GmAOC3基因转化载体构建及转化大豆的初步研究

选用内切酶SacⅠ和MluⅠ切割目标基因和载体,构建了携带bar基因的重组载体p BA002-Gm AOC3,选用2个大豆品种(Jack和南农88-1)和2种外植体(子叶节和整个子叶节),研究大豆品种和外植体对根癌农杆菌介导的子叶节转化Gm AOC3基因的影响。结果表明:外植体为整个子叶节的大豆品种Jack的出芽率和转化率最高,分别为79.5%和2.27%。经Quick Stix PAT/bar基因试纸条检测、目标基因的分子检测和草丁膦抗性鉴定,共得到转Gm AOC3基因的T0代阳性株系3株,T1代阳性转基因大豆6株,其中5株以Jack品种为受体的转基因大豆Gm AOC3基因的表达量均显著高于非转基因植株,荧光定量拷贝数检测结果显示,4株转基因单株为单拷贝,2株为双拷贝。     

影响农杆菌介导的大豆基因转化因素的研究

通过农杆菌介导法用含NPT－Ⅱ和Barnase基因导入到大豆幼胚，大豆子叶节叶，然后经过含Kan的筛选培养基中连续筛选，获得一批Barnase转基因植株。经PCR扩增结果，证实此基因已整合到大豆中，在当代植株中生物学特性观察，花粉粒不育率在98％。     

RNA干扰高油GmPEPc基因转入大豆研究

通过构建GmPEPc基因的植物RNAi双元表达载体,并通过农杆菌介导的大豆子叶节遗传转化方法将控制油脂和蛋白合成途径的相关基因GmPEPc转入受体品种沈农9号中,通过抑制大豆内源GmPEPc基因的表达,增加油脂积累,从而获得高油的转基因大豆新品种。在大豆组织培养过程中,共切取大豆外植体407块,获得T0代转化苗35株,转化率8.9%。通过对转基因后代中目的基因的整合及表达情况进行分子鉴定。获得23株T_1代转基因后代,其中高抗草丁膦除草剂(喷施浓度300 mg·mL~(-1))14株,通过PCR检测结果表明其中12株为PCR阳性;PCR和Southern杂交检测表明,GmPEPc基因已经成功插入到转基因大豆植株基因组DNA中。对T_1代测定结果显示,转基因大豆籽粒的平均含油量比对照高9.51%,平均蛋白质含量下降5.44%。这些研究结果为筛选高油脂含量的转基因大豆新株系提供了依据,为下一步高油新品种的选育提供了种质基础。     

基因枪共转化法获得玉米转基因植株的研究

通过基因枪共转化方法将磷高效利用phy基因、钾高效利用AlHAK1基因和筛选标记bar基因等外源基因导入玉米自交系H99的1 500块愈伤组织中。经PCR分析,获得79株阳性植株,其中53株为AlHAK1阳性,49株为phy阳性,23株为AlHAK1和phy阳性,单基因转化率为5.27%,双基因转化率为1.53%,共转化整合比率达到29.11%。     

BcWRKY1转录因子基因花粉管通道法转化玉米的初报

采用花粉管通道法,以LH1037、合344、05-2044等7份优良玉米自交系为试材,转化转录因子BcWKKY1基因,并优化了遗传转化体系。结果表明,当转化DNA溶液浓度为200μg/mL、授粉后9 h转化,结实率和转化率最高;转基因玉米的发芽率均低于非转基因对照。共获得T0代PPT抗性植株89株,PCR阳性植株55株,其中36株结实。T1代转基因植株的PCR和PCR-Southern阳性率为86.7%。     

用基因枪法将小麦病程相关蛋白基因TaPR-1导入小麦的研究

为验证小麦病程相关蛋白基因TaPR-1的功能,利用gus基因瞬间表达技术对基因枪法转化小麦幼胚的两个重要参数金粉用量和轰击距离进行了优化.研究结果表明,金粉用量为250.0 μg,轰击距离9cm的轰击参数最为理想,GUS瞬间表达率和平均蓝点数分别可达89.8%和33.7个.采用此轰击参数,将TaPR-1基因导入扬麦158幼胚,经3～5 mg/L Bialaphos筛选获得49株抗性再生植株,经PCR、Southern杂交分析,其中2株为阳性植株,转化率为0.11%.对转基因小麦植株进行苗期离体叶段和田间成株白粉病接种鉴定,初步结果表明,转基因植株对白粉病抗性与对照相比无明显差别.     

农杆菌介导法将Bt(cryⅠA)基因导入大豆的研究

构建含有Bt(cry Ⅰ A)基因的植物表达载体pCAMBIA3300-Bt,以大豆子叶节为受体,通过农杆菌介导法将Bt基因导人大豆品种黑农37中,获得转基因植株.并进行人豆的再生和遗传转化系统优化的研究,以获得较高的转化率.结果表明:在6-BA浓度为1.7 mg·L-1时,丛生芽分化率最高,确定该品种大豆在从生芽分化阶段的草铵膦筛选浓度为3.5 mg·L-1获得转化质粒pCAMBIA3300-Bt的转基因植株,其中T1代PCR阳性植株19株.采用real-time PCR的方法对T1代抗性植株进行Bt基因的转录水平的分析,初步证明Bt基因已整合到受体大豆的基因组内.     

抗虫基因Cry8-like在大豆中的遗传转化及抗虫性初步鉴定

利用无缝克隆技术把抗虫基因Cry8-like重组到pCAMBIA3301载体上,成功构建了含有抗草铵膦筛选标记的植物表达载体pCAMBIA3301-RSP-Cry8-like-nos。将该载体通过农杆菌介导法转入两个受体大豆品种吉农28和吉农42中,经PCR检测,吉农28和吉农42分别获得了17株和13株T_1阳性植株,36株和25株T_2阳性植株。T_2转基因植株Southern杂交结果显示,目的基因以单拷贝的形式整合到大豆基因组中。荧光定量PCR检测结果表明,Cry8-like在转基因植株幼根得到了表达,而非转化受体对照未检测到荧光信号。室内抗暗黑鳃金龟幼虫鉴定结果表明,转基因植株提高了抗暗黑鳃金龟幼虫的能力。     

农杆菌介导大豆子叶节转化cry1Iem基因

利用农杆菌介导法,以东北地区种植的9个大豆基因型和国外品种Williams82的子叶节为外植体,将抗虫基因cry1Iem转入栽培大豆品种中。共转化外植体1 459个,获得84棵再生植株。采用除草剂叶片涂抹法、PCR及Bar蛋白试纸条检测法对得到大豆再生植株进行鉴定,转cry1Iem基因大豆T_0阳性植株为61株。对部分T_1转基因植株的遗传分析表明,外源基因能够稳定遗传到下一代。通过对部分T_1阳性转基因植株进行Southern blot分析,证明目的基因片段均已整合到受体大豆的基因组DNA中,单拷贝率为22%左右。对获得的部分T_2材料进行了室内抗大豆食心虫鉴定,有2份转基因材料抗性较对照显著提高。     

农杆菌介导的大豆植株整体转化

农杆菌介导的植物整体转化法主要以植物的分生组织和生殖器官作为外源基因导人的受体,通过真空渗透法、浸蘸法及注射法等方法使农杆菌与受体材料接触,以完成可遗传细胞的转化,通过抗生素筛选和分子检测鉴定转基因植株后代.以大豆幼苗的顶端生长点和叶腋生长点为靶点,通过农杆菌介导的整体转化法--注射法进行转录因子DREB1C基因的遗传转化,最终获得6株T0代PCR阳性转基因植株,转化率为9.2%.T1代植株的PCR、Southern blot和RT-PCR鉴定结果表明获得1株阳性植侏,DREB1C基因以单拷贝形式整合于大豆基因组中,在200 mmol·L-1NaCl胁迫条件下在转录水平得到成功表达.该方法克服了大豆组织培养再生难、转化率低的缺点,是一种高频、简单、快捷的非组培遗传转化途径.     

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety (Digu) by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 (pZH01-tp309) and three different expression constructs, pCB-Pi-d25.3kb (pCB5.3kb), pCB-Pi-d26.3kb (pCB6.3kb) and pZH01-Pi-d22.72kb (pZH01-2.72kb) of Pi-d2, driven by Pi-d2 gene's own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.     

Recent Advances in Development of Herbicide Resistant Transgenic Hybrid Rice in China

In addition to weed control in direct seeding field of hybrid rice, herbicide resistance genes were used by Chinese scientists to increase and identify the purity of hybrid seeds, and to realize the mechanization of hybrid seed production. The elite restorer lines, such as Minghui 63, R752, T461, R402, D68 and E32 were transformed directly with herbicide resistance genes, in which D68 and E32 are restorer lines of two-line system and the others are of three-line system. Because almost all of important restorer lines are indica varieties and are recalcitrant in transformation, many herbicide resistant near-isogenic restorer lines were developed by sexual hybridization of indica and japonica varieties and backcross with indica restorer lines later, such as Ce 64, Minghui 63, Teqing, Milyang 46, R402 and 9311, in which 9311 is a restorer line of two-line system. The elite photoperiod-sensitive/thermo-sensitive genic male sterile lines, such as Pei'ai 64S, P88S, 4008S and 7001S, were transformed with herbicide resistance genes. A few herbicide resistant male sterile lines were developed through sexual hybridization and subsequently systemic selection, such as Bar1259S, Bar2172S, 05Z221A and 05Z227A. With the employment of herbicide resistant male sterile lines or herbicide resistant restorer lines, a few herbicide resistant hybrid rice combinations were developed, such as Xiang 125S/Bar 68-1 and Pei'ai 64S/Bar 9311. Based on herbicide resistance, the research was marching on to investigate the parental lines of hybrid rice with insect resistance, drought tolerance, etc.     

转Bar基因抗除草剂两系杂交早稻恢复系Bar 68-1的培育研究

通过基因枪转化方法将抗除草剂基因Bar转入早籼稻两系恢复系D68中,育成了转基因抗除草剂恢复系Bar 68-1和杂交早稻组合香125S/Bar 68-1;Southern杂交检测和除草剂抗性鉴定结果显示,它们具有抗除草剂特性并能稳定遗传。转基因抗除草剂杂交稻组合香125S/Bar 68-1和对照香两优68(香125S/D68)相比,株高显著降低,直链淀粉含量极显著降低,其它农艺、品质和产量性状没有明显变化。     

Allelic Analysis for bar Gene from Different Rice Transformation Events

Abstract

Mechanism(s) of gene transformation and integration in rice (Oryza sauva L.) is/are not currently well understood. This research was conducted to determine whether a transgene is inserted into the rice genome specifically or randomly. Seven homozygous transgenic Taipei (T) 309 and Nipponbare plants with the bar transgene from different rice transformation events were crossed. The segregation of F2 and F3 populations from a total of 21 crosses was studied in a greenhouse and field to determine if the genes were allelic or non-allelic. Five genomic locations appeared to be involved among the seven transgenic plants. An additional 20 homozygous transgenic T309 plants, with the bar transgene from different transformation events, were crossed reciprocally with the previous seven plants. One hundred and fifteen crosses made during 1999 and 2000 were analyzed for allelism. In some combinations, the genes were allelic, but most of them were non-allelic, with two or more pairs of genes being expressed. Twenty loci among the 27 transgenic plants were involved and some plants had several inserted genes expressed. Genes in nine out of 27 transformed plants were allelic. We concluded that the functional foreign (bar) gene was restrictively/preferentially inserted into the rice genome in some cases and was not completely randomly inserted and expressed in the rice genome. If the mechanism(s) for preferential insertion were identified, rice researchers could possibly control insertion sites of transgenes to optimize gene expression.     

不同转化方法培育无抗性选择标记转基因水稻效率的比较

 为研究不同的转化方法培育无抗性选择标记转基因水稻的效果,以4个籼粳稻品种为受体材料,比较了用农杆菌介导的双菌双载体和双T DNA单载体两种共转化技术的共转化频率及随后获得的无抗性选择标记基因转化子的效率。结果表明,在双菌双载体介导的转化中,4个水稻品种的平均共转化频率为10.1%,其中55.6%的共转化植株自交后代中可筛选出无抗性选择标记的转基因水稻植株。在单载体双 T DNA介导的共转化中,平均共转化频率为45.0%,从其中600%的共转化植株自交后代中可获得无抗性选择标记的转基因植株,即双T DNA单载体法的去选择标记的效率（270%）要较双菌双载体法（5.6%）高。     

CbDREB1A基因表达载体构建及转化水稻光温敏核不育系的研究

为了增强荠菜DREB1A基因在转基因水稻中的表达,构建了由Ubi启动子驱动的植物表达载体pUΩCbDREB3300.通过基因枪转化法将CbDREB1A基因导入水稻光温敏核不育系4008S,获得5个抗干旱胁迫的再生植株.运用PCR及Southern杂交技术对抗性再生植株进行鉴定,结果显示CbDREB1A基因已整合到水稻基因组中.干旱胁迫后转基因水稻植株脯氨酸含量显著高于对照,显示耐旱性增强.     

云南地方稻种抗稻瘟病基因Pi ta和Pi b的鉴定

 根据抗病等位基因Pi ta、Pi b与pi ta、pi b感病等位基因在DNA序列上的差异,合成Pi ta引物YL155/YL87和YL183/YL87,Pi b基因引物Pi bdomF/Pi bdomR和Lys145F/Lys145R。利用这两套分子标记检测了176份云南地方稻种抗性基因Pi ta和Pi b的分布。结果表明,供试材料中72份含有Pi ta基因,占40.9%,其中99份粳稻中有28份含有Pi ta基因,占28.3%;77份籼稻中有44份含有该基因,占57.1%;抗性基因Pi ta在粳稻和籼稻品种中的分布频率存在明显差异,籼稻高于粳稻;Pi ta基因主要分布于玉溪、保山、临沧、思茅等地区。而176份供试材料中仅有33份含有Pi b基因,占18.8%,其中99份粳稻中有18份含有该基因,占18.2%;77份籼稻中有15份含有该基因,占19.5%;Pi b基因主要分布在滇西部的怒江、保山和昭通等地区。分别选取具有代表性的18及17份材料在温室进行Pi ta和Pi b基因抗性鉴定,结果与分子鉴定结果基本相符。     

Transgene Transfer to United States Commercial Rice Cultivars via Conventional Breeding Techniques

Abstract

Breeding programs can benefit from transfer of a foreign gene from one transgenic plant to commercial cultivars through continuous backcrossing, especially to cultivars in which it is difficult to transfer a foreign gene directly through biotechnology. In this study, two homogeneous transgenic plants, T-28 and T-64 (from Taipei 309), and one homogeneous transgenic plant, N-84 (from Nipponbare), were used as donors of Bar gene. Commercial cultivars, Cypress and Laffite, were used as the recurrent parents. Three to five backcrosses were made using the transgenic plants as donor parents and the commercial cultivars as recurrent parent. The results from selected progeny rows, and two-years of yield tests with selected lines, indicated that the target Bar gene could be transferred to lines from homozygous transformants in 2–3 years of backcrossing, giving lines similar to the recurrent parents in phenotype and yield potential.     

水稻显性半矮秆突变体及其衍生系的广亲和性鉴定

分别以水稻显性半矮秆突变体Y98149及其衍生系D3、D4、D5与4个籼稻和4个粳稻品种测交进行广亲和性鉴定。结果表明,D5与标准籼稻测验种(IR36、南京11号)测交F1结实率均值为78.7%,与标准粳稻测验种(巴利拉、秋光)测交F1结实率均值为77.9%,具有较好的广亲和性;且D5与籼、粳品系测交在产量性状上均表现出较强的杂种优势,而株高较矮,具有负向超亲优势。     

转Bt基因水稻克螟稻杂交转育后代农艺性状的研究

 对转Bt基因水稻与常规品种的杂交后代进行了GUS活性的组织化学分析和农艺性状考察。结果表明，报告基因gus与抗虫基因 cry1Ab是连锁遗传和协同表达的，利用GUS组织化学染色法可快速筛选抗虫杂种植株；无论是籼粳交，还是粳粳交后代，抗虫和感虫的两类植株间在株高、穗长、单株分蘖数、播始历期和千粒重等主要农艺性状上无显著差异，这无疑为利用Bt水稻培育优良的抗虫品种提供了科学依据。