Research on Frequency of Exogenous Gene Flow from Marber-free Insect-resistant Transgenic Rice to Conventional Rice Varieties

[Objective] This study aimed to investigate the frequency of exogenous gene flow to non-transgenic conventional rice cultivars and assess the potential risks of marker-free of insect-resistant transgenic rice to agricultural ecological environment. [Method] Insect-resistant transgenic rice variety HUAHUI No.1 was planted as the experimental material and surrounded by several non-transgenic conventional rice cultivars. F1 non-transgenic rice seeds were collected according to different distances and identified by using PCR technology, the frequency of exogenous gene flow from insect-resistant transgenic rice to non-transgenic conventional rice cultivars was counted and analyzed. [Result] The average frequency of exogenous Bt gene flow to P13381 and CHUNJIANG063 was 0. Transgene flow occurred to varying degrees from insect-resistant transgenic rice HUAHUI No.1 to several non-transgenic rice lines including HEX122-2, TIANXlANG, MINGHUI63 and Pl157, with the maximum average gene flow frequency of 0.875%. The frequency of gene flow was gradually reduced with the increase of distance, and the average transgene flow frequency de- creased to 0 in all the sampling points 7 m away from transgenic rice material. [Conclusion] This study revealed that the exogenous gene flow frequency of insect-re- sistant transgenic rice variety HUAHUI No.1 was very low, leading to very small risk to the eco-environment. Rational distribution in the field for physical isolation, keeping the appropriate distance and scientific farming arrangement to avoid the synchronization of flowering can effectively control the exogenous gene flow from transgenic rice and reduce he ecological risks caused by transgene escape.

Genetic Transformation of NGc Insect-resistant Gene in Hi-Ⅱ Maize

In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with CryNGc insect-resistant genes were cultured by explant infection,co-culture and differentiation screening to study the genetic expression and resistance of exogenous genes in the offspring.The results showed that the infection effect was the best when the size of young maize embryo was 1.2-1.8 mm.Ten positive transformed plants with CryNGc insect-resistant genes were successfully obtained,and the transformation efficiency was 1.428‰.

Construction of Double Right-Border Binary Vector Carrying Non-Host Gene Rxo1 Resistant to Bacterial Leaf Streak of Rice

Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxol, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.

Classification with binary gene expressions

Microarray gene expression measurements are reported, used and archived usually to high numerical precision. However, properties of mRNA molecules, such as their low stability and availability in small copy numbers, and the fact that measurements correspond to a population of cells, rather than a single cell, makes high precision meaningless. Recent work shows that reducing measurement precision leads to very little loss of information, right down to binary levels. In this paper we show how properties of binary spaces can be useful in making inferences from microarray data. In particular, we use the Tanimoto similarity metric for binary vectors, which has been used effectively in the Chemoinformatics literature for retrieving chemical compounds with certain functional properties. This measure, when incorporated in a kernel framework, helps recover any information lost by quantization. By implementing a spectral clustering framework, we further show that a second reason for high performance from the Tanimoto metric can be traced back to a hitherto unnoticed systematic variability in array data: Probe level uncertainties are systematically lower for arrays with large numbers of expressed genes. While we offer no molecular level explanation for this systematic variability, that it could be exploited in a suitable similarity metric is a useful observation in itself. We further show preliminary results that working with binary data considerably reduces variability in the results across choice of algorithms in the preprocessing stages of microarray analysis.

Incorporation of Resistance Gene into Tomato using Binary Vector System

Exoplants of tomato(Lycopersicon esculentum) leaf were transformed with Ti plasmids using binary vector system.After screening.with selection culture, kanamycin-resistant seedling were obtained from callus. Molecular hybridization proved the integration of Km gene into plant cell genome via A.tumefaciens. Higher activity of Nos-NPTase was demonstrated in the transformed plant,thus confirming the successful expression of the resistance gene in recipient cells.

基于F-score和二进制灰狼优化的肿瘤基因选择方法

针对肿瘤基因数据维度高、噪声多、冗余性高的现状,结合Spearman相关系数改进F-score算法,在此基础上优化二进制灰狼算法,提出了一种基于改进F-score和二进制灰狼算法的肿瘤基因选择算法.首先,考虑特征之间的相关性,计算每个特征的F-score值和特征之间的Spearman相关系数的绝对值;然后,计算权重系数得出各个特征的权重值,依据重要性进行排序,选出初选特征子集;最后,通过收敛因子的衰减曲线和初始化方法优化二进制灰狼算法,调整全局搜索和局部搜索所占比例,增强全局搜索能力并提高局部搜索速度,有效节省时间开销,提升特征选择的分类性能和效率,得到最优特征子集.在9个肿瘤基因数据集上测试所提算法,在分类准确率和筛选特征数目两个指标上进行仿真实验,并与4种其他算法进行对比,实验结果证明所提算法表现良好,可有效降低基因数据维度,并具有较好的分类精度.

Cotton Plants Transformed with the Activated Chimeric Cry1Ac and API-B Genes

A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.

Indication of Genetic Linkage Map for Sunflower(Helianthus annuus L.) by SSR Markers

In order to investigate the possibility and efficiency of exogenous gene spread in nature and potential ecological risk of transgenic rice, as well as analyze the effect of exogenous Bt gene insertion on ecological fitness of transgenic rice plants, a experiment was carried out with three insect-resistant Bt transgenic rice cultivars Bt63, R1 and R2 and one conventional rice line 11-838 as experimental materials, the insect-resistant transgenic and non-transgenic rice plants were inter- cropped pair-wisely under high and low insect-infestation pressures, and the vegeta- tive growth, seed-setting and the resistance to rice stem borers were compared be- tween transgenic and non-transgenic lines. According to the experimental results, both the tiller number and fresh weight of Bt transgenic rice plants under low insect- infestation pressure showed no significant differences compared with the control, but the plant height, spike length and spike weight were all lower than those of non- transgenic rice plant, and Bt63 and R2 were significantly different compared with the control. On the contrary, under high insect-infestation pressure, the tiller number, spike length and spike weight of three Bt transgenic rice cultivars were significantly higher than those of the control, while the plant height showed different fitness ef- fects among various transgenic rice cultivars, which might be related to the charac- teristics of the receptive cultivars. The individual filled grain number and 1 000-grain weight of three transgenic rice cultivars showed no significant difference compared with the control under two different insect-infestation pressures, suggesting that the effect of exogenous Bt gene on seed setting was not significant. Under insect-infes- tation pressure, the resistance of three Bt transgenic rice cultivars against rice stem borer was significantly superior to non-transgenic rice, indicating that the effect of exogenous Bt gene on insect resistance of receptive plants was distinctly. Further- more, experimental results showed that the fitness cost of Bt transgenic rice was rel- atively low, which implied that exogenous Bt gene in insect-resistant transgenic rice might escape under certain environmental conditions, but this risk was very low.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

Supervised Learning for Gene Regulatory Network Based on Flexible Neural Tree Model

Gene regulatory network (GRN) inference from gene expression data remains a big challenge in system biology. In this paper, flexible neural tree (FNT) model is proposed as a binary classifier for inference of gene regulatory network. A novel tree-based evolutionary algorithm and firefly algorithm (FA) are used to optimize the structure and parameters of FNT model, respectively.The two E.coli networks are used to test FNT model and the results reveal that FNT model performs better than state-of-the-art unsupervised and supervised learning methods.

基于全基因组重测序挖掘大白-长白二元母猪产仔数性状候选基因

【目的】产仔数是种用母猪的重要经济指标,挖掘调控母猪产仔数性状相关的候选基因,探索调控二元母猪为代表的商品猪繁殖力遗传机制,为进一步应用分子选育奠定基础。【方法】采用全基因组重测序对极端高产与极端低产的两组大长二元母猪进行比较分析,并利用选择消除分析方法取F_(st)和θ_(π)信号交集为高选择区域,进行基因注释和候选基因筛选。【结果】在两组样本中共发现8040367个SNP,50.6%的SNP位于基因间区域,45.2%的SNP位于内含子区域,在外显子区域、非编码区3’端、5’端的SNP分别仅占1.0%、1.1%、0.6%,其中位于基因组外显子区域的SNP中,有28879个为非同义变异,占比35.0%。提取位于外显子区域、内含子区域、非编码区3’端和5’端的SNP位点进行基因注释,共筛选到两组样本间的差异基因有1136个。对差异基因进行KEGG与GO富集分析,筛选得到可能影响生殖性能和产仔数性状相关的候选基因共10个,包括EPHA4、SMAD7、GLI2、LRP1B、WT1、BAX、BCAT2、IL1B、FAF1,基因功能主要涉及胚胎发育、细胞凋亡等。【结论】大长二元母猪在产仔数性状上经历了显著的人工选择,初步挖掘出影响二元母猪产仔数性状的候选基因,这些基因在猪繁殖性能相关的生理活动中发挥作用,主要在母猪卵细胞成熟凋亡时期及妊娠初期参与调控。

Integration and inheritance stability of foreign Bt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.

转双价抗蛀干害虫基因杨树的获得及其抗虫性鉴定

Transgenic poplar(Populus alba×P.glandulosa cv`84k’) plants with the coleopterous insect resistant genes (BtCry3A and OC-I)were obtained. The transgenic nature of these plants was confirmed by PCR amplification and dot hybridization. The transgenic poplar’s toxicity towards the Anoplophora glabripennis larvae was assessed on two year-old selected plants in laboratory conditions. The results indicated that the transgenic lines BOGA-38 and BOGA-39 were deleterious for A. glabripennis larvae, and BOGA-5、BOGA-31、BOGA-38、BOGA-39 could inhabit the growth of A. glabripennis larvae. BOGA-39 was the most toxic one among the transgenic lines, with (41.18 %) corected mortality rate and 78.90 % growth inhabit rate for the larvae.

油菜PEP基因的克隆及PEP反义基因的构建

丙酮酸羧化酶（ Ｐ Ｅ Ｐ）是控制油菜蛋白质／油脂含量比例的一个关键酶⒚本研究用 Ｐ Ｃ Ｒ 法扩增出了 Ｐ Ｅ Ｐ 基因片段，并将其 克隆到 ｐ Ｂ Ｓ Ｓ Ｋ＋ 的 Ｓｍ ａ Ⅰ位点⒚ Ｄ Ｎ Ａ 序列分析表 明克隆片段 的长度为 ５３０ｂｐ，其序列与报道序列相同⒚将该 Ｐ Ｅ Ｐ基因 片段反向插入 ｐ Ｉ Ｇ１２１ 质粒，构建了带 Ｐ Ｅ Ｐ 反义基因的超级双元载体并进行油菜的转化。

转双价抗虫基因BmkIT-Chitinase玉米株系的获得

通过超声波辅助花粉介导法,将双价抗虫基因BmkIT-Chitinase分别导入以玉米自交系昌7-2及郑58的花粉为受体的不同基因型的自交系中。本研究共处理玉米雌穗1072穗,获得T0代种子1563粒,经卡那霉素初筛,T1代～T4代PCR及SouthernBlot杂交分子跟踪检测共获得20个转化株系,田间抗虫性鉴定表明共有16个转化株系与对照在抗虫性方面有显著差异,且此抗性随着各代稳定遗传。农艺性状调查结果表明,所获得的转基因玉米株系中大部分材料的农艺性状与对照无显著差异,除了N55材料及N20-1材料。N55材料的穗位高度与对照相比略低6±0.5cm,而穗粒数增加75±5粒。而N20-1材料百粒重增加5±0.5g。因此,转入此双价抗虫基因对玉米农艺性状影响不是很大。经过分子检测、田间抗虫性鉴定及农艺性状调查我们最终选育了9个转双价抗虫基因昌7-2自交系优良株系,6个郑58转双价抗虫基因自交系优良株系。

双价抗虫植物表达载体p3300-bt-pta转化苜蓿的初步研究

以中苜1号无菌苗子叶为外植体,采用农杆菌介导的叶盘法,将含有CryIA(a)/CryIA(c)基因、半夏凝集素(pta)基因和bar基因的双价抗虫植物表达载体p3300-Bt-pta导入苜蓿子叶中,在含除草剂的筛选培养基中连续筛选,获得抗性转化植株。研究了农杆菌菌液浓度、浸染时间、共培养时间等因素对苜蓿转化效率的影响,结果表明:各因素对苜蓿转化率均有不同程度的影响,当转化的菌液浓度OD600为0.6、浸染时间为10min、共培养时间为3d时转化效率较好。载体的抗性基因为除草剂草丁膦抗性的Bar基因,对子叶外植体的最佳筛选浓度为8mg/L;抑制农杆菌所用Carb的有效工作浓度为400mg/L,以后继代逐次减少用量到100mg/L;按此方法以根癌农杆菌菌株EHA105介导,将双基因Bt和pta导入紫花苜蓿品种"中苜1号",最终获得122棵除草剂草丁膦(ppt)抗性的植株,经过初步的PCR及RT-PCR分子生物学检测,有30棵检测到特异性条带,表明外源基因已成功整合到苜蓿基因组中,并在转录水平得到表达。

植物凝集素在抗刺吸式害虫转基因工程中的应用

植物凝集素是一类具有高度特异性糖结合活性的蛋白,含有1个或多个可与单糖或寡聚糖特异可逆结合的非催化结构域,在农业生产上应用前景广阔。本文简要概述了几种植物凝集素:雪莲凝集素、天南星科植物凝集素、苋菜类植物凝集素在抗刺吸害虫基因工程上的应用,并简单介绍了转双价抗虫基因方面的研究。

豌豆早期结瘤素基因ENOD12A的克隆及与PsLectin基因双元表达载体的构建

采用PrimStar HS DNA聚合酶从豌豆基因组DNA中扩增出了豌豆早期结瘤素基因PsENOD12A.序列分析结果表明,所克隆的基因与GenBank公布的序列仅相差1个碱基,但推导的氨基酸序列没有改变.将PsENOD12A基因和豌豆凝集素基因psl重组进了双元表达载体.

双价抗虫植物表达载体p3300-bt-pta的构建及转基因烟草的初步检测

用限制性内切酶HindⅢ酶切分别含有CryIA(a)和CryIA(c)基因的pGEM-4zf质粒得到Ubiquitin(玉米泛素)基因启动子驱动的CryIA(a)和CryIA(c)基因表达盒,将它们分别插入到用HindⅢ开环的pCAMBIA3300(含编码抗除草剂草丁膦的bar基因)载体上形成中间载体p3300-bt。采用Pfu高保真DNA聚合酶用PCR的方法从质粒pBI121-pta上扩增得到含CaMV35S启动子和NOS终止子的pta基因表达盒,然后插入到用SmaⅠ切开的中间载体p3300-bt中,获得带有抗性基因的双价抗虫基因表达载体p3300-bt-pta。通过根癌农杆菌介导的叶盘法转化烟草品种百日红,获得一批抗除草剂的转基因烟草植株。

豌豆早期结瘤素基因PsSYM10的克隆及其表达载体的构建

采用Pri mStar HS DNA聚合酶从豌豆基因组DNA中扩增出了豌豆早期结瘤素基因PsSYM10.序列分析结果表明,所克隆的基因与GenBank公布的同源基因在碱基和氨基酸序列上的相似性分别为98.6%和99.7%.将PsSYM10基因与结瘤相关基因PsENOD12、凝集素基因PsLectin串联组合,构建成了双元表达载体.