转甜瓜CmSAMDC基因拟南芥的获得及其耐盐性研究

该研究以哥伦比亚生态型野生拟南芥为材料,将甜瓜CmSAMDC基因构建到植物双元表达载体pCAMBIA1304上,采用农杆菌介导法转入拟南芥,在含有50mg/L潮霉素(Hyg)MS固体培养基上筛选转基因后代,并利用T3代转基因幼苗进行耐盐性分析。结果显示:(1)成功构建了植物超表达载体35S∷CmSAMDC,并经农杆菌介导法转化拟南芥,潮霉素抗性筛选后获得了转CmSAMDC基因拟南芥T3代植株。(2)转CmSAMDC基因拟南芥T3代幼苗在含100、150、200mmol/L NaCl培养基中,侧根长势比野生型植株更为健壮;在200mmol/L NaCl浇灌处理后,转CmSAMDC基因T3代植株仍能维持正常生长,而野生型植株的生长明显受到抑制;在400mmol/L NaCl浇灌处理后16d,野生型植株逐渐死亡,而转基因植株仍能继续存活;对盐胁迫后植株的脂质过氧化程度(MDA)测定显示,野生型植株MDA水平较转基因植株上升更为明显。研究表明,过表达甜瓜CmSAMDC基因增强了转基因拟南芥的耐盐性。     

TaNHX2基因植物表达载体的构建及在拟南芥中的功能分析

将TaNHX2基因重组于质粒pBIN438的CaMV 35S启动子下游,构建含TaNHX2基因的植物双元表达载体pBIN438-TaNHX2。采用根癌农杆菌介导的真空渗透法转化拟南芥,得到T0代转基因拟南芥种子。经含Kan的平板筛选及PCR鉴定,获得54株阳性植株,选取生长一致的转基因阳性植株进行耐盐、耐旱分析,结果表明TaNHX2能够提高转基因植株的耐盐性和耐旱性。     

拟南芥AtJ2基因定位表达载体的构建

AtJ2是拟南芥中的一种分子伴侣,参与了许多重要的生命活动,但其具体的作用机制还不清楚。为进一步研究该蛋白的功能,我们构建了该基因的定位表达载体,拟对该基因在拟南芥中的定位进行研究。将该基因构建到带有绿色荧光蛋白(GFP)基因的质粒表达载体pB inGFP中,并将此重组质粒通过农杆菌介导转入拟南芥中,得到了转基因植株,为后续该基因的定位研究奠定基础。     

利用RNAi技术抑制拟南芥NHX1基因家族的表达

采用RNAi抑制NHX1基因家族的表达,并观察其对拟南芥耐盐性和耐旱性的影响.根据从拟南芥(Ara-bidopsis thaliana)cDNA中扩增出编码Na /H 反向转运蛋白基因AtNHX1长度为210 bp高度保守序列作为RNAi的靶标区,并正反2个方向插入载体pHANNIBAL中,2个片段用intron连接;将RNAi表达框连入具有NPTⅡ筛选标记基因的表达载体pART27中,构建以拟南芥NHX1基因家族为靶标的RNAi载体.采用农杆菌介导的真空渗透法转化拟南芥,得到T0代转基因拟南芥种子.对转基因阳性植株进行RT-PCR检测以及耐盐性和耐旱性分析.结果表明,利用本实验构建的NHX1基因家族RNAi载体,拟南芥NHX1基因家族表达被成功地抑制;耐盐和耐旱分析表明RNAi技术对基因表达沉默是有效的.     

棉花乙烯合成基因促进拟南芥和烟草不定根发生的研究

从棉花纤维cDNA中克隆获得乙烯合成基因GhACO3,构建了植物过量表达载体p35S::GhACO3.通过花序侵染法和叶盘法分别转化拟南芥和烟草,利用卡那霉素筛选及分子检测获得转基因阳性拟南芥和烟草植株.结果表明,GhACO3基因已整合到拟南芥和烟草基因组中;经过纯合筛选后获得转基因T2代拟南芥植株;与野生型拟南芥相比,GhACO3基因对拟南芥不定根发生具有显著促进作用;与野生型烟草植株相比,转GhACO3基因烟草不定根发生得到了显著的促进.研究表明,GhACO3基因的过量表达能够促进拟南芥和烟草不定根的形成发育,为进一步探讨GhACO3的生物学功能和进行转基因育种奠定了基础.     

小麦耐逆基因-TaLEA2转化拟南芥的研究

研究小麦第3组LEA基因中T aLEA2对耐旱和耐盐性能的影响.将小麦第3组LEA基因T aLEA2连接在双元表达载体pB I121 C aM V 35S启动子下游,构建了能在植物中高效表达的载体pB I121-T aLEA2.通过农杆菌介导的真空渗透法,将其转入野生拟南芥中,经抗性筛选及PCR验证,获得T0代转基因植株,并用不同浓度的PEG 4000和N aC l对转基因拟南芥的耐逆性进行检测.结果表明,这些转基因植株可明显改进拟南芥在10%PEG及0.8%N aC l培养基上的生长状态.在实验条件下,转基因拟南芥的耐旱性及耐盐性均有所提高,提示T aLEA2基因在植物水分调节方面有重要作用.     

转移拟南芥CBF1基因引起水稻植株脯氨酸含量提高

利用农杆菌介导的转基因技术,成功地将拟南芥抗冻转录激活因子基因CBF1转入粳稻中花11中,并获得了T1代转基因植株。CBF1基因及筛选基因HPT（潮霉素抗性基因)均在T1代中检测到,呈现单位点的孟德尔式遗传。常温和低温处理之后,T1代植株体内的脯氨酸含量均比野生型明显提高,同时,耐低温表型也在T1-1株系中出现。     

转拟南芥ICE1基因增强烟草抗寒性的研究

ICE1是CBF冷响应通道的上游转录调控因子,通过与CBF启动子中MYC顺式作用元件的结合激活CBF3基因表达.采用RT-PCR方法,从拟南芥获得AtICE1基因,将AtICE1导入pCAMBIA1301构建35S:AtICE1植物表达载体.通过根癌农杆菌GV3101,将AtICE1基因导人烟草,T1代植株经潮霉素抗性筛选,PCR、RT-PCR检测,结果表明AtICE1基因已经整合到烟草基因组中,并在转录水平表达;在正常生长条件下,转基因烟草与对照烟草的生长未见明显区别,而在瞬时低温冻害下,转基因烟草存活率明显高于对照烟草植株,说明Atl-CEI基因可以提高低温敏感作物的耐寒性.     

拟南芥谷氨酸受体基因的亚细胞定位

为明确拟南芥谷氨酸受体1.3基因(AtGLR1.3)的亚细胞定位,该实验以拟南芥(Arabidopsis thalianaCo-lumbia ecotype)为材料,运用PCR方法从其基因组中扩增得到了AtGLR1.3的启动子和基因序列,将其连接到载体pBIsGFP上,构建成AtGLR1.3基因与绿色荧光蛋白基因融合的植物表达载体,通过农杆菌介导的花序浸润法将重组载体转化拟南芥野生型,转基因植株通过激光共聚焦扫描显微镜观察显示,GFP荧光信号存在于细胞质膜上,表明AtGLR1.3为细胞膜蛋白.该结果为进一步研究AtGLR1.3的作用机理奠定了基础.     

拟南芥PR-1基因启动子的克隆与植物表达载体的构建

根据已报道的拟南芥（Arabidopsis thaliana L.）病程相关蛋白基因（PR-1）序列设计引物,通过PCR技术从拟南芥中扩增得到水杨酸诱导表达的PR-1基因启动子片段,序列分析表明,该启动子含910bp核苷酸,与已报道的序列比较,核苷酸的同源性为99．7％：将该启动子构建到植物表达载体pB1121上,获得病程相关蛋白基因（PR-1）启动子驱动的GUS报告基因的植物表达载体pBI-prlp,将其转入根癌农杆菌GV3101,通过农杆菌介导转化拟南芥,获得转基因拟南芥植株,为深入研究寄主-病原物相互作用的分子机理奠定基础。     

A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Phenotypic instability of transgenic tobacco plants and their progenies expressing Arabidopsis thafiana small GTP-binding protein genes

Chimeric genes consisting of the cauliflower mosaic virus 35S promoter, a CDNA encoding a small GTP-binding protein from Arabidopsis thaliana (ara-2 or ara-4) and the terminator of the nopaline synthase gene were cloned into a binary vector. Tobacco leaf tissues were transformed with this plasmid via Agrobacterium-mediated transformation. Transgenic plants possessing either ara-2 or ara-4 occasionally showed morphological abnormalities in leaves and other organs. However, such alterations were not always associated with co-transferred characters, such as kanamycin tolerance, and they arose in no more than 10% of the transgenic plants. Such phenomena were also observed in the progenies of the primary transgenic plants. Despite such unusual inheritance of the phenotypic abnormalities, GTP-binding activity of the inserted ara gene products was detected in all plants tested.     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

蕙兰CfCIN基因克隆及其功能分析

以蕙兰(Cymbidium faberi Rolfe)为试验材料,采用RT-PCR技术克隆了TCP家族的CIN同源基因,开放阅读框长1 161bp,编码386个氨基酸,将其命名为CfCIN(GenBank登录号为KJ956809)。为进一步分析CfCIN的功能,构建了植物表达载体,采用农杆菌介导法转化非洲紫罗兰叶片,获得了转化植株并对转基因植株进行了性状分析。结果显示:与野生型非洲紫罗兰叶片相比,转基因植株的叶片更大,由圆形变为卵圆形,叶缘由平整光滑变为有缺刻且稍向后卷曲,叶脉明显,叶柄红,花器官形状变化不明显。研究表明,CfCIN可能参与调控植物叶片的形态建成。     

小麦盐华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达迫相关基因的克隆与表达分析

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

砂藓RcPLD基因的抗旱功能分析

砂藓(Racomitrium canescens)是一种具有极强耐脱水性的苔藓植物,编码磷脂酶D的基因RcPLD能够在砂藓的脱水和复水过程中产生显著的表达响应,它可能参与了砂藓的强耐脱水性功能。该研究使用已克隆的RcPLD编码序列构建拟南芥(Arabidopsis thaliana)过量表达转基因株系rcpld-oe,初步考察过表达株系的干旱胁迫耐受能力及其相关的生理生化指标,分析RcPLD增强拟南芥抗旱性的机制。结果表明:(1)利用已克隆的RcPLD编码序列构建了植物中的过表达载体,成功构建了RcPLD的过表达转基因拟南芥株系rcpld-oe,并获得了多个T_3代rcpld-oe纯合体株系。(2)在正常生长条件下,rcpld-oe株系T_3代纯合体植株比野生型拟南芥植株体积小,但营养生长期较长,抽薹较晚,莲座叶衰老速率较慢;在干旱处理条件下,rcpld-oe株系表现出比野生型拟南芥更强的干旱耐受能力。(3)在干旱胁迫处理过程中,rcpld-oe株系莲座叶的水分散失速率降低,可能在一定程度上降低了干旱对膜完整性的损伤和光合作用的抑制,但其渗透调节物质含量的变化相对较小。研究发现,在干旱胁迫条件下,rcpld-oe植株莲座叶的水分散失速率和光合作用抑制程度显著降低,从而表现出明显强于野生型的干旱耐受能力,这为后续RcPLD功能的深入研究和更多砂藓抗旱功能基因的挖掘奠定了基础。     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。