Pi-ta+基因和几丁质酶基因双价基因导入水稻的研究

为了提高‘云资粳41’和‘云资粳43’的抗稻瘟病能力,利用农杆菌介导法将二价表达载体pCAMBIA1300-Pi-ta⁺-Bchi转化到水稻愈伤组织中。经组织培养获得再生苗,再通过氯酚红显色法、PCR检测和抗稻瘟病鉴定法获得抗稻瘟病的转基因植株,为进一步创建持久、广谱抗稻瘟病水稻新材料奠定基础。结果显示:(1)抗性愈伤组织经分化和生根培养后,共获得T⁰代水稻再生苗137株,其中‘云资粳41’14株,‘中花11’82株,‘云资粳43’41株。(2)经氯酚红显色法和PCR对再生苗检测,‘中花11’、‘云资粳41’、‘云资粳43’的转化苗阳性率分别为66%、43%和55%。证明2个外源基因已经整合到水稻基因组中。(3)对转化阳性植株温室接种稻瘟病病菌66b鉴定结果显示,转基因植株较非转基因植株对稻瘟病的抗性明显增强,而且转Pi-ta⁺基因和几丁质酶基因双价的水稻植株比转单价Pi-ta⁺基因或几丁质酶基因的水稻植株抗稻瘟病能力强。（4）氯酚红检测结果存在一定的假阳性,PCR检测结果更真实可靠,但氯酚红显色法方便、快速,结果观察直观,可对大量的转基因植株进行初步筛选。研究表明,转Pi-ta⁺基因和几丁质酶基因双价基因的水稻植株具有更高的抗稻瘟病能力。     

导入TaNHX2基因提高了转基因普那菊苣的耐盐性

我国部分地区土地盐碱化的日益严重,对作物的生长和生态环境产生了显著影响,因此通过植物基因工程手段培育耐盐碱的转基因作物品种对改善作物的生存能力和生态环境,提高作物产量具有重要的意义。采用农杆菌介导法将来自小麦(Triticum aestivum Linn)的Na⁺ /H⁺逆向转运蛋白的基因(vacuolar Na⁺/H⁺ exchanger or antiporter,简称NHX、NHE或NHA),对普那菊苣(Cichorium intybus L.cv.Puna)植株进行了遗传转化。经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株。用不同浓度NaCl溶液对普那菊苣野生型和T₀代种子、愈伤组织和幼苗生长情况胁迫的研究,结果表明:转TaNHX2基因普那菊苣植株表现出一定的抗性,比野生型明显提高。在300 mmol/L NaCl胁迫下转基因植株种子的出芽率、外植体出愈率和分化率是野生型植株的2-4倍,而500 mmol/L NaCl浓度为野生型和转基因外植体能否生长的临界点。在此临界值下野生型外植体或不能形成愈伤组织、或幼苗不能正常生根、或已生根幼苗不能正常成长,而转基因外植体可以继续形成愈伤组织并正常生根生长。同时对500 mmol/L NaCl胁迫下野生型和转基因普那菊苣幼苗其体内丙二醛含量(MDA)、过氧化氢酶(POD)和超氧化物歧化酶(SOD)活性进行测定,结果表明 转基因植株比野生型植株的MDA含量降低了1-3倍,POD活性提高了1-3倍,SOD活性提高了2-3倍,分析发现普那菊苣的耐盐性与其体内的丙二醛含量(MDA)、过氧化氢酶(POD)和超氧化物歧化酶(SOD)活性密切相关。     

人核糖核酸抑制因子基因在人脐血干细胞中的转染表达及对小鼠黑色素瘤基因治疗的研究

探讨人核糖核酸抑制因子 (hRI)基因在人脐血干细胞中的转染及表达情况 ,及转染后对小鼠B16黑色素瘤生长的影响。用免疫磁珠分离系统 (MACS)分离纯化人脐血CD34⁺ 细胞后 ,用制备的含hRI基因的逆转录病毒上清转染脐血CD34⁺ 细胞 ,采用克隆形成法和PCR法检测转染效率 ,Western blot和免疫荧光法检测基因表达 ,同时观察RI对荷瘤C57BL小鼠B16黑色素瘤生长的影响。应用MACS能高度纯化人脐血CD34⁺ 细胞 ,使分选后的脐血CD34⁺ 细胞纯度平均达96.15%。hRI基因能够转染到脐血CD34⁺ 细胞上 ,转染效率达 35% ,Western blot和免疫荧光检测转染后CD34⁺ 细胞hRI基因有阳性表达。经转hRICD34⁺ 细胞治疗 ,使小鼠B16黑色素瘤的生长速度减慢 ,成瘤率和瘤重降低 ,成瘤潜伏期延长。     

过量表达AtNHXS1新基因提高烟草耐盐性的研究

拟南芥液泡膜上的Na⁺/H⁺逆向转运蛋白是由 AtNHX1 基因编码的一种重要的植物耐盐性因子。 AtNHXS1 是利用DNA改组(DNA shuffling)技术对 AtNHX1 基因进行定向分子进化获得的新基因。利用农杆菌介导的叶盘法将该基因转入烟草中,经过潮霉素和PCR鉴定,得到了10个独立的转基因株系。对其中两个PCR阳性株系进行Southern blot 鉴定,确定 AtNHXS1 以单拷贝的形式成功地插入到烟草的基因组中。荧光定量PCR分析表明, AtNHXS1 基因可以利用烟草的转录体系正确转录。在盐处理下,随着盐浓度的提高,植株不同组织部位 AtNHXS1 基因的表达均有不同程度的提高,其中叶片上调趋势最明显。耐盐性试验结果表明,盐处理下,转基因烟草的长势明显优于野生型。400 mmol/L NaCl 处理下,野生型烟草完全死亡,转基因烟草生长受到抑制,但是仍然能够正常生长。研究结果表明, AtNHXS1 新基因能够显著提高烟草的耐盐性。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

番茄复三螺旋基因响应外源激素和非生物胁迫的研究

复三螺旋(double trihelix)基因在植物形态建成和植株抗逆性方面发挥关键作用。该研究以番茄自交品种AC⁺⁺为试验材料,运用生物信息学方法与qRT PCR技术对5个复三螺旋成员(SlGTL1~SlGTL5)在番茄体内不同器官的表达模式、以及基因对激素与非生物胁迫的响应进行表达分析,以探讨番茄复三螺旋基因的功能。结果表明：(1)生物信息学分析显示,番茄中含有5个复三螺旋基因(SlGTL1~SlGTL5);进化树分析表明,番茄复三螺旋基因具有物种特异性。(2)qRT PCR分析显示,番茄SlGTL3基因在根和茎中特异表达,其他4个基因均在果实中较高表达,表明不同番茄复三螺旋基因的表达具有组织特异性。(3)激素诱导表达结果显示,SlGTL1只响应ABA(1种)激素,而SlGTL5基因可响应4种激素,且速度较快。(4)非生物胁迫诱导证实,SlGTL3、SlGTL5基因可响应盐胁迫,SlGTL3~SlGTL5基因可响应极端温度,SlGTL3和SlGTL4基因可响应机械损伤;SlGTL1、SlGTL4和SlGTL5可响应脱水胁迫。研究认为,SlGTL3的功能可能与植株形态建成和非生物胁迫有关,其他4个基因的功能可能与果实的发育有关;推测SlGTL1可能与ABA信号途径有关,SlGTL5快速响应多种激素,可能位于信息传递的节点,其功能可能与信号传递有关。     

猪2型圆环病毒ORF1与ORF2基因和伪狂犬病毒基因的重组与表达的研究

根据GenBank发布的猪2型圆环病毒(PCV2 )序列(AY0 35 82 0 ) ,设计两对特异性引物,采用PCR方法,分别扩增了猪2型圆环病毒ORF1和ORF2基因。将ORF1和ORF2基因的PCR产物回收并酶切后,依次插入到伪狂犬病毒gE gI双缺失通用转移载体pIECMV中,构建了猪2型圆环病毒_伪狂犬病毒重组中间转移质粒pIEORF1-ORF2。采用脂质体介导法,将重组中间转移质粒pIEORF1_ORF2与伪狂犬病毒TK^- gE^- LacZ⁺ 基因组共转染IBRS_2细胞,待发生细胞病变后收集病毒液进行空斑纯化,利用检测PCV2ORF1基因和ORF2基因的PCR方法筛选重组病毒TK^- gE^- gI^- ORF1-ORF2⁺ ,用Southernblotting鉴定重组病毒,并用Westernblotting检测ORF1_ORF2融合蛋白的表达情况,在此基础上也测定了重组病毒在不同细胞上的增殖滴度。结果表明,外源基因ORF1和ORF2已成功插入到TK^- gE^- LacZ⁺ 亲本株的基因组中,并获得了表达,表达的蛋白可与PCV2阳性血清发生反应。同时发现ORF1和ORF2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当。     

通过表达ACC脱氨酶基因控制番茄果实的成熟

乙烯在跃变型果实的成熟过程中起着触发呼吸跃变和促进果实成熟的作用。细菌来源的1-氨基环丙烷-1-羧酸(ACC)脱氨酶能降解乙烯的直接前体ACC,从而抑制植物体内乙烯的合成。我们用PCR方法从假单孢杆菌中克隆到ACC脱氨酶基因并通过农杆菌介导的方法将其转入番茄(Lycopersicun esculentum)中。再生植株经Southern blot检测证明,ACC脱氨酶基因已整合到番茄基因组中并稳定表达。转基因番茄果实成熟期的推迟时间与体内乙烯的抑制程度有相关性。转基因番茄植株乙烯的合成降低80%左右,果实在离体条件下可保鲜75d左右。研究ACC脱氢酶基因在植物体内的作用可阐明高等植物体内乙烯的作用机理并为培育耐贮藏果蔬品种打下基础。     

An insight into functional genomics of transgenic lines of tomato cv Rio Grande harbouring yeast halotolerance genes

Tomato cv Rio Grande plants were transformed with yeast halotolerance genes (HAL I or HAL II) using pPM7HAL I or pJRM16HAL II, with p35GUSINT as control. Transformation efficiency varied in the three constructs, with highest transformation found with p35GUSINT. Final selection of the transgenic plants was made on the basis of PCR. Transgene integration and copy number were assessed by Southern hybridisation. The primary transformants were allowed to self-pollinate and the expected Mendelian ratios were studied in second-generation progeny. Five independent homozygous lines each of HAL I and HAL II, as well as the control, were characterised to study inter-transformant expression variability. The transformants showed considerable variability in expression of the respective genes, as shown by salt tolerance assays, chlorophyll content and peroxidase activity. The transgene expression in transgenic lines was analysed by semi-quantitative RT-PCR. In response to different salt concentrations, transgenic plants over-expressing HAL I and HAL II had significantly (α=0.05) better performance than the control This study presents the comparative responses of the three constructs under the same transformation conditions and suggests possible mechanisms governed by yeast HAL I and HAL II genes, which seem to work in a coordinated manner by relatively decreasing osmotic and oxidative shock at different rates. Our results suggest that the yeast HAL I increases K(+) /Na(+) selectivity and has a more functional role than HAL II in improving salt tolerance of the tomato cv Rio Grande grown in Pakistan.     

The yeast HAL1 gene improves salt tolerance of transgenic tomato

Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K(+) concentration and decreasing intracellular Na(+) during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K(+) to Na(+) ratios showed that transgenic lines were able to retain more K(+) than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectivity under salt stress

The yeast HAL1 gene facilitates K⁺/Na⁺ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K⁺ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K⁺/Na⁺ selectivity under salt stress.     

HAL1 mediate salt adaptation in Arabidopsis thaliana

INTRODUCTIONSalinity is a major environmental stress that isa substantial constraint to crop production both fordry land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitatehigh-output crop production. To overcome theselimitations and improve production efficiency in theface of a burgeoning world population, more salt tolerant crops must be developed. In contrast with traditional breeding, the direct ill…     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

Comparative physiological responses of the yeast halotolerance genes expressed in transgenic lines of tomato cv Rio Grande under saline conditions

We previously analyzed the transgenic lines of tomato cv Rio Grande over-expressing the yeast HAL I and HAL II genes for their response to salt stress under in vitro conditions. In this study, six homozygous tomato lines harbouring the yeast HAL I or HAL II genes with highest expression level were selected for exploring their physiological responses against different salt stresses in the field. These transgenic plants showed significant growth and improved water content in comparison with control under 100 and 150 mM salt stress conditions. The HAL I and HAL II lines showed better Ca²⁺ content than their control counterparts. Furthermore, the transgenic lines exhibited lower values of relative electrical conductivity and improved resistance against the fungal pathogens Fusarium oxysporum and Alternaria solani when tested by detached leaf and agar tube dilution assays. Physiological analyses carried out in this study suggest an involvement of multiple mechanisms in transgenic tomato plants harbouring yeast genes to confer biotic and abiotic tolerance under stress conditions.     

The expression of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> HAL1 gene increases salt tolerance in transgenic watermelon [<Emphasis Type="Italic">Citrullus lanatus</Emphasis> (Thunb.) Matsun. &; Nakai.]

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.     

The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato ( Solanum lycopersicum ) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1 ) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na⁺ accumulation in leaves, being moreover the Na⁺ homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na⁺ transport to the shoot to long term was even shown in low accumulation of Na⁺ in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na⁺ homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na⁺ accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 m M NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na⁺ levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na⁺ exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.