根癌农杆菌介导转Bt基因苎麻的获得及其抗虫鉴定

采用本研究室建立的根癌农杆菌介导的高效遗传转化体系,转化苎麻优良品种芦竹青,获得了转Bt基因苎麻候选植株。通过PCR和Southern杂交等分子检测,证明Bt基因已经整合到部分候选植株基因组中。选取PCR和Southern杂交均为阳性的部分株系(T₀)种植在大田,对这些植株进行室内抗虫鉴定并考察其主要农艺性状和品质性状,次年对T₁代植株进行PCR和Southern杂交检测。结果表明, 与对照相比,T₀代转Bt基因苎麻植株的抗虫性均强于对照,部分株系的抗虫性显著强于对照植株;且基本保持了亲本的优良性状;T₁代植株中也含有Bt基因,表明Bt基因能稳定遗传,且T₁代植株在大田的抗虫性明显强于非转基因植株。     

转Xa23基因水稻的白叶枯病抗性及其遗传分析

在分离克隆抗白叶枯病基因Xa23研究中获得大量转基因水稻材料。为了系统研究转Xa23基因水稻的抗病稳定性和遗传模式, 本文通过逐株进行抗白叶枯病接种鉴定、PCR和Southern blot分子检测, 对一批转Xa23基因水稻植株进行了T₀代到T₂代的跟踪分析。结果表明, Xa23基因的整合和表达, 使感病受体品种牡丹江8号获得抗病性。由于Xa23基因插入受体基因组的位点不同, 同是单拷贝插入的转基因T₀代抗病植株, 其抗病程度有明显差异。T₀代植株的抗病程度, 可以准确、稳定地遗传到T₁代和T₂代。单拷贝转基因植株分离群体的抗感植株分离比接近3∶1, 表明转Xa23基因遵循孟德尔单基因遗传模式。已获得2个纯合的单拷贝转基因抗病株系, 它们的抗病程度稍有差别, 将用于外源基因插入位置效应分析和杂交稻抗病育种。     

转GbVe1基因在棉花基因组中的整合与定位分析

【目的】明确转GbVe1基因棉花的插入位点序列特征。【方法】Southern杂交筛选低拷贝基因插入的转基因棉花株系,以hiTAIL-PCR(Polymerase chain reaction)获取其T-DNA侧翼序列,然后根据获得的T-DNA侧翼序列设计特异PCR引物,验证插入位点的准确性。【结果】Southern杂交候选了T-DNA低拷贝插入的3个转基因棉花株系,hiTAIL-PCR分离到RB端侧翼序列(119~1 018 bp)、LB端侧翼序列(243~516 bp);侧翼序列的AT碱基含量在63%以上。转基因株系7/100826-152和12/100826-393插入位点都位于Gohir.D01G157600.1内含子上。转基因株系1/w-ch14插入位点分别位于Gohir.D01G157600.1内含子和A12染色体的基因间隔区中。T-DNA在Gohir.D01G157600.1内含子的插入事件造成了21 bp碱基的基因组序列缺失。T-DNA到侧翼序列的PCR产物证明Gohir.D01G157600.1上的插入位点真实可靠。【结论】hiTAIL-PCR获取了转GbVe1基因棉花的T-DNA侧翼序列,提供了T-DNA插入位点位于Gohir.D01G157600.1基因内含子的特异性检测引物。     

抗纹枯病、赤霉病的转TaPIEP1基因小麦的分子鉴定与选育

小麦纹枯病(主要病原为禾谷丝核菌)和赤霉病(主要病原为禾谷镰刀菌)已成为我国小麦生产的重要病害。TaPIEP1是从小麦中分离到的1个病原诱导的基因,其编码蛋白是可与GCC-box顺式元件结合、转录激活型的ERF转录因子。本研究以8个转TaPIEP1基因小麦株系的T₄和T₅代植株为试材,进行了外源转TaPIEP1基因的PCR检测、Southern杂交、RT-PCR与Q-RT-PCR的分析以及纹枯病菌、赤霉病菌接种与抗性鉴定。结果表明,外源TaPIEP1基因在转基因小麦中能够稳定遗传,以单拷贝或双拷贝整合到7个转基因小麦株系基因组的不同位点;外源TaPIEP1基因在转基因小麦中能超量表达;与受体扬麦12相比,TaPIEP1表达水平高的8个转基因小麦株系对纹枯病抗性显著提高,4个株系中一些材料对赤霉病抗性显著提高,3个株系中一些材料兼抗纹枯病和赤霉病,说明TaPIEP1正向参与了小麦对纹枯病和赤霉病抗性反应,利用该基因通过基因工程可创制抗纹枯病、赤霉病的小麦新种质。     

海岛棉两种转Bt基因方法的研究

 以新疆海岛棉军海1号为受体,利用农杆菌菌液喷雾法（Floral spray）和质粒注射法（Plasmid injection）导入外源Bt基因。经过对转化植株T₁和T₂代大田筛选和PCR、Southern检测,并经孟德尔遗传规律符合度分析,结果表示：农杆菌菌液喷雾法T₀代成铃率比质粒注射法高8.3百分点;两种方法均可获得转基因植株,农杆菌菌液喷雾法较质粒注射法转化率高3.4百分点;农杆菌菌液喷雾处理后代较符合孟德尔遗传规律且单拷贝插入几率高。因此,农杆菌菌液喷雾法优于质粒注射法。     

水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。     

转基因高油酸油菜T-DNA插入拷贝数及整合位点分析

为获得转基因高油酸油菜T-DNA插入拷贝数及整合位点相关信息,本研究应用地高辛标记的NPTII基因片段为探针,与经BamHI酶切的转基因甘蓝型油菜高油酸株系W-4的T2代单株的基因组DNA进行Southern杂交。结果显示:W-4的T2代单株的基因组含有一个T-DNA拷贝。用3到4个根据载体pCNFIRnos序列设计的嵌套特异性引物分别与简并引物组合进行TAIL-PCR反应,扩增得到转基因油菜T-DNA插入位点的左、右边界旁侧序列。经分析右边界旁侧序列长度为470bp,其中180bp为载体序列,290bp为W-4的基因组序列;左边界旁侧序列长度为641bp,其中365bp为W-4的基因组序列,276bp为载体序列。序列比对结果发现该转基因事件中,T-DNA左边界序列完全整合到油菜基因组中,仅有1个碱基由G转换成了A。而右边界则缺失了包括RBborder在内的62个碱基。结果表明:转基因高油酸油菜T-DNA的整合是一次无其他额外载体序列的整合。blast分析获得的与左右边界相连的油菜基因组序列,未检索到与之高度同源的序列,推测T-DNA插入位点可能位于油菜基因组非编码区。综上所述,本研究分析了转基因油菜W-4基因中T-DNA拷贝数、整合特点和旁侧序列,研究结果为转基因油菜的生物安全性评价以及转基因高油酸油菜的检测提供重要的基础信息。     

抗根腐病的转GmPGIP3基因小麦扬麦18的获得与鉴定

GmPGIP3是大豆的一种多聚半乳糖醛酸酶抑制蛋白, 能够特异性地抑制部分病原真菌内切多聚半乳糖醛酸活性, 从而减弱病原菌对植株的侵害。利用基因重组技术构建了GmPGIP3基因的单子叶植物表达载体pA25-GmPGIP3, 通过基因枪介导法将pA25-GmPGIP3转入小麦品种扬麦18中。对转GmPGIP3基因扬麦18的T₀至T₂代植株进行PCR、Southern杂交、半定量RT-PCR和荧光定量Q-RT-PCR分析, 并对根腐病进行抗性鉴定。结果表明, GmPGIP3已转入扬麦18, 并在转基因小麦中遗传、转录和表达;比受体材料相比, 5个GmPGIP3过表达的转基因小麦株系对根腐病的抗性有明显提高。     

大丽轮枝菌致病缺陷型T-DNA插入突变体的筛选与鉴定

【目的】从大丽轮枝菌T-DNA突变体库中筛选鉴定致病缺陷型突变体并分析致病相关基因。【方法】将落叶型大丽轮枝菌菌株Vd991的294个T-DNA插入突变体在寄主棉花上进行致病力测定。利用Southern杂交和hiTAIL-PCR(High-efficiency thermal asymmetric interlaced PCR)技术分别对筛选获得的9个致病力显著降低的突变体进行拷贝数和侧翼序列分析。【结果】与接种野生型Vd991的棉花相比,接种突变体的棉花的病情指数极显著降低。Southern杂交表明其中1个突变体中T-DNA为双拷贝插入,其余8个突变体均为单拷贝插入。生物学特性分析发现T-DNA的插入导致这些突变体在生长速率和产孢量等方面与野生型Vd991相比均有不同程度的降低。Blast比对分析明确了这些突变体中T-DNA的插入位置和在基因组上的分布,并从野生型菌株Vd991中成功克隆得到了这些可能影响大丽轮枝菌致病力的相关基因。【结论】筛选和鉴定T-DNA插入突变是在全基因组范围内快速获得致病相关基因信息的1种有效方法,极大的促进了大丽轮枝菌的致病分子机制等研究,为进一步培育抗病棉花品种奠定了基础。     

叶绿体型转昆虫抗冻蛋白基因烟草的耐寒性

根据已构建的大豆叶绿体表达载体pJY01,设计特异性引物,将昆虫抗冻蛋白基因MpAFP149插入此载体中构成叶绿体表达载体pJY01-MpAFP149,利用基因枪轰击法转化烟草,经壮观霉素筛选获得4株叶绿体型转抗冻蛋白基因烟草株系。PCR和PCR-Southern结果显示外源基因已整合至烟草叶绿体基因组中但同质化水平不高,RT-PCR结果也表明昆虫抗冻蛋白基因已发生了转录。将野生型烟草、叶绿体型转抗冻蛋白基因烟草及核转化T₁代转抗冻蛋白基因烟草(pCAMBIA1302- MpAFP149)于–1℃低温处理3 d,观察耐寒表型及测定相对电导率。结果表明, 叶绿体型转基因烟草的耐寒表型优于野生型烟草,但与核转化的T₁代转抗冻蛋白基因烟草无显著差异。处理3 d时,叶绿体型转基因烟草和T₁代转抗冻蛋白基因烟草的电导率分别为39.2%和38.2%,而野生型烟草已达73.7%。本实验获得的异质化转叶绿体抗冻蛋白基因烟草与转核基因烟草的耐寒力无差异。     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Insect-mediated seed-set evaluation of 21 soybean lines segregating for male sterility at 10 different loci

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T₂ lines obtained from T₁ PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T₁ PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.     

利用子房滴注法获得转高亲和性钾离子转运蛋白基因(AlHAK1)棉花植株

棉花植株再生困难、基因型依赖性强和转化周期长等因素一直制约着棉花遗传转化的发展。本研究利用1种不依赖组织培养的转化方法 ,即子房滴注转化方法将獐茅高亲和性钾离子转运蛋白基因(Al HAK1)导入棉花基因组中。结果表明在1006株转化幼苗中有44株为卡那抗性植株,其中35株经PCR检测为阳性(T0代),转化率为3.5%。Southern与Northern杂交结果进一步表明外源基因已整合至棉花基因组中并在转录水平上表达。在提供外源0.05 mmol·L-1 K+水平下,T1转基因棉花叶片中K+含量约为对照植株的2倍,在根中约为野生型植株的1.5倍;而在2.5 mmol·L-1 K+的正常水平下,转基因棉花与野生型植株K+含量差异不明显。在50~200 mmol·L-1 Na Cl胁迫条件下,转基因棉花的种子发芽率明显高于野生型植株,尤其是在150mmol·L-1Na Cl胁迫条件下,转基因植株种子的发芽率是野生型的2.8倍左右。本研究为子房滴注转化体系在生产实践中的广泛应用提供了理论依据,并为培育适应土壤钾素匮乏及盐渍化环境下生长的棉花新品种提供了新的种质资源。     

兼抗全蚀病和白粉病小麦新种质的创制与鉴定

TaLTP5是从小麦中分离到的一个脂质转移蛋白编码基因。利用基因枪介导法将TaLTP5表达载体pA25-TaLTP5转入抗白粉病的小麦品种扬麦18 (含抗白粉病基因Pm21)中, 旨在选育兼抗全蚀病和白粉病的小麦新种质。对转基因小麦T₀~T₃代植株中引入TaLTP5基因进行分子检测和抗病性鉴定。PCR检测、Southern杂交分析结果表明, 外源TaLTP5基因已转入、整合到3个转基因小麦株系的基因组中, 并能稳定遗传; 荧光定量RT-PCR的分析以及全蚀病菌的接种与鉴定结果表明, 与受体小麦扬麦18相比, 这3个转基因小麦株系中TaLTP5表达量显著提高, 其对全蚀病的抗性也明显增强。对3个转基因株系的Pm21分子标记和白粉病抗性鉴定表明, 外源TaLTP5基因的导入没有影响受体小麦对白粉病抗性, 说明这些转基因株系为兼抗全蚀病和白粉病小麦新种质。     

Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids

To evaluate the possible antiaphid function of Amaranthus caudatus agglutinin (ACA) in allogenetic plants, transgenic cotton plants expressing the ACA gene under the control of a phloem‐specific promoter were generated via Agrobacterium‐mediated gene transformation. Based on the results of Southern blot analyses, six plants with single or lower copy transgene and favourable agronomic traits were selected for further studies. ACA expression levels ranged from 0.02% to 0.45% of total soluble protein as determined by Western blot analysis in the six selected transgenic plants. Insect bioassays using nymphs of cotton aphid (Aphis gossypii Glover) showed that five of the six transgenic plants significantly inhibited the population growth of cotton aphid, with the highest inhibition rate of 64.5%. These results shed some new light on the antiaphid function of the ACA gene as well as the promising application of the gene for obtaining aphid‐resistant transgenic cotton plants to reduce the yield loss and honeydew contamination of fibre by aphids.     

转AcAMP-sn基因抗全蚀病小麦新种质的创制与鉴定

抗菌肽是一类具有广谱抗菌性的小分子多肽,在植物防卫反应中起着重要作用。本研究人工合成了洋葱抗菌肽基因AcAMP-sn,利用基因重组技术构建了该基因的单子叶植物表达载体pAHC25::AcAMP-sn,使AcAMP-sn基因的表达受玉米Ubiqutin启动子控制。采用基因枪介导法,将AcAMP-sn基因导入小麦品种扬麦18,利用PCR、半定量RT-PCR及荧光实时定量RT-PCR检测、分析转基因小麦T0~T4代目的基因及其表达量,并对转基因植株进行全蚀病的抗性鉴定。PCR检测结果表明,导入的AcAMP-sn基因能够在转基因小麦中遗传。与受体扬麦18相比,在5个转基因小麦T4代株系中,AcAMP-sn基因的表达量及全蚀病抗性均显著提高,且全蚀病菌的相对含量明显下降,说明AcAMP-sn基因的过表达可以增强转基因小麦对全蚀病的抗性。     

The Suaeda liaotungensis kitag betaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment

In this research we established a particular vector-free and marker-free plant transformation system of maize to overcome the obstacles of biosafety limits. The BADH gene was introduced into maize by pollen-tube pathway, using the principle of minimum linear length of the transformation element, which was composed of only the BADH gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T₁ generation was 1.3%. Further Southern blotting results indicated that the BADH gene was integrated into maize genome. Transgenic lines of progeny were examined for tolerance to NaCl by induced salt stress with 250 mM NaCl Hoagland solution. After 15 days of treatment, 73.9–100% of the transgenic seedlings survived and grew well, whereas most wild-type seedlings wilted and showed loss of chlorophyll. Only 8.9% of the wild-type plants survived but gradually died after salt stress. The electrical conductivity of the transgenic line of progeny after salt stress was lower than wild type. The transgenic progeny had higher glycinebetaine and Chlorophyll content than wild type after salt stress.