拟南芥AtGDPD-like6和AtGDPD-like7基因突变体鉴定

[目的]鉴定拟南芥AtGDPD-like6和AtGDPD-like7基因的突变体。[方法]以拟南芥AtGDPD-like6和AtGDPD-like7为研究对象,采用半定量RT-PCR法进行分析,并用3引物PCR扩增法进行筛选和鉴定。[结果]各得到2个AtGDPD-like6和AtGDPD-like67独立T-DNA插入纯合突变体。半定量RT-PCR结果显示,4个纯合突变体中目标基因转录表达都被完全敲除。[结论]AtGDPDlike6和AtGDPD-like67突变体材料的获得为生理功能的鉴定提供了反向遗传学材料。     

根癌农杆菌介导致病疫霉转化体系的建立

为建立低成本、快速、高效的根癌农杆菌介导的致病疫霉转化体系,以菌株 HK0919为材料,从抗生素筛选浓度、共培养转膜方式、乙酰丁香酮（AS）浓度、共培养时间和共培养温度等方面对致病疫霉的转化体系进行了研究。综合各因素优化结果,建立的转化体系条件为：以1．0μg/mL的潮霉素B进行转化子筛选,采用玻璃纸作为共培养介质,AS浓度为200μmol/L,培养6 d,温度为22℃。在此条件下的转化效率为每106个游动孢子获得50～60个转化子。随机选取10个转化子,利用特异性引物对潮霉素抗性基因hph进行PCR扩增,转化子均能扩增出800 bp左右的预期条带;同时,利用根癌农杆菌vir基因特异引物对转化子进行 PCR扩增,排除了转化子被农杆菌污染所致的假阳性;转化子继代培养5代后,仍能在含潮霉素 B的黑麦培养基上生长。说明外源T DNA已成功整合到致病疫霉基因组中,并能稳定遗传。     

Production of Transgenic Plants： More, Better, and Faster

Over the last two decades, transgenic plants have moved from being solely laboratory vehicles for basic research work to providing new varieties grown on large areas throughout the world. A number of plant     

Identification and characterization of T-DNA inserts by T-DNA fingerprinting

A T-DNA fingerprinting method is presented based on amplified fragmentlength polymorphism with an anchored polymerase chain reaction step. Thismethod allows discrimination between different T-DNA inserts in stablytransformed plants. The technique was evaluated by analyzing 51 transgenicArabidopsis lines that had been characterized in detail by genomicblotting. Comparison of the obtained fingerprints with the availableintegration information demonstrated that fingerprints were correlated tothe predicted patterns, except for the inverted repeat junctions and forthose inserts with large deletions at the left or right border. Ourexperiments show that by using T-DNA fingerprinting multi-copy transgeniclines can be eliminated efficiently so that the technique can be used toenrich a population of transgenic plants for putative single-copytransformants.     

T-DNA插入产生的水稻多分蘖突变的遗传分析

在筛选和鉴定水稻T-DNA(含Basta抗性基因Bar)插入纯合体的过程中，观察到一个多分蘖的突变体。其分离群体中多分蘖和正常分蘖两种植株类型的分离比率为3∶1，符合1对基因的显性遗传。Basta 抗性检测、PCR分子检测及Southern杂交证实，该突变体是由单一T-DNA插入引起的，多分蘖性状与T-DNA共分离。该突变材料可用于插入座位的     

PSORA：一种基于高通量测序的T-DNA插入位点分析方法

【目的】建立一种批量分析T-DNA插入位点的简单、有效的方法。【方法】提供一种基于高通量测序技术的T-DNA插入位点的分析方法,将其命名为PSORA：Parallel sequencing of one round amplicons。首先对一轮交错式热不对称PCR（TAIL-PCR）的扩增产物进行高通量测序,随后通过生物信息学分析T-DNA插入位点,该方法降低了TAIL-PCR过程中对特异性扩增的要求。PSORA中使用的引物一侧为简并引物,另一侧为T-DNA特异性引物。在特异性引物的5’端设计6 nt的样品标签（Barcode）,用于标记不同的转化事件。所使用的5个转基因烟草株系（L1、L6、L9、L15和L19）由农杆菌介导质粒pBI121转化获得。此外,通过标准PCR对PSORA的结果进行验证。【结果】利用PSORA对5个转基因株系的T-DNA插入位点进行分析,结果显示,L6含2个插入位点（NW_015801367的36 316 bp处和NW_015950898的42 202 bp处）,L9、L15和L19各含1个插入位点（L9的插入位点为NW_015943682的235 969 bp处;L15的插入位点为NW_015802951的60 529 bp处;L19的插入位点为NW_015863435的12 188 bp处）,L1的插入位点未能成功获取。对生物信息学分析结果进行PCR验证,含不同插入位点的转基因株系之间可互为阴性对照,野生型（WT）作为空白对照,结果表明,在各转基因株系中均得到与预期一致的特异性扩增,该结果验证了PSORA的有效性。【结论】PSORA是一种分析T-DNA插入位点的有效方法,可以同时分析多个插入事件,相对于传统的染色体步移方法更简便、快速。     

质粒拯救型T-DNA标签质粒的构建

[目的]为了高效率地获得转基因植物的旁邻序列。[方法]以pUC18及pWM101为出发质粒,通过用限制性内切酶EcoRⅠ和HindⅢ双酶切质粒pUC18,得到含大肠杆菌复制起点及氨苄青霉素抗性基因的pUC18大片断,并将这段DNA整合到pWM101的T-DNA之中构建重组质粒。[结果]该质粒利用根癌农杆菌转化植物后,可利用潮霉素筛选转化细胞,拯救质粒则可转化大肠杆菌后,以氨苄青霉素进行筛选。[结论]该研究为以后的用T-DNA标签研究植物功能奠定了基础。     

Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

Rice production and quality are seriously affected by the lepidopteran pest, striped stem borer (SSB), in Northeast China. In this study, a synthetic cry1C gene encoding Bacillus thuringiensis (Bt) δ-endotoxin, which is toxic to lepidopteran pest, was transformed into a japonica rice variety (Jigeng 88) in Northeast China by Agrobacterium-mediated transformation. Through molecular detection and the Basta resistance germination assay, a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1C. Finally, four cry1C-transgenic lines (JL16, JL23, JL41, and JL42) were selected by evaluation of the Cry1C protein level, insect-resistance and agronomic traits. The cry1C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic (NT) control plants. T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1C gene was inserted into the intergenic region of chromosome 11, indicating that its insertion may not interfere with the genes near insertion site. In summary, this study developed four cry1C-transgenic japonica rice lines with high insect resistance and high yield. They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.     

玉米大斑病菌ATMT突变体库的构建及其分析

【目的】利用农杆菌（Agrobacterium tumefaciens）介导的遗传转化技术,对玉米大斑病菌（Setosphaeria turcica）进行转化,构建ATMT突变体库,为从分子水平上揭示病菌的致病机理奠定基础。【方法】以带有重组双元载体的农杆菌对玉米大斑病菌进行转化,利用潮霉素B进行筛选,对抗性稳定的转化子进行PCR检测,构建玉米大斑病菌ATMT突变体库;从突变体库中随机选取一定数量的突变体,对其菌落形态、菌丝及分生孢子发育、致病性等进行分析。【结果】获得了1 265株玉米大斑病菌T-DNA插入突变体;从中随机选取36株突变菌株,对其进行抗性筛选和PCR检测,发现潮霉素磷酸转移酶基因已整合进入野生型菌株的基因组中,且能够稳定遗传;与野生型菌株相比,在供试的菌株中,大部分菌株菌落形态和生长速率没有发生明显改变。生长速率明显减慢的菌株占总数的13.8%,明显加快的菌株占16.7%;发现了2株分生孢子形态发生明显改变的菌株,占菌株总数的5.6%;产孢量明显增大的菌株占5.6%,产孢量减少的菌株占13.5%;分生孢子萌发率发生明显改变的菌株占16.6%;发现了1株致病性明显增强的菌株,占菌株总数的2.8%。【结论】构建了玉米大斑病菌ATMT突变体库,并对突变体库进行了初步分析,将为克隆玉米大斑病菌生长发育和致病性相关基因奠定基础。     

农杆菌介导尖孢镰刀菌西瓜专化型转化体系的建立及突变体筛选

以尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum)FO-11-06菌株为受体,采用农杆菌介导的遗传转化方法,实现了农杆菌AGL1(含pATMT1)介导的西瓜枯萎病菌的转化,并研究了转化介质和pH值对转化效率的影响。结果表明:在25℃下,采用乙酰丁香酮(AS)浓度为200μmol/L、农杆菌OD600=0.15、尖孢镰刀菌分生孢子含量为1×106个/mL、pH值为5.1～5.8时可实现T-DNA的插入转化。当转化体系pH值为5.3时,转化效率最高,达到每106个分生孢子产生553个转化子。不同共转化介质对转化效率影响明显,玻璃纸和硝酸纤维素滤膜转化效率较高,每106个分生孢子获得551个和549个转化子,显著高于滤纸。通过转化,获得尖孢镰刀菌西瓜专化型转化子1 989个,对生长速度、菌落颜色和产孢量等性状进行分析,发现有46个性状特异的突变体。研究结果为进一步研究尖孢镰刀菌西瓜专化型的生长发育、致病机制和相关基因的功能奠定了基础。