细菌人工染色体文库及其应用

细菌人工染色体是1992年以来发展起来的一种新型克隆载体，用于构建人、动物、植物核基因组DNA大片段插入文库。细菌人工染色体在文库构建中具有转化效率高、构建文库简单、嵌合体少、插入片段容易回收、在寄主细胞中插入片段稳定性好、插入片段较大等优点。细菌人工染色体文库的构建对于基因组较大的真核生物基因组学研究具有重要作用，该文库可用于真核生物重要性状基因及全基因组的物理作图、重要农艺性状基因的图位克隆、基因结构及功能分析等研究。本文主要综述了细菌人工染色体文库的构建及其在图位克隆基因、荧光原位杂交、全基因组物理作图等研究中的应用进展。     

棉花细菌人工染色体文库构建的方法优化

以我国抗病、优质、丰产的棉花优良品种中棉所12号为材料,对棉花细菌人工染色体(BAC)文库构建过程中的一些关键技术,如plug的制备、DNA的部分消化、酶切片段的选择、插入片段与载体的摩尔比值、连接产物的浓缩等进行了研究,建立了构建棉花BAC文库的适宜方法体系。依照该方法初步构建了含有38000个克隆的棉花BAC文库。经检测,插入片段平均大小约为120kb,蓝斑率小于0.5%,空载率小于1%。插入片段与载体的最适摩尔比为1∶15,一次转化可以获得约2000个克隆,cfu·μl 1高达4。该方法为进一步构建中棉所12号多倍基因组的BAC文库、从而进行棉花基因组有关研究奠定了基础。     

棉花细菌人工染色体文库构建方法探讨

 细菌人工染色体(Bacterial artificial chromosome, BAC）文库是开展基因组测序、基因图位克隆、分子标记、物理作图等研究的重要基因组资源。本文在构建了二倍体野生棉阿非利加棉（Gossypium herbaceum var. africanum）BAC文库的基础上,就棉花细菌人工染色体基因组文库构建过程中高分子量基因组DNA的提取、部分酶切片段选择、DNA的回收、连接转化以及BAC文库的保存等过程中一些细节和注意事项进行了比较详细的分析比较,希望能为棉花BAC文库的构建提供一些可供借鉴的经验。     

云南疣粒野生稻细菌人工染色体（BAC）文库的构建与分析

我国有3种野生稻,即普通野生稻(oryza rufipogon)、药用野生稻(O.officinalis)、疣粒野生稻(O.granulata).野生稻由于长期处于野生状态,经受了各种灾害和不良环境的自然选择,抗逆性较强,是天然的基因库,保持有栽培稻不具有或已经消失了的遗传基因,它在水稻育种中具有独特的作用,是水稻育种的宏厚物质基础.     

籼稻品种H359基因组可转化人工染色体(TAC)文库的构建

村建能够通过农杆菌介导转化植物的人工染色体文库可以加快突变基因的精细定位和克隆的速度。为了克隆水稻突变基因的需要，我们利用pYLTAC7载体构建了籼稻品种H359基因组的TAC文库。该文库包含41088个克隆，保存在107块384孔板中。插入片段大小在50—100kb之间，平均插入大小为77kb，推测该库覆盖水稻基因组接近7．4倍。     

长片段小麦细菌人工染色体DNA亚克隆文库的构建

为了加快小麦基因组的测序,以Langdon库中的一个BAC克隆(BAC790O10)为材料,利用HydroShear DNA剪切仪将其打断,产生许多大小不同的DNA随机片段,回收其中3～5 kb的片段,并将这些片段随机克隆入TOPO载体,构建了适合于鸟枪法测序的亚克隆文库.     

长果种黄麻单染色体未克隆DNA文库的构建

单染色体分离、扩增和克隆技术对植物的遗传研究（尤其对那些遗传研究基础薄弱的植物而言）具有重要的应用价值。本研究用玻璃针显微分离法成功分离出长果种黄麻（Corchorus olitorius L.）品种闽引一号的单染色体80条。用LAM-PCR或DOP-PCR对单染色体DNA进行两轮扩增,所包含的DNA片段长度分别为250～2 000 bp和100～600 bp。共建立了50个单染色体未克隆文库。Southern杂交表明,获得的扩增片段确实来自黄麻基因组。对单染色体DNA文库应用的有关问题进行了讨论。     

陆地棉雄性不育恢复系18R的BAC文库构建

 18R雄性不育恢复系农艺性状优良,恢复性状稳定,对不育系能够100%恢复,是研究棉花三系互作的重要材料。本研究以pCC1BAC(BamHI)为载体,构建了18R的细菌人工染色体（BAC）文库。建立的文库包含139,200个BAC克隆。分析结果表明, BAC文库DNA插入片段为50～200 kb,91%的克隆插入片段为80～150 kb,平均102 kb,空载率<2%,覆盖6.3倍基因组。     

山羊基因组文库的构建研究

构建了山羊基因组文库,为筛选山羊乳蛋白基因,构建山羊乳蛋白基因定点整合的转基因敲入载体奠定基础。全血提取基因组DNA,Sau3AΙ部分酶切,分级分离回收15~23 kb片段,将片段插入Lambda BlueSTAR BamHΙ vector,连接并包装后,NotI酶切鉴定。结果表明,文库滴度为3.4×106;共得到1.7×106个有效克隆,插入片断长度在15~23kb范围,成功构建了西农萨能奶山羊基因组文库。     

甘蓝型油菜基因组文库的构建及与硼高效基因相连锁克隆的筛选

以甘蓝型油菜宁RS-1为材料,构建了含有82944个克隆的甘蓝型油菜的BAC基因组文库.从文库中随机挑取克隆进行DNA长度检测,BAC克隆平均插入片段大小为80 kb,覆盖甘蓝型油菜基因组的5.1倍.随机挑取108克隆进行继代培养100代,分离质粒酶切检测表明不存在插入片段丢失现象,表明该文库的克隆在大肠杆菌中稳定存在;以与硼高效基因相连     

甘蓝枯萎病抗性基因FOC1序列分析及抗性相关变异位点分析

旨在发掘甘蓝抗枯萎病基因FOC1中与抗性相关的特异性单核苷酸多态性(SNP),为进一步开发FOC1特异分子标记提供理论依据。在对11份甘蓝自交系材料枯萎病抗病表型鉴定基础上,通过基因测序,对每份材料中FOC1等位基因及相应的氨基酸序列变异进行分析。结果表明,FOC1等位基因序列之间共存在92处SNP变异和2处Indel变异,其中包含C381A/G等18个在抗、感材料中表现出特异性差异的SNPs。此18个特异的SNP中转换型比率为86.11%、颠换型比率为13.89%,4种碱基变异率以T/C、G/A最高,分别占47.22%和38.89%,而C/G和A/C变异率共占13.89%。本研究也发现抗、感材料中存在4个特异的SNP,可造成3个氨基酸的变化。     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

NaCl胁迫对甘蓝、白菜和油菜种子萌发的影响

为研究蔬菜种子吸收或外排Na+能力与其耐盐性的关系,采用非损伤微测技术检测了628甘蓝、青杂中丰白菜、苏州青油菜、茼蒿、上海鸡毛菜种子外Na+流速的动态变化.结果表明,在10mmol/L NaCl溶液处理下,甘蓝和茼蒿种子外排Na+,而白菜、油菜和鸡毛菜种子吸收Na+,其中,甘蓝、白菜和油菜种子对Na+吸收或外排的效果明显.随后以甘蓝、白菜和油菜种子为材料,在不同NaCl浓度下对种子萌发进行了实验,结果表明,随着NaCl浓度升高,甘蓝、白菜和油菜种子的发芽率、相对发芽率、发芽势和发芽指数都呈明显下降趋势.当NaCl浓度达到300mmol/L时,甘蓝、白菜和油菜种子的发芽率分别为37.33％、14.67％和10.00％,耐盐半致死浓度分别为323.73,241.68mmol/L和200mmol/L.初步说明种子可以通过增加Na+的外排,减弱高浓度Na+对种子的毒害作用.     

原生质体非对称融合获得花椰菜与Ogu CMS甘蓝型油菜种间杂种

甘蓝型油菜Ogu CMS系幼叶原生质体经剂量分别为0.025 0,0.075 0,0.315 0,0.468 0和0.960 0 J/cm2的紫外线辐照后,与花椰菜下胚轴原生质体通过PEG法诱导融合,其中0.025 0~0.468 0 J/cm2获得再生植株68株,0.960 0 J/cm2辐射剂量下没有获得再生株。通过根尖染色体计数、流式细胞仪倍性鉴定、同工酶酶谱分析、RAPD及Ogu CMS线粒体基因orf138 STS(Sequence Tagged Site)标记引物扩增等方法对再生株进行了鉴定。结果表明,其中32株为杂种植株。同时也发现,紫外线辐照剂量的增加对供体染色体的丢失程度影响不大。     

Identification of Brassica oleracea germplasm with improved resistance to Verticillium wilt

Two hundred and ninety‐nine accessions representing 11 cultivar groups of Brassica oleracea and eight additional accessions of the wild species B. cretica, B. incana, B. insularis and B. villosa were screened for resistance to Verticillium wilt. A disease index (DI) was calculated for each accession, and a correction of the DI was carried out to compensate for a fluctuating infection level between 11 independent trials. A total of 235 , or 77% of the accessions tested, had a DI_corr less or equal to the oilseed rape cv. ‘Express’ (DI_corr= 2.81), the reference cultivar. Only one accession of the wild species, B. incana, showed an enhanced level of resistance (DI_corr= 2.01). Twenty‐four accessions, distributed over eight cultivar groups of B. oleracea were selected for subsequent crosses involving B. rapa. Hybrid plants with 14 accessions were resultant and seed was obtained from crosses where the cultivar groups acephala, alboglabra, botrytis, capitata, gemmifera, italica and sabellica were used as female parents. When progeny of the produced resynthesized rapeseed lines were evaluated for Verticillium wilt resistance, three lines showed a significantly lower disease index (P ≥ 0.01) compared with the cv. ‘Express’. This source of resistance is now being crossed to advanced breeding material of oilseed rape.     

A cytogenetic study of the progenies of hybrids between Brassica napus and Brassica oleracea, Brassica bourgeaui, Brassica cretica and Brassica montana

In this cytogenetic study the progeny of all crosses were investigated in F₁, F₂ and backcross (BC₁) hybrids. Brassica napus and F₁ hybrids between B. napus and B. oleracea, and between B. napus and three wild relatives of B. oleracea (B. bourgeaui, B. cretica and B. montana). Each of the wild relatives has 18 somatic chromosomes. Interspecific F₁ hybrids were obtained through ovary culture mean. These had 28 and 37 chromosomes and their mean pollen fertility was 10.7% and 93.0%, respectively. Many F₂ and BC₁ seeds were harvested from the F₁ hybrids with 37 chromosomes after self‐pollination and open pollination of the F₁ hybrids, and backcrossing with B. napus. Many aneuploids were obtained in the F₂ and BC₁ plants. It is evident from these investigations that the F₁ hybrids may serve as bridge plants to improve B. napus and other Brassica crops.     

甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析

自交不亲和性(self-incompatibility,SI)是指植株的雌蕊对自身花粉和异体花粉进行识别从而抑制自身花粉萌发的特性, PUB (Plant U-Box)蛋白在植物抗逆及信号转导过程中起着重要的作用。本研究通过分析甘蓝自花授粉0～60min的柱头转录组数据,筛选到1个受自花授粉诱导上调表达的PUB蛋白编码基因BoPUB9。BoPUB9 cDNA序列长度为1368 bp, gDNA序列全长1720 bp,含有1个臂重复结构域和1个U-box结构域。荧光定量PCR结果显示, BoPUB9基因在甘蓝的不同组织中均有表达,在萼片中的表达量最高,花瓣、雄蕊、柱头表达量次之,在花粉花蕾中表达量相对较低,同GUS染色结果保持一致。BoPUB9基因在自花授粉0～30 min内快速上调表达, 15 min后为异花授粉后表达量的20多倍,在30min后达到差异最大,自花授粉后的表达量为异花授粉的40多倍。亚细胞定位结果显示,BoPUB9蛋白在细胞核和细胞质中均有表达,且BoPUB9蛋白在大肠杆菌中被成功诱导表达,相对分子质量为51 kD,与预测结果一致。酵母双杂交与poll-down结果显示...