筛选出十六个与病毒颗粒高度亲和结合的噬菌体克隆。在草鱼肾组织细胞系中,测定GCHV873的半数细胞培养感染剂量（TCID50）,发现六个阳性克隆能使该病毒的TCID50下降5个指数,通过对抑制病毒繁殖的特异性短肽编码基因序列分析,这六个能强烈抑制病毒复制的阳性克隆的核苷酸序列完全一致,推导其相应短肽氨基酸顺序为（NH2-Leu-Trp-Val-GlY-GlY-GlY-Arg-Asn-Ala）。（2）通过随机RNA库与完整的GCHV873颗粒快速体外亲和循环筛选,将每一轮筛选获得的RNAs逆转录为DNA,再转录成RNA进行下一轮筛选。经过8轮筛选,检测经循环筛选获得的RNAs与病毒亲和能力以及抗病毒感染效果。发现RNAS在细胞培养系统中,使GCHV873的TCID50下降四个指数等级。本研究结果提示,肽库技术及RNA库技术能够应用于抗病毒方面的研究,本实验方法不需要了解病毒复杂的蛋白质结构及病毒感染过程,,筛选出的短肽及RNAs在细胞培养系统中能够直接中和草鱼出血病病毒、抑制病毒复制的,为抗病毒研究提供一条可借鉴的有效途径。
(Wang Bing)
　　Grass Carp Hemorrhage Virus (GCHV873)
is a new member in reoviridae family. We used the methods of gene engineering
to construct a random nonapeptide epitope libr-ary displayed on FUSE5 and used automated chemical solidstate
synthesis to construct a large RNA pool of random sequences. (1) After 3
rounds of Biopanning (affinity selection immuno-screening), 16 specific
clones which had higher stable affinity with GCHV were selected from 300 clones of phage displayed
peptide library using CsCl gradient purified-biotinylated intact virion
of GCHV873 by ELISA. Six of the sixteen inhibited the replication of GCHV
in CIK cells in vitro and decreased the TCID50 of GCHV from 10 (-10) to
10 (-5). After sequenced DNA of phage clones and deduced the sequences
of amino acid of the inserted foreign fragments. The results show that
the sequences of six clones which could strongly inhibit GCHV replication
have no difference, they are NH2-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala. (2)
Using intact viral particles to select from the sequence pool
the RNAs that bound specifi-cally to GCHV in vitro. By reserve transcription and
PCR, the selected RNA molecule -s could be amplified
and subjected to further fast affinity selection. After eight cycles of
such selection and amplification led to the isolation of the best target-binding RNA molecules from the library, and subsequently
by screening the selected molecules for the ability to neutralized GCHV
through determining the TCID50 of GCHV in CIK cells line in vitro. The
result showed that the selected specific RNAs could neutralize GCHV particles
and the TCID50 of GCHV was reduced four grades of the index. The results showed that these techniques can apply
to anti-viral research. Without knowledge of the structures of viral proteins
and the procedure of the comp-lex infection, the anti-viral peptides and RNAs that
bound with the surface active sites of GCHV related to the infection, could
be isolated systematically and effecti -vely respectively,
as result the infection was been inhibited. These findings suggested that the selection of random pool in vitro provided
a potential way for antiviral research.
论文题目：圈卷产色链霉菌swa4及samf1基因研究及基因组物理图谱的构建
博士研究生：刘钢
专&&& 业：遗传学
导&&& 师：谭华荣
&&& 圈卷产色链霉菌(Streptomyces
ansochromogen-es)7100是从我国东北土壤中分离到的一株尼可霉素（Nik-komycin）产生菌。它在以葡萄糖－天冬素为碳源的基本培养基上具有典型的链霉菌的分化过程。
链霉菌发育分化控制启动子－PTH4下游基因proX的24个碱基间隔处存在一个部分开放阅读框(ORF)，序列分析推测为丝氨酸蛋白酶的一部分。本文以此部分序列为探针，在圈卷产色链霉菌7100中克隆到了含有链霉菌分化有关的新基因saw4的一个4.6kb的DNA片段。序列测定及分析表明，在测定的3.2kb的DNA序列中有两个完整的开放阅读框架(ORF1和ORF2)。ORF2由789个核苷酸组成，推测编码一个263个氨基酸的蛋白产物，翻译起始密码子为GTG，终止密码子为TGA。在计算机蛋白文库中进行了同源性比较研究，结果表明，263个氨基酸的蛋白产物与Caulobacter
crescentus的依赖于ATP的丝氨酸蛋白酶有44.7%的同源性，其中存在一个丝氨酸的保守区（GPSAG），该基因称为saw4(Streptomyces
ansochromogenes whi gene)。基因破坏结果表明，该基因与圈卷产色链霉菌孢子形成中分隔和色素合成有关，而与Nikkomycin的生物合成没有关系。ORF1位于ORF2上游，由639个核苷酸组成，编码一213个氨基酸的蛋白质，翻译起始位点为GTG，终止密码子为TGA。在计算机蛋白文库中进行的同源性比较表明，该阅读框所编码的213个氨基酸的蛋白产物与红球菌3羟苯丙基丙酸(3H
-PP)代谢的调控基因hppR具有54%的同源性，该基因称为samf1(Streptomyces
ansochromogenes mycelium formation gene)。基因破坏结果表明，samf1基因与菌丝的形成有关，而与Nikkomycin的生物合成没有关系。
&&& 本文通过脉冲场凝胶电泳首次证明了圈卷产色链霉菌的基因组大小为7900kb，与天蓝色链霉菌基因组8000kb相近。同时，以在链霉菌中具有稀有切点的限制性内切酶AseI和DraI构建了圈卷产色链霉菌7100基因组的物理图谱。12个AseI酶切片段和7个DraI酶切片段被分别定位于圈卷产色链霉菌7100的基因组上。与圈卷产色链霉菌分化相关的4个基因(saw1,
saw2, saw3和saw4)也被分别定位在基因组物理图谱的不同位置上。此外，在脉冲场凝胶电泳过程中，还发现了一个350kb的巨型质粒。
(Liu gang)
　　Streptomyces ansochromogenes,
a producer of nikkomycin, is isolated from the soil of Northeast China.
It undergoes a complex cycle of morphological differentiation on minimal
medium containing glucose-asparagine as carbon source.
&　　The downstream of proX is a
partial open reading frame(ORF). This fragment(about 400bp) was made as a probe, a homologous DNA fragment
was cloned from total DNA library of Streptomyces ansochromogenes
by Southern hybridization. The results showed that a 4.6kb DNA fragment inserted into Bluescript
M13- vector exhibited a positive signal. After deletion and subclon, a 3.2kb DNA
fragment was sequenced.Nucleotide sequencing analysis indicated that the 3.2kb DNA
fragment contained two complete open reading frames (ORF1 and ORF2). In search
of databases, the deduced product of ORF2 contained 263 amino acids. It was homologous
to the ATP-dependent serine protease and contained a conserved serine-catalytic
active site(GPSAG), the identity of amino acids is about 26.7%. It was designated
as saw4 (Streptomyces ansoch-romogenes whi gene).
The deduced product of ORF1 contained 213 amino acids, is homo-logous to the HppR of Rhodococcus globerulus,
the identity of amino acids is about 38%. It was designated as samf1
(Streptomyces ansochromogenes mycelium formation).
　　The functions of the two genes were
studied using the strategy of gene gisruption.The mycelium septum could not be formed and pigments
overproduced when saw4 was disrupted. The aerial mycelium could
not form normally when samf1 was disrupted. The production of nikkomycin
did not change in these disruptants. Therefore, saw4 may be related
to the formation of septum and the production of pigment, samf1
may be related to the formation of aerial mycelium.
　　A physical map of the Streptomyces
ansochromogenes genome was constructed by ordering the macrorestriction fragments generated from
the genomic DNA with the restriction enzymes AseI and DraI. The 12 AseI
and 7 DraI fragments were aligned as a single chromosome of about 7900kb. Four cloned genes (saw1,
saw2, saw3 and saw4), which are related to the differentiation
of Streptomyces ansochromogenes, was located on the chromosome by
Southern hybridization. A large plasmid (about 350kb) was found in Streptomyces
ansochromogenes.
论文题目：抗肿瘤血管基因工程抗体的研究
博士研究生：汤健
专&& 业：微生物学
导&& 师：田波
　　血管内皮生长因子是与肿瘤的生长及转移密切相关的有丝分裂原，特异作用于血管内皮细胞。抗VEGF的抗体可以阻断VEGF与其受体的结合，从而抑制肿瘤生长与转移。我们利用大肠杆菌表达VEGF165，经纯化后作为抗原，从人源噬菌体抗体库中筛选出可与VEGF165特异结合的单链抗体，命名为hv37。它是由786个核苷酸组成，蛋白分子量为32KD。我们用突变引物将hv37基因克隆至pET21a表达载体上，在大肠杆菌BL21中获得了高效表达，表达量约为90mg/L培养物，并对表达产物单链抗体hv37进行了纯化及复性。免疫检测表明hv37可与不同来源的VEGF特异结合，并且能够识别胃肠道肿瘤组织中的肿瘤细胞及血管。鸡尿囊膜实验表明，抗体hv37可封闭VEGF刺激血管增生的作用。
　　肿瘤组织内IV型胶原酶表达和活性升高与肿瘤转移密切相关。抑制CoIV活性的物质能够抑制或降低肿瘤的转移。我们从人抗体库中筛选出一株CoIV特异单链抗体，命名为hCo4。该抗体分子量约为27KD。免疫检测证明hCo4能够与CoIV特异结合，并有中和活性。随后，我们将hCo4基因克隆到Pichia
pastoris酵母分泌表达载体pPIC9上，转化受体菌GS115，免疫检测表明Pichia
-oris 分泌表达人抗coIV单链抗体。
　　抗体hv37和hCo4可以用于肿瘤与血管生成相互关系的基础研究并可望成为肿瘤免疫治疗的新型靶向药物。
(Tang jian)
　　Vascular endothelial growth factor
(VEGF) is an endothelial cell specific mitogen which is related to tumor growth and metastasis. The
VEGF165 was expressed in E.coli and was then purified and used as an antigen
to select scFv from a human semi-synthetic phage-antibody library. A scFv capable of
binding to VEGF165 was obtained and named as hv37. Hv37 gene consisted
of 786 nucleotides and its molecular weight was 32 kD. Hv37 gene was cloned
into pET21a expression vector using mutagenized primers and the expression yield is 90mg/L culture. The
hv37 was then purified and renatured. Immuno-assays showed that hv37 could
bind specifically to VEGF from various origins and recognize tumor cells
and vasculature in the tumor tissues of gastric carcinoma. CAM assay showed
the activity of VEGF was blocked by hv37.
　　The overexpression of collagenase IV
in tumor tissues is closely related to tumor metastasis. We screened a coIV specific single chain
antibody from human antibody library, named hCo4. The molecular weight
of hv37 is 27 kD. Both ELISA and immuno-dot blot analysis verified that hCo4 could specifically
bind to coIV and block its activity. We cloned the hCo4 gene into the yeast
Pichia pastoris secretive expression vector pPIC9. The recombinant construct was transformed
into the host cell GS115.ELISA and Western blot analysis indicated that
only one of 40 clones of Pichia pastoris expressed and secreted
human anti-coIV single chain antibody.
&&& Antibodies hv37 and hCo4 may be applied
in the theoretical and clinical studies on the relationship between tumor
and angiogenesis.
论文题目：水稻黄矮弹状病毒的分子生物学M和G基因的序列及病毒转录物的新结构
博士研究生：罗宗礼
专&& 业：遗传学
导&& 师：方荣祥
　　水稻黄矮病毒是一种含单链、负极性RNA基因组的植物弹状病毒。我们前已克隆并测定了基因组3′端部分的leader、N基因、NS基因、基因3及5′端部分的基因6、
L基因、trailer的序列。本研究利用基因3和基因6末端片段为探针，通过朝未知基因方向步进的方法，从RYSV基因组RNA的cDNA文库中筛选得到了覆盖RYSV
M基因和G基因序列的克隆，并测定了它们的核苷酸序列。M和G基因序列的测定促成了RYSV基因组的全序列分析：RYSV基因组全长14042个核苷酸，共含有七个基因，其中基因6是在植物弹状病毒中发现的一个新基因。
　　M基因共含913个核苷酸（nt），包括17
nt的5′非编码区、789 nt的编码区和107 nt的3′非编码区。M基因编码的蛋白质含262个氨基酸，计算分子量29,125
Da。血清学实验证明M基因编码RYSV的M蛋白。RYSV的M蛋白与其它弹状病毒M蛋白无明显的序列同源性，但它们的氨基酸组成及结构特点却很相似，如含有较多的碱性氨基酸、酸性氨基酸区与碱性氨基酸区相间排列等。
　　G基因全长2158 nt，包括45
nt的5′非编码区、2010 nt的编码区及103 nt的3′非编码区。G基因编码的蛋白质分子量75,358
Da, 含669个氨基酸，具有典型的弹状病毒糖蛋白的结构特点，N端有一个由32个氨基酸组成的信号肽，C端含有一疏水性氨基酸组成的跨膜区，另外还存在10个糖基化位点和一些可能的磷脂结合域。
　　用3′RACE和 5′RACE的方法确定了RYSV所有基因的转录终止位点及部分基因的转录起始位点，证明RYSV各基因间存在保守的基因间隔序列并发现了基因转录物的新的结构特征：1）M、G、L基因和基因6的转录物的5′端存在非病毒来源的核苷酸序列；2）正链leader
RNA 3′末端带有poly（A）尾。
(Luo zongli)
　　Rice yellow stunt virus (RYSV) is a
plant rhabdovirus which contains a non-segmented, single-stranded negative sense RNA genome. The
nucleotide sequences of the leader、N gene、NS gene and gene 3 at the 3′part of the
RYSV genome and the gene 6、L gene and trailer at the 5′part of the genome
have been previously determined. In this study, by walking along the genome towards the sequence-unknown
region, we have now identified and sequenced clones covering the M and
G genes, thus completing the sequence analysis of the entire RYSV genome:
it consists of 14,042 nucleotides (nt) and contains seven genes，in which
the gene 6 is a novel gene in plant rhabdoviruses.
　　The M gene is 913-nt long, comprising
a 17-nt untranslated 5′region, a 789-nt open reading frame (ORF) encoding
a polypeptide with a calculated molecular mass of 29,125 Da, and a 107-nt
untranslated 3′region. Serological analysis confirmed that the M gene
encodes the M protein of RYSV. Comparisons of the amino acid sequence of
the RYSV M protein with those of other rhabdoviruses revealed no significant
homologies. However, it showed some similar features with
other rhabdoviral M proteins, for instance, it contains more basic amino
acids and the acidic and basic stretches in the protein alternate along
the sequence.　　The G gene is 2158-nt long and comprises
a 45-nt untranslated 5′region, a 103－nt untranslated 3′region and a 2010-nt ORF encoding
a protein of 669 aa with a calculated molecular mass of 75,358 Da. The amino acid sequences
deduced from the RYSV G gene showed some typical features shared by other
rhabdovirus glycoproteins: it contains an amino -terminal signal peptide
of 32 aa, a carboxyl terminal hydrophobic transmembrane domain, 10 putative
glycosylation sites and some putative phospholipid-binding domains.
　　The transcription termination site
of each gene and transcription initiation site of some of the genes of
RYSV were defined by 3′or 5′RACE. The conserved intergenic sequences were verified to exist between the RYSV
genes and the following novel structures of the RYSV gene transcripts were
found: 1) Some non-viral nucleotides exist at the 5′ends of mRNAs of M、 G、 L genes
and gene 6; 2) The plus-strand leader RNA is polyadenylated.
论文题目：人促血小板生成素CDNA克隆、表达、纯化及生物活性的研究
博士研究生：樊云祯
专&& 业：微生物学
导&& 师：田波
　　人血小板生成素(hTPO)是含有332个氨基酸的糖蛋白，主要存在于肝脏中。以人胎肝组织总RNA为模板，RT-PCR方法扩增出TPO
cDNA，将扩增得到的cDNA以平端形式克隆到pUC19的SmaI位点，酶切鉴定和序列分析表明得到cDNA确为TPO
cDNA，且与已知TPO cDNA高度同源，同源性高达99.5%，其中N-端EPO类似区域cDNA序列没有变化，而C-端的第497bp、595bp、767bp和795bp碱基分别由T、G、T、T替代了已知的G、A、G和C，从而导致了166、199、256位的氨基酸由已知序列的Ser、Lys和Gly分别变为Phe、Glu和Val，而265位并没有导致氨基酸的变化。
　　在克隆并分析了TPO cDNA的基础上，将全长hTPO cDNA克隆到原核表达载体pET-15b和pBV220中得到了表达，并且以pBV220为载体探讨了SD序列与AUG之间的距离对cDNA表达效率的影响；为了进一步提高表达效率，将hTPO
cDNA 5?端在不改变氨基酸的前提下修饰起始密码子ATG下游核苷酸，PCR方法得到hTPO功能区域cDNA172，在原核表达载体pET-15b中得到较高效率表达；进一步优化表达条件，功能片段的表达量占菌体总蛋白的15%左右；确定了表达蛋白主要以可溶形式存在于菌体细胞中，通过离子交换和分子筛层析两步纯化了表达蛋白，纯度达到90%以上。
　　SDS-PAGE和N-端氨基酸序列分析等理化性质表明表达蛋白为hTPO及其功能片段；免疫学分析也证明表达蛋白能与兔抗人TPO多抗特异性结合；鼠血小板生成试验证明表达的hTPO功能片段具有明显的体内促进循环血小板生成作用，而且随TPO给药剂量的上升，血小板数量也同样升高，与对照组相比，25ng、50ng和100ng给药7天的试验组平均血小板计数分别升高了10.51%、24.64%和48.55%，具有明显的体内生物学活性，为基因工程生产TPO及其临床应用奠定了基础。
(Fan yunzhen)
　　Human thrombopoietin(hTPO), a ligand
for the proto-oncogene c-mpl, is the major humoral regulator of megakaryocytopoiesis
and platelet production by affecting the proliferation and maturation of
committed cells. Recently hTPO gene and its cDNA were cloned from human
genome and cDNA libraries. In this report, hTPO cDNA was synthesized by
reverse transcriptase-polymerase chain reaction from Chinese fetal liver
and cloned into pUC19. The full-length cDNA was subcloned and sequenced.
Sequence of this cDNA showed a high extent of homology with hTPO cDNA in
the Gene-bank data base(accession nos. L36052) except that base
substitution occurred at four sites(497,595,767 and 795bp), which led to
the change of three amino acid residues in the predicted protein.
　　Expression vectors pET-TPO and pBV-TPO
were constructed to express full-length TPO cDNA in E.coli. Influence
of SD-AUG and promotors on the expression level was studied. Truncated
form of human thrombopoietin cDNA172, containing the Epo-like domain was
synthesized by PCR and cloned into pET-15b after modification of its nucleiotides
downstream of initial codon AUG and expressed in E.coli BL21(DE3)
(pLysS). Inducing by 1.0mmol/L of IPTG, the expression level of rhTPO172
was about 15% of total bacteria protein. The expressed protein is mainly
soluble, which was purified by cation ion exchange and gel chromatography.
The purity is more than 90%.
　　Physical and chemical features, including
SDS-PAGE, amino acid analysis etc., and western blot analysis indicated
that the expressed proteins are full-length hTPO and its truncated form
hTPO172, which have immunological activity. In mouse thrombopoie -sis assay,
BALB/c mice treated with daily intraperitoneal injections of 25ng, 50ng
and 100ng of purified rhTPO172 had 10.51%, 24.64% and 48.55% increase in
circulating platelet levels respectively, after seven days when compared
to control animals receiving control buffer.
论文题目：双生病毒的分子生物学
博士研究生：刘玉乐
专&& 业：微生物学
导&& 师：田波
　　本研究完成了巴基斯坦棉花曲叶病毒（CLCuV-PK）全长基因组单体和双体DNA克隆，证明了CLCuV-PK是一个单组分双生病毒。分子杂交、PCR、序列和酶切分析表明：在CLCuV-PK侵染的烟草和番茄中除CLCuV-PK全长基因组存在外，还存在一系列的与CLCuV-PK基因组相关的小环状DNA分子，而且不同的侵染植株有不同的小环状DNA分子。小环状DNA分子可分为三类：第一类为CLCuV-PK的亚基因组，第二类为几个CLCuV-PK基因组片段重排后形成，第三类小环状DNA分子除含有CLCuV-PK部分DNA序列外还含有非病毒DNA序列；这是首次发现双生病毒重排现象、CLCuV-PK的亚基因组现象和非病毒序列插入病毒序列的现象。本文首次报道了双生病毒相关的小环状DNA分子可经粉虱传播的现象。本研究表明寄主植物以及侵染时间均对小环状DNA分子的产生及其大小有一定的影响；嫁接实验初步表明一些小环状DNA分子的存在可加重CLCuV-PK在番茄植株上引起的症状。
　　本文用二种方法（ELISA和PCR）检测CLCuV-PK，证明CLCuV-PK与引起巴基斯坦秋葵曲叶和秋葵黄化叶脉花叶的病毒有很近的亲缘关系。此外，发现5类不同于CLCuV-PK的粉虱传双生病毒，一些病毒可能为新病毒。
　　根据大量的序列分析结果，提出了CLCuV-PK与秋葵曲叶病毒实质相同，且均起源于秋葵黄化叶脉花叶病毒的新见解。序列分析表明至少存在二类CLCuV-PK,
二类CLCuV-PK的 AC1基因以及基因间隔区左半部分有较大差异，而基因间隔区右半部分、AV2基因和CP基因几乎完全相同，表明至少一类CLCuV-PK的产生与病毒间重组有关。
　　本文表明乌干达严重木薯花叶病流行不仅与非洲木薯花叶病毒有关，还与一种新病毒有关，这种新病毒由EACMV和ACMV基因组间重组产生。杂种病毒可能也存在多种类型，这是二个已知的植物病毒基因组重组产生新病毒的首次报道。同时表明乌干达严重木薯花叶病非流行区的木薯花叶病为ACMV引起。
　　克隆和部分序列分析表明中国和非洲塞内加尔的番茄黄化曲叶病毒分别为不同的粉虱传双生病毒，且均为新的双生病毒。
(Liu yule)
　　The monomer and dimer clones of full-length
genome of cotton leaf curl gemini-virus from Pakistan(CLCuV-PK) were constructed , and
it was shown that CLCuV-PK could be a geminivirus with monopartite genome.
The results from southern blots, PCR, DNA sequencing and patterns of enzyme
digestion showed that there are a series of small circular DNA molecules
except full-length genomes of CLCuV-PK in plants infected with CLCuV-PK,
and the small circular DNA molecules are different in different plants infected with CLCuV-PK. These small circular
DNA molecules could be grouped into three categories according to the source
and form of their sequences: (1) subgenomic DNA of CLCuV-PK, (2) which
consist of several pieces of CLCuV-PK genome with rearrangements, (3) small
circular DNA molecules with short sequences not found in the genome of
CLCuV-PK. This is the first report of rearrangement within geminivirus
DNA, subgenomic DNA of CLCuV and naturally-ocuured insertion of non-viral
sequence into viral replicon separately. it is also first reported that
small circular geminivirus DNA molecules can be transmitted from a plant
to another plant by whiteflies. it was shown that production and size of
small circular DNA molecules were affected by plant species and individual
plant. Small circular DNA molecules were also producted following a long
time of growth of plants only with fullth-length of genome of CLCuV-PK.
The grafting experments showed that the symptom became more severe if some
small circular DNA molecules were present in tomato plants.
　　The thesis decribes two methods of
detecting CLCuV-PK: TAS-ELISA and PCR. it is shown that CLCuV-PK is very
closely related to viruses causing yellow vein disease or leaf curl disease
of okra in Pakistan. In addition, Five additional whitefly-transmitted geminiviruses were found in Pakistan, and some
of them could be new species of geminiviruses.
　　It was suggested that there is same
entity between CLCuV-PK and okra leaf curl virus (OLCV), and both CLCuV-PK
and OLCV could be originated from okra yellow vein mosaic virus. It was
shown that there are at least two types of CLCuV-PK, which have big difference
in of AC1 gene and 5’part of large intergenic region, but have nearly identical sequences in
3’part of large intergenic
region, AV2 gene and coat protein gene, and these results showed that at
least one of two types of CLCuV-PK was producted by recombination between
　　It was shown that the epidemic of an
extremely sever form of cassava mosaic disease in Uganda is not only associated with ACMV, but
also associated with a new virus. this kind of new virus is caused by recombination
between ACMV and EACMV, and there could be a lot of types of recombinant
The cassava mosaic disease before the epidemic was caused by isolates of
　　The results from DNA sequencing and
ELISA showed that tomato yellow leaf curl virus from Senegal and PR China
is different new whitefly-transmitted geminivirus separately.
论文题目：(一)分子伴娘60（Chaperonin）的分子生物学研究
(二)二株嗜盐嗜碱古生菌以16S rRNA序列为基础的系统发育学地位研究
博士研究生：徐毅
专&& 业：微生物学
导&& 师：周培瑾
论文摘要(一)
&&& 首先从一株嗜碱芽孢杆菌alkaliphilic
Bacillus sp. strain C－125中分离出分子伴娘60 groEL基因，克隆到大肠杆菌宿主细胞中，亚克隆后进行测序。C－125菌株分子伴娘60基因的测序结果表明，该基因编码一544个氨基酸肽链的产物，该基因的上游含有一个缺失5?
－端不完整的groES基因。由DNA序列推测的氨基酸序列表现出与B. subtilis
( 87.5 % ), B. stearothermophilus ( 85.4 % ) 和E. coli (
60.9 % )很高的同源性。
&&& C－125菌株的GroEL蛋白在E. coli中高表达以后，通过一系列步骤纯化，得到单一组分的GroEL蛋白聚合体。体外实验表明，这一纯化的GroEL蛋白在高温环境中可以保护a-glucosidase避免不可逆的凝聚沉淀。Mg－ATP的加入对于释放有活性的天然态a-glucosidase是必须的。体外实验还表明，E.
coli的GroES蛋白可与C－125菌株的GroEL蛋白合作，从而加速a-glucosidase的复性。
&&& 当嗜碱芽孢杆菌alkaliphilic Bacillus
sp. C－125菌株在亚致死温度下（52℃）预培养一段时间，可以导致GroEL蛋白表达量的提高，同时，使该菌株获得一定的耐热性，从而可在致死温度下（54～55℃）存活和生长。
Abstract(一)
　　The groEL gene of an alkaliphilic Bacillus
sp. strain C-125 was cloned in Escher-ichia coli and its nucleotide sequence was determined.
The groEL gene encoded a pol-ypeptide of 544 amino acids and was preceded by the
incomplete groES gene lacking its 5'-end. The derived amino acid sequence
exhibited high sequence identity to those of B. subtilis ( 87.5
% ), B. stearo-thermophilus ( 85.4 % ) and E. coli ( 60.9
　　The GroEL protein was overexpressed
in E. coli. Purified GroEL protested yeast α -glucosidase from irreversible
aggregation at high temperature and addition of Mg-ATP was essential for reactivation of the a-glucosidase.
E.coli GroES accelerated there activation, indicating that the alkaliphilic
Bacillus C-125 GroEL could function in coordination with E. coli GroES.
　　Exposure of the strain C-125 to a sublethal
temperature of 52℃ lead to a large increase of GroEL production and in
meantime exhibit an acquired thermotolerance response, thus, survived at
a lethal temperature of 55℃.
论文摘要(二)
　　新的嗜盐嗜碱古生菌的发现和描述正在以前所未有的速度发展，旧的分类体系已不能够适应这种快速的发展势头。因此，就需要根据新的分类标准对这些古生菌进行重新分类。分子生物学技术的发展和应用使得在嗜盐古生菌中建立真正的系统发育学关系成为可能。二株嗜盐嗜碱古生菌（A33和GA33）的16S
rRNA序列已经测定，以16S rRNA序列为基础的系统发育树通过于其它嗜盐古生菌成员16S
rRNA序列的多序列匹配排列而建立。这二株嗜盐嗜碱古生菌的极性脂组成进行了分析，它们的其它表型特征也被确定。实验结果表明，这二株嗜盐嗜碱古生菌（A33和GA33）应被分成一个新属，暂时成为Natronorubrum
bangense gen. nov., sp. nov. （菌株A33）和Natronorubrum
tibetense gen. nov., sp. nov.（菌株GA33）。
Abstract(二)
　　The discovery and description of novel
haloalkaliphilic archaea are continuing at an accelerated pace, and the
old taxonomical system has been unable to adapt to such rapid strides,
so, it is necessary to reclassify them on the basis of new standards. Molecular biological techniques are allowing true
phylogenetic relationships within halobacterial taxa to be established.
The nucleotide sequences of the 16S rRNA genes from two strains of haloalkaliphilic
archaea, A33 and GA33, were supplied by the analysis of the cloned rDNAs.
Phylogenetic subtrees for subfamily containing core diether lipids of C20-C25
were produced by the alignment of the 16S rRNA sequences of the two strains with those of other halobacteria,
polar lipid compositions were analyzed, and some phenetic properties were
determined. The results indicates that the strains A33 and GA33 should
be classified into a new genus, tentatively called Natronorubrum
gen. nov. as Natronorubrum bangense ( strain A33) and Natron－orubrum tibetense ( strain GA33).
论文题目：黄瓜花叶病毒卫星RNA及其在抗病毒基因工程中的应用
博士研究生：叶寅
专&& 业：微生物学
导&& 师：田波
　　从患香蕉花叶心腐病香蕉病叶分离的黄瓜花叶病毒中,发现一种新的卫星RNA（Ba-RNA）。合成并克隆了该卫星cDNA,序列分析结果表明，Ba-Sat由390个核苷酸组成。分析了Ba-Sat的二级结构，并与其它株系卫星RNA进行了同源性比较。生物学实验证明该Ba-RNA具有致弱卫星RNA的特性。
　　通过反转录合成和克隆方法获得了黄瓜叶病毒JV株系外壳蛋白基因（CP），构建了包含CWV
CP和卫星（Sat）的双基因植物转化载体pRCPl，经遗传转化获得转双基因植株。攻毒实验证明转双基因植株中的CMV浓度只相当于未转对照的0-5%，而单独表达Sat的转基因植株相当于对照的10-20%；抗性分析结果表明，转双基因植株比单转Sat或CP基因植株的抗性高一倍以上。
　　以烟草NC89品种单倍体植株作为转化受体，获得3株抗性水平高且能同时表达CP和Sat的转基因单倍体植株，人工加倍后直接获得转基因NC89纯合新品系。大田试验结果表明，转基因纯合品系第二、三代表现出一致且稳定遗传的抗性，田间相对防护效果达到80%以上，且转基因材料品种特性未变。在主要经济性状上，转基因品系与原NC89相比有明显改善，上等烟叶比例和产量均有较大提高。
　　One satellite RNA was found associated
with cucumber mosaic virus (CMV) from banana (Ba Sat). The full-length
cDNA was synthesized and sequenced. The data showed that Ba sat had 390
nucleotides in size and had high extent homology with other CMV satellite
RNAs in both 3* and 5* end.
　　A chimaeric vector was constructed
to express CMV satellite RNA (Sat) and coat protein (CP). Transgenic lines
of tobacco cultivar G-140 expressing both Sat and CP were obtained. These
lines had high resistance to CMV. Resistance was about twice that conferred
by Sat or CP gene alone in transformed plants.
　　A procedure for the fast production
of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from another
cultures were transformed with the chimaeric vector pRCPI. Five transgenic
haploid plants showing no symptoms 30 days after inoculation with high
concentration of CMV (200mg/ml) were diploidized by colchicine treatment.
Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The
three transgenic lines were further tested under field conditions. The
results showed that the progenies of this transgenic lines were homozygous
and were highly resistance to CMV under natural field infection and manual
inoculation conditions.