第三节、基因克隆策略

一、目的与意义 Isolation of gene enables the determination of its nucleotide sequence. determining –intron number and position、 promoter elements，etc. A comparison of DNA sequences between genes can lead to insights in gene evolution. Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to a determination of the structure of the protein product of the gene, and from this knowledge, the function of the gene can be inferred. 2

A gene can be moved from one organism to another A gene can be moved from one organism to another. An organism containing a "foreign" gene via genetic engineering process is called transgenic. Transgenic organisms can be used either for laboratory research to advance our understanding of biological processes or for specialized industrial applications – for examples: 转基因用，分子机理研究 Development of insect resistant crops Production of human insulin in transgenic bacteria carrying appropriate human gene 3

二、常用的克隆策略 图位克隆法 标签法 同源序列法 功能克隆法 mRNA差异比较 利用EST数据库和更新的SAGE法 基因芯片 RNA-seq 基因文库及目的基因分离

1.图位克隆（Map-based cloning）法（未知序列的基因克隆） 图位克隆(Map-based cloning)又称定位克隆(positional cloning)，1986年，剑桥大学的Alan Coulson提出，用该方法分离基因是根据目的基因在染色体上的位置进行基因克隆的一种方法。 在利用分子标记技术对目的基因进行精确定位的基础上，使用与目的基因紧密连锁的分子标记筛选DNA文库，从而构建目的基因区域的物理图谱，再利用此物理图谱通过染色体步行逼近目的基因或通过染色体登陆的方法最终找到包含该目的基因的克隆，最后通过遗传转化和功能互补验证最终确定目的基因的碱基序列。 图位克隆方法分离基因的优点是无需预先知道基因的DNA顺序，也无需预先知道其表达产物的有关信息。 在已知基因组序列的基础上可以不构建物理图谱。仅仅进行精细定位，最后用PCR的方法克隆基因。 5

图位克隆法步骤 ٭ 90年代初发展起来的基因克隆技术。很多分离克隆的基因都是通过这种方法获得的。 ٭ 图位克隆法是基于基因在遗传图谱上的位置而分离克隆基因的方法。 ٭ 图位克隆法包括以下步骤： (a) Target gene mapping: a high resolution genetic map with average distance is less than 5 cM. 水稻 1cM=270kb, 人类 1cM=1000kb (b) Physical mapping: a map of the locations of identifiable landmarks on DNA, regardless of inheritance. Distance is measured in base pairs other than genetic recombination (in cM). 6

图位克隆法步骤 (c ) Chromosome walking and landing: -- Chromosome walking relies on isolation of a DNA fragment at or near an end of a cloned insert for use as a probe to screen the library and identify more clones. -- Chromosomal landing: It is based on the principle that the expected average between-marker distances can be smaller than the average insert length of a clone library containing the gene of interest (d) Gene identification: Genetic complementation through transformation.

主要技术环节 遗传作图 近等基因系 BSA 筛选连锁的分子标记 目的基因的精细定位和作图 目的基因分离 目的基因鉴定

遗传作图 Genetic Map Genetic map – in this case, the maps of potato and tomato are aligned, and the markers align perfectly except for a few inversions (indicated by arrows)

物理图谱（physical map) 物理图谱 基因所在DNA区域的物理特征 限制性酶切图谱 跨叠克隆群(contig)图谱 菌落杂交 PCR筛选 Fiber-FISH DNA序列图谱

近等基因系（near iso-genic lines, NILs） 除了目标基因所在座位的局部区域外，基因组DNA序列的其余部分都是相同的。 BSA (Bulked segregant analysis)将分离群体中研究的目标性状根据其类型(如抗病、感病)分成两组，将每组内一定数量的植株DNA等量混合，形成两个池，这两个池除在目标性状(抗病性)上有差异外，其余遗传背景均相同。利用分子标记技术寻找两个池的扩增谱带的差异，这种多态性极可能与目标基因连锁。

目的基因分离 染色体步移法 在鉴定出紧密连锁的分子标记所在的大片段克隆以后，以此为起点进行染色体步移，逐渐靠近目标基因。可能中断或遇重复序列转向。 染色体登陆 找出与目标基因的物理距离小于基因组文库插入片段的平均距离的分子标记，筛选文库直接获得含有目标文库的克隆。

图位克隆示意图 Chr 构建作图群体（F2、BC1F1、RIL、DH或NIL） 分子标记作区段高密度遗传连锁图 基因X i g f h e d c b a 1.8cM 3.5cM 0.5cM 1.2cM 0.8cM 构建作图群体（F2、BC1F1、RIL、DH或NIL） 分子标记作区段高密度遗传连锁图 以距基因最近的两侧分子标记筛选文库（BAC,PAC or YAC 文库），分离片段末端进行染色体步查（chromosome walking) RIL: recombinant inbrid line. DH: doubled haploid NIL: near isogenic line PAC 载体以F 因子和噬菌体P1 为基础构建,兼有二者的特点，通过电激穿孔转化可将PAC 导入大肠杆菌。其特点是插入的外源DNA 没有明显的嵌合和缺失现象，PAC 载体可以插入约300 kb 的外源片段，可以稳定遗传及高效扩增。 YAC可以克隆 200-500kb 在RFLP作图中，连锁距离是根据重组率来计算的， 1cM（厘摩）相当于1%的重组率。人类基因组中， 1cM≈1000kb；拟南芥菜中，1cM≈290kb；小麦中， 1cM≈3500kb。 g e f 1.8cM 0.5cM X gene walking landing 13

图位克隆示意图 文库片段测序 Genscan和Blast，候选基因确定 转基因功能验证 在RFLP作图中，连锁距离是根据重组率来计算的， 1cM（厘摩）相当于1%的重组率。人类基因组中， 1cM≈1000kb；拟南芥菜中，1cM≈290kb；小麦中， 1cM≈3500kb。 14

Map-based cloning of Bph14, a resistant gene to rice brown planthopper Du et al. Proceed. Natl. Acad. Sci. USA 2009, 106:22163-22168

哪些园艺植物适合做图位克隆？ 你认为图位克隆最快捷的方法是什么？需要注意哪些问题？

2. 标签法 转座子或T-DNA标签构建成质粒载体； 载体导入目标植物 筛选群体 表型突变 扩增标签边上的基因

T-DNA插入引起的后果 （Krysan et al. 1999）

利用T-DNA突变体分离克隆基因

侧翼序列（Flanked-sequence tag, FST）的分离方法 热不对称交错PCR（Thermal Asymmetric Interlaced PCR，TAIL-PCR） 反向PCR（Inverse PCR，IPCR） 质粒拯救（Plasmid rescue） 接头PCR （Adaptor PCR）

TAIL-PCR AD primer：任意简并引物 （Arbitrary Degenerate Primer）

反向PCR

质粒拯救

接头PCR

3. 同源序列法 PCR 基因同源性分析 保守序列 简并引物 PCR获得基因片段 或是RT-PCR获得cDNA片段 RACE法获取全长 基因同源性分析 保守序列 简并引物 PCR获得基因片段 或是RT-PCR获得cDNA片段 RACE法获取全长 cDNA片段做探针，从cDNA文库钓取全长

巢式PCR 多重PCR 反向PCR Touchdown PCR

4. mRNA差异比较为基础的分离法 Mutant and wild-type 表型差异： 不同基因型目标基因在细胞总DNA中所占比例很小。mRNA仅代表那些在基因组中能够表达的基因顺序。 同一材料不同时空背景、不同环境响应下mRNA表达可能不同 The mRNA Differential Display technique works by resolving the 3' terminal portions of mRNAs on a DNA sequencing gel. Using a primer designed to bind to the 5' boundary of a poly A tail for the reverse transcription, followed by PCR amplification with additional upstream arbitrary primers, mRNA sub- populations are visualized by denaturing polyacrylamide gel electrophoresis. This allows direct side by side comparison of the mRNAs between two or more related cells. The Differential Display method has the potential to visualize all the expressed genes (about 10,000 to 15,000 mRNA species) in a mammalian cell by using multiple primer combinations. More importantly, the new method enables a researcher to recover their sequence information and use them as probes to isolate their cDNA and genomic DNA for further molecular characterizations. Because of its simplicity, sensitivity, and reproducibility, the mRNA Differential Display method is finding wide rang and rapid application in developmental biology, cancer research, pathology, plant physiology and other biological systems

4. mRNA差异比较为基础的分离法 差异显示(mRNA differential display) DDRT-PCR (difference display RT-PCR) 差减杂交Subtractive　Hybridization 代表性差异分析（Representational Difference Analysis，RDA ） 抑制性差减杂交（Suppression Subtractive Hybridization， SSH 竞争性杂交（ Competitive Hybridization） The mRNA Differential Display technique works by resolving the 3' terminal portions of mRNAs on a DNA sequencing gel. Using a primer designed to bind to the 5' boundary of a poly A tail for the reverse transcription, followed by PCR amplification with additional upstream arbitrary primers, mRNA sub- populations are visualized by denaturing polyacrylamide gel electrophoresis. This allows direct side by side comparison of the mRNAs between two or more related cells. The Differential Display method has the potential to visualize all the expressed genes (about 10,000 to 15,000 mRNA species) in a mammalian cell by using multiple primer combinations. More importantly, the new method enables a researcher to recover their sequence information and use them as probes to isolate their cDNA and genomic DNA for further molecular characterizations. Because of its simplicity, sensitivity, and reproducibility, the mRNA Differential Display method is finding wide rang and rapid application in developmental biology, cancer research, pathology, plant physiology and other biological systems

Differential Display ①3’端有12种组合锚定引物 ②10核苷酸引导第2链合成 ③两引物扩增产物差异 CAAAAAAAAAAAA-An TAAAAAAAAAAAA-An GAAAAAAAAAAAA-An 5’-AAGCTTTTTTTTTTTG-3’(H-T11G) dNTPs MMLV reverse transcriptase Reverse Transcription CAAAAAAAAAAA-An GTTTTTTTTTTTCGAA 5’-GCTTGATGCC-3’(H-AP-1 Primer) 5’-CTTTTTTTTTTTG-3’(H-T11G) dNTPs a-[33P-dATP] Taq DAN polymerase PCR Amplification GCTTGATGCC The mRNA Differential Display technique works by resolving the 3' terminal portions of mRNAs on a DNA sequencing gel. Using a primer designed to bind to the 5' boundary of a poly A tail for the reverse transcription, followed by PCR amplification with additional upstream arbitrary primers, mRNA sub- populations are visualized by denaturing polyacrylamide gel electrophoresis. This allows direct side by side comparison of the mRNAs between two or more related cells. The Differential Display method has the potential to visualize all the expressed genes (about 10,000 to 15,000 mRNA species) in a mammalian cell by using multiple primer combinations. More importantly, the new method enables a researcher to recover their sequence information and use them as probes to isolate their cDNA and genomic DNA for further molecular characterizations. Because of its simplicity, sensitivity, and reproducibility, the mRNA Differential Display method is finding wide rang and rapid application in developmental biology, cancer research, pathology, plant physiology and other biological systems GTTTTTTTTTTTCGAA Denaturing Polyacrylamide Gel ①3’端有12种组合锚定引物 ②10核苷酸引导第2链合成 ③两引物扩增产物差异 X Y

Subtractive Hybridization ①生物素标记 ； ②cDNA与RNA复合体 Samples of Interest mRNA Extraction Reverse transcription cDNA RNA Hybridization cDNA cDNA RNA RNA

Suppression Subtractive Hybridization (SSH) 抑制消减杂交

2次PCR 1st接头外侧（5‘）作引物； 2nd：接头内侧（3‘）作引物

应用的几个关键点： ①tester与driver配对密切，最好双方是同源细胞株，这样双方才具有高度可比性，筛选出的差异表达基因才可能与表型关系密切．从而减少工作量，减少下一步工作的肓目性。 ②在选用限制酶时应尽量选用对于基因组DNA中酶切位点较少的限制酶，使酶切后产生的片段尽量大—些。例如使用Rsa I可产生600 bp左右的片段，这样既可防止过长的cDNA片段影响杂交效率，也可提高每个基因的代表性。 ③接头的设计上。接头要含有酶切仿点以连于平端cDNA上，重要的是要在其末端含有一段反向重复序列，这使得在PCR反应中能选择性扩增目的cDNA片段，同时抑制非目的cDNA片段的扩增，即抑制PCR技术。

优点： 特异性强 假阳性率低 高敏感性，检出低丰度mRNA 速度快，效率高 缺点 mRNA用量高 得到的是cDNA片段 研究材料之间不能差异太大