拟南芥AtCAO和AtHEMA1基因共表达载体的构建及转烟草研究

增加作物的叶绿素含量,尤其是叶绿素b的含量,是增强作物耐弱光性的可能途径.将拟南芥叶绿素合成的关键酶——谷氨酰tRNA还原酶(glutamyl-tRNA reductase,HEMAl)的基因和促进叶绿素b合成的关键酶——叶绿素酸酯a加氧酶(chlorophyllide a oxygenase,CAO)的基因,构建于同一个植物双元表达载体,经农杆菌介导整合进烟草基因组.结果显示,弱光下转基因烟草比野生烟草的叶绿素总量增加不显著,但叶绿素b含量增加16％～17％、叶绿素a/b比值降低9％ ～12％、光合作用增强42％ ～65％,均达到显著水平,而且生长发育比野生烟草快.为今后改良作物的耐弱光性奠定了基础.     

4种化学调控剂对烟草幼苗耐弱光性及光合特性的影响

弱光环境严重影响烟草植株的生长发育、物质代谢和烟叶品质。该研究通过对烟草幼苗喷施不同浓度的氯化钙[3.0(C_1)和6.0(C_2)mmol/L CaCl_2]、赤霉素[100(G_1)和200(G_2)mg/L GA_3]、水杨酸[1.0(S_1)和2.0(S_2)mmol/L SA]和多效唑[100(P_1)和200(P_2)mg/L PP_(333)]进行预处理,研究了这4种化学调控剂预处理对弱光胁迫(80μmol·m~(-2)·s~(-1))下烟草幼苗的生长发育、光合性能及耐弱光性的影响,并用隶属函数法进行了综合评价。结果显示:(1)与喷施蒸馏水对照(CK)相比,4种化学调控剂预处理提高了烟草幼苗的单株干重、根系活力、烟叶总叶绿素、脯氨酸含量及光合性能,改善了烟苗的整体素质。(2)当烟苗经弱光胁迫6d后,与CK相比,4种化学调控剂预处理使烟苗单株干重、根系活力、烟叶总叶绿素含量及其光合性能相对增加,同时降低了烟叶电解质渗透率及MDA含量,延缓了烟苗在弱光胁迫下相对生长速率及干物质积累速率的降低速度,进而增强了烟苗对弱光胁迫的耐受性。(3)4种化学调控剂处理对烟草幼苗的壮苗综合效果为S_1S_2C_1C_2P_2G_2P_1G_1,且SA和CaCl_2处理对改善烟苗品质的效果优于GA和PP_(333);经弱光胁迫6d后,不同浓度4种化学调控剂预处理对缓解烟草幼苗弱光胁迫的作用大小依次为S_1C_1S_2P_1P_2G_2G_1C_2。研究表明,4种化学调控剂预处理可在一定程度上改善烟苗生长,提高烟苗的成苗素质,并在一定程度上提高弱光胁迫下烟苗的耐弱光性及光合性能,并以1.0mmol/L SA预处理综合效果最好。     

5-氨基乙酰丙酸对苹果叶片耐弱光能力的影响

为了探讨5-氨基乙酰丙酸(ALA)对苹果耐弱光性的影响,以‘润太2号’和‘郑优3号’两个品种苹果为材料,设置露天对照(CK)、轻度遮荫(LS)和重度遮荫(SS)3种光照条件,通过根际浇灌法研究了10mg·L~(-1) ALA处理对弱光条件下苹果叶片活性氧代谢与叶绿素荧光特性的影响。结果显示:(1)与CK相比,弱光胁迫显著降低了两品种苹果叶片的超氧化物歧化酶(SOD)与过氧化物酶(POD)的活性,增大了超氧阴离子(O_2~(-·))产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量,且‘郑优3号’的活性氧产生速率及MDA含量在弱光下的升高幅度更大;ALA处理显著提高了弱光胁迫下两品种的保护酶活性,降低了活性氧产生速率和MDA含量,并以耐弱光性较差的‘郑优3号’的变化更显著。(2)在弱光胁迫下,苹果叶片的叶绿素含量和叶绿素b/a升高,而ALA处理使二者进一步显著升高。(3)弱光胁迫下,苹果叶片的叶绿素荧光参数V_J、M_o、DI_o/RC显著升高,而ψ_o、ψE_o、ψR_o、PI_(ABS)、PI_(CS)和PI_(total)显著降低;ALA处理抑制了叶绿素荧光参数在弱光胁迫下的变化,甚至使其达到优于对照的水平。研究表明,弱光条件下苹果的抗氧化能力较差,受到明显的氧化伤害,且‘郑优3号’的耐弱光能力比‘润太2号’差;ALA处理提高了苹果在弱光下的抗氧化能力,降低了弱光对苹果叶片的氧化伤害,同时还提高了弱光条件下苹果叶片捕捉、传递和转化光能的效率,改善了光合电子传递情况,增大了苹果叶片在弱光胁迫下的光能利用效率,改善了光合性能;根灌ALA可以明显提高苹果的耐弱光性。     

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分别导入烟草基因组, 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。     

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。     

低温胁迫对转新疆雪莲sikRbcs2基因烟草光合作用的影响

研究分布于新疆的雪莲(Saussurea involucrata)中的sikRbcs2基因在低温条件下对植物光合作用的影响,以16、10、6、4℃温度梯度处理非转基因型和转sikRbcs2基因型烟草,每个温度处理72 h,比较研究其叶绿素荧光特性和光合特性。实验分析结果:低温胁迫下,转基因型烟草叶片叶绿素a(Chla)、叶绿素b(Chlb)、叶绿素a+b(Chla+Chlb)、类胡萝卜素(Car)含量都显著高于非转基因型烟草。叶绿素荧光参数分析:低温胁迫下,转基因烟草PSⅡ最大光化学效率(Fv/Fm)、q P(光化学猝灭系数)、ETR(电子传递效率)都显著或极显著高于非转基因型,光合参数测定:随着低温胁迫程度的加剧,转基因烟草和非转基因型烟草净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、胞间CO_2浓度(Ci)总体都呈下降趋势,但转基因烟草的净光合速率有一个明显的动态变化,先急剧下降后有一个稳定上升的趋势。通过对生长指标的测定发现:在低温处理后和恢复后转基因烟草生物量的积累都显著高于非转基因型烟草。研究结果表明:转新疆雪莲sikRbcs2基因的烟草在低温条件下具有较高的Fv/Fm、q P、ETR,对光合机构的损伤小,降低了低温胁迫效应,提高了烟草在低温胁迫条件下的耐受性。     

两种绣线菊耐弱光能力的光合适应性

研究彩色叶花灌木金山绣线菊(Spiraea bunmalba‘Goldmound’)、金焰绣线菊(Spiraea bunmalba‘Goldflame’)对弱光胁迫的光合适应能力,分析其弱光处理及恢复过程中光合特性的变化,探讨其对弱光环境的光合生理适应性变化,为绣线菊在城市园林不同光照环境中得到科学应用提供参考.2种绣线菊具明显光合日变化规律,光强为自然光照60％-65％、40％-45％时光合速率日变化为双峰型曲线,第1峰值高于第2峰值,有明显光合“午休”现象,光强为自然光照20％-25％时光合速率日变化为单峰型曲线,无明显光合“午休”现象;2种绣线菊最大净光合速率、光补偿点、光饱和点、最大表观量子效率和暗呼吸速率随处理光强减弱而减小;叶绿素(a+b)、类胡萝卜素含量随处理光强减弱而增加,叶绿素a/b值随处理光强减弱而降低;随处理光强减弱2种绣线菊叶片变薄,上、下表皮细胞变小,栅栏组织、海绵组织厚度变薄,海绵组织细胞间隙变大,2者比值减小;2种绣线菊弱光胁迫均产生低分子量(45.0-66.2 kDa)特异表达蛋白,推测为弱光胁迫诱导蛋白.2种绣线菊经60％-65％、40％-45％弱光处理后具一定恢复能力,光强仅为自然光照20％-25％处理时其恢复能力较弱.栅栏组织与海绵组织厚度比值、叶绿素a/b值和最大净光合速率是评价2种绣线菊耐弱光能力的重要指标,叶绿素a/b值与耐弱光能力显著负相关,栅栏组织与海绵组织厚度比值、最大净光合速率与耐弱光能力显著正相关,2种绣线菊耐弱光能力按隶属函数值排序为金焰绣线菊＞金山绣线菊.     

30个不结球白菜品种的耐弱光性鉴定

用人工遮荫技术对不同来源的30个不结球白菜品种进行弱光处理,以干物质减少百分率、叶片厚度减少百分率和叶绿素a/b减少百分率3个指标作为评价标准,用系统聚类分析方法,将30个供试不结球白菜品种按其耐弱光性分为3类,即耐弱光性强、耐弱光性中等和不耐弱光品种。其中耐弱光性强的品种6个,占全部样品的20%;耐弱光性中等的品种15个,占50%;不耐弱光的品种9个,占30%。方差分析结果表明,3类品种间的耐弱光性指标差异极显著。其中,品种暑绿、正大抗热青3号、热优2号、抗热605、矮王、华王属于耐弱光品种。     

干旱胁迫对转BnDREB1-5基因烟草光合特性的影响

检测了干旱胁迫对烟草(Nicotiana tabacum)叶片光合特性、叶绿素、类胡萝卜素和可溶性糖含量的影响.结果显示,干旱胁迫后转基因烟草的初始荧光(Fo)、非光化学猝灭系数(NPQ)增加幅度明显低于野生型烟草;其它荧光参数降低幅度明显小于野生型烟草;虽然干旱胁迫后两个烟草样品的光合参数、叶绿素含量都有所降低,但转基因烟草的降低幅度明显小于野生型烟草;干旱胁迫后,转基因烟草可溶性糖含量明显高于野生型烟草.表明BnDREB1-5转录因子具有抗干旱胁迫的作用.     

转C4光合酶基因水稻的CO2交换和荧光特性

用转PEPC、PPDK、NADP-ME、PEPC+PPDK酶基因水稻(Oryza sativa L.)及原种为材料 ,研究了光合作用对光照、温度、CO2的响应和光抑制条件下的叶绿素荧光特性,结果如下: 1.转C4光合酶基因水稻的饱和光合速率比原种高,其中转PEPC、PEPC+PPDK双基因水稻的光饱和点比原种高200 μmol*m-2*s-1,饱和光合速率比原种分别高51.6%和 58.5%;转PEPC基因水稻的羧化效率比原种高49.3%,CO2补偿点降低26.2%;在高温(35 ℃)下,转PEPC基因水稻的光合速率比原种高17.5%.2.经光抑制处理8 d后,转PEPC、PEPC +PPDK酶基因水稻的PSⅡ光化学效率(Fv/Fm)和光化学猝灭(qP)下降20%- 30%,非光化学猝灭(qN)增加了约30%;但原种的Fv/Fm和qP下降了5 0%多,qN变化不明显,表明转C4光合基因水稻耐光抑制能力增强.这些结果为用生物技术提高水稻光合效率研究提供了新的依据和途径.     

Resistance to Potato Virus X Infection in Transgenic Tobacco Plants with Coat Protein Gene of Virus

A major commercial cultivar of tobacco was transformed via Agrobacterium mediated procedure. Tobacco leaves started to form shoots on shoot inducing medium containing kanamycin after infected by Agrobacterium containing the plasmid with PVX CP gene. Regenerated plants were obtained in two weeks on hormone-free MS medium containing kanamycin. The transgenic tobacco plants were identified with nopaline detection,enzyme-linked immunosorbent assay and western blot analysis, symptom appearance was significantly delayed and virus accumulation was either absent or reduced in PVX CP gene transformed plants. Progenies of transgenic tobacco plants also gained resistance to PVX infection to a certain degree. These experiments demonstrate that CP protection is effective against PVX.     

谷胱甘肽转移酶基因过量表达能加速盐胁迫下转基因拟南芥的生长

盐地碱蓬谷胱甘肽转移酶基因(glutathione s-transferase gene,GST)克隆到植物表达载体pROKⅡ35s启动子的下游,通过农杆菌介导,利用花絮浸泡法转化拟南芥.转化子在含有卡那霉素的培养基上经过筛选以后,将初步验证为阳性的转基因植株通过PCR-Southem进一步证实.经过选育,筛选并分离到卡那霉素的抗性并且遗传稳定的T3代纯合子转基因拟南芥品系.通过Northern杂交证实外源基因在转基因拟南芥中表达.在盐胁迫条件下,通过测量转基因植株(GT)和野生型植株(wY)的生物量和谷胱甘肽(氧化型:GSSG;还原型:GsH)发现:转基因植株的生物量较野生型有一定程度的提高;GssG含量在转基因品系中比野生型的含量明显高.因此,过量表达GsT能够提高转基因植株在盐胁迫条件下的生长,而且这很可能是由于还原型谷胱甘肽被氧化的结果.     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene <Emphasis Type="Italic">PvSR2</Emphasis>

PvSR2 (Phaseolus vulgaris stress-related gene) has been cloned from French bean and shown to be expressed specifically upon heavy metal treatment. In order to investigate the role of PvSR2 in plant, PvSR2 gene under the control of cauliflower mosaic virus 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens LBA4404. The regenerated plantlets were selected on medium with 100 mg/L kanamycin. PCR and Southern blot analysis showed PvSR2 gene was integrated in tobacco genome. Gus and Northern blot analysis indicated PvSR2 gene was expressed in transgenic seedling. The heavy metal resistance assay showed that the transgenic tobacco seedlings with the PvSR2 coding sequence exhibited higher tolerance to Cd compared with wild-type (WT) under Cd exposure. The Cd content accumulated in root between transgenic and WT seedlings had no obvious difference at lower Cd external concentration (0.05-0.075 mmol/L CdCl2), whereas transgenic plant showed a lower root Cd content than the control at higher external Cd concentration (0.1 mmol/L CdCl2). These results suggested that the expression of PvSR2 can enhance the Cd tolerance, and PvSR2 may be involved in Cd transportation and accumulation at the test concentration of 0.1 mmol/L Cd.     

转星星草金属硫蛋白基因烟草的获得及其抗重金属镉能力分析

用农杆菌介导法将星星草金属硫蛋白基因（MD转化烟草,PCR及PCR—Southern检测的结果表明,该基因已导入烟草中。Real-TimePCR检测显示,该基因在转基因后代中的转录水平高于非转基因植株。Cd2＋胁迫下,转基因植株能够正常生长,鲜重、株高、叶绿素含量、Cd2＋含量和SOD活性均高于非转基因植株,表明MT基因的过量表达可提高转基因烟草的抗Cd2＋能力。     

Allene Oxide Cyclase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Improves Tolerance Against Low Temperature and Salt Stress in Tobacco and Bacteria

Allene oxide cyclase (AOC, E 5.3.99.6) is an essential enzyme in jasmonate (JA) biosynthetic pathway. An AOC gene (defined as CaAOC, Database Accession No. AY863428) had been isolated from Camptotheca acuminata in previous work. Real-time quantitative PCR analysis indicated that mRNA expression of CaAOC was induced by salt stress (120 mM NaCl) and low temperature (4 degrees C). In order to further investigate the role of AOC gene in the processes, CaAOC was introduced into tobacco via Agrobacterium tumefaciens, and the transgenic lines were subjected to the examination of tolerance against salt stress and low temperature. Under salt stress, the chlorophyll content in transgenic tobacco was higher than that of in the wild plants. The electrolyte leakage test revealed that transgenic tobacco plants were more resistant to low temperature over control. Furthermore, 5'-truncated CaAOC was inserted into pET30 and then expressed in Escherichia coli strain BL21DE3 (pLysS). Interestingly, the transformants could grow on 2YT agar containing 400 mM NaCl. Although these mechanisms are not clear yet, this study suggested that CaAOC could not only be a potential target gene in the engineering of plants and bacteria for improved endurance against salt stress, but also be quite useful in enhancing plant tolerance to cold.     

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2

Heavy metal pollution such as Cd, Hg, Pb, As and Se is an increasing environment problem worldwide. These metals and metalloids have toxic effect on both plants and animals, which are strongly poisonous to metal-sensitive enzymes, resulting in growth inhibition and death of the organism[1]. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, and heavy-metal tolera…     

转popW基因烟草的构建及相关表型分析

PopW是克隆于青枯劳尔氏菌Ralstonia solanacearum ZJ3721中的一种新的编码harpin蛋白的基因,原核表达的PopW蛋白能够诱导烟草对TMV的抗性、促进烟草生长、提高烟草品质。将popW基因连接到植物表达载体pBI121上,构建成重组转基因载体pB-popW,通过冻融法转化根癌土壤杆菌EHA105,获得阳性转化子。再采用叶盘法转化三生烟Nicotiana tobacum cv.Xanthi nc.,经卡那霉素抗性筛选、PCR检测、RT-PCR分析获得21个株系的T3代阳性植株。PCR及RT-PCR检测结果表明popW基因已经整合到烟草基因组中,并在转录水平正常表达。GUS染色进一步证明popW基因在翻译水平上进行了表达,且不同株系之间表达存在差异。对烟草花叶病毒(TMV)的抗病性测定结果表明,转基因烟草对TMV的抗病性增强,防效最高达54.25%。转基因烟草在生长上也具有一定优势,生长15 d的根长最高为野生型的1.7倍,移栽后60 d的株高、鲜重、干重最高分别为野生型烟草的1.4、1.7和1.8倍。     

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root‐predominant gene promoter (SbUGT) to drive the expression of a tryptophan‐2‐monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild‐type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild‐type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.