转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。     

Capnographic documentation of nasoesophageal and nasogastric feeding tube placement in dogs

Objective: To evaluate the ability of capnography to document proper placement of nasoesophageal (NE) and nasogastric (NG) feeding tubes. This study was conducted in 3 phases. Phase I of this study was designed in order to test the efficacy of capnography to distinguish placement of a feeding tube in the alimentary tract versus the respiratory tract. Phase II was designed in order to document that carbon dioxide (CO₂) could be measured through a polyvinyl chloride (PVC) feeding tube. Phase III was performed in order to evaluate the technique of continuous monitoring during insertion of the feeding tube into the esophagus and stomach as would be performed during a clinical‐tube placement. Design: Prospective study. Setting: Research laboratory. Animals: 24 adult dogs. Interventions: In Phase I, sedated dogs were instrumented with an intratracheal catheter and an 8 French feeding tube placed nasally into the distal esophagus and later advanced into the stomach. In Phase II, dogs were anesthetized and an 8 French feeding tube was placed down the endotracheal tube, then into the esophagus and later advanced into the stomach. In Phase III, sedated dogs were instrumented with an 8 French feeding tube inserted intranasally and then advanced to the level of the nasopharynx, distal esophagus and, lastly, the stomach. Fluoroscopy was used in order to determine location of the feeding tube. Measurements and main results: Phase I measurements included respiratory rate and CO₂ from the trachea, esophagus, and stomach and pH of gastric fluid sample. Phase II measurements included respiratory rate and CO₂ from the endotracheal tube, feeding tube in the endotracheal tube, feeding tube in the distal esophagus, and feeding tube in the stomach. Phase III data collection included respiratory rate and CO₂ as the tube was passed through the nasal cavity, nasopharynx, esophagus and stomach. Phase I fluid samples were collected from 5 of the 9 dogs and had pH values from 1.68 to 4.20. In both phases, values for the respiratory rate and CO₂ from the esophagus and stomach were 0 ± 0, significantly lower (P < 0.001) than the values from the trachea. In Phase II, there was no significant difference between the respiratory rates (P = 0.886) and CO₂ (P = 0.705) readings obtained from the endotracheal tube compared to readings from the feeding tube in the endotracheal tube. In Phase III, there was a significant difference (P < 0.001) between the respiratory rates and CO₂ readings obtained from the nasal cavity and the nasopharynx when compared to those readings obtained from the esophagus and stomach. Measurement of CO₂ and respiratory rate resulted in a reading of 0 every time the feeding tube was in the esophagus or stomach. Conclusions: Capnography may be used in order to detect airway placement of NE and NG tubes.     

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.     

水稻卷叶突变体rlm1的基因克隆

叶片是植物光合作用的主要场所,优良的叶片形态有利于塑造理想的株型,提高光合效率。为了研究叶片形态建成的分子机制,自水稻T-DNA插入突变体库中筛选获得1个叶片半卷曲的卷叶突变体(roll leaf mutant,命名为rlm1),突变体rlm1主要特征为成熟叶片沿中脉向内卷,叶片最终卷成直立半圆筒状,叶片卷曲度达0.64,叶片直立参数达95,且光合效率显著优于野生型。通过图位克隆技术,确定突变体rlm1突变位点位于LOC_Os03g06654基因的第1个内含子,LOC_Os03g06654基因编码黄素单氧化酶(flavin-containing monooxygenase),RT-PCR表达分析表明LOC_Os03g06654基因因T-DNA插入而导致完全失活。该基因与已报道的水稻卷叶基因Os COW1(Constitutively Wilted 1)为等位基因,而且突变体rlm1所表现的农艺性状均佳,可期待利用该突变体进行高光合的育种实践。     

Morphological Basis of Resistance in Cotton to the WhiteflyBemisia Tabaci

The morphological basis of resistance to the whiteflyBemisia tabaci Genn. (Aleyrodidae: Hemiptera) was studied. The plant characters examined were leaf area, thickness of leaf lamina, hair density, hair length, angle of insertion of leaf hair, and density of gossypol glands. Hair density and leaf thickness were positively correlated with the population ofB. tabaci, and a positive correlation was obtained between the adult whitefly population and gossypol glands on stem internodes. Cotton genotype USA-22 (sparsely hairy) was found to be more tolerant toB. tabaci than was genotype USA-13 (velvety hairy). The use of thinner and glabrous leaved cotton varieties is suggested to minimize the whitefly menace in cotton.     

稻瘟病菌T-DNA插入的突变表型分析

PCR技术检测28个形态发育和致病性相关T-DNA插入突变体，结果所有突变体均含磷酸转移酶基因序列。对这些突变体展开进一步生物学性状观察，发现15个颜色异常，8个菌落生长缓慢，2个分生孢子形态异常，2个附着胞形态异常，3个不能形成附着胞。致病性测定结果，9个突变体完全不能导致抗瘟（C101）和感瘟（日本晴）水稻苗致病，病害级别均为0级。用标准菌株1528和P131测定突变体有性世代的形成能力，结果发现， Y34-0211、Y34-1469和Y34-0635 3个突变体完全丧失产生有性世代的能力。     

发根农杆菌(Agrobacterium rhizogenes) Ri T-DNA遗传转化丝石竹(Gypsophila paniculata)的研究

近年来,已发展出遗传转化高等植物的一些新技术,其中有些技术如脂质体融合,微注射技术和电击导入都是基于动物细胞培养方面的工作,而另一些技术是来自于植物界独特的天然转化系统,其中包括已知能遗传转化高等植物的农杆菌(Agrobacterium)的二个种,即致瘤农杆菌(A.tumefaciens)和发根农杆菌(A.rhizogenes),这二个种都能够将其致病质粒Ti或Ri所携带的DNA序列(T-DNA)插入到双子叶植物细胞核基因组中。pTi诱发寄主产生根基肿瘤,pRi诱发寄主产生毛状根。二者的差别可能是毛状根可以从毛状根培养物获得具有完整的T-DNA序列的有生育能力的再生植株,而从致病农杆菌(A.tumefaciens)菌株所诱发的肿瘤很难获得再生植株。因此,利用发根农杆菌(A.rhizogenes)pRi作为遗传转化高等植物的基因克隆载体的研究和应用日益受到重视。     

一例金毛犬胫骨斜骨折的髓内针逆向插入法联合钢丝内固定术治疗

通过对一例金毛犬胫骨斜骨折进行内固定术,详细介绍了髓内针逆向插法联合钢丝进行内固定术的手术要点,为同行进行犬胫骨斜骨折内固定术治疗提供参考。     

转基因植物中T-DNA整合的分子特征及表达

植物中不同转基因方法转化外源基因的T-DNA整合特征既具有共性,又具有特性,使得转基因的遗传在各独立转化体间呈现多样性,另外多种遗传因子和限制因素使受体植物中外源基因的表达存在下降,甚至出现基因沉默等复杂现象。本文主要对农杆菌介导及裸露DNA直接转化转基因植物中T-DNA的分子特征和转基因表达的影响因子进行了介绍和概述。转化体中转基因的遗传稳定性和表达主要取决于转基因在植物基因组中的整合位置、拷贝数及组成结构。因而,通过对具有表达水平各异的转化体进行深入的遗传分析和分子生物学研究以及转化体之间进行的比较研究,将对转基因技术自身的完善、定点整合以及更有效的利用转基因技术都具有十分重要的意义。     

葡萄炭疽病菌T-DNA插入突变体的表型及致病性变异分析

胶孢炭疽菌(Colletotrichum gloeosporioides)引起的葡萄炭疽病是葡萄生产中的一种重要病害。本研究对从2100个T-DNA插入突变体库中挑选的11株PDA培养特征变异较大的突变体进行表型和致病性分析，旨在为葡萄炭疽病菌产孢与致病基因网络调控研究奠定基础。结果表明：突变体M112、M247、M1288、M1325、M1386和M1430不能产生分生孢子，其余的5株突变体产孢量降低；突变体M247、M1288和M1325丧失了致病性；突变体M112、C1094、M1386和M2013突变体致病力增强。此外，突变体C1094孢子萌发率明显降低，其余4株产孢突变体的孢子萌发率与野生型菌株WS15无明显差异。