论文题目 ：小球藻病毒的分子生物学性质及其外壳蛋白基因5’侧翼区顺式作用元件的分析
博士研究生：张远征
专 业：病毒学
导 师：田 波（院士）
论文摘要 ：

以小球藻NC64A株系为寄主,从17个省市采集的数百个水样中分离到了11个病毒分离物。这些病毒具有许多相同的性质,如均为球形多角体,基因组为300kbp左右的dsDNA。由于小球藻病毒与寄主的关系具有严格的专一性,所以这11个病毒分离物均属于NC64A小球藻病毒。然而,对病毒的生物学性质的研究结果表明,这些病毒间存在着许多差异:如11个病毒分离物的大小不同;基因组DNA限制性酶切图谱和结构蛋白的组成不同（除BJ-1和BJ-2相同外）; 基因组的甲基化碱基含量不同等。所以,它们应属于同一类病毒的不同的病毒株系。在11个病毒分离物中除FJ-1的主要外壳蛋白的分子量小于54KD外,其它10个病毒分离物与PBCV-1一样,主要外壳蛋白的分子量均为54KD。Westernblot分析的结果显示, 除FJ-1外其它病毒分离物与PBCV-1的抗血清有较强的免疫交叉反应。说明这些病毒与PBCV-1的同源性较高,而且病毒同源性的高低并不受地理位置的影响。在11个病毒分离物中,FJ-1具有一些特殊的性质。Southernblotting分析的结果表明, FJ-1的主要外壳蛋白基因位于基因组EcoRI酶切图谱的第十条带上,而PBCV-1的主要外壳蛋白基因则位于第4条带上。

通过PCR扩增了PBCV-1主要外壳蛋白（Vp54）基因5’侧翼区（5’FLR）,并将其克隆在质粒载体pUC19上。序列分析表明5’FLR长为632bp,和已知序列进行比较发现,第616、623和625的碱基由A突变为C。对5’FLR的启动活性的研究表明,该侧翼区在大肠杆菌中具有很强的启动活性,在酵母菌中也具有启动活性。这说明Vp54基因的启动子在原核和真核生物中均具有启动活性。然而,和小球藻病毒腺嘌呤甲基化酶基因的启动子一样,Vp54基因5’FLR也没有真核或原核启动子的典型cis作用元件的结构,说明小球藻病毒的启动子具有特殊的结构。

为了深入研究Vp54 5’FLR cis作用元件的结构和功能, 利用Exo Ⅲ法产生了5’FLR的系列缺失突变体,并测定了其核苷酸序列。筛选到的7个突变体在大肠杆菌中均具有启动活性。在7个突变体中最小的突变体只含有19bp核苷酸片段,它的启动活性也是最低的。所以,推测19bp片段含有核心启动子元件。5’FLR经HindⅢ酶切产生1-336bp和337-632bp两个片段,分别用同位素标记制成探针, 与大肠杆菌DNA结和蛋白相互作用进行凝胶阻滞试验。结果表明,在1-336bp和337-632bp片段上均存在有大肠杆菌DNA结合蛋白的结合位点。证明在两个片段中均包含有Vp54基因启动子的cis作用元件。由于最大的缺失片段（514bp）在大肠杆菌中的启动活性是19bp片段的3倍,所以推测在1-336bp片段上存在有远端增强子元件。

Abstract:
An initial survey was done acrossthe whole country.Our studies showed that Chlorella viruses are widelydistributed over China.Eleven viruses have been isolated,which lysis theChlorella sp.（strain NC64A）.These viruses have several common properties,suchas polyhedral morphology,and linear dsDNA genomes.However,they have moredifference in the restriction pattern of viral DNA,the concentration ofm5dC and m6dA in viral DNA,and the composition ofviral structure protein.It was suggested that they should belong to thesame virus group,but different strains.All of viral major capsid proteinsare 54KD except FJ-1,which major capsid protein is smaller than 54KD .Westernblot analysis showed strong immunological cross reaction among all of viralproteins with PBCV-1 antiserum except FJ-1.The results indicated that therewas a high homology between some chinese chlorella viruses and PBCV-1,which was not influenced by geographical position. Compared with other10 viruses,FJ-1 has some specific properties.The southern hybridizationanalysis showed that the major capsid protein gene was located at the 10thband of electrophoretic separation patterns of the FJ-1 DNA EcoRI digest,butPBCV-1’s at the4th band.

The 5’flanking region （5’FLR）of the major capsid protein （Vp54）gene of PBCV-1 was amplified by PCR,andcloned into pUC19.The nucleotide sequence of 5’FLR is 632bp,which is the same as the published sequence except the A mutatedto C at the site of the 616th,623th and 625th.The promoter function ofthe 5’ FLR indicatedthat the 5’ FLRhas high activity in E.coli and works in Yeast.It was demonstrated thatthe Vp54 gene promoter has activity in both prokaryotes and eukaryotes.However,likeadenine methyltransferase gene promoter of Chlorella virus,the 5’FLR lacks a canonical structure of cis acting element of both eukaryoticand prokaryotic promoter.It was suggested that Chlorella viruses’promoters have specific structure.

In order to analyze the structureof cis acting element of the Vp54 gene 5’FLR,a series of deletion mutants was generated using the ExoⅢ protocolsand sequenced.All of the seven deletion mutants have activity in E.coli.Amongthe seven mutants,the smallest mutant only containing 19bp fragment hasthe lowest activity in E.coli,but it has the function which promotes thebasal transcription.It was implied that the 19bp fragment contains thecore promoter element.

An interaction of the Vp54 gene 5’FLR with E.coli DNA bind proteins was analyzed by mobility shift assay.Both1-336bp and 337-632bp fragments of 5’FLR have the binding sites of E.coli DNA binding proteins.It indicatedthat both of them contain the cis acting elements of Vp54 gene pormoter.Sincethe activity of the bigest deletion mutant （514bp） is the three timesof 19bp fragment’s,adistal enhancer element may locate at the upstream of 5’FLR.

论文题目:细菌无毒信号基因avrD和avrRxv介导的抗病转基因烟草
博士研究生: 刘国胜
专 业: 病毒学
导 师: 田 波（院士）
论文摘要:
本实验构建了4个由病原菌特异诱导型启动子与病原菌无毒基因组成的植物表达载体,分别含PAL-avrD、CHS-avrD、Pill-avrD和Pill-avrRxv嵌合基因。通过土壤农杆菌LBA4404介导分别转化烟草品种K326和BS。SouthernBlot和Northern Blot分析证明,无毒基因已整合到烟草基因组中并表达。在温室和田间通过接种不同种类的病原菌--烟草赤星病菌、青枯病菌和野火病菌,对转基因植株的R0和R1代的抗病性进行了鉴定。结果表明,尽管在不同的基因--品种组合中抗病性存在差异,但是每种转基因植株的抗病性都有了较大提高,并且某些植株可以抵抗2种病菌的侵害。转基因植株和对照植株PO和PAL酶的活性在病原菌侵染后的动态变化类型上也存在显著差异。上述结果支持了以下假说:某些信号基因不但在相应的寄主植物上而且也能够在非寄主植物上激发主动的防御反应。通过本策略获得的抗病转基因烟草具有广谱、高效和持久的特点。

Abstract:
Four chimeric gene fusions,eachof those was composed of a pathogen-inducible promoter and an avirulencegene of pathogen,were constructed and introduced into tobacco or tomatoby Agrobacterium-mediated transformation.The gene fusions are PAL-avrD,CHS-avrD,PiII-avrDand PiII-avrRxv.Integration and expression of the genes into the plantgenome were confirmed by Southern and Northern Blot.The disease resistanceof the transgenic plants at R0 and R1 generationwere evaluated through inoculating with different kinds of pathogens suchas Alternaria alternata,Pseudomonas solanacearum and P.syringaepv tabaci in the condition of greenhouse and field.The results showedthat the disease resistance was strongly enhanced in each transgant fromR0 to R1 generation although there existed slightdifference among gene-variety combinations,and some plants could retardthe development of two or three sorts of fungal or bacterial diseases,whichsupported for the hypothesis that some signal genes could trigger the regulationand expression of a series of programmed defense genes not only in theircorresponding host plants but also in certain nonhost plants.The differenceof change patterns of two enzymes-phenylalanine ammonia-lyase（PAL）andperoxidase （PO）were also observed among transgenic and control plants.Theresistance of the engineered plants by this novel strategy might be expectedto be wide,high and stable.

论文摘要:TMV-L运动蛋白基因植物表达载体的构建及其与番茄Tm-22基因的互作
对含有烟草花叶病毒番茄株系（TMV-L）部分基因组cDNA的克隆进行缺失改造,得到了含有TMV-L运动蛋白（MP）基因的克隆。MP片段分别与组成型启动子CaMV35S和病原物特异诱导型启动子CHS、PiII和BG体外重组,构建成植物表达载体p35S-30K,pCHS-30K,pPiII-30K和pBG-30K。通过农杆菌LBA4404介导,分别转化含Tm-22抗性基因的番茄品种Geneva80。转化p35S-30K的基因工程植株在苗期观察到了部分坏死和黄化现象,初步证明TMV-LMP与Tm-22存在基因对基因的互作关系。诱导型启动子控制下的MP转基因番茄已得到部分潮霉素抗性愈伤组织,为进一步获得广谱高效抗病植株奠定了基础。

Abstract:
A clone harboring movement proteingene （MP）was obtained by deletion of plasmid containing partial TMV tomatostrain （TMV-L）genomic cDNA.The MP fragments were then fused with constitutiveexpression promoter CaMV 35S.and pathogen-inducible promoters CHS,Pilland BG,respectively,which led to the construction of plant expression vectorsp35S-30K,pCHS-30K,pPill-30K and pBG-30K.Tomato cultivar Geneva 80 whichpossessed resistance gene Tm-22 was separately transformed bythe mediation of Agrobacterium tumefaciens LBA4404 with the vectors.Someof the transgenic plants derived from p35S-30K transformants showed yellowishand necrotic symptoms at seedling stage,but none in control plants.suggestingthat there exist gene-for-gene relationship between TMV-L MP and Tm-22.Somehygromycin-resistant calli were also obtained through transforming tomatowith MP gene under the control of pathogen-inducible promoters.which wouldlay a foundation for us to develop transgenic tomatoes with high efficacyand broad-spectrum disease resistance.

论文题目:（黑曲霉葡萄糖氧化酶基因的克隆及其介导的转基因烟草）
活性氧在植物抗病过程中起着重要作用。真菌来源的葡萄糖氧化酶（GO）基因能够降解植物中的葡萄糖产生过氧化氢。本研究用PCR方法扩增了黑曲霉的GO基因已整合到烟草基因组中。KI和淀粉显色法检测到该基因在烟草中具有一定的生物活性。烟草赤星病菌定量接种测定结果表明,转基因植株的抗病性有了明显提高。

Abstract:
Active oxygen species （AOS）playa important role in the plant disease resistance.The fungal glucose oxidase（GO）gene could catalyze the glucose in plant and produce H2O2.Thegene encoding GO in Aspergillus niger was cloned from PCR amplificationsand constructed in a plant expression vector pROKll,and introduced intotobacco cultivar K326 by the mediation of Agrobacterium tumefaciens.SouthernDot Blot demonstrated that the gene had been integrated into tobacco genomes.KIand starch assay detected out its biological activity in the transformants.Detachedleaves test with Alternaria alternata showed that the disease resistancein transgenic plants enhanced obviously.

论文题目: 凝乳酶第二个β-发夹结构中β-转角的功能的研究
博士研究生: 黎红晔
专 业: 生物化学
导 师: 杨开宇（研究员）
论文摘要:
根据蛋白质结构统计学分析和计算机模拟,设计将野生型凝乳酶第二个β-发夹结构中的β-转角（Gly-Thr-Pro-Pro）突变为I’型β-转角（Gly-Gly,Ser-Gly）和II’型β-转角（Gly-Ser）。利用DNA片段转换法,将合成的突变寡核苷酸片段直接替换野生型中相应的序列,获得了三个凝乳酶原突变基因pMCGG、pMCSG和pMCGS。并经序列测定证实发生了预期的突变。

pMCGG和pMCSG突变凝乳酶原基因,均能在大肠杆菌DH5α、JM105和BL21（DE3）plysS中进行表达。表达产物GTPP（21-24）GG和GTPP（21-24）SG均以包含体形式存在,且表达水平与野生型相当。在上述三种宿主菌中,两突变凝乳酶原基因在BL21（DE3）plysS表达水平最高,约为32-34%;JM105次之,为25-29%;DH5 α最差,只有不到15%。而pMCGS突变凝乳酶原基因在大肠杆菌中却没有表达,其确切原因尚不清楚。

pMCGG和pMCSG的表达产物经过变性溶解、复性后,均能正确折叠,形成类似于野生型凝乳酶原的空间结构。两突变凝乳酶原在DEAESepharose CL-6B柱上的行为,荧光光谱,CD光谱以及荧光淬灭常数均与野生型的相类似。二级结构含量估算结果表明它们都是以β-折叠为主的蛋白质。但它们的荧光强度下降了16-22%,在负谷中的最大椭圆值增加了13-16%,表明突变确实给凝乳酶原的空间结构造成了微扰。

GTPP（21-24）GG和GTPP（21-24）SG都具有自我活化的能力。在pH2,它们的折叠效率,（即对酶原活化产物进行SDS-PAGE电泳及凝胶扫描求出所产生的假凝乳酶占凝乳酶原的百分比）,分别为86.0%和80.4%,仅比野生型8.4%-14.0%。在pH5.5,它们的假凝乳酶亦能活化为凝乳酶,且活化效率与野生型的基本一致。这些结果表明突变对凝乳酶原的自我活化功能影响不大。

但是突变导致蛋白质的稳定性明显下降。在尿素诱导的去折叠平衡实验中,两种突变凝乳酶原的稳定自由能下降了5-6KJ/mol。两种突变凝乳酶原在50℃中温育10分钟完全丧失潜在的凝乳活性,而野生型凝乳酶原在60℃中温育10分钟才完全失活。两突变凝乳酶原碱变性的中点较野生型的下降了0.3pH单位。

突变体假凝乳酶更不稳定,对pH十分敏感。在37℃,天然假凝乳酶在pH1.5至6.5范围内比较稳定,而突变体在pH1.5至pH5完全失活。即使在对三种酶都比较稳定的pH2的条件下,所测出的失活温度也有显著的区别。野生型为65℃,GTPP（21-24）GG为50℃,GTPP（21-24）SG为45℃。在pH8,最大荧光发射波长开始发生红移,野生型由335nm移至337nm,两突变体移至339nm。在不同pH中测定335nm荧光强度发现两突变体在pH2-11范围内均较野生型低,GTPP（21-24）GG下降约20%,GTPP（21-24）SG下降了50%。荧光下降程度似与稳定性高低有对应关系。

动力学分析表明,突变导致凝乳比活力和降解酪素效率的下降,但对酪素的亲和力无明显变化。

根据以上一系列结果,有理由认为凝乳酶第二个β-发夹结构的β-转角在决定凝乳酶原和假凝乳酶的稳定性中起至关重要的作用。突变后的肽链虽然能折叠成有活性的构象,但突变确实导致了构象的明显变化从而降低稳定性。此项研究可能有助于阐明蛋白质稳定性的机制和建立理性的分子设计以改进蛋白质的稳定性。

Abstract:
According to the statistic analysisof protein database and computer-modeling,the β-turn（Gly-Thr-Pro-Pro）inthe second β-hairpin of wild-type chymosin was designed to be replacedwith type I’β-turn（Gly-Gly or Ser-Gly）and type II’β-turn（Gly-Ser）.By using the method of DNA fragment replacement,e.g.,substitutingthe duplex mutated oligonucleatide fragment for the wild-type,three mutantgenes,pMCGG,pMCSG and pMCGS were generated.Their sequences were confirmedby DNA sequencing.

pMCGG and pMCSG mutant genes canbe expressed in E.coli DH5 α,JM105,BL21（DE3）plysS.Their products,GTPP（21-24）GGand GTPP（21-24）SG were accumulated in the form of inclusion bodies andtheir expression levels were comparable with that of the wild-type gene.Likethe wild-type gene,the mutated genes exhibit different expression levelsin different host strains with the highest （estimated to be 32-34% ofthe total cellular protein）in BL21（DE3）plysS,the moderate（25-29%）in JM105 and the lowest in DH5α(Less than 15%).Surprisingly,pMCGS cannot be expressed in all these three stains at all.The exact reason forthat remains unclear.

The expression products of pMCGGand pMCSG were capable of correctly refolding into a conformation similarto that of the wild-type prochymosin after solubilization of inclusionbodies and renaturation.The refolded prochymosin mutants bear similarityto the native prochymosin in behavior on DEAE Sepharose CL-6B column,fluorescencespectra,far-UV CD spectra and the accessibility of fluorophore to the quencher（Ksv）.Thecompositions of their secondary structures indicate that they are proteinsconsisting β-Sheet predominantly.However,their fluorescence emission intensitiesdecreased by 16-22% and the ellipticity values at the negative trough increasedby 13-16% for the mutants,indicating that the conformation of prochymosinwas perturbed after mutation.

Activation studies demonstrate that theprochymosin variants were able to undergo autocatalytic activation.At pH2,therefolding efficiencies, calculated as the conversion percentage from prochymosininto pseudochymosin,judged by SDS-PAGE and gel scanning after activation,were86.0% and 80.4% respectively, showing only 8.4-14.0% less than that ofthe wild-type prochymosin.At pH 5.5,their pseudochymosin analogues alsodisplayed an ability to convert into chymosin with an efficiency comparableto that of the native pseudochymosin.All these results demonstrate thatmutation does not cause substantial influence on the function of prochymosinand pseudochymosin with respect to autocatalytic activation.

On the other hand,mutation results ina dramatic reduction of protein stability,The equilibrium studies of urea-induceddenaturation reveal that the stability energy was decreased 5-6KJ/mol forprochymosin mutants.Their potential milk-clotting activities were completelyinactivated at 50℃ for 10min.,whereas the wild-type prochymosin lost itsactivity at 60℃ for 10min.The mid-point corresponding to the transitionof the alkaline-induced unfolding decreased by 0.3 pH unit.The pseudochymosinanalogues showed even less stability,especially being sensitive to pH.At37℃,the native pseudochymosin was stable at pH range from 1.5 to 6.5,whereasits mutants were completely inactivated at pH 1.5 and 5.Even at pH2,therelatively stable pH for all these three enzymes,the inactivation temperatureswere determined to be 65℃,50℃ and 45℃ for the native,GTPP（21-24）GG,GTPP（21-24）SGpseudochymosin respectively.At pH 8 the maximum fluorescence emission wavelengthwas red-shifted from 335nm to 339nm for two mutants and to 337nm for theircounterpart.At pH values from 2 through 11 the fluorescence intensitiesof GTPP（21-24）GG and GTPP（21-24）SG were consistently decreased by about20% and 50% respectively,compared with the wild-type one.It seems thatthe decrease of fluorescence intensity is correlated with the decreaseof stability.

Kinetic analysis revealed thatmutation results in a remarkable reduction both in the specific activitytowards milk and in the catalytic efficiency towards casein although nosignificant change to the casein binding ability was observed.

Taking all the results mentionedabove into consideration it is reasonable to conclude that the β-turnin the second hairpin of chymosin play a crucial role in determining thestability of prochymosin and pseudochymosin. Mutation cause a significantmodification of their conformation,leading to a substantial destabilizationalthough an active conformation is formed after refolding.The study maybe helpful to elucidating the mechanism of

protein stability and to establishinga rational design to improve protein stability.

论文题目: 真菌α-淀粉酶和葡萄糖淀粉酶的基因克隆、测序及其在大肠杆菌和啤酒酵母中的表达
博士研究生: 王永吉
专 业: 生物化学
导 师: 张树政（院士）
论文摘要:
一般酒精酵母只能利用三碳以下的糖,以淀粉为原料进行发酵生产时,都必须先进行淀粉的液化和糖化。目前,通常采用在发酵前添加酶（如α-淀粉酶,葡萄糖淀粉酶等）来分解转化淀粉。利用基因工程技术构建能直接利用淀粉的酵母工程菌将对酵母发酵工业带来突破性的进展。

1.西方许旺酵母α-淀粉酶基因克隆及其表达

以E.coli/yeast穿梭质粒YCEp1为载体构建西方许旺酵母部分基因组文库,在大肠杆菌中扩增后提取混合质粒DNA,经电转化非缺陷标志酒精酵母(Saccharomycescerevisiae) AS.2.1364,在YPDS平板(含1%葡萄糖和1%可溶性淀粉)上用淀粉水解活力筛选含水解淀粉的阳性转化子,从阳性转化子中分离重组质粒证实含5.0kb的插入片段,用α-淀粉酶基因两端序列设计的引物PCR扩增及扩增片段序列分析证实该片段中含有α-淀粉酶全部编码序列。该片段能在酒精酵母中表达α-淀粉酶,说明该片段带有α-淀粉酶基因5’-上游序列和3’-端下游序列。该转化子在YPDS平板30℃培养48小时形成清晰可见的水解淀粉圈。48hrs发酵液（YPDS）可获得0.74-0.78u/ml产酶活力及高的生物量。PAGE证实发酵液有α-淀粉酶蛋白带,占胞外总蛋白含量的6.3%以上。说明许旺酵母α-淀粉酶基因自身启动子（promotor）和终止子（terminator）在酒精酵母AS.2.1364中具有活性,并可使该基因高效表达。

将α-淀粉酶结构基因插入带有双向启动子Gall-10的质粒pBM262的EcoRI位点,构建了表达质粒pBM262-AMY1,并转化啤酒酵母,表达了α-淀粉酶。α-淀粉酶基因在pBM262-AMY1质粒上可作为非诱导型的、显性筛选标志基因用来克隆外源基因。

将α-淀粉酶编码序列插入质粒pKK223-3的EcoRI位点,构建了原核表达质粒pKK223-3-α,并转化大肠杆菌JM105,诱导表达了许旺酵母α-淀粉酶,使宿主菌淀粉水解活力提高了320%。

2.泡盛曲霉葡萄糖淀粉酶基因克隆与表达

从泡盛曲霉中分离完整的总RNA,并以总RNA逆合成mRNA-cDNA杂合链为模板进行PCR扩增。获得了约2.0kb的DNA片段。将该片段克隆进pGEM测序质粒进行测序,证明与泡盛曲霉葡萄糖淀粉酶I基因序列完全一致。说明我们用RT-PCR技术克隆了泡盛曲霉葡萄糖淀粉酶I基因。

用E.coli/yeast穿梭质粒作载体,将葡萄糖淀粉酶I基因置于酵母烯醇酶启动子和终止子之间,构建了表达载体pACI-GA,用以转化酵母,获得了具有水解淀粉能力的阳性转化子,而阴性对照并没有淀粉水解能力。这说明酵母转化子不仅表达了该酶,且有效地分泌到胞外,从而水解培养基中的淀粉,进一步测定酵母转化子48hrs发酵液的葡萄糖淀粉酶活力为8.4u/ml,培养基中的淀粉已完全水解,其生物产量较高。PAGE证实发酵液有α-淀粉酶蛋白带,占胞外总蛋白含量的4%以上。实验测定pACI-GA在酒精酵母AS.2.1364中的丢失率仅为3%。

用葡萄糖淀粉酶基因插入带有双向启动子Gall-10的质粒pBM262的HindⅢ位点,构建了表达质粒pBM262-GA。葡萄糖淀粉酶基因在该质粒上可作为诱导型的、阳性筛选标志基因用来克隆外源基因。

3.红曲霉葡萄糖淀粉酶抗血清的制备和cDNA文库的构建及基因克隆

用制备电泳纯化红曲霉葡萄糖淀粉酶E3和E4,免疫家兔制备兔抗血清。WesternBlotting分析表明抗E3和抗E4血清能相互识别结合,而且抗E3和抗E4血清与葡萄糖淀粉酶的各种酶型都有免疫结合反应。说明红曲霉葡萄糖淀粉酶的各种酶型具有相同的抗原免疫原性。可用E3或E4的抗血清来进行文库筛选。

实验由总RNA中分离mRNA,经逆转录合成cDNA,并实现cDNA与表达载体λgtllEcoRI臂连接,经包装构建了红曲霉的cDNA文库。检测文库滴度为2.2×106pfu/ml。用原位免疫筛选获得了具有免疫结合反应的阳性克隆,测定诱导表达的融合蛋白,具有淀粉水解活力。可能说明该阳性克隆含有葡萄糖淀粉酶cDNA或催化域的cDNA片段。

Abstract:
Saccharomyces cerevisiae,anorganism widely used in brewing,baking and ethanol production process isnot able to hydrolyze starch.It is of great commercial interest to createnew yeast strains which are able to convert starch to fermentable sugars.Thisstudy is dealt with cloning of α-amylase and glucoamylase genes from fungaiand then the construction of starch digestible strain of yeast,Saccharomycescerevisiae AS.2.1364,which is the ethanol -tolerated yeast strain withoutauxotrophic marker used in fermentation industry.

1.Cloning of the Schwanniomyces occidentalisα-amylase and high expression in S.cerevisiae.
The E.coli / yeast shuttle plasmid YCEplpartial library of Schwanniomyces occidentalis DNA was constructedand α-amylase gene fragments were screened in Saccharomyces cerevisiaeby amylolytic activity.Several transformants with amylolysis were obtainedand one of the fusion plasmids has about 5.0 kb inserted DNA fragment.Itcontains the upstream and downstream sequences of α-amylase gene fromS.occidentalis.It is confirmed by PCR and sequence determinationthat this 5.0 kb DNA fragment contained the whole coding sequence of α-amylase,thePCR primers were synthesized according to the 3’-terminaland the 5’-terminalof α-amylase gene.The amylolytic tests showed that when this transformantwas incubated on plate of YPDS medium containing 1% glucose and 1% starchat 30℃for 48 hrs zones of starch degradation could be visualized by stainedwith iodine vapour.Meanwhile,α-amylase activity of the culture filtratateis 0.74-0.78u/ml and PAGE shows that the yeast harboring fusion plasmidsefficiently secreted α-amylase into the medium.These results showed thatα-amylase gene can be highly expressed and efficiently secreted in S.cerevisiaeAS.2.1364 and the promotor and the terminator of α-amylase gene fromS.occidentalis work well in S.cerevisiae AS.2.1364.

This 5.0 kb α-amylase gene fragmentfrom YCEpl-α was inserted into a plasmid pBM262 at a site of EcoRI,anda expression plasmid pBM262-AMY1 was constructed.It then was introducedinto the yeast S.cerevisiae AS 2.1364 by electrosporation.The transformantcontaining α-amylase gene was able to express and secrete the recombinantα-amylase to hydrolyze the starch in the medium.It was suggested thatthe AMY1 of plasmid pBM262-AMY1 was able to be applied for cloning of novelgenes in S.cerevisiae as a dominant screening marker,which was expressedwithout any galactose inducer.

The α-amylase gene from the sequencingplasmid pGEM containing α-amylase gene was inserted into pKK223-3 at asite EcoRI.The expression plasmid pKK223-3-α was constructed,and thenwas introduced into E.coli JM105.The recombinant α-amylase was expressedby addition of IPTG to medium as inducer,which was able to hydrolyze thestarch.The activity of the recombinant α-amylase from the strain pKK223-3-αwas higher 320 percent than that from non-recombinant plasmid pKK223-3.Thesedemostrated that the S.occidentalis α-amylase was expressed inprokaryotic host E.coli JM105.

2.Cloning of the Aspergillusawamori glucoamylase I and expression in S.cerevisiae

We isolated the total RNA fromA.awamori and synthesized the mRNA-cDNA used total RNA as templateand oligo(dT)18 as primer,the 2.0 kb DNA fragment was obtainedby RT-PCR,This 2.0 kb DNA fragment was inserted into pGEM and the sequencewas determined.The results showed that the nucleode sequence of this fragmentis the same as that of the published A.awamori glucoamylase I gene,Itproved that we have got the A.awamori glucoamylase I gene by RT-PCR.

We used the E.coli /yeastshuttle plasmid pAC1as vector.The glucoamylase I gene was inserted intothe pAC1 plasmid,and a expression plasmid was constructed,then it was usedto transform the yeast strain,S.cerevisiae AS2.1364,without auxtrophicmarker,The amylolytic tests showed that when this transformant was incubatedon plate of YPDS medium containing 1% glucose and 1% starch at 30℃ for48 hrs zones of starch degradation could be visualized by stained withiodine vapour.Meanwhile,glucoamylase activity of the culture filtratateis 8.4u/ml and PAGE shows that the yeast harboring fusion plasmids efficientlysecreted glucoamylase into the medium.

The 2.0 kb glucoamylase gene fragmentwas also inserted into a plasmid pBM262 at the site of Hind ,and a expressionplasmid pBM262-GA was constructed.It was suggested that the glucoamylasegene of plasmid pBM262-AMY1 was able to be applied for cloning of novelgenes in S.cerevisiae as a dominant screening marker,which was expressedat existence of the galactose inducer.

3.Praparation of rabbit antiserumof the Monascus rubigillisus glucoamylase and construction of cDNAlibrary and its immunological sreening.

Glucoamylase E3 and E4 from Monascusrubigillisus was purified by preparation PAGE,and then was used toimmunize rabbits to obtain the glucoamylase-rabbit antiserums.Western Blottinganalysis with the antiserum showed that E3/or E4-rabbit antiserums wasable to bind to E3 and E4 antigens.Meanwhile,they were able to bind toall in vitro and in vivo multiforms of glucoamylase.These implied thatthe main forms of glucoamylase have the common antigen determinants andthe E3/or E4-rabbit serum was able to be applied for the immunologicallyscreening cDNA library.

mRNA was isolated from the M.rubigillisustotal RNA,then cDNA was synthesized by reverse transcriptase,used oligo(dT)12-18as primer and mRNA-cDNA as template.The EcoRI adaptors were added to a5’-and 3’-terminolsof the cDNA.The cDNA with adaptors at its terminols was linked with theEcoRI λgtII arms.The ligation mixture were added to the packaging proteinsystem.The titration of the packaging mixture was up to 2.2×106.The5 positive recombinants were obtained by the in immunological screening.Oneof them was able to produce the fusion protein by the IPTG inducer,whichhave an amylolytic activity.These implied that this positive recombinantcontained the whole cDNA/or cDNA fragment of the Monascus rubigillisusglucoamylase catalytic domaim.

论文题目: 凝乳酶定位突变研究
博士研究生: 潘学峰
专 业: 生物化学
导 师: 杨开宇（研究员）
论文摘要:
为了研究牛凝乳酶C端结构域中一对二硫键C206-C210在凝乳酶变性、再折叠、以至对三维空间结构稳定维持中的作用的有关信息,特开展了本工作。
首先利用Kunkle氏掺尿嘧啶模板突变法对它进行了系列的改造。在此过程中发现:
1.含有待突变部位的凝乳酶基因片段,依亚克隆方式不同,在M13噬菌体载体上表现出显著的稳定性差异。采用SmaI/SalI双酶切亚克隆比采用EcoRI单酶切亚克隆稳定性要差得多。原因可能与前者在插入片段两端形成的发夹结构有关。同时,在EcoRI亚克隆中发现发夹介导的高频缺失,缺失达102bps。序列分析确定了缺失体的接头。发现缺失与PstI限制性内切酶识别序列有关。缺失的机制可以用DNA聚合酶“滑－－托”假说进行解释(DNApolymerase Slippage hypothsis)。

2.为了获得C210S突变体,我们设计了C210S引物,利用这一引物探讨了模板性质对定位突变操作的影响。比较了引物与模板在快、慢退火时异源双链合成、转化出斑、突变率等方面的差异;发现快退火比慢退火更有利于合成含突变的,可转化的异源双链。并发现ATP分子在慢退火操作过程中,对异源双链的合成有明确的辅助作用,而在快退火体系中,对合成的影响不明显。同时通过快退火或慢退火并辅加ATP等手段,得到了凝乳酶C210S突变体基因。

3.C210S突变引物3’端恰好位于模板206位的一个发夹茎内,如果在退火过程中,发夹先于引物与模板结合而预先形成,则引物3’端引导第二条链的能力丧失。针对这一情况,我们利用ssDNA模板和引物延伸,设计并建立了一个可测定单个发夹结构Tm值的方法。利用这一方法测得凝乳酶cDNA206位发夹Tm为19℃,比Suggues法高9℃,弥补了Rychlink方法对该发夹Tm无能为力的缺点。这一方法可以帮助确定定位突变过程中的Ta和Ts值。

4.在求得206位发夹Tm为19℃的基础上,参照Rychlink等引物与模板退火在Tm值附近取值的方法,在进行C206定位改造过程中,选定Ta值为22℃、合成体系温度为22℃，利用快退火、并在合成体系中补加过量的ATP（1mM到2mM）等手段,有效地合成了分别含有C206D、C206S突变的异源双链,转化E.coli感受态细胞后,从所得噬菌休中以25%和72%的突变率获得了C206S和C206D突变体。同时发现,C206D突变亚克隆在E.coliTG1、Rz1032中表现出小噬菌斑的表型。

5.利用设计的C206S和C206D引物,探讨了快、慢退火对定点突变异源双链合成的影响。以T4DNA聚合酶为探针,发现C206S引物5’端3bp序列以及C210S引物3’端序列可以在快退火条件下与模板完全结合,而在慢退火时不能与模板完全结合,明确了快退火操作在凝乳酶206位定位突变过程能够有效合成异源双链的原因。

6.获得了凝乳酶C206S/C283A、C206D/C283A、C210S/C283A、C206S/C210S/C283A突变体基因,构建了各自的E.coli IPTG诱导表达载体。在E.coli TG1 和JM105中,表达了这些凝乳酶突变体基因。与野生型的表达水平相比,C206S/C283A和C206S/C210S/C283A略低,C206D/C283A和C210S/C283A基本上没有发生变化。突变体C206S/C283A、C206D/C283A、C210S/C283A在包含体形成能力上与野生型相比不明显,而C206S/C210S/C283A形成包含体能力下降。上述突变体包含体蛋白经过变性、复性后初步结果表明C206D/C283A似乎能够自活化为假凝乳酶,尚待进一步实验肯定。而C206S/C283A、C210S/C283A、C206S/C210S/C283A均不能自活化为假凝乳酶。表明其不能正确折叠。

Abstract:
The aim of these studies is toclarify the functional and structural roles of the C206-C210 disulfidebond in the C domain of bovine prochymosin B in the renaturation in vitroand in maintaining the 3D structure.
With Kunkle’smethod,mutagensis were performed and the following results were obtained:
1.The target fragment,when subclonedby different patterns in M13 phagemid,showed great differencein their structural stabilities.The SmaI/SalI double digested fragmentis much less stable than that the fragment obtained with EcoRI digestion.Thepossible reason for this instability is primarily addressed with the existenceof two hairpines in each ends of SmaI/SaII subclone.Other instability wasalso found in EcoRI subclone which concerned the hairpin-mediated highfrequency deletion.By sequencing the mutant,the end-junction of this deletionwas determined and showed:the end-junction of the deletion is concernedwith an restriction enzyme PstI.The mechanism for this deletion can beelucidated with DNA polymerase slippage model.

2.To obtain the C210S mutant,amutagenic primer was designed.Using this primer,the effects of differentannealing ways on heteroduplex DNA synthesis efficiency,plague formationand mutation frequency were investigated. Results show,the fast annealingis superior to the slow annealing. The presence of ATP was helpful to theextension of the hybridized primer after slow annealing, but no apparenteffect was found in the fast annealing system.On these bases,the C210Smutant was obtained by fast annealing and by slow annealing with ATP supplementrespectively.

3.The 3’terminal of the mutagenic primer for changing the cystein residue to serineat postion 210 is just located in the hairpin structure around position206,thiswill result in the failure of the second strand extension if this hairpinis formed prior to the annealing.For this,a practical method to determinethe melting temperature of this hairpin was designed and estabilished.Bythis method,the hairpin’sTm was 19℃,9℃ higher than that calaculated bythe Suggues’smethod,and compensable to Rychlinke’smethod.This method can be used effectively in determining the Ta and Tsin performing site-directed mutagenesis.

4.According to the Tm value ofhairpin around position 206 and Rynchlinker’srule,we have chosen Ta=22℃,Ts=22℃ By using fast annealing, the transformableheteroduplex DNA was obtained.After transformation,we obtained the expectedmutants C206S and C206D with mutation percentage of 25% and 72% respectively.Duringthis work,we found the C206D mutant with the three bases change of TGTto GAC,resulted in the small plague phenotype both in E.coli TG1 and inRZ1032.

5.By using the C206S and C210Sprimers,the effects of the fast and the slow annealing on the heteroduplexsynthesis were studied.With the aid of T4 DNA ligase,it was found thatthe 5’ terminalof the primer C206S and the 3’terminal of C210S primer could well combine to the template by fast annealingoperation but not after slow annealing.By this,the reason for that: Fastannealing is helpful in the heteroduplex synthesizing in prochymosin 206,waselucidated.And the C206S,C206D,C206S/C210S were also obtained.

6.Based on the above studies,C206S/C283A,C206D/C283A,C210S/C283A and C206S/C210S/C283A mutants were obtained, and the correspondingIPTG-inducted expression vectors were also constructed.The expressionsof the above mutants in TG1,JM105 showed less level with C206S/C283A,C206S/C210S/C283A,andalmost identical level with C206D/C283A,C210S/C283A compared with the wildtypegene.With the exception of C206S/C210S/C283A’s,theother three mutants C206S/C283A,C206D/C283A,C210S/C283A,were accumulatedas the form of inclusion bodies.After denaturation and renaturation,C206S/C283A,C210S/C283A and C206S/C210S/C283A mutants did not show milk-clotting activity.Itseems that C206D/C283A exhibits some milk-clotting,but it remains to beconfirmed.

论文题目:促生长激素释放刺激因子融合蛋白基因的构建、表达、纯化和活性测定
博士研究生:高 彤
专 业: 病毒学
导 师: 田 波（院士）
论文摘要:
1.GRF蛋白是一种在医疗上有重要作用的小分子蛋白。根据GRF编码区基因序列,挑选大肠杆菌偏爱的码子,利用基因合成仪,全序列合成GRF基因。再通过“搭桥”法合成GRF双链,克隆于pUC19载体上,构建成pUG0质粒并测序。同时,根据已报导的人CAII基因序列,合成GAII基因两端引物,应用RT-PCR技术从人胚肝总RNA中调出CAII编码区cDNA,克隆于pUC19载体,构建pUCA质粒。测序后,同美国已发表的从胚肝CDNA库中得到的CAII基因序列相比较,发现核苷酸水平只有562位一个碱基的差别,编码的氨基酸同源性为100%。将两片段分别切下接到pUC19载体上,构建成pUCG质粒并测序。至此,融合基因构建完成。

利用pUC19上XbaI位点,在CAII基因下游“制造”一个终止密码子,插入原核表达载体pBV220中,构建了表达融合蛋白的基因工程载体。同时,将CAII-GRF融合基因插入质粒pBV220,构建成表达CAII-GRF融合蛋白载体。

2.将表达载体转入工程菌中进行一系列表达,纯化,变性,复性研究。CAII-GRF融合蛋白摇瓶时的表达量接近50%,发酵时的表达量为25%,经过一步PBS（pH8.0）的洗涤纯化可达80%纯度,再经分子筛层析即可达到95%左右的纯度。碱性条件下,融合蛋白的复性率为18%。

3.一系列物理性质研究表明,我们表达的蛋白为CAII-GRF融合蛋白,生物学活性测定表明融合蛋白与经酶切后的GRF蛋白几乎有同样的活性,免疫学活性测定也证实了表达的蛋白为CAII-GRF,并且证实融合蛋白及酶切后GRF单体与GRF抗体有很强的结合能力。

Abstract:
1.Growth hormone releasing factoris a small polypeptide with 44 amino acid residue,which plays an importantrole in medical engineering.In order to express it in E.coli,a newexpression vector was constructed with allow the overproduction in Escherichiacoli of tripartite proteins consisting of human carbonic anhydraseII（CAII）,a peptide linker containing an enterokinase cleavage site andGRF.CAII is stable in E.coli and expresses as inclusion body.Theenterokinase cleavage site was engineered into the construct to allow accurateand efficient release of the target protein.The three part of fusion geneswere inserted downstream λphage PLPR promoter.

2.Recombinant CAII-GRF fusion proteinproduced as inclusion bodies in genetically engineered E.coli.Quantityof expressed fusion protein was reached 50% in whole bacterial protein,Purityof it was nearly 95% by washing the inclusion body with PBS buffer（pH8.0）anddenaturing with 8 mol/L Urea and then followed by gel chromatography onSephacryl S-200.The recovery of purified fusion protein was nearly 18%.

3.A series of research work onphysical chemical character demenstrated that what we expressed is CAII-GRFfusion protein.Biological and immunological activety also get the samedecision.

论文题目:抗蚜转基因烟草的研究
博士研究生:周 岩
专 业:遗传学
导 师:田颖川（研究员）
论文摘要:
采取PCR方法,从雪花莲（Galanthusnivalis）幼苗的总DNA中克隆到雪花莲外源凝集素（Galanthus nivalisagglutinin,GNA）前体蛋白基因GNA34和成熟蛋白基因GNA12。序列分析表明GNA12和GNA34基因的核苷酸序列及其导出的氨基酸序列,与以往报道的LECGNA克隆存在着明显差异,可能是其多基因家族的新成员。

前体蛋白基因GNA12插入pET11d质粒,构建成大肠杆菌表达载体pEGna12。在IPTG诱导下,pEGna12在E.coli.BL21（DE3）中表达出与预期分子量相应的蛋白。Westernblot分析表明此蛋白能与GNA抗血清发生特异免疫反应,占可溶性总蛋白的11.6%。

含双倍增强子的CaMV35S启动子、TMV-RNAcDNA“Ω”片段的双元表达载体pBin438介导外源基因组成型表达;含竹节花黄色斑驳病毒（Commelinayellow mottle virus,CoYMV）启动子的植物表达载体pBcop1介导外源基因韧皮部特异表达。GNA12和GNA34基因插入到pBin438和pBcop1启动子下游,分别构建成植物表达载体pBGna12、pBGna34、pBCGna12和pBCGna34。采用叶盘法,分别将含有以上表达载体的根瘤农杆菌LBA4404转化烟草K326,获得了一批转基因植株。转基因烟草的PCR和Southernblot分析表明外源GNA基因整合到烟草DNA中。转基因植株的Western blot 分析发现pBGna34和pBCGna34的转基因植株能有效地表达外源GNA,占可溶性总蛋白的0.08-0.15%,而pBGna12和pBCGna12的转基因植株几乎检测不到外源GNA的表达。转基因烟草高效表达外源GNA需要前体GNA基因的5’-信号序列和3’-末端序列的存在。转基因烟草的桃蚜（Myzuspersicae）虫试结果显示,有效表达外源GNA的pBGna34和pBCGna34转基因植株具有较强的抗蚜活性,能够抑制45-60%蚜口密度,有的高达90%以上。pBGna12和pBCGna12的转基因烟草也有较弱的抗蚜活性,抑制蚜口密度为12-20%。我们还证实在转基因烟草中含双倍增强子的CaMV35S启动子与CoYMV启动子介导外源GNA基因表达具有相似的强度。

为了提高所克隆的GNA34基因在双子叶植物中的抗蚜活性,我们对GNA34基因进行了改造。在没有改变氨基酸序列的前提下,通过定点突变,GNA34中的9个双子叶植物的最稀有密码子XCG和XTA被改造成优化密码子,改造后的GNA34基因的转基因烟草工作正在进行。

Abstract:
The Snowdrop（Galanthus nivalis）precursorand mature lectin genes were cloned from DNA extracted from Snowdrop seedlingsby PCR,and characterized.Sequence analysis revealed that the characterizedgenes GNA12（Galanthus nivalis agglutinin）which encode mature lectinsand GNA34 which encode precursor lectins are obviously different from previouslyreported LECGNA2 and other LECGNA cDNA with regard to their nucleotidesequence and deduced amino-acid sequence.Therefore it is postulated thatthese GNA genes should be new members of the multiple gene family of mannose-bindinglectins of GNA.

A expression vector of E.coli,pEGna12,wasconstructed by insertion of a GNA12 gene into pET 11d vector.Expressionof the GNA 12 gene in E.coli BL21（DE3）produced a protein as expected,andaccounted for 11.6% of total soluble proteins as determined by Westernblot analysis.

GNA12 and GNA34 genes were inserteddownstream of 35S promoter with double enhancer and “Ω”fragment of TMV-RNAcDNA in the binary vector pBin438,which directs foreign gene in a constitutiveexpression,and CoYMV promoter in the vector pBCop1,which directs foreigngene in a phloem-specific expression,to construct the plant expressionvectors, pBGna12,

pBGna34,pBCGna12 and pBCGna34 respectively.Leavestripes of tobacco plant var.K326 were transformed with A tumefaciensLBA4404 harboring the above expression vectors respectively.PCR and Southernblot analysis showed that foreign GNA genes were inserted into the genomicDNAs of tobacco plants.Western blot analysis showed that the transgenictobacco plants of pBGna34 and pBCGna34 could express GNA efficiently upto 0.08-0.15% of total proteins,while those of pBGna12 and pBCGna12 werehardly detected by Western blot immunoassay. It seems that 5’-signaland 3’-extensionsequences of GNA gene are necessary for expression foreign GNA with highlyefficiency in transgenic tobacco plants.Insect assay with peach potatoaphid（Myzus persicae）showed that the transgenic plants of pBGna34and pBCGna34 expressing GNA efficiently were highly aphid-resistant by45-60% against M.persicae population growth on control and transgenicplants,with the highest up to over 90%,while those of pBGna12 and pBCGna12had lower aphid-resistant qualities by 12-20%.In addition,it was evidentthat 35S promoter with double enhancer and CoYMV promoter had a similarabilities while they directed GNA gene to express in transgenic tobaccoplants through our work.

To enhance the aphid-resistant,geneGNA34 was directly mutagenized to revert 9 of XCGs and XTAs of the leastfavored codons to preferred codons in dicots without changing amino acidcomposition.Transformation of tobacco plants with the modified GNA34 geneis being carried out.

论文题目:九肽库中IV型胶原酶活性肽及抗体库中抗红细胞生成素单链抗体的研究
博士研究生:李传昭
专 业:病毒学
导 师:田 波（院士）
论文摘要:
采用丝状噬菌体表达载体fUSE5,构建了表达于丝状噬菌体fUSE5尾丝蛋白pIIIN-末端的九肽随机序列肽库。首先将丝状噬菌体fUSE5的单链DNA转化到大肠杆菌雌性菌株K802,大量繁殖转化菌后提取其复制形式DNA（RFDNA）用作建库载体。人工合成编码随机序列九肽的寡核苷酸片段,利用与片段两端酶切位点及保护序列相对应的引物,通过PCR反应得到其双链片段,经Bg1I酶切消化,与经SfiI酶切消化的载体连接,连接产物电击转化大肠杆菌MC1061,四环素抗性和插入复活筛选得到插入阳性转化子,转化子培养后从上清中提取表面展示有外源九肽的重组噬菌体粒子。转染单位分析表明库容量达1010个克隆。随机挑取12个克隆测序分析,结果12个克隆的序列都不相同。表位九肽肽库的构建为进一步应用奠定了基础。

以IV型胶原酶为靶蛋白,采用亲和纯化筛选模式,从九肽库中筛选出IV型胶原酶结合肽。首先将IV型胶原酶生物素化,再将生物素化IV型胶原酶固定在包被有亲和素的塑料平皿上,加入滴度为1012的噬菌体表位随机序列九肽肽库进行三轮“结合-洗脱-扩增”亲和筛选,获得IV型胶原酶的结合克隆。进一步ELISA检测筛选出能与IV型胶原酶特异结合的20个阳性克隆。序列分析发现一组肽含有WDXXD的共同序列,一组含有WVGXXR的共同序列。其中WDXXD的序列与IV型胶原酶单链抗体可变区序列同源。IV型胶原酶切割IV型胶原的活性检测表明,噬菌体肽有影响或抑制IV型胶原酶切割IV型胶原的活性。

从噬菌体人抗体可变区单链抗体库中筛选了几株抗人红细胞生成素（EPO）的FV单链抗体,并克隆了该单链抗体的基因。将其基因转入不含琥珀抑制基因的大肠杆菌HB2151中,从而获得了可溶性人抗EPO单链抗体。ELISA、Dotblot、Western blot检测均证明,人抗EPO FV单链抗体能够与人红细胞生成素特异结合。DNA序列分析表明,该FV单链抗体是由708个核苷酸组成,其中重链可变区为339个核苷酸,轻链可变区为324个核苷酸,中间为45个核苷酸的接头序列。

以红细胞生成素EPO为靶蛋白,采用亲和纯化筛选模式,从噬菌体表位九肽库中筛选了EPO特异结合肽。三轮“结合-洗脱-扩增”筛选获得了滴度为8×108噬菌体克隆。随机扩增单个克隆576个,ELISA测定得到阳性克隆39个,其中E201和E220两个克隆的ELISA结果最高。序列分析发现,这两个克隆的外源肽的基因相同。与EPO单链抗体比较,肽的ELISA结果比单链抗体低,该肽与单链抗体没有同源序列。将该噬菌体肽与Sepharose4B偶联制备亲和层析柱,初步亲和层析试验中得到回收蛋白,表明筛选出的肽配体可以用于亲和层析。

结果表明,多肽库和抗体库是筛选蛋白特异结合肽、活性肽和单链抗体的有力工具,表位九肽库的构建和亲和纯化筛选方法的建立为进一步应用筛选具有高亲和力的特异结合肽、活性肽或酶抑制剂、抗体等奠定了基础。

Abstract:
A phage display nonapeptide libraryexpressed on the surface of N-terminal of the filamentous phage fd minorcoat protein pIII was constructed with the vector fUSE5.In the processof construction,the E.coli female strain K802 was first transformedby the ssDNA extracted from phage fUSE5,then the replicative form （RF）DNAof the transformant clone was extracted and digested with Sfi I as theexpression vector.Degenerate oligonucleotides which encoded random nonapeptideswere synthesized,and were used as template in the preparation of the double-strandinserts by using PCR with the primers complementary to the both ends ofthe template oligonucleotide.The PCR products were digested with Bg1 I.Theinserts were obtained from the digestion products removed the fragmentsof both cleaved ends.The inserts and Sfi I digested vector were ligated.Theligation products were transformed into E.coli MC1061 by multi-electroporation.Therecombinant phage particles that formed the library were extracted andpurified from the culture supernatants. Transfection tests indicated thatthe contents of the library is up to 1010 clones.12 clones wereselected randomly to be sequenced,their sequences are different.The constructionof the nonapeptide library forms the bases of the further research andapplication.

Affinity selection has been usedto identify potent ligands for the target protein,human type IV collagenaseand a family of type IV collagenase binding bacteriophage peptides wereobtained.In the selection,human type IV collagenase was biotinylated andfixed on the surface of petri dishes coated with streptavidin,1012phage clones were panned three rounds “binding-washing and eluting-amplifying”,morethan 108 collagenase binding clones were obtained in the thirdelution. ELISA results showed 20 positive clones that could bind the typeIV collagenase specifically.The type IV collagenase enzymatic activitywas tested with the presence of phage displayed peptides specific for collagenase.Theprimary results showed that the peptides had the activities of hinderingor inhibiting to the collagenase.

From the phage display human antibodyV gene segment library,phages were isolated with binding activities tohuman erythropoietin（EPO）.The gene of the single chain Fv fragment wascloned into the phagemid pCANTAB5E and transformed into E.coli HB2151which allowed to express soluble scFv proteins by inducing of IPTG.ELISA、Dotblot、Western blot showed that the soluble scFv specifically bound to EPO.DNAsequence analysis showed that the scFv gene consists of 708 bp in whichVH is 339 bp.VL is 324 bp,there are 45bp linker betweenVH and VL..

Affinity purification has beenused to identify potent peptide ligands for EPO.A family of 108clones had been obtained after three rounds of panning.576 clones wererandomly selected and amplified for ELISA selection.39 positive cloneswith binding activity were obtained,among which the ELISA signals of E201and E220 were strongest.DNA sequence analysis showed the genes of E201and E220 are similar.Compared with the scFv for EPO,the binding activityof the phage peptide is low,there is not homologous sequences in both genes.wecoupled the peptide displayed on the phage to the CNBr activated Sepharose4B and made a column for affinity chromatography.In the primary purificationexperiments we had got the eluted protein.The results showed that the selectedpeptide ligands could be used in the affinity chromatography.

The results showed that the peptidelibrary and antibody library are powerful tools for the selection of bindingpeptides、active peptides and scFv for poteins.The construction of nonapeptidelibrary and the method of affinity purification forms the bases to selectthe specific peptides、peptide inhibitors、single chain antibodies forfurther research and application.

论文题目 :中国黑粉菌科的分类研究
博士研究生:郭 林
专业 :微生物学
导师 :郑儒永（研究员）
论文摘要 :
在1985-1995年期间,作者赴新疆阿尔泰山和巴尔鲁克山、陕、甘、川省交界的秦岭、陕、川、鄂等省交界的大巴山、内蒙古阿尔山、云南玉龙雪山和西双版纳、四川卧龙自然保护区、河北小五台山和北京附近山区采集了大量黑粉菌标本。除了研究以上标本外,还研究了保藏在本所真菌标本馆和外国标本馆（BPI,LE,UPS）的中国标本,并对比研究了一些有关的外国模式标本。确认在12科寄主植物上的中国黑粉菌科有15属136种,其中包括4个新种,15个新组合,2个中国新记录属,30个中国新记录种,其中2个同时为亚洲新记录种。新种是:细叶蒿草炭黑粉菌（Anthracoideafilifoliae L.Guo）、小孢炭黑粉菌（Anthracoidea microspora L.Guo）、稗刺疣黑粉菌（Ustilagoechinochloae L.Guo Wang）和野青茅黑粉菌（Ustilago deyeuxiaeL.Guo）。新组合是:薏苡皮特黑粉菌[Franzpetrakia okudairae （Miyabe）L.Guo,Vanky Mordue]、水蔗草孢堆黑粉菌[Sporisorium apludae（H. P.Sydow）L.Guo]、荩草孢堆黑粉菌[Sporisoriumarthraxone（Patouillard）L.Guo]、野古草孢堆黑粉菌[Sporisorium arundinellae（H. P.Sydow）L.Guo]、菅大孢孢堆黑粉菌[Sporisorium benguetense（Zundel）L.Guo]、广州孢堆黑粉菌[Sporisoriumcantonense（Zundel）L.Guo]、细柄草孢堆黑粉菌[Sporisorium capillipedii（Ling）L.Guo]、香茅粒孢堆黑粉菌[Sporisoriumcymbopogonis-distantis（Ling）L.Guo]、菅小孢孢堆黑粉菌[Sporisorimexsertum（McAlpine）L.Guo]、海南孢堆黑粉菌[Sporisorium hainanae（Zundel）L.Guo]、大孢孢堆黑粉菌[Sporisoriummacrospora（Yen C.S.Wang）L.Guo Y.B.Li]、芒孢堆黑粉菌[Sporisoriummiscanthi（Yen）L.Guo]、黄茅粒孢堆黑粉菌[Sporisorium moniliferum（Ellis Everhart）L.Guo]、戴氏孢堆黑粉菌[Sporisorium taianum（H.Sydow）L.Guo]和鸭嘴草孢堆黑粉菌[Sporisoriumtanglinense（Tracy Earle）L.Guo]。我国新记录属是:皮特黑粉菌属（FranzpetrakiaThirumalachar Pavgi）和裂孢黑粉菌属（Schizonella Schroeter）。我国新记录种是:角孢炭黑粉菌[Anthracoideaangulata（H.Sydow）Boidol Poelt]、石竹叶苔黑粉菌（Anthracoideacaryophylleae Kukkonen）、柄囊苔炭黑粉菌（Anthracoidea eleocharidisKukkonen）、林氏炭黑粉菌[Anthracoidea lindebergiae（Kukkonen）Kukkonen]、迷炭黑粉菌（Anthracoideamisandrae Kukkonen）、尼泊尔炭黑粉菌（Anthracoidea nepalensisKakishima Ono）、黍状苔炭黑粉菌（Anthracoidea paniceae Kukkonen）、范基炭黑粉菌（Anthracoideavankyi Nannfeldt）、莠竹皮特黑粉菌（Franzpetrakia microstegiiThirumalachar Pavgi）、苔草裂孢黑粉菌[Schizonella melanogramma（DC.）Schroeter]、荞麦轴黑粉菌（Sphacelotheca fagopyri H.,P.Sydow Butler）、荩草孢堆黑粉菌[Sporisorium arthraxone（Patouillard）L.Guo]、特氏楔孢黑粉菌（Thecaphoratrailii Cooke）、山野豌豆楔孢黑粉菌（Thecaphora viciae-amoenae Harada）、米勒粘孢黑粉菌[Tolyposporiummuellerianum（Thuemen）McAlpine]、波斯尼亚黑粉菌（Ustilago bosniacaBeck）、拂子茅黑粉菌[Ustilago calamagrostidis（Fuckel）Clinton]、戴维斯黑粉菌（Ustilagodavisii Liro）、 草黑粉菌（Ustilago echinata Schroeter）、雅致黑粉菌（Ustilagoelegans Griffithus）、喜马拉雅黑粉菌[Ustilago himalensis（Kakishima Ono）Vanky Oberwinkler]、伊朗黑粉菌（Ustilago iranicaH.Sydow）、长花黑粉菌（Ustilago longiflora Mundkur）、酢浆草黑粉菌[Ustilagooxalidis Ellis Tracy],其寄主所属的酢浆草科（Oxalidaceae）植物为我国黑粉菌的新记录科,还有皮氏黑粉菌（Ustialgopiperii Clinton）、网状黑粉菌（Ustilago polygoni-alati Thirumalachar Pavgi）、斑疱黑粉菌[Ustilago pustulata（DC.）Winter]、网疣黑粉菌（Ustilagoscrobiculata Liro）、田中一郎氏黑粉菌（Ustilago tanakae S.Ito）和撒克斯特黑粉菌（Ustilagothaxteri Zundel）。角孢炭黑粉菌和米勒粘孢黑粉菌也是亚洲的新记录种。

通过分子生物学的研究,对疑难种进行了澄清。长期以来,黑粉菌学者在寄生于蓼科植物子房上的几种近似黑粉菌的分种界限上有分歧。美国黑粉菌学家Fischer（1953）和Duran（1987）将科尔达黑粉菌（Ustilagocordae Liro）和网孢黑粉菌（Ustilago reticulata Liro）视为Ustilagoutriculosa（Nees）Unger的同种异名。凌立（1953）将柳叶刺蓼黑粉菌（Ustilagobungeana Yen）作为科尔达黑粉菌的异名。作者（Guo,1988）曾把科尔达黑粉菌合并于异形黑粉菌（Ustilagoanomala Kunze ex Winter）中。Vanky Oberwinkler（1994）认为异形黑粉菌仅局限于寄生在Bilderdykia属[此属在刘慎谔（1959）植物分类系统中放在蓼属（Polygonum）的蔓蓼组（Sect.Tiniaria）],而科尔达黑粉菌,柳叶刺蓼黑粉菌和网孢黑粉菌是不同的种。分子生物学的研究证明,核糖体脱氧核糖核酸（rDNA）的变异区域对于区分相似种是非常有用的（Whiteet al.,1990）。作者在试验中所用的5份国内、外标本都经过了详尽的形态学研究,并与有关种的原始描述作过核对,其中4号分别代表了科尔达黑粉菌、网孢黑粉菌、柳叶刺蓼黑粉菌、以及异形黑粉菌的4个种,另外1号则为作者过去定为“异形黑粉菌”的标本。以这些标本为材料,用玻璃片压碎黑粉孢子,提取rDNA,通过聚合酶链式反应（PCR）,对其转录间区（ITS2）进行了扩增,经核苷酸序列测定和分析,表明异形黑粉菌与作者原定为“异形黑粉菌”、以及科尔达黑粉菌、柳叶刺蓼黑粉菌和网孢黑粉菌的rDNA在ITS2区域中差别很大,在被测定的190个碱基对中,差别达69个位点以上（26.3-36.3%）,异形黑粉菌与这三种黑粉菌亲缘关系较远。作者原定名为“异形黑粉菌”的种与在欧洲取材的科尔达黑粉菌相似程度较高,因此将作者原定名为“异形黑粉菌”的种更正为科尔达黑粉菌,而柳叶刺蓼黑粉菌与科尔达黑粉菌的rDNA的ITS2片段近似程度很高。在形态上,这两个种的黑粉孢子壁的厚度,网眼大小和网脊的高度都很接近,因此将柳叶刺蓼黑粉菌作为科尔达黑粉菌的异名。

本研究还对前人的工作进行了订正研究。通过研究模式标本,将新疆蓼黑粉菌（Ustilagosinkiangensis Wang）更正为皮氏黑粉菌（Ustilago piperii Clinton），将大油芒轴黑粉菌（Sphacelothecapekingensis Wang）更正为大油芒孢堆黑粉菌[Sporisorium abramovianum（Lavrov）Karatygin]。发现Sphacelothecabothriochloae Wang（1962）是Sphacelotheca bothriochloae Zundel（1939）的晚出异物同名,其正确名称是Sporisoriumcapillipedii（Ling）L.Guo。此外,还将某些错定标本进行订正,例如,将原来错定为莎草穗黑粉菌（Cintractialimitata Clinton）的标本更正为球柱草团黑粉菌（Sorosporium kuwanoanumTogashi Maki）。

Abstract:
From 1985 to 1995,the author collecteda large number of specimens of smut fungi from different places of China.Thesematerials were identified together with all specimens of the Ustilaginaceaeof China kept in HMAS(Herbarium Mycologicum Instituti Microbiologi AcademiaeSinicae),as well as some of the Chinese specimens deposited in BPI(USA),HUV(Germany),

LE(Russia) and UPS(Sweden).Onehundred and thirty-six species belonging to 15 genera of the family Ustilaginaceaeare recognized from China,including 4 species new to science,15 new combinations,2genera new to China,and 30 species new to China with 2 species also newto Asia.These species are parasitic on 12 host families,in which Oxalidaceaeis new to China.
The 4 species new to science are:1)Anthracoideafilifoliae L. Guo, 2) Anthracoidea microsporaL.Guo,3)Ustilago deyeusiae L.Guo,and 4)Ustilago echinochloaeL.Guo Wang.The 15 new combinations are: 1) Franzpetrakia okudairae(Miyabe)L.Guo,Vanky Mordue,2)Sporisorium apludae(H. P.Sydow) L.Guo,3)Sporisoriumarthraxone(Patouillard)L.Guo,4)Sporisorium arundine-llae(H. P.Sydow)L.Guo,5)Sporisorium benguetense(Zundel)L.Guo,6)Sporiorium cantonense(Zundel) L.Guo,7)Sporisorium capillipedii(Ling)L.Guo,8)Sporisorium cymbopogonis-distantis (Ling) L.Guo,9)Sporisoriumexsertum (McAlpine) L.Guo,10)Sporisoriumhainanae(Zundel) L.Guo, 11) Sporisorium macrospora(Yen C.S.Wang) L.Guo L.B.Li,12)Sporisorium miscanthi(Yen)L.Guo,13)Sporisorium moniliferum(Ellis Everhart)L.Guo, 14) Sporisorium taianum(H.Sydow)L.Guo and 15)Sporisorium tanglinense (Tracy Earle) L.Guo. The 2 genera new to China are:FranpetrakiaThirumalachar Pavgi and Schizonella Schroeter.The 30 species new to China are: 1) Anthracoidea angulata(H.Sydow)Boidol Poelt,2)Anthracoidea caryophylleae Kukkonen,3)Anthracoidea eleocharidis Kukkonen,4)Anthracoidealindebergiae(Kukkonen) Kukkonen,5)Anthracoidea misandrae Kukkonen,6) Anthracoidea nepalensis Kakishima Ono,7)Anthracoideapaniceae Kukkonen, 8)Anthracoidea vankyi Nannfeldt,9)Franzpetrakiamicrostegii Thirumalachar Pavgi,10)Schizone- llamelanogramma (DC) Schroeter, 11) Sphacelotheca fagopyri H.,P.Sydow Butler,12)Sporisoriumarthraxone(Patouillard) L.Guo,13)
Thecaphora trailii Cooke,14)Thecaphoraviciae-amoenae Harada,15)Tolyposporium muellerianum
(Thuemen) McAlpine,16)Ustilago bosniaca Beck,17)Ustilagocalamagrostidis (Fuckel) Clinton,18)Ustilagodavisii Liro,19)Ustilago echinata Schroeter, 20)Ustilagoelegans Griffithus,21)Ustilago himalensis (Kakishima Ono)Vanky Oberwinkler,22)Ustilago iranica H.Sydow,23)Ustilagolongiflora Mundkur,24)Ustilago oxalidis Ellis Tracy,25)Ustilagopiperii Clinton, 26)Ustilago polygoni-alatiThirum. Pavgi,27)Ustilago pustulata(DC.) Winter,28)Ustilagoscrobiculata Liro,29) Ustilago tanakae S.Ito and 30)Ustilagothaxteri Zundel.Among which,Anthracoidea angulata and Tolyposporiummuellerianum are also new to Asia.

Sequence analyses in ITS2 of rDNAof some species of Ustialgo on Polygonaceae have been made in orderto clarify the taxonomy of these disputed taxa,Since opinions about theirlimits were different in different ustilaginologists historically.For example,thespecies Ustilago cordae and U.reticulata were treated asconspecific by Fischer(1953) and Duran(1987). U.cordae was listedas a synonym of U.anomala by Guo(1988).The species U.bungeanawas treated as a synonym of U.cordae by Ling(1953).Vanky and Oberwinkler(1994)considered that Ustilago anomala parasitizes the genus Bilderdykiaonly,and Ustilago bungeana,U.cordae and U.reticulatawere distinct species. All these species are morphologically similar, dis-tinguishing from each other by mesh size,muri heightand thickness of wall of ustilospores.Ribosomal DNA is useful in analysingclosely related taxonomic species.Sequences of the internal transcribedspacer 2(ITS2) were used to clarify these species.The rDNA of species studiedwas extracted from the herbarium specimens misidentified as “Ustilagoanomala”and identified as U.reticulata from China,and U.anomala,U.cordaeand U.reticulata from foreign countries which are depositedin HMAS (Herbarium Mycologicum Instituti Microbiologi Academiae Sinicae).Driedustilospores were crushed to powder between two glass slides.ITS2 regionwas amplified by PCR method with 5.8SR and ITS4 primer pairs.A total of190 bp of the sequences of these 4 species have been determined.

Results of the research indicatethat rDNA sequence of Ustilago anomala parasitic on Bilderdykiadiffers significantly from the so-called“Ustilago anomala”on Polygonumand Ustilago bungeana, U. cordae and U.reticulata.More than69(26.3-36.3%) variable positions of 190 bp in this sequences occur betweenUstilago anomala and the others.Morphologically U.anomalaon Bilderdykia has lower muri(ca1μm) of ustilospores than “U.anomala”on Polygonum and U.cordae(ca1-1.5μm).Hence,many specimensoriginally identified as “U.anomala”from China by the author shouldbe transferred to U.cordae.These sequences also show that U.bungeanais closely related to U.cordae.Morphologically both species havesimilar thickness of wall,mesh size and muri height of ustilospores.Basedon molecular and morphological characteristics,the author considers thatU.bungeana is synonymous with U.cordae.