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可转化人工染色体（TAC）

可转化人工染色体（TAC）

2010/08/17 08:45:53

Liu等结合PAC和双元载体的特点，构建了TAC载体2pYLTAC7，TAC载体具有P1复制子和Ri质粒pRiA4复制子，能在大肠杆菌和农杆菌中穿梭复制。pYLTAC7在其T2DNA右边界处插入了植物选择标记潮霉素磷酸转移酶基因（hpt）和Pnos启动子。在TAC载体中重组筛选标记是卡那霉素抗性基因和_sac_B基因。Liu等以pYLTAC7为载体构建了拟南芥的基因组文库，并把部分克隆通过农杆菌介导转化拟南芥，获得了转化植株并能稳定遗传。为了证明TAC载体系统在图位克隆中的可利用性，用与拟南芥重力感应基因_sgr_1紧密连锁的标记筛选文库，构建了_sgr_1的跨叠克隆群，并把含有_sgr_1基因的2个TAC克隆转化到_sgr_1突变体中，使其恢复了重力感应。由于Pnos启动子在单子叶植物中的启动能力较弱，Liu等对pYLTAC7进行了改造，选用水稻肌动蛋白_Act_Ⅰ强启动子，把植物选择标记基因_hpt_替换为_bar_基因，构建了TAC载体pYLTAC17。_bar_基因编码对磷化麦黄酮（PPT）和双丙胺类（bialaphos）除草剂的抗性，适合用作禾本科作物转化体的选择。Liu等用pYLTACl7载体构建了普通六倍体小麦的基因组文库，对部分较大插入片段（150kb）克隆的遗传稳定性分析表明TAC也存在插入子重组现象。用_bar_基因作为筛选标记基因,在候选克隆的功能互补转化实验中发现,存在筛选困难，假阳性率高,有基因型限制等问题。为了提高外源基因的表达及利于筛选，针对不同的受体材料对TAC载体进行改进，构建了载体pYLTAC27，并构建了水稻“明恢63”的基因组文库。该载体以_Act_Ⅰ为启动子，_hpt_为筛选标记基因，该载体系统可以高效地用于单子叶植物的基因图位克隆及功能分析。

TAC载体与BIBAC载体一样具有克隆大片段DNA和借助于农杆菌直接转化植物的功能。TAC和BIBAC载体除具有BAC载体的优点外还具有以下优越性：①具有大肠杆菌和农杆菌的复制子，是一个穿梭质粒，在大肠杆菌和农杆菌中均保持稳定。②可通过农杆菌介导直接进行基因功能互补实验。TAC载体与BIBAC载体相比，首先大肠杆菌中的复制起点不同，TAC源于P1噬菌体；而BIBAC源于F因子。其次，TAC载体中具有P1裂解子（lytic replicon），可以在IPTG的诱导下产生5-20个拷贝，提高了质粒产量。目前已经构建了水稻、小麦、拟南芥、大豆、金鱼草等植物的TAC基因组文库，并从拟南芥中分离出了_filamentous flower_基因、_kor1_基因等。

General information of the TAC vector

The structure of the TAC vector pYLTAC7 is shown in Fig. 1a. The TAC vector is available from the Plant Cell Bank of the RIKEN Gene Bank (Tsukuba, Japan; e-mail, pcbank@rtc.riken.go.jp). The entire sequence of the vector is also available in GenBank (accession number, AB020028).

Structural features of the TAC vector

For preparing genomic DNA libraries;

  1. Stable maintenance of inserted fragments in Escherichia coli

The TAC vector contains the P1 bacteriophage replicon, which maintains the vector in a single copy, and therefore renders foreign DNA fragments stable, in E. coli _cells. The vector also contains the pRiA4 replicon of the Ri plasmid, which ensures a vector copy number of 1 in _Agrobacterium tumefaciens. The kanamycin resistance marker gene (NPTI), modified by removal of the Hind III site, is included in the vector to allow selection of clones in both E. coli and A. tumefaciens by culture in the presence of kanamycin.

  1. Cloning sites for library preparation

Unique Hind III and Bam HI cloning sites for the preparation of genomic DNA libraries are included in the TAC vector between the sacB gene and its promoter. The efficient ligation of Hind III cohesive ends ensures that large-scale genomic DNA libraries can be readily prepared with the use of the Hind III site. However, Hind III fragments of relatively small size may not encompass entire genomes. The use of the Bam HI site is thus recommended for preparation of another genomic DNA library from the same DNA source. The Bam HI site also may be used for preparing libraries with small (<30 kb) Sau 3AI-Mbo I fragments.

  1. Positive selection of E. coli cells harboring plasmids with insert DNA

Insert-bearing clones can be selected on sucrose-containing agar plates. The insertion of a DNA fragment at the cloning sites between the sacB gene and its promoter reduces the production of levansucrase, which is encoded by the sacB gene, in E. coli. Given that the production of this enzyme is lethal for E. coli in the presence of 5% sucrose, culture of transformed cells in the presence of this sugar eliminates those harboring the empty vector.

_For analysing TAC clones _

  1. Plasmid preparation from E. coli

The low-yield disadvantage for DNA preparation of P1 replicon-mediated maintenance of the TAC plasmid in a single copy in E. coli can be overcome by releasing suppression of the P1 lytic replicon with isopropyl-b-D-thiogalctopyranoside (IPTG). The resulting plasmid amplification ensures that several micrograms of TAC plasmid DNA can be obtained from a 3-ml culture grown in the presence of 0.5 mM IPTG (Protocol 1). (Note that 0.3 to 0.5 mM IPTG is sufficient for releasing suppression in E. coli strain DH10B.)

  1. Isolation of end fragments of inserted DNA

The TAC vector includes TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction) primer sequences (R1, R2, R3, L1, L2, and L3) flanking the cloning sites (Fig. 1b). The TAIL-PCR protocol, which requires three specific primers for the vector sequence and an arbitrary degenerate primer, but not an insert-specific primer, has been applied successfully for the isolation of end fragments of inserted DNA from many TAC clones in our and other laboratories.

Reference for the TAIL-PCR protocol:

Liu, Y.-G. and Whittier, R.F. (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674-681

For transfering large DNA fragments into plant genomes by Agrobacterium-mediated protocols

  1. Cis elements for gene transfer

The TAC vector contains left (LB) and right (RB) borders, the cis sequences required for Agrobacterium-mediated gene transfer in plants. The plant-selectable marker gene HPT, which encodes hygromycin phosphotransferase, is also included in the vector under the control of the nopaline synthase gene (nos) promoter (Pnos) and upstream of the nos terminator (Tnos). The HPT construct is included together with the cloning sites and sacB between the LB and RB sequences.

TAC plasmids containing large (~100 kb) inserts are transferred efficiently into A. tumefaciens by electroporation (Protocol 1). The pRK2 oriT sequence, which is used for delivering plasmids from E. coli _to _A. tumefaciens by the triparental-mating method, is therefore not included in the TAC vector.

For Southern analysis of transgenes

  1. The I-SceI sites for cutting the whole insert fragment out

The TAC vector contains two I-Sce I sites, one upstream of the Pnos sequence and the other downstream of the cloning sites, to allow Southern blot analysis of the integrity of large transgenes. Thus, the entire insert sequence together with the HPT tag can be released from the plant genome by digestion with I-Sec I and be detected by Southern analysis with an HPT gene probe. Given that I-Sce I recognizes an 18-bp sequence, recognition sites for this enzyme should occur only once per 6.9 x 1010 bp of random DNA sequence. Most plants should therefore not contain a I-Sce I site. Protocols describing the preparation of high molecular weight genomic DNA and I-Sce I digestion for Southern analysis are included in Protocol 2.

  1. Multiple unique sites for preparation of nested deletion clones

Unique sites for Asc I, Sfi I, Srf I, Fse I, and Not I, each of which recognizes rare 8-bp sequences, are included in the TAC vector flanking the Hind III and Bam HI cloning sites. After a TAC clone containing a large genomic DNA fragment is shown to complement the mutation of interest, these sites can be used to create a series of deletion clones in order to identify the target gene by further complementation tests.

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