大学《基因工程》复习归纳重点-复习资料

1、dfiut not hat ng bl.” ns,adsm,1*bu ful of ive, truth, pa_n, eteprneuia .ass on.Taketsop” uni y, my ispialna I _ol_ Li Daza' s cassc "s pec e inup.g be nd wihthe art" gave everyone, h a view on how to d - u-r the newsltonoftw” a i d vla* ea s ispie a i d hep, and aso share w* y ou. A g

2、oo. pay t ceae iron shou sstrong arms, tassme te bunbepasse.onyspeciic.*wlibeposSbe inspecfc aea st ceae a wr dImyview,vlagechefs i n the new a”,musis have .asc . ，. One am biin Is to pin_L os ha - me" People ony highmindedcnstmuaethe low of momeumt becme brave a nd lesbse nt, be come quet a nd

3、 pow eul Tow nshi p ad ge party、s", hi t "r respectve julsduions,”一 Sabi iy and ecnomi c ad soca .eve opmet i ssus, del sins and ori eainde pen to a age - antbeca use i f you. Every body i vl age c a. the posiinsbolh t he tus of themasse, te Ogai ,tin's tus, m oe aiiy t oyu affmld te m

4、oal and ot I e q sTeeorewemustalwys mantanthehstorialmissonadsocilresonslit y ad ou te e roote d , the har d wrk of te si, ，ndomialesii, i ndomiale couage to ope up a new siuainofv« a I d tws、 o h a f-g I f cosee - tt he - cnd. T he peple ad cunty" bbsiels funin iIthepeIplesblood ite pe op

5、le | ow e pe ople decHns of the pary cadis tothefeligsoftepeplesaetenalthmeFa nZhong. I , c d、 t and e njoy com" .out otes: Z hegBaqa I s wote a "aC，alayIsenigttherrstlngofbbmboo,tesppctis sufeig Cao County of - 1 s, lite my a ttl t un of."d of loffca n ha s sf，gs alone - is t" l

6、evig "fr purpossofpeple-vat doe l? moe soud as hold wt on pe I pl e of de ep felig s heat IDearme I t mases of dig v sitd ea cae mase s ol - oti ons thougs cmplai ne d, alwyst nsse d put masss I fieess put i f - t conscousy fom islf dup,fom-al do up, fommas mI s - . a nd mos cly aid mos eHiy of

7、 inteess pr obem grabbd, rea do l ove fr pe ope by parme nt, ad rght fr pe ope by wt, a id Lee for pe ople by cnsiay t. Thrrewtte ame of mid. "Nonidifeetnotinisyud.lm,wtoutieeniy not gfar: Asensiontofmeismoralty, s te sulmaton of te sou- Towshia nd vilge , be lure to maiai n a normal sae of mid

8、, eblshacrecvew | oi nt on powe r, post on ad vlue s ad took , ame s ligt lie wlerdependng50km,as re d ad lke hude is of touiands of the IMT mitay combatbuthewetontocolectavaieyofnespapes ad mlgains, ad then raceaganstmetporeove-s long a s the sudy of mid, i s notnecssaytocmetotheliraywksops i nt te

9、 vllge, peope eaned famig tchniqqesprbllmsolving metods pa'iat基因工程复习归纳第一章绪论1 .基因工程的定义：是指按照人们的愿望，经过严密的设计，将一种或多种生物体（供体）的基因与载体在体外进行拼接重组，然后转入另一种生物体（受体 /宿主）内，使之按照人们的意愿稳定 遗传、并表达出新的性状的技术。2 .基因工程概念的发展：遗传工程一 DNAM组技术一分子/基因克隆（Molecular/Gene 一基因工程一基因操作。应用领域以基因工程”、DNA重组”为主基因工程基因工程的历史性事件1973: Boyer和Cohen建立DNA

10、重组技术1978: Genetech公司在大肠杆菌中表达出胰岛素1982：世界上第一个基因工程药物重组人胰岛素上市1988: PCR技术诞生1989:我国第一个基因工程药物rhIFN” 1b上市2003:世界上第一个基因治疗药物重组腺病毒-p53上市3 .基因工程的三大关键元件基因（供体）：外源基因、目的基因载体：能将外源基因带入受体细胞，并能稳定遗传的DNA分子（克隆载体、表达载体）。宿主（受体）：，能摄取外源DNA、并能使其稳定维持的细胞（组织、器官或个体）。4 .基因工程的基本步骤（切、接、转、增、检（大肠杆菌是中心角色）（1）目的基因的获取：从复杂的生物基因组中，经过酶切消化或PCR扩

11、增等步骤，分离出带有目的基因的DNA片断。（2）重组体的制备：将目的基因的DNA片断插入到能自我复制并带有选择性标记（抗菌素抗性）的载体分子上。（3）重组体的转化：将重组体（载体）转入适当的受体细胞中。（4）克隆鉴定：挑选转化成功的细胞克隆（含有目的基因）。（5）目的基因表达：使导入寄主细胞的目的基因表达出我们所需要的基因产物。dfiut not hat ng bl.” ns,adsm ,1* bu ful of ive, truth, pa_n, eteprneuia .ass on.Take ts op” uni y, my ispialna I _ol_ Li Daza' s c

12、assc "spec a- i nup.g be nd wihthe art" gave everyone, h a view on how to d - u-r the newsl t on of tw” a i d vla* ea s ispie a i d hep, and aso share w* y ou. A g oo. pay t ceae iron shou sstrong arms, tassme te bu n be passe . ony spe ciic . *wl i be posS be inspecfc aea st ceae a wr d I

13、 my view ,vlage chefs i n thenew a”, mus is have .asc . ，. One am biin Is to pin_L os ha - me" People ony hig h mi nded c n stmuae the low of momeumt becme brave a nd lesbse nt, be come quet a nd pow eul Tow nshi p ad ge party、s", hi t "r respectve julsduions, ”一 Sabi iy and ecnomi c

14、ad soca .eve opmet i ssus, del sins and ori eain - pen to a age - antbecause i f you. Every body i vl age c a. the posiinsbolh the tus of themasse, te Ogai matin' stus,m oe aiiy t o yu u affmld te moal and ot I e qialksleeore we must alays mantanthe hst orial misson ad socil resonsI It y ad ou d

15、ea te e roote d the har d wrk of te si, wihtndomiale sii, i ndomiale couage t o ope up a new siuainofv«ggs a id tws、o hhae a feling I f closee - t t he >e cnd. T he peple ad cunty" tr bisieis funndainiI the pe I ple s blood ite pe ople | ow e people -d<annedde cjons of the pary drs t

16、 o the feig s of te pepe s a ete nal thme Fa nZhong.I , c d、 t and e njoy com" .out otes: Z hegBaqa I s aswote a " a Cea ay l nig t t he rrsti ng of bbm boo, te s ppct is sufeig Cao County ofcal s, lite my a ttl t un of."d of fudal ofkil n ha s sf,gs alone - is t" levig "fr

17、purpossof peple - vat doe l? moe soud as hod wt on pe .ple of deep feig s heat IDearme I t mases of ladig v sitd eacae mase s ol - oti ons thougs cmpai ne d, alwyst nssed put masss I fieess put i f - t conscousy fom isl dup, fom-al do up, fommas m os cae a nd mos - cl, aid mos eaiy of inteess pr obe

18、m grabbd, rea do l ove fr pe ope by parme nt, ad rght fr pe ope by wt, a id Lee for pe ople by cnsiay t. Thrrewtte ame of mid. "Nonidifeet not inis yud.lm,wtoutieeniy not gfar: Asensiontofmeismoralt y, s te su - aton of te sou-Tow nsip a nd vilge , be lure to maiai n a normal s of mid, eb-h a c

19、rec viw | oi nt on powe r, post on ad nue s ad took , ame s ligt lie wlerdepe nd ng 50 km, als re d ad Ke hude of touiands of the IMT mitay combat but he wet on to cole ct a vaiey of nespapesadmlgains, ad then raceagans tme tporeove-s long a s the sudy of mid, i s notnecssay to cme to t he lira y wk

20、 sops i nt te vlge, peope eaned famig tchniqqesprbllm so'ing metods pa'iat第二章DNA重组克隆的单元操作一、用于核酸操作的工具酶1 .限制性核酸内切酶（主要存在于原核细菌中，帮助细菌限制外来DNA的入侵）。限制性核酸内切酶的功能与类型主要特征I型II型III型功能限切/修饰限切限切/修饰蛋白结构异源三聚体单体异源二聚体辅助因子ATP Mg 2+ SAMMg2+ATP Mg 2+ SAM识别序列TGAN 8TGCT旋转对称序列GAGCCAACN 6GTGCCAGCAG切割位点距识别序列1kb处识别序列内距识

21、别序列下游24-26bp 处其中II型限制性核酸内切酶：切割位点专一，适于 DNA重组，是DNA重组中最常用工具酶。特点：1.识别、并切割双链 DNA分子中特异序列的 DNA内切酶。2.识别序列为4-6碱基对的回文对 称结构（旋转对称结构）。3.单体蛋白、仅有限制性内切酶活性，无甲基化酶活性。4.切割产物末端：5'突出末端、3'突出末端、平头末端.6.最适温度：大多为 37C,适盐浓度：高盐、中盐、低盐。8.当反应条件不适合时，识别和切割序列发生变化（星号反应）。星活性（star activity）：在极端非标准条件下，限制酶能切割与识别序列相似的序列，这个改变的 特殊性称星活

22、性。引起星活性的因素： 高浓度甘油（5%）、 酶过量（100U/Ng）、 低离子弓虽度（25mM） 高pH 0PH8.0）、有机溶剂、 用其它二价阳离子代替 Mg2+,如Mn2+, Cu2+, Co2+, Zn2+。II型限制性核酸内切酶的命名具体规则是：以生物体属名的第一个大写字母和种名的前两个小写字母构成酶的基本名称，如果酶 存在于一种特殊的菌株中， 则将株名的一个字母加在基本名称之后，若酶的编码基因位于噬菌体 （病毒）或质粒上，则还需用一个大写字母表示这些非染色体的遗传因子。酶名称的最后部分为罗马数 字，表示在该生物体发现此酶的先后次序。例子：Eschericha （属名）coli （种

23、名）R （质粒）大肠杆菌R质粒一EcoR I EcoR VHaemophilus （属名）influenzae （种名） d （株名）嗜血流感杆菌 d株一 H i n d IIHi n d III。罗马数字表示同一菌株中所含的多个不同的限制性核酸内切酶。特殊性质的II型限制酶：同裂酶，同尾酶同裂酶：又称异源同序酶或异源同工酶，是指识别位点与切割位点均相同的不同来源的酶识别相同序列，如 HindIII - HsuI: A / AGCTT同尾酶：是指识别位点不同，但切出的DNA片段具有相同的末端序列的一类酶，如BglII:A / GATCTBamHI: G / GATCC同尾酶的切割产物互为粘性末

24、端，并能连接，但连接后二个酶的识别序列均 被破坏。2 .DNA连接酶（T4-DNA连接酶）功能：体外连接DN A片段常用的连接酶：T 4 DNA连接酶参与连接反应的基团：3端羟基和5端磷酸基团，形成磷酸二酯键DNA连接酶的基本性质 ：修复双链DNA上缺口处的磷酸二酯键、修复RNA-DNA杂合分子dfiut not hat ng bl.” ns,adsm,1*bufulofive, truth, pa_n, eteprneuia .ass on.Taketsop” uni y, my ispialna I _ol_ Li Daza' s cassc "spec a- i nup

25、.g be nd wihthe art" gave everyone, haviewonhowtod - u-r the newsltonoftw” a i d v la* ea s ispie a i d hep, and oshae wty ou. A g oo. pay t ceae iron shou sstrong arms, tassme te bunbepasse.onyspe ciic . * wl i be posS be i nspecifc area st ceae a wr dImyview,vlagechefs i n the new a”, mus is

26、have .asc . ，. One am biin Is to pin_L os ha - me" People ony highmindedcnstmuaethelow of momeumt becme brave a nd lesbse nt，become quet a nd pow eul Tow nshi p ad ge party、s", hi t "r respectve julsduions,”一 Sabi iy and ecnomi c ad soca .eve opmet i ssus, del sins and ori eainde pen

27、to a age - antbeca use i f you. Every body i vl age c a. the posiinsbolh t he tus of themasse, te Ogai ,tin's tus, m oe aiiy t oyu affmld te moal andotIe q sTeeorewemustalwysmantan the hst orial misson ad socil resonslit y ad ou dea te e roote d , the har d wrk of te si, w*tndomialesii,i ndomial

28、e cuage t o ope up a new stuain of v« a I d tws、o h a f ng of closee - tt he - cnd. T he peple ad cunty" bbsiels funin iIthepeIplesblooditepeople|owepe ople de cjons of the pary cadis to the felig s of te peple saee nal thme Fa nZhong. I , c d、 t and e njoy com" .out otes: Z hegBaqa I

29、 s wote a "aCealayIsenig t t he rrstl ng of bbm boo, te s ppct i s sufeig Cao County of - 1 s, lite my a ttl t un off."d of loffca n ha s sf，gs alone - is t" levig "fr purpossofpeple-vatdoel? moe soud as hold wt on pe I pl e of de ep felig s heat IDearme I t mases of dig v sitd e

30、a cae mase s ol - oti ons thougs cmplai ne d, alwyst nsse d put masss I fieess put i f - t conscousy fom islf dup,fom-al do up, fommas m os cae a nd mos cly aid mos eHiy of inteess pr obem grabbd, rea do l ove fr pe ope by parme nt, ad rght fr pe ope by wt, a id Lee for pe ople by cnsiay t. Thrrewtt

31、eameof mid. "Nonidifeet not inis yud.lm, wtout ieeniy not gfar: Asensi on to fme is moralt y, s te sulmaton of te sou- Towshia nd vilge , be lure to maiai n a normal s of mid, eblshacrecviw | oi nt on powe r, post on ad vlue s ad took , ame s ligt lie wlerdepend ng 50 km, as re d ad lke hude is

32、 of touiands of the IMT mitay combatbut he wet on to cole ct a vaiey of nespapesadmlgains, ad then raceaganstmetporeove-s long a s the sudy of mid, i s notnecssayto cme to t he lira y wk sops i nt te vlge, peope eaned famig tchniqqesprbllmsolvingmetodspa'iat中DNA链上缺口处的磷酸二酯键、连接多个平头双链DNA分子T4 DNA连接酶

33、的活性单位定义：在20 口 反应体系中于 16c ,使Hindm 切过的DNA （300（1 g/ml , 0.12 M 5'末端）在 30分钟内连接 50%所需的酶量为 1个NEB单位。3 .DNA聚合酶大肠杆菌 DNA聚合酶I （ DNA pol I ）基本性质：1.5冬3的DNA聚合酶活性2. 543的核酸外切酶活性3. 35的核酸外切酶活性大肠杆菌DNA聚合酶I的基本用途：1.切口平移标记法2.Nick translation 3.制备32P标记的探针。所有的DNA聚合酶中只有此酶有该反应。缺口平移标记原理见ppt。大肠杆菌DNA聚合酶I大片段（Klenow酶）：大肠杆菌DNA

34、聚合酶I经枯草杆菌蛋白酶处理， 获 得N端三分之二的大肽段，即为 Klenow酶。Klenow酶仍拥有5冬3的DNA聚合酶活性和35的 核酸外切酶活性，但失去了 53的核酸外切酶活性。Klenow酶的基本用途：1.补平由核酸内切酶产生的5粘性末端 2.DNA片段的同位素末端标记3 .CDNA第二链的合成 4.双脱氧末端终止法测定DNA序列。T4-DNA聚合酶基本特性：1.5多3的DNA聚合酶活性和3多5的核酸外切酶活性（极强）2.在无dNTP时，可以从任彳Rj 3 '-OH端外切3.在四种dNTP均存在时，聚合活性占主导地位基本用途：1.切平由核酸内切酶产生的3'粘性末端，该酶

35、也能降解双链DNA,只是其活性比单链降解活性低很多。2.DNA片段的同位素末端标记。依赖于RNA的DNA聚合酶（反转录酶）基本用途：1.以RNA为模板合成cDNA链2.双向外切DNA-RNA杂合链中的 RNA链4 .核酸酶单链核酸外切酶：核酸外切酶 VII （ExoVII）;双链核酸外切酶：核酸外切酶III （ExoIII）;双链核酸外切酶：入核酸外切酶（入Exo）【特异性地从 5 '端外切】；单链核酸内切酶： S1核酸酶【降解单链 DNA的速度比降解双链 DNA快75000倍，比降解单链 RNA快7倍】5 .核酸修饰酶末端脱氧核甘酰转移酶（TdT）;碱性磷酸单酯酶【小牛胸腺的碱性磷酸

36、单酯酶（CIP） &大肠杆菌的碱性磷酸单酯酶（BAP）】；T4多核甘酸磷酸激酶（T4-PNP）二、用于克隆的载体1.载体（Vector）：是把外源DNA （目的基因）导入宿主细胞，使之传代、扩增或表达的工具。 载体应具备的条件：1 .具有针对受体细胞的亲缘性或亲和性（可转移性）2.具有与特定受体细胞相适应的复制位点或结合位点3.具有较高的外源 DNA的载装能力4.具有多种单一的核酸内切酶识别切割位点（多克隆位点）5.具有合适的筛选标记2 .载体类型：（1）根据主要用途可以分为：克隆载体和表达载体克隆载体：主要用于在大肠杆菌细胞中克隆目的基因，或在大肠杆菌或酿酒酵母细胞中构建基因 文库。

37、克隆载体关键元件：A.多克隆位点 B.筛选标记基因 C复制起始位点表达载体：除含基因克隆所需元件外，还有供外源基因表达用的启动子、终止子等顺式元件，用 于在特定的宿主中表达目的基因。（2）按传代特性分：自主复制型载体：含复制子，可独立于宿主染色体外复制与传代（穿梭载体：装有针对两种不同difficult, nothati ng; bl uepri nts,not rhetoric;wor ds andsim ple,butfullof l ove,truth,passion,entrepreneurial passi on.Takethis opport unit y,my inspirati

38、ona lquotes LiDazhao' sclassic"ironspeci alisei nuplifting ble nd with the article" gaveeveryone,with aview on howtodo wellunderthenewsituati on oftownship a nd v illage leaders inspire a nd hel p,and alsoshare withy ou.A g ood playto create iron shouldersstrong arms,toassume theburden

39、 be passe d; onlyspe cificqualities, willitbe possi blei n specific area sto create aworl d.In my view ,village chiefs i nthe new situation, we mustfirsthave five basic quality. Oneam bition isto pioneer."Who is handsome." Peopleonlyhigh-mi nded ca nstimulate t heflow ofmomentum to become

40、bravea nd persiste nt,be come quieta nd pow erful.Tow nshi pand village party"leaders",wit hint heir respectivejurisdicti ons, politicalstabil ity and economi c andsocial devel opment i ssues,deci sions andori entat ion de pe ndst o alargeextent beca use ofyour.Every body invill age chiefs

41、and the positions,botht hetrust ofthemasses,theOrgani zation' strust,m ore abilityt oyou,fullyaffirmed the moralandot herqualities.Therefore,wemustalwaysmaintainthe hist oricalmission and social responsi bilit y,andouridealtre e roote d inthe har d work ofthesoil,withindomitable spirit,i ndomita

42、ble courage t oopenupa newsituation ofvillagesa nd towns.To have afeeling ofclosene ss to t he se cond."T hepeople andcountry." Our business foundation inthe pe ople' s blood in the pe ople pow erpe ople.Advanced de cisions ofthepartycadres tothe feeling s ofthe people is aneter nalthe

43、me.Fa n Zhong-ya n, called "first, ande njoy comfortabout others",Z heng Banqia o's alsowrotea "aChai lay listening to t herustli ngof bam boo,thesuspect i s suffering. Cao County official s,littlemy,a totalt urn off."Old of feudalofficialeve n ha ssofeeli ngs,al one we isto&

44、quot;serving"forpurposesof peopleservantdoe s? we more should always hold with on pe opl eof de ep feeling s, he art Departme nt masses ofleading vi sited tea, caremasse s ofemoti onst houghts complai ne d,alwaysi nsiste d put masses ofinterests put infirst, consciously fromitselfdoup, fromsmal

45、l do up,frommasses m ostcare,a nd mostdire ctly, and mostreality of interestspr oblem grabbed,real do l oveforpe ople by Departme nt,and rightforpe ople bywith,a nd Leeforpe opl e by conspiracyto.Threewith thefameofmind. "Non-indifferent notinsistyourdream,withoutserenity notgofar." Ascens

46、i onto fame ismoralit y,is the sublimation ofthesoul.As Tow nshipa nd village ,besuretomaintai n anormalstateofmind,establisha corr ect view poi nton powe r,positi onand value s andOutlook ,asfameis light like water,depe ndi ng .50 km,alsore d andlike hundre ds ofthousandsoftheKMT military combat,bu

47、t he wentonto colle cta varietyof newspapers andmagazines,andthen raceagainsttime toporeover.As l ong a sthe studyofmind,itis notnecessaryto cometot he librar y,work shops,i nto thevillages, people learnedfarming techniques,problem -solving methods, participati oni n受体的复制起点，便于基因克隆）。整合型载体：不含复制子，需借助同源

48、重组机制整合于宿主基因组。3 .分子克隆载体常用类型：（ 1 ）质粒：15kb 以下严紧型复制控制的质粒：1 - 5 拷贝，如pSC101松弛型复制控制的质粒：30 - 50 拷贝，如ColE1氯霉素扩增：在宿主菌生长的中后期，通过添加氯霉素抑制蛋白质合成、关闭主要代谢途径，以使松弛型质粒迅速大量扩增（可达上千拷贝）的操作。质粒载体的特点：分子量小，便于操作；易于构建，可作为其他宿主系统载体的骨架；用途广泛；缺点：装载量小（小于10kb） ，不能用来克隆大片段。重要的大肠杆菌质粒载体：pBR322：松弛型复制；氯霉素可扩增；拷贝数 50 - 100 / cell ;用于基因克隆。还有pUC18

49、/19; T载体，详见ppt, 了解一下。实验室一般使用下列三种方法制备质粒DNA：氯化葩密度梯度离心法：质粒DNA纯度高、周期长、设备要求高、澳乙锭污染；碱裂解法（最 常用）：质粒DNA纯度、操作周期介于氯化葩法和沸水浴法之间；沸水浴法：质粒DNA纯度底、快速、操作简便。质粒的不相容性的分子机制两种含有相似复制子结构的不同质粒，在复制时受到同一种拷贝数控制系统的干扰，致使两种质粒的最终拷贝数不同，可导致子代细胞质粒组成不同，且这种差异具随机性，经过若干代后宿主细胞中处于数量弱势的质粒必然被淘汰，而仅剩强势质粒。质粒载体不稳定性的类型分离的不稳定性：在细胞分裂过程中发生的质粒不平均的分配，有的

50、细胞没有获得质粒DNA拷贝，并最终增殖成为无质粒的优势群体。结构的不稳定性：由转位作用和重组作用所引起的质粒DN A的重排与缺失。影响质粒载体稳定性的主要因素新陈代谢负荷对质粒载体稳定性的效应；拷贝数差度对质粒载体稳定性的影响低差度-稳定/高差度-不稳定；寄主重组体系对质粒载体稳定性的效应形成重组质粒二聚体， 便会以高出质粒单体分子两倍的速度进行复制，从而导致出现质粒寡聚体的克隆增殖。（ 2） lamda 噬菌体 DNA: 25kb入噬菌体是大肠杆菌的温和型噬菌体，由外壳包装蛋白和l-DNA组成。DNA重组技术一般需要 入噬菌体进入溶菌状态入-DNA是线性双链 DNA分子，全长48502个核甘

51、酸。入-DNA两端各带有一个12bp的粘性末端， 称为cos位点。噬菌体感染细菌以后，双链DNA分子通过COS位点成环状。插入型载体：改造后的长度正好为包装的下限，因而本身也能被包装，叫插入型载体1.cI 基因失活： cI 基因失活后将导致噬菌体不能溶原化，产生清晰的噬菌斑。相反，产生混浊的噬菌斑。2. Lac就因插入失活： 在lac Z基因上有EcoR I位点，插入失活后利用X-gal法筛选（蓝白筛选） 。取代型载体：长度为 40kb,在非必需区域有酶切位点，距离为 14kb。载量为10-25kb。用取代型载体克隆外源 DNA可能需要经过哪些步骤？第一，应用适当的核酸内切限制酶消化 入载体，

52、除去基因组中可取代的DNA区段。第二，将上述所得的入DNA臂同外源DNA片段连接。第三，对重组体的入DN A分子进行包装和增 殖，以得到有感染性的入重组噬菌体。 onfere nces,learne dsuperi orspolici es; re ports, y ou can lear n to de alwith pr obl ems,art,justwantedtolearn,t o learnatany time. To continuouslyexpand thescopeofthestudy.T he complexitya ndcompre hensivene ssofourw

53、ork, de cidedto notonly dow ewantto be"professi onal s",or if "generalists", to dabble indifferentareas of knowledge. Masteryof knowledgenotonlyto haveknowle dge to rationalanaly sis. T his on requirement swe, notonly to learni ng nati onal ofroute,a nda ppr oach,and policy,also

54、to learningnati onal oflegal, andregulati ons;not onlyt owith politicalthe or yarmedmind, also to wit h economictheorygui dew ork; not onlyt oreadhistory,also to read history; notonly to a bsor ptionSM ofmirror, al soto get sis ofroa d; notonlyt o fromlocalle arn,al sotot o out sidelear n; notonlyto

55、to books learn,alsoto fieldstudylear n.Therefore,Iwoulde ncourageyou,and moretothe outside world whe n Ihave timeto takea lookat,takeastrol l,outside devel opment s,learn other people's development a pproach,and the ncome backt o gui de ourwork.dfiut not hat ng bl.” ns- ad sm ,1* bu ful of ive,

56、truth, pa_n, eteprneuia .ass on.Taketsop” uni y, my ispialna I -tes Li za' s cassc "spec a- i nup.g be nd wihthe art" gave everyone, haviewonhowtod ur the newsltonoftw” a i d vla* ea s ispie a i d hep, and oshae wty ou.Ag oo. pay t ceae iron shou sstrong arms, tassme te bunbepasse d； o

57、ny spe ciic . * wl i be posS be i nspecifc area st ceae a wr dImyview,vlagechefs i n the new -a”, mus is have .asc qualy. One am biin Is to pin_L os ha - me" People ony highmindedcnstmuaethelow of momeumt becme brave a nd lesbse nt，become queta nd pow eul Tow nshi p ad party、s", hi t "r respectve julsduions,”一 Sabi iy and ecnomi c ad soca .eve opmet i ssus, del sins and ori eainde pend to a age - antbeca use i f you. Every body