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1.
PtLFY, a LEAFY (LFY) gene, was cloned from Populus tomentosa (LM50) by PCR. Sequencing analysis indicated that PtLFY was 2 629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice
acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the
PtLFY gene in genomic DNA of male and female P. tomentosa (LM50 and 5082). The pBI121-Ptalfy (reverse)-intron-Ptlfy-GUS-nos was constructed using RNA interference (RNAi) technique
and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained
by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type
ones and some phenotypic differences were observed.
[Supported by the National Natural Science Foundation of China (Grant No. 30371175) and Postdoctoral Foundation of China (Grant
No. 2002032041)] 相似文献
2.
毛白杨PtLFY在花芽发育中的表达模式与花芽形态分化 总被引：3，自引：0，他引：3
以毛白杨花芽为材料,采用RT-PCR技术分离克隆毛白杨PtLFYcDNA序列,测序结果表明该序列全长1314bp,包含1个开放阅读框,编码377个氨基酸。Alignment分析显示该基因与拟南芥等物种LFY/FLO同源基因所编码的氨基酸相似性达到68%～75%。蛋白结构预测分析表明,在PtLFY蛋白N-端和C-端具有2个高度保守的区域,其中PtLFY-C端由7个α-helix组成Helix-turn-helix结构。采用Real-time qRT-PCR技术检测PtLFY在雌雄花芽发育过程中的表达模式,结果显示从9月13日到翌年1月25日,PtLFY在毛白杨雌雄花芽中持续稳定表达,到2月25日表达量少许下调,但该基因在雄花芽中的相对表达量明显高于雌花芽。解剖分析结果表明,雄花芽形态分化进程明显早于雌花芽,这种差异可能与PtLFY在雌雄花芽发育过程的差异表达存在密切联系。研究结果对于阐明PtLFY在毛白杨雌雄花芽发育和开花中的分子作用机制具有重要的理论意义,为进一步开展毛白杨开花调控研究奠定基础。 相似文献
3.
To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions. 相似文献
4.
Wang Dong-mei An Xin-min Zhang Zhi-yi Key Laboratory of Genetics Breeding in Forest Trees Ornamental Plants Ministry of Education Beijing Forestry University Beijing P. R. China 《中国林学(英文版)》2005,(3)
To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions. 相似文献
5.
A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems. 相似文献
6.
Shiokawa T Yamada S Futamura N Osanai K Murasugi D Shinohara K Kawai S Morohoshi N Katayama Y Kajita S 《Tree physiology》2008,28(1):21-28
We report the isolation and characterization of CjNdly, a homolog in Japanese cedar (Cryptomeria japonica D. Don) of the FLORICAULA/LEAFY (FLO/LFY) genes. We determined the entire nucleotide sequence of CjNdly, including short 5'- and 3'-untranslated regions. The deduced amino acid sequence was similar to those of the products of the FLO/LFY genes from other species. The nucleotide sequence showed the closest homology to that of the NEEDLY gene in Pinus radiata D. Don. Although no proline-rich region has been reported previously in homologous gene products from gymnosperms, we found such a region at the amino-terminal end of the deduced amino acid sequence encoded by CjNdly. We detected the expression of CjNdly in both reproductive and vegetative tissues and organs of C. japonica. Heterologous expression of CjNdly in transgenic tobacco plants induced precocious flowering of regenerating shoots on agar-solidified medium and flowers with an abnormal phenotype, namely, petal-like stamens. Our findings suggest that the CjNdly gene may have important roles in flower development in Japanese cedar, resembling those of its angiosperm homologs. 相似文献
7.
Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol 总被引：2，自引：0，他引：2
The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance. 相似文献
8.
对转豇豆胰蛋白酶抑制剂基因的三倍体毛白杨杂种 [(毛新杨×毛白杨 )×毛白杨 ]的可溶性总蛋白和胰蛋白酶抑制剂蛋白的含量进行测定 .结果发现 ,与未转基因植株相比 ,所有供试转基因株系叶片中的总可溶性蛋白含量明显增加 ,但老叶的含量高于嫩叶 ,这表明转基因株系可溶性总蛋白含量增加可能是CpTI基因表达的结果或由于外源基因导入后引起杨树基因组中某些自身基因的表达所致 .转基因株系叶片中有较高含量CpTI,而对照叶片则检测不到CpTI,这进一步证实了CpTI基因已在转基因株系中得到稳定表达 .进一步比较发现 ,在供试的 5个无性系中 ,TG0 7、TG0 4和TG71在总蛋白含量和CpTI含量增加较为明显 .另外 ,聚丙烯酰胺凝胶电泳胶分析发现 ,与未转基因植株相比 ,在所有供试转基因株系叶片中均出现一条分子量为 11.3kD的清晰蛋白带 相似文献
9.
以毛白杨为转基因受体材料,利用根癌农杆菌介导法转化来源于益母草的阳离子抗菌肽基因LJAMP2。经卡那霉素筛选,共获得50株抗性植株。GUS组织染色和PCR检测显示有30株抗性植株呈阳性,初步证明外源目的基因已整合到毛白杨基因组中。RT-PCR证实抗菌肽基因LJAMP2在转基因植株中能大量表达。离体抗病性试验表明:转基因毛白杨细胞粗提液的抑菌能力明显强于非转基因植株。进一步将溃疡病菌接种在转基因和野生型毛白杨茎段上培养30天,转基因植株的病级指数均低于非转化植株。上述抗性试验结果表明:在毛白杨中超量表达益母草抗菌肽基因LJAMP2能显著提高其溃疡病抗病性。 相似文献
10.
Zhang Qian Lin Shanzhi Lin Yuanzhen Zhang Zhiyi Liu Haijun Zou Yening Wang Zeliang College of Biological Sciences Biotechnology Beijing Forestry University Beijing P. R. China College of Resources Environment Beijing Forestry University Beijing P. R. China 《中国林学(英文版)》2004,6(3)
The putative transgenic hybrid triploid poplars [(P. tomentosa P. bolleana) P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar. 相似文献
11.
12.
Ferritin, a universal intracellular protein, can store large amounts of iron and improve plant resistance to abiotic and biotic stress. In this study, a ferritin gene(TaFer) from Tamarix androssowii Litv. was transferred into Populus tomentosa Carr. cv 'BJR01' via Agrobacterium. Six independent transgenic lines were obtained with a tolerance to kanamycin and three were randomly selected for further analysis. The PCR and RT-PCR results indicate that the TaFer gene had been integrated into the poplar genome. The effect of the gene on abiotic stress tolerance was tested, and the results show that transgenic plants improve growth, had higher chlorophyll and lower MDA contents, and higher relative electrical conductivity,fewer changes of SOD and POD activities, higher iron content, higher root ferric reductase activity and lower levels of ROS accumulation and cell death in response to drought, Fe-insufficient or Fe-excess tolerance. These results indicate that the TaFer gene can improve abiotic stress tolerance in transgenic Populus tomentosa. 相似文献
13.
Li Min Wang Hua-fang Yin Wei-lun He Si-jie Chen Shou-yi College of Biological Sciences Biotechnology Beijing Forestry University Beijing P. R. China Institute of Genetics Developmental Biology Chinese Academy of Sciences Beijing P. R. China 《中国林学(英文版)》2006,8(4):20-24
Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene (mtl-D) into the Populus tomentosa Carr. genome. An Agrobacterium tumefaciens strain (AGL1), constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg·L-1 kanamycin and 500 mg·L-1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium containing 30 mg·L-1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the transformation plants of the P. tomentosa genome. The mannitol content in control plant was 69μg·g-1 FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5μg·g-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCl. 相似文献
14.
15.
【目的】为了揭示热激蛋白90(Heat shock protein 90,HSP90)基因在杨树抗溃疡病中的功能,克隆获得毛白杨HSP90基因的全长序列,以期明确HSP90基因的表达与溃疡病菌侵染间的关系。【方法】采用RT-PCR技术和Gateway克隆的方法,获得了毛白杨的HSP90基因序列,并利用相关软件对该基因编码的蛋白的理化性质、疏水性、结构域、功能及亚细胞定位等进行了生物信息学分析。【结果】毛白杨的HSP90基因序列(NCBI登录号:AGU99972.1)全长为2 100 bp,其中A+T占52.90%,C+G占47.10%,共编码699个氨基酸。毛白杨HSP90基因的生物信息学分析结果显示,该基因编码的蛋白相对分子质量为80.08 kDa,理论等电点为4.96。毛白杨HSP90蛋白主要位于细胞核和细胞质膜中,且在N端含有1个基因保守结构域HATPase_c,可与ATP结合,具有内源ATPase活性,属于HSP90家族,为亲水性蛋白,可能具有离子通道的功能。同时,对毛白杨的HSP90基因进行系统进化分析,发现毛白杨的HSP90基因与毛果杨(gi 539331543)、大豆(gi 358248990和gi 356552478)的亲缘关系较近,而与毛果杨(gi 224124864)的亲缘关系相对较远,说明同一物种的HSP90进化可能有所不同。【结论】首次从毛白杨中成功克隆得到与抗溃疡病相关的HSP90基因,并对其序列和生物学信息进行了分析,为下一步研究HSP90基因在杨树抗溃疡病中的功能奠定基础,也为进一步研究HSP90基因在杨树抗逆胁迫中的生理功能提供了理论支持。 相似文献
16.
杨树皮储藏蛋白基因启动子的克隆和功能研究 总被引：17，自引：0，他引：17
杨树树皮储藏蛋白BSP是类似种子储藏蛋白的氮素储藏物 ,冬季在韧皮部薄壁细胞中大量积累 ,是落叶树氮代谢中的重要成分。为了研究BSP基因启动子在转基因植物中的表达特性 ,探索其在植物基因工程研究中潜在的应用价值 ,我们用PCR方法从美洲黑杨基因组中DNA扩增得到了BSA启动子片段。与GUS基因融合构建中间载体后 ,转化烟草 ,获得了一批PCR检测为阳性的转化再生植株。经GUS组织化学检测 ,发现若干转基因烟草的茎和叶柄韧皮部以及叶脉都呈GUS染色阳性 ,初步证明杨树BSP基因启动子确有韧皮部表达特性 ,可介导GUS基因在转基因烟草韧皮部特异表达。 相似文献
17.
To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for
microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target
microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both
1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates
of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions
of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type
of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope.
The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis,
in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic
poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.
[Supported by the National Project in Transgenic Plant and Application (Grant No. J2002-2003)] 相似文献
18.
多基因转化是基因工程研究热点之一。本研究应用DNA重组技术,将两个抗病机制不同,抗菌谱较广的抗病基因(天麻抗真菌蛋白GAFP和兔防御素NP1基因)构建在一个植物表达载体pBin35SGAFP-NP1上,两者具有各自的CaMV35S启动子和Nos终止子。通过根癌农杆菌介导,采用叶盘法转化烟草,PCR和PCR-Southern分析证明已将NP1和GAFP基因整合到烟草基因组中。离体抑菌实验表明转基因植株对真菌和细菌表现出一定的抗性。以上结果表明通过该表达载体进行遗传转化可获得含双价抗病基因植物,并能有效表达,提高转基因植物抗病能力。 相似文献
19.
糖基化磷脂酰肌醇锚定蛋白(GPIAP)因其结构和功能的多样性,决定了它在各种生物学过程中都发挥着重要作用。采用同源克隆的方法从绿竹(Bambusa oldhamii)中获得一个GPIAP同源基因,命名为BoGPIAP,cDNA全长1 772 bp,其中包括1 356 bp的开放阅读框,编码一个451 aa的的蛋白。蛋白结构分析表明,该蛋白包含1个典型的GPIAP家族保守区域(47-211)和1个CCVS结构域,在N-端和C-端分别具有1个跨膜信号肽和1个GPI锚定信号肽,属于GPIAP家族。构建BoGPIAP与GFP融合的表达载体,在洋葱表皮细胞中瞬时表达,结果显示BoCOBL::GFP融合蛋白定位于细胞膜上,证明BoGPIAP基因编码的蛋白为膜蛋白。分别构建BoGPIAP的正义、反义表达载体并转化烟草(Nicotiana tabacum)。PCR检测结果表明,BoGPIAP已转入烟草。与野生型相比,转反义基因植株细弱,纤维细胞壁明显变薄;而转正义基因植株粗壮,纤维细胞壁明显变厚。表明BoGPIAP可能对竹子纤维细胞壁的发育具有调控作用。 相似文献