几种白菜类蔬菜卡那霉素抗性的研究

标记基因的利用是筛选和鉴定基因转化的细胞、组织和转基因植株的有效方法。针对目前遗传转化中常用的卡那霉素(Km)抗性基因,对几种白菜类蔬菜的卡那霉素抗性作了较系统的探索,发现5.0 mg/L Km可完全抑制大白菜、小白菜及菜心子叶的再生;幼苗Km抗性测验表明各品种均表现出随着Km浓度的提高,子叶黄化率升高、根茎变短、鲜重减轻的趋势。白菜类蔬菜的卡那霉素抗性检测为遗传转化和阳性后代筛选奠定了基础。     

转基因植物中标记基因的安全性新策略

转基因植物中的除草剂和抗生素抗性标记基因潜在的生态环境和食用安全性令人担忧。解决转基因植物中抗性标记基因安全性问题有两种途径：一是转化时仍使用抗性标记基因,转基因植物再生成功后,在释放大田前将标记基因剔除;二是发展安全性标记基因用于植物遗传转化。本文综述了三种标记基因剔除系统和几种安全性标记基因在植物转化中的应用进展。     

芥菜型油菜抗虫转基因植株及其后代株系的研究*

带有1～2mm子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加10～20mg/L卡那霉素的MS选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含30～50mg/L卡那霉素的选择培养基上继代培养,再转移到含20mg/L卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行Southern blot分子杂交,得到阳性结果。PCR分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系。     

芥菜型油菜抗虫转基因植株及其后代株系的研究

带有１ ～２ ｍ ｍ 子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加１０ ～２０ ｍｇ／ Ｌ卡那霉素的 Ｍ Ｓ 选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含３０ ～５０ ｍ ｇ／ Ｌ 卡那霉素的选择培养基上继代培养,再转移到含２０ ｍｇ／ Ｌ 卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行 Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分子杂交,得到阳性结果。 Ｐ Ｃ Ｒ 分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系     

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。     

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载体pYP1202并转化烟草,受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较仅有26.7％,在200mg/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。     

转基因植物中的卡那霉素抗性

在转基因植物中一个经常使用的标记基因就是卡那霉素抗性基因。对转基因植物的安全性估价将对于现代植物育种的普及和接受十分有利。对于这个标记基因的生物安全性进行估测认为,没有必要对含这个基因的转基因植物进行注册的限制。     

转石蒜凝集素基因烟草的抗蚜虫性

利用农杆菌介导法将质粒pBILRA转化烟草(NicotianatabacumL.),该质粒含有由花椰菜花叶病毒35S启动子(CaMV35S)引导的筛选基因新霉素磷酸转移酶基因(nptII)及石蒜凝集素基因(lra)。通过卡那霉素筛选获得了25株独立转基因烟草植株。Westernblot分析表明,石蒜凝集素蛋白在不同转基因植株中表达量不同。对转基因T1代植株的遗传分析表明,lra基因在大多数独立转基因植株后代中以孟德尔31的分离比方式遗传。抗虫试验表明,表达较高水平石蒜凝集素蛋白的转基因烟草对桃蚜种群的生长具有明显的抑制作用。首次报道了表达石蒜凝集素基因的烟草对蚜虫具有抗性。石蒜凝集素基因可用于植物抗虫基因工程研究及应用。     

转石蒜凝集素基因烟草的抗蚜虫性

利用农杆菌介导法将质粒pBILRA转化烟草(Nicotianatabacum L.),该质粒含有由花椰菜花叶病毒35S启动子(CaMV35S)引导的筛选基因新霉素磷酸转移酶基因(nptⅡ)及石蒜凝集素基因(lra).通过卡那霉素筛选获得了25株独立转基因烟草植株.Western blot分析表明,石蒜凝集素蛋白在不同转基因植株中表达量不同.对转基因T1代植株的遗传分析表明,lra基因在大多数独立转基因植株后代中以孟德尔3:1的分离比方式遗传.抗虫试验表明,表达较高水平石蒜凝集素蛋白的转基因烟草对桃蚜种群的生长具有明显的抑制作用.首次报道了表达石蒜凝集素基因的烟草对蚜虫具有抗性.石蒜凝集素基因可用于植物抗虫基因工程研究及应用.     

拟南芥甲基结合蛋白基因AtMBP11启动子在种子形成和萌发中的功能鉴定

为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.     

Ploidy levels in transgenic tomato plants determined by chloroplast number

We determined germline ploidy of primary tomato transformants by counting meiotic chromosomes. We then determined the number of chloroplasts in stomatal cells by cytological staining. A correlation of these values indicated that diploid transformants had significantly fewer chloroplasts than tetraploid transformants. By maximum likelihood, we estimate that less than 1% of diploid transformants will have chloroplast values in the tetraploid range. Transformed plants generally had more chloroplasts than plants derived from seed. Also, there was more variability between transformed than seed derived plants. Less than 5% of transformed plants were chimeric when comparing leaf and pollen ploidy levels. Of 129 transgenic plants examined, 29 (22%) were polyploid.     

The indole alkaloid tryptamine produced in transgenic Petunia hybrida

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine, an anti-insect compound. This TDC cDNA was transformed and expressed in transgenic Petunia hybrida under the control of the strong and constitutive 35S promoter from cauliflower mosaic virus. Kanamycin screening and Southern hybridization with the TDC cDNA confirmed plant transformation. Northern analysis indicated greater TDC mRNA accumulation in transgenic plants compared to non-transformed plants. Additionally, eight-fold more tryptamine accumulated in leaves of kanamycin resistant transgenic plants compared to non-transformed plants. Flower petals from the transgenic plants contained lower tryptamine levels than their leaves. Because tryptamine titers were higher in transformed plants compared to controls, over-expression of the TDC enzyme may partially overcome endogenous tryptamine catabolism and/or other negative biosynthetic regulation. Future alteration of tryptamine breakdown in Petunia may further increase total endogenous tryptamine concentrations, potentially discouraging insect reproduction on these transgenic plants.     

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。     

Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato

Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).     

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。     

Transgenic Tobacco plants with T-DNA Phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.     

Transformation of wheat with the HMW-GS 1Bx14 gene without markers

High molecular weight (HMW) glutenin polypeptides are critical contributors to the visco/elastic properties responsible for the processing characteristics and utilizations of wheat flour. In order to improve bread making quality of flour and produce transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang 19 by microprojectile bombardment. The transformants were selected by PCR instead of herbicidal markers. Seven transgenic plants were identified from a total of 1219 transformants, yielding a transformation frequency of 0.28%. An SDS-PAGE analysis confirmed that the 1Bx14 gene was expressed in three T1 seeds of the transgenic plants. Our results demonstrated that it is feasible to obtain marker-free transformants using the linear-expression-cassette-transformation approach coupled with PCR selection.     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

蓖麻毒蛋白A链基因RNAi转化研究

通过基因沉默技术调控蓖麻毒蛋白A链基因的表达,以期获得低毒蓖麻新材料.利用基因克隆技术获得蓖麻毒蛋白A链基因762 bp片段,命名为RTA基因.进一步利用该基因构建了植物RNAi表达载体pBI-RTA-S-AS,通过农杆菌介导法转化蓖麻子叶节,用卡那抗性筛选转化再生植株,PCR进一步鉴定转基因植株.结果表明:克隆得到目的基因长762 bp,与预期结果一致;卡那抗性筛选和PCR鉴定结果显示,获得了3株转基因阳性植株.