铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用。使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用,利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导人烟草基因组,采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明,整合到烟草基因组的外源基因多为单拷贝基因,也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明,外源基因已整合到烟草基因组中,并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明,转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系,而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛（MDA）含量和过氧化物酶（POD）活性等生理性状也发生了明显变化,表现为转大豆铁蛋白基因株系的叶绿素含量明显增加,POD活性明显增强,MDA含量明显降低：而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力,而铁蛋白抑制表达则降低了烟草耐低铁能力。     

转NtFer1基因粳稻空育131抗Fe~(2+)胁迫能力分析

旨在研究烟草铁蛋白基因NtFer1功能,提高粳稻抗逆性等。利用农杆菌介导法将NtFer1基因转入粳稻空育131,经抗性筛选、PCR、Southern杂交、Northern杂交和RT-PCR检测等,表明外源基因已整合到空育131基因组中,并能够在子代稳定遗传表达。在缺铁和铁过量条件下培育T2代转基因植株,缺铁条件下转基因株系抗氧化酶活性(SOD、POD)、叶绿素相对含量、叶片总铁含量和根系生长量均显著高于对照,而丙二醛(MDA)含量显著低于对照;铁过量条件下转基因株系抗氧化酶活性(SOD)、叶片总铁含量、糙米总铁含量和根系生长量均显著高于对照,而MDA含量和稻瘟病叶瘟指数显著低于对照。表明铁蛋白基因NtFer1能够提高转基因植株抗氧化胁迫能力和稻瘟病抗性,增加植株铁离子储藏能力,增强植株缺铁胁迫的耐性和铁过量胁迫的抗性。     

转铁蛋白基因烟草中内源铁蛋白基因的差异表达及含铁量的变化

铁蛋白是一种由24个亚基组成的高分子贮藏蛋白质,可以储存多达4500个铁原子,在动植物及微生物的新陈代谢中起着非常重要的作用。有研究表明,外源铁蛋白的大量表达可以提高植物储存铁离子的能力。为了明确外源铁蛋白基因转化植物中内源铁蛋白基因差异表达与植物含铁量的关系,本研究在成功获得2个烟草铁蛋白基因的全长cDNA克隆NtFerl（登录号：ay083924）和NtFer2（登录号：ay141105）的基础上,以烟草品种SR-1（Nicotiana tabacum cv．Petit Havana SR-1）为受体,培育了转铁蛋白基因烟草。将双元载体pBI121中的GUS基因用来自大豆的铁蛋白基因SoyFer1（登录号：m64337）置换,利用农杆菌介导法转化烟草叶盘,获得在CaMV35S启动子驱动表达的大豆铁蛋白基因转化烟草植株。Northern杂交和Western杂交分析表明外源铁蛋白基因在转基因烟草中得到了正确表达。比较转基因烟草和非转基因烟草的内源铁蛋白基因表达强度、叶片铁含量、根系铁还原酶活性、株高和鲜重表明,外源铁蛋白基因不但促进了NtFer1的表达,提高转基因植株的储存铁的能力和根系铁还原酶活性,而且促进植株的生长速度。以上结果说明,外源铁蛋白基因转化烟草中内源铁蛋白基因的表达、铁离子的还原吸收及光和作用都得到了进一步的提高。     

转金属硫蛋白基因(MT_1)烟草耐NaCl胁迫能力

为明确柽柳(Tamarix sp.)金属硫蛋白(MT1)基因过量表达对提高植物耐NaCl能力的作用,对转MT1因烟草进行分子检测和生理特性分析,结果表明具有卡那霉素抗性的转基因植株经RT-PCR Southern杂交均表现为阳性,说明外源MT1基因已整合到烟草基因组,并且得到了表达。金属硫蛋白基因的过量表达提高了转基因烟草植株的耐NaCl能力,表现为在含有150mmol/L和300mmol/L NaCl的MS培养基上,转基因植株的株高和鲜重均明显优于非转基因株系;在生理性状上表现为转基因植株丙二醛(MDA)含量明显低于非转基因株系,而超氧化物歧化酶(SOD)、过氧化物酶(POD)活性比非转基因株系明显增加。     

甲基紫精胁迫下转TheIF1A基因烟草的活性氧代谢

真核翻译起始因子e IF1家族基因具有一定的抗逆调节能力。前期研究表明柽柳The IF1A基因能提高转基因植物的抗旱耐盐胁迫能力。本研究旨在对该基因的抗氧化能力进行分析,探讨The IF1A基因是否具有抗氧化能力。组织化学染色结果显示,甲基紫精胁迫下转The IF1A基因烟草叶片、保卫细胞、根尖积累的活性氧明显少于野生型烟草(WT),H2O2含量测定结果也表明转基因株系的H_2O_2含量显著低于非转基因株系。此外,甲基紫精胁迫下转The IF1A基因烟草的CAT活性、POD活性显著高于WT株系,表明过表达The IF1A可能通过提高保护酶活性来调节体内活性氧清除能力进而改善植株活性氧积累,提高抗氧化能力。     

启动子RD29A对转雪莲SikCDPK1基因烟草抗逆性的影响

探究逆境诱导启动子RD29A对转雪莲SikCDPK1基因烟草抗逆性的影响,为SikCDPK1基因在植物遭遇低温、干旱时更好发挥作用奠定基础。采用基因重组技术构建RD29A启动子驱动SikCDPK1基因的植物表达载体,通过农杆菌介导法遗传转化烟草,分别观察、测定和比较分析低温、干旱处理后,RD29A::SikCDPK1转基因烟草、35S::SikCDPK1转基因烟草和非转基因烟草的表型、POD活性、SOD活性、叶绿素含量、MDA含量和相对电导率的差异。结果显示,干旱和低温胁迫后,RD29A::SikCDPK1转基因烟草的生长状况优于35S::SikCDPK1转基因烟草,更优于非转基因烟草;同时,RD29A::SikCDPK1转基因烟草的POD活性、SOD活性、叶绿素含量显著高于35S::SikCDPK1转基因烟草,极显著高于非转基因烟草;但MDA含量与相对电导率显著低于35S::SikCDPK1转基因烟草,极显著低于非转基因烟草。表明启动子RD29A可通过减缓叶绿素降解速率、提高抗氧化系统酶活性、减小膜通透性,使转基因烟草表现出更强的干旱、低温耐受性。     

利用转基因标记NPTⅡ快速、规模化纯合转基因番茄

利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3∶1的孟德尔分离比例,T2代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T3代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。     

利用转基因标记NPTⅡ快速、规模化纯合转基因番茄

利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3:1的孟德尔分离比例,T₂代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T₃代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。     

褪黑素合成酶基因转化烟草及转化植株抗氧化系统变化

通过根癌农杆菌(含植物表达载体YXu55)介导的转化技术,将褪黑素生物合成酶-芳烷基胺N-乙酰转移酶(Arylalkylamine N-acetyltransferase,AANAT)与羟基吲哚O-甲基转移酶(Hydroxyindole O-methyltransferase,HIOMT)基因导入到烟草(秦烟95)中。对所获得的庆大霉素抗性烟草株系进行Southern blotting和RT-PCR分子生物学检测,结果表明,AANAT-HIOMT基因已成功地整合到烟草基因组中,并且可以在mRNA水平上进行转录。用反相高效液相色谱法(RP-HPLC)测定转化株系的褪黑素含量表明,转AANAT-HIOMT基因烟草株系的褪黑素含量均明显高于pZP122(不含AANAT和HIOMT基因的空白质粒)转基因株系和未转基因的对照植株,证明AANAT-HIOMT基因在转基因植株中的表达增强了褪黑素的合成能力。对不同株系抗氧化系统的部分指标进行了测定,并与其亲本对照植株比较,发现AANAT-HIOMT基因在转基因植物中的表达引起超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性增加,谷胱甘肽(GSH)浓度...     

表达SmERF1调节烟草种子大小及耐盐性

【目的】ERF转录因子生物功能广泛,在调控植物生长发育和响应胁迫中发挥重要作用。前期研究显示,丹参SmERF1参与植物响应胁迫反应。该研究旨在进一步明确SmERF1潜在的生物功能,并为药用植物抗性及种子发育研究奠定基础。【方法】该研究采用农杆菌介导的方法在模式植物烟草中异源表达了丹参SmERF1基因;通过抗性相关酶活性变化检测,对转基因植株抗性进行评价;通过酶联免疫方法和qPCR方法分析GA和ABA等激素含量及合成途径关键酶基因的表达变化。【结果】(1)表达SmERF1的烟草植株在幼苗期表现出生长缓慢、生物量和叶绿素减少,而在其他生长阶段与对照植株没有明显差异;此外,表达SmERF1的烟草植株种子比野生对照的种子小、轻;(2)在盐处理下,转基因烟草株系中脯氨酸含量、SOD和POD活性高于对照株系,而MDA含量低于对照株系,转基因烟草株系表现出较高的耐盐性;(3)在转烟草株系中,ABA含量上调,GA水平降低。实时定量PCR结果显示,表达SmERF1调节了与植物激素生物合成相关的关键酶基因的表达,如NtSDR、NtGA20ox、NtACO和NtACS。【结论】SmERF1通过ABA依赖途径...     

Differential Expression of Endogenous Ferritin Genes and Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Soybean Ferritin Gene

For studying the effects of endogenous ferritin gene expressions (NtFer1, GenBank accession number ay083924; and NtFer2, GenBank accession number ay141105) on the iron homeostasis in transgenic tobacco (Nicotiana tabacum L.) plants expressing soybean (Glycine max Merr) ferritin gene (SoyFer1, GenBank accession number m64337), the transgenic tobacco has been produced by placing soybean ferritin cDNA cassette under the control of the CaMV 35S promoter. The exogenous gene expression was examined by both Northern- and Western-blot analyses. Comparison of endogenous ferritin gene expressions between nontransformant and transgenic tobacco plants showed that the expression of NtFer1 was increased in the leaves of transgenic tobacco plants, whereas the NtFer2 expression was unchanged. The iron concentration in the leaves of transgenic tobacco plants was about 1.5-folds higher than that in nontransformant. Enhanced growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weights significantly greater than those in the nontransformant. These results demonstrated that exogenous ferritin expression induced increased expression of at least one of the endogenous ferritin genes in transgenic tobacco plants by enhancing the ferric chelate reductase activity and iron transport ability of the root, and improved the rate of photosynthesis.     

超量表达MrCN基因提高烟草植株对TMV的抗性

利用农杆菌介导的遗传转化法将含有普通烟草Ubi．U4启动子驱动MrCN基因表达的元件导入TMV敏感烟草品种K326中,对筛选鉴定出的T0代转基因植株接种TMV,测定其接种前后不同时期的理化指标,以接种TMV的野生型植株为对照。结果显示,转基因植株和野生型植株接种TMV前叶绿素（Chl）和MDA含量YLSOD、POD和CAT酶活力均无显著差异,但接种TMV后野生型植株Chl含量逐渐降低,转基因植株Chl含量先增后降,在接种TMV后5d时转基因植株叶片Chl含量显著高于野生型植株。接种前期（0-3d）转基因植株MDA含量略低于野生型植株,但差异不显著;但接种后期（3～5d）前者的MDA含量显著低于后者。接种TMV后转基因植株中SOD、POD及CAT酶活性变化幅度较野生型植株高,以CAT的变幅最为显著。另外,Real-timePCR分析结果表明,接种TMV后3d时转基因植株删蹦、PR-1α及NrCN表达量均显著高于野生型植株。以上结果表明,通过转基因技术提高烟草抗病基NNrCN的表达量,能提高防御酶活性及病程相关基因的表达量,从而延缓植株感染TMV的发病时间,增强敏感植株对TMV的抗性。     

Iron accumulation in tobacco plants expressing soyabean ferritin gene

High iron-content transgenic tobacco plants have been produced by transfer via Agrobacterium tumefaciens of soyabean ferritin cDNA under the control of a CaMV 35S promoter. Immunoblot analysis of protein from transgenic tobacco plants suggested mature ferritin subunits are produced by cleavage of transit peptides. The expressed ferritin was observed in the tissues of leaves and stems. The maximal iron content of transformant leaves was approximately 30% higher than leaves from non-transformants. The increased iron content of each transformant was correlated with increases in ferritin content. These results demonstrate the potential of breeding high iron content crops by introduction of the ferritin gene     

镉胁迫下大豆生长发育的生理生态特征

采用土壤盆栽试验方法,研究了不同浓度Cd2+胁迫对大豆整个生长发育周期的生长以及叶片叶绿素含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性和丙二醛(MDA)含量的生理生态适应性变化过程。结果表明,(1)Cd2+胁迫对大豆整个生活周期的叶绿素含量、POD活性、SOD活性及MDA含量的影响都是极显著的(P0.01);(2)短时间、低浓度的Cd2+胁迫对大豆植株的生长发育有刺激效应,高浓度、长时间的Cd2+胁迫对大豆植株构成明显的抑制效应;大豆株高增长开始受到抑制的Cd2+浓度为1.00mg·kg-1,远低于大豆生物量的增长开始受抑制的Cd2+浓度(2.50mg·kg-1);(3)当Cd2+浓度超过一定水平时,大豆植株生物量和株高的抑制程度与外源Cd2+浓度呈极显著的正相关(P0.01),对土壤Cd2+污染程度具有指示作用,且大豆植株高度与其生物量相比,株高对Cd2+污染具有更好的指示作用;大豆幼苗期叶绿素含量对镉的敏感性高于开花结荚期和成熟期的敏感性;(4)大豆POD、SOD活性的增加,能在一定程度上减轻Cd2+胁迫引起的膜脂过氧化造成的伤害作用;在Cd2+达到2.50mg·kg-1水平时,植物保护性酶系统活性的提高已经不足以弥补因Cd2+胁迫对大豆植株造成的伤害;大豆幼苗期和花荚期叶片的POD活性对土壤Cd2+污染程度具有较好的指示作用,而大豆花荚期和成熟期叶片的SOD活性对土壤Cd2+污染程度具有较好的指示作用;在Cd2+胁迫下大豆MDA含量增加,表明细胞膜脂过氧化作用加强。     

过表达ZmSKIP基因提高烟草抗低温胁迫的能力

以野生型和过表达ZmSKIP基因烟草为试材, 研究了低温胁迫下过表达ZmSKIP对烟草抗氧化能力的影响。测定了不同低温处理时间下过表达ZmSKIP转基因烟草T3代植株和野生型植株抗氧化酶如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性和丙二醛（MDA）含量以及相对电导率, 结果表明, 低温下, 相对于野生型植株, 转基因烟草具有较高的抗氧化酶活性和较低的相对电导率和MDA含量, 说明过表达ZmSKIP提高了转基因植株的耐低温胁迫能力。     

铬对烟草组培苗生长和某些生理指标的影响

以烟草无菌苗为材料,在培养基中添加0、50、100、150、200、300μmol.L-1浓度的重金属铬离子,进行组织培养,35d后分析植株生长和相关生理变化。结果表明,低浓度(50μmol.L-1)铬对烟草组培苗生长有促进作用,株高、鲜重、叶绿素含量、蛋白质含量、SOD活性呈现上升趋势;高浓度铬处理则抑制生长,株高、鲜重、叶绿素、蛋白质含量、SOD活性都下降,相反,POD活性、MDA含量和细胞膜透性显著增加。     

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.