水稻转座子Pong RNAi载体的构建和农杆菌介导的遗传转化

为探讨水稻转座子Pong的功能及其对水稻基因组甲基化的影响,构建了水稻转座子Pong的RNAi表达载体TPAI和TPAⅡ,并利用这两个载体采用农杆菌介导法转化水稻(品种松前)的成熟胚愈伤组织,获得PCR结果阳性的再生植株.同时通过不同的农杆菌侵染途径、侵染浓度和侵染时间等,建立并优化了水稻的遗传转化程序.结果表明:农杆...     

农杆菌介导转化水稻方法的改进

采用培矮64S和9311两种籼稻为材料,研究了影响农杆菌介导转化的几种因素．研究结果表明,预培养4d的幼胚最适宜作为农杆菌介导转化的受体,其次是来源于幼胚和成熟胚的生长状态良好的胚性愈伤组织．以AAM培养基作为共培养培养基,对于农杆菌EHA105（pUblm）来说,菌液浓度为OD600值=0．8时,愈伤组织的转化率最高．在农杆菌侵染愈伤组织过程中,通过负压处理可明显提高愈伤组织的转化频率．在愈伤组织浸染后,以含一定浓度的Ca^2＋和表面活性剂Tween20的溶液处理可明显提高水稻愈伤组织的转化频率．     

农杆菌介导的小麦成熟胚愈伤遗传转化研究进展

小麦是中国重要的粮食作物之一.传统的育种方法存在周期长、改良慢等缺点,基因工程育种现已成为小麦遗传改良的研究热点.因此,小麦成熟胚愈伤组织的遗传转化在小麦遗传改良研究中具有重要意义.农杆菌介导的小麦成熟胚愈伤组织的遗传转化以其自身具有的优势,得到了广泛的关注.介绍了农杆菌介导的小麦成熟胚愈伤遗传转化体系的各种影响因素,如小麦愈伤组织的诱导、分化、转化效率、农杆菌菌株和转化载体等,并对小麦成熟胚愈伤组织遗传转化的发展方向进行了展望.     

农杆菌介导转化水稻成熟胚影响因素研究

实验对蜀恢881、蜀恢527、中花9号等7个水稻品种的成熟胚为受体材料，研究了影响农杆菌转化水稻频率的几个重要因素，实验结果表明：CC培养基是籼稻成熟胚愈伤组织的最适诱导培养基，而NB培养基是粳稻品种成熟胚的最适诱导培养基；诱导培养基中添加ABA并不能显著提高籼稻和粳稻成熟胚愈伤组织的诱导率，但在ABA为3mg／LN可以明显改善愈伤质量；适当的低温预处理可以不同程度提高诱导率；农杆菌菌株EHA105对水稻愈伤组织的转化能力优于LBA4404和AGL1；在分化时，采用适宜的激素配比有利于提高分化率。     

农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

采用携带卡那霉素抗性基因nptII和GUS基因的ubiquitin启动子驱动的表达载体pBI121／DREB1A的根癌农杆菌AGL1, 对多花黑麦草幼胚来源的胚性愈伤组织进行了遗传转化,并优化了各种影响因素。胚性愈伤组织经根癌农杆菌感染和共培养后,用50mg／L巴龙霉素筛选抗性愈伤组织,待抗性愈伤组织在IB分化培养基上分化成苗后用25mg／L卡那霉素进一步筛选再生植株, 获得了部分抗性植株。抗性植株的总DNA用DREB1A基因的特异引物进行PCR检测,转化频率为2.14％,PCR-Southern blot进一步验证了转化植株基因组中含有该外源基因。各种影响转化效率因素的优化实验表明,当转化时菌液浓度的OD600为2.0、侵染时间为1h、共培养时间为2d、共培养温度为21℃及在共培养期间使用乙酰丁香酮等,均可明显提高转化频率。     

农杆菌介导的胆碱脱氢酶基因(betA)转化小麦及影响转化效率的因素

用LBA4404／pCDH，Agl 1／pUNN2和Ag;P(无质粒)3种菌株分别转化小麦品种济南177、99P、核生3号幼胚诱导的愈伤组织以及济南177的幼胚．其中以胚性愈伤组织为外植体，获得了转基因植株．PCR和PCR-Southem分析证实转化植株中包含了外源基因．通过染色体分析，发现胚性愈伤组织在农杆菌LBA4404侵染后形成的染色体削减比经Agl1侵染和未经感染的对照形成的染色体削减程度更大，甚至发现较多的染色体断片．分析了农杆菌菌株，材料的基因型，外植体类型，培养时间和温度以及筛选的时间和周期对于转化效率的影响．     

影响根癌农杆菌转化水稻频率的因素研究

根癌农杆菌与来自水稻成熟种子的愈伤组织共培养,将外源基因导入水稻愈伤组织,并获得了转基因植株．通过比较影响根癌农杆菌转化频率的各种因素,表明在转化过程中酚类化合物和单糖的加入使农杆菌的转化频率提高了０．９％～１７．４％．在共培养时农杆菌的稀释方式也是影响农杆菌转化频率的重要因素     

农杆菌介导东方百合“西伯利亚”遗传转化体系建立

以东方百合"西伯利亚"(Lilium seberia)为材料,建立了百合再生和农杆菌介导的遗传转化体系.采用LBA4404和GV3101菌株对鳞片来源的愈伤组织进行侵染,并以鳞片作为对比转化材料,两种菌株携带有相同的双元载体pCAMBIA1301.研究结果表明,农杆菌侵染愈伤组织外植体转化率较高;乙酰丁香酮(AS)的添加能大大提高根癌农杆菌转化百合的效率.另外,共培养时间、不同菌株均对转化效率有一定的影响.筛选后获得的抗性植株经PCR检测和GUS组织化学染色证实外源基因已经整合到基因组DNA中并获得表达,转化率为5%～8%.     

不同温度培养对根癌农杆菌生长及水稻幼胚再生的影响

设计26,30,34,36,38,40℃6个温度对根癌农杆菌进行培养.结果显示:根癌农杆菌在38℃和40℃条件下无法生长;将水稻幼胚经26℃诱导获得的愈伤组织分别放置在农杆茵不能耐受的38℃和40℃环境中培养.并设计了7,14,21,28d不同培养时间,同时以26℃常规培养温度作为对照,分析在较高温度条件下培养对水稻愈伤组织再生的影响.结果显示:水稻幼胚诱导的愈伤组织经过38℃所有时间及40℃部分时间(7,14d)下模拟抗性筛选培养,仍然能够正常生长,而且经38℃、14d继代培养的愈伤组织植株再生率比26℃常规培养提高63.17%,统计分析两者达显著差异.     

根癌农杆菌介导的绿色荧光蛋白基因在水稻植株中的表达

将改良的绿色荧光蛋白（EGFP）基因插入到植物表达载体中,构建了ubi启动子驱动下的植物表达载体p13UEGFP．通过根癌农杆菌介导转化水稻的胚性愈伤组织,经潮霉素筛选,获得抗性愈伤组织和再生植株．对T2代植株进行PCR分析、激光共聚焦显微镜检测和RT—PCR分析,结果表明,绿色荧光蛋白基因已经在转基因植株中稳定表达．     

An active DNA transposon family in rice

The publication of draft sequences for the two subspecies of Oryza sativa (rice), japonica (cv. Nipponbare) and indica (cv. 93-11), provides a unique opportunity to study the dynamics of transposable elements in this important crop plant. Here we report the use of these sequences in a computational approach to identify the first active DNA transposons from rice and the first active miniature inverted-repeat transposable element (MITE) from any organism. A sequence classified as a Tourist-like MITE of 430 base pairs, called miniature Ping (mPing), was present in about 70 copies in Nipponbare and in about 14 copies in 93-11. These mPing elements, which are all nearly identical, transpose actively in an indica cell-culture line. Database searches identified a family of related transposase-encoding elements (called Pong), which also transpose actively in the same cells. Virtually all new insertions of mPing and Pong elements were into low-copy regions of the rice genome. Since the domestication of rice mPing MITEs have been amplified preferentially in cultivars adapted to environmental extremes-a situation that is reminiscent of the genomic shock theory for transposon activation.     

Mobilization of a transposon in the rice genome

Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analysing the genome structure of grasses. To practice efficient rice breeding through genetic engineering techniques, it is important to identify the economically important genes in this crop. The use of mobile transposons as gene tags in intact plants is a powerful tool for functional analysis because transposon insertions often inactivate genes. Here we identify an active rice transposon named miniature Ping (mPing) through analysis of the mutability of a slender mutation of the glume-the seed structure that encloses and determines the shape of the grain. The mPing transposon is inserted in the slender glume (slg) mutant allele but not in the wild-type allele. Search of the O. sativa variety Nipponbare genome identified 34 sequences with high nucleotide similarity to mPing, indicating that mPing constitutes a family of transposon elements. Excision of mPing from slg plants results in reversion to a wild-type phenotype. The mobility of the transposon mPing in intact rice plants represents a useful alternative tool for the functional analysis of rice genes.     

三种水稻转基因技术体系的比较研究

研究比较了外源基因导入水稻的三种技术体系,即ＰＥＧ介导法,基因枪法和农杆菌介导法．综合考虑转化频率、转化周期、转化难度和转化成本等因素,认为在水稻转基因研究中,最经济、有效和易行的技术体系是农杆菌介导法．     

农杆菌介导GNA基因对水稻的遗传转化

在ｐＣＡＭＢＩＡ３３００质粒中插入带有ＲＳｓ－１启动子的ＧＮＡ基因,并利用农杆菌介导的遗传转化将其导入水稻的微不定芽受体,得到了再生植株。ＰＣＲ,Ｓｏｕｔｈｅｒｎ ｂｌｏｔ和Ｗｅｓｔｅｒｎ ｂｌｏｔ的检测结果初步证明ＧＮＡ基因已经整合到水稻的基因组中并得到表达。     

Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene

Southern blot analysis indicated that mtlD gene (encoding mannitol-1-phosphate dehydrogenase) and gutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated by Agrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

尖镰胞菌细胞色素P450 55a1基因超表达载体的构建和水稻日本晴遗传转化

要将细胞色素P450 55a1基因克隆到镰刀菌素植物超表达载体pCAMBIA1302中,构建了pCAMBIA1302-cyp55a1-gfp1植物超表达载体,以水稻日本晴为遗传转化的受体对象,通过农杆菌介导侵染方法进行了遗传转化.结果表明：成功构建了细胞色素P450 55a1基因超表达载体,获得了多个细胞色素P450 55a1基因超表达的阳性植株,并以RT-PCR技术分析了阳性植株中细胞色素P450 55a1基因的表达水平.     

Salt tolerance of transgenic rice (Oryza sativa L.) withmtlD gene andgutD gene

Southern blot analysis indicated thatmtlD gene (encoding mannitol-1-phosphate dehydrogenase) andgutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated byAgrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.