PSAG12-ipt基因转化植株研究进展

叶片衰老是一种程序性死亡过程;ipt(isopentenyl transferas)基因转化植株,可以催化调控内源细胞分裂素合成,延缓转化株叶片衰老.SAG12基因启动子能够控制ipt基因在植株下部衰老叶片中表达.介绍了ipt基因和SAG12基因启动子的来源和应用,以及PSAG12-ipt基因的产生和转化植株在国内的研究概况.     

转ipt和反义ACO基因番茄的叶片衰老相关特性

以ipt和反义ACO转化的两类转基因番茄纯系为材料,研究在植株不同生长发育阶段,不同叶位中,与叶片衰老相关的生理生化指标.结果表明:两类基因导入番茄后,均可增强内源iPA和IAA表达水平,增加或保持番茄叶片的叶绿素含量、提高光合效率,进而明显地延缓植株的叶片衰老,提高单株果实产量.但它们调控叶片衰老的途径不同,ipt主要通过提高CTK的水平延缓叶片衰老,而反义ACO则主要是通过抑制乙烯生成,间接提高IAA的水平来实现.     

转叶片衰老延缓基因PSAG12-IPT小麦株系光合性能的研究

以导入叶片衰老延缓基因PSAG12-IPT小麦西农1376的T5代株系为材料,于灌浆期测定了它们功能叶片的净光合速率和叶绿体色素含量.结果表明:经过早代鉴定的转PSAG12-IPT基因小麦株系的净光合速率均高于受体对照西农1376,部分株系达到极显著水平;转基因株系叶绿体色素含量的平均值也显著高于对照.可见,转叶片衰老延缓基因PSAG12-IPT小麦株系灌浆期功能叶片的光合性能得到显著改善.     

紫叶水稻光温敏核不育系及其杂种生育后期几种与叶片衰老相关生理指标的变化

测定紫叶水稻光温敏核不育系‘桂紫-(?)S’及其配组杂种生育后期与叶片光合及衰老相关生理指标、杂种产量和农艺性状的结果表明:‘桂紫-(?)S’剑叶的叶绿素含量、可溶性蛋白含量,光合速率和超氧化物歧化酶(SOD)活性均比对照绿叶水稻不育系‘GD-(?)S’高,丙二醛(MDA)含量和过氧化物酶(POD)活性则较低,显示其未表现出早衰现象;其配组的杂种间叶片衰老生理指标差异较大,部分优于对照品种‘博优253’,也未出现早衰:籼粳中间型父本所配杂种的产量和主要农艺性状显著优于对照品种‘博优253’。     

大豆生长素响应因子GmARF16参与调节叶片衰老进程

生长素响应因子(auxin response factors,ARFs)通过调节下游靶基因广泛参与植物生长发育过程,但ARFs如何调控植物叶片衰老的分子机制还不清楚。该文首先利用实时荧光定量PCR(q PCR)技术,分析大豆生长素响应基因Gm ARF16在叶片自然衰老、人工黑暗诱导衰老、外源植物生长素IAA处理条件下的表达模式,结果表明,该基因与叶片衰老调控密切相关,并且属于生长素的原初响应基因。为了进一步验证Gm ARF16基因的功能,采用农杆菌转化方法分别获得基因敲减(Gm ARF16-RNAi)和抗降解表达(m Gm ARF16)的转基因大豆植株。与非转基因对照相比,Gm ARF16-RNAi转基因大豆植株的叶片叶绿素含量和最大光量子效率(Fv/Fm)显著提高,叶片衰老标记基因(Gm CYSP1)的表达受到抑制,而m Gm ARF16转基因大豆植株则呈现出与Gm ARF16-RNAi转基因大豆植株相反的叶片生理表型。结果表明大豆生长素响应因子Gm ARF16正调节叶片的衰老进程。该研究表明,Gm ARF16在植物生长发育进程中发挥着重要作用。     

外源ipt对叶片衰老分子调控研究进展(综述)

综述了叶片衰老的分子机理,并介绍了将外源异戊烯基转移酶（ipt,isopentenyl transferase)基因转入植物以获得抗衰老植株的研究进展。     

转PSAG12-ipt基因水稻叶片中叶绿体超微结构的变化

研究了转PSAG12-ipt基因水稻和对照植株发育过程中叶片中的叶绿体结构的变化。发现水稻发育到乳熟期,转基因植株叶片中的叶绿体与对照植株开始出现明显的差别。对照叶绿体中嗜锇体体积增大,数目增多,大部分基粒的类囊体膜膨胀、裂解,片层结构解体。而转基因植株叶片中的叶绿体结构变化不大,嗜锇体相对有所增加,但体积较小,大部分基粒类囊体片层结构仍然排列整齐,少数类囊体垛叠化丧失。     

转10kD玉米醇溶蛋白基因甘薯蛋白质及农艺性状分析

目的:检测转10kD玉米醇溶蛋白基因甘薯蛋白质及农艺性状的变化,初步证明外源目的基因在转基因甘薯中能够表达。方法:以转基因甘薯和非转基因甘薯无菌苗及田间栽培苗为试材,对两者叶片、叶柄、茎、根不同器官醇溶性和水溶性蛋白质含量,以及田间栽培中植株农艺性状的变化作了比较研究。结果:离体培养20d转基因植株根中醇溶性蛋白质含量较对照增加了330.43%,是对照的4.30倍;田间栽培45d转基因植株叶片、茎、根中醇溶性蛋白质含量分别是对照植株的8.38倍、4.60倍、5.19倍,较对照分别高出738.30%、360.26%、418.60%;田间栽培中转基因植株的形态及生长状况分析表明,转基因植株中外源遗传物质的存在对植株生长状况影响不显著。结论:初步证实了转基因甘薯中外源目的基因能够特异表达。     

转山菠菜胆碱单加氧酶基因(AhCMO)水稻创制及其耐盐性研究

胆碱单加氧酶(Choline monooxygenase,CMO)是高等植物体内合成甜菜碱的一个关键酶.利用载体pCAMBIA3300与玉米泛素(Ubiquitin)启动子构建了AhCMO基因的过表达载体.采用基因枪轰击愈伤组织细胞将AhCMO基因转化东稻3号(OryzasativaL.subsp.japonica Kato),经抗性筛选,获得再生苗18株,选取农艺性状与野生型基本一致的其中7株进行PCR与Southern杂交检测,获得3株转AhCMO基因阳性植株;RT-PCR的检测结果表明外源AhCMO基因能正常转录;草铵膦叶片涂布试验结果表明,Bar基因在转AhCMO基因水稻植株中能正常表达;对T1代转基因再生植株进行0.5%NaCl高盐处理,结果表明,4号、7号转AhCMO基因水稻耐盐性强于非转基因对照,说明外源AhCMO基因能提高东稻3号的耐盐性.     

云南生态条件下基因枪法转溶菌酶基因水稻潜在变异性

在云南2种生态条件下,对基因枪法获得的转溶菌酶基因水稻高代品系D2-1-2和受体品种中花9号的农艺性状、稻米蛋白质含量、氨基酸含量和11种矿质元素进行了对照分析。结果表明转基因品系和受体对照在生育期农艺性状上无明显的差异,但转基因水稻结实率和株高明显低于受体品种;转基因水稻糙米蛋白质含量、氨基酸含量以及9种矿质元素含量都有所增加,其中钙、铁和锌等元素增加幅度较大。认为基因枪法转基因植物可能会产生一些潜在的遗传变异,因此,建议应对转基因植物进行多年多点的环境风险评价。     

通过转PSAG12—IPT基因培育延缓叶片衰老水稻

利用农杆菌介导的遗传转化方法将PSAG12 _IPT导入籼稻品种明恢 6 3。在获得的 6 1个独立的转基因植株中 ,有一些表现出叶片衰老显著延缓 ,对选择的两个纯合转基因株系的小区试验的结果显示 :(1)转基因植株倒三叶的持绿性极显著延长 ;(2 )两个纯合转基因株系比原品种的结实率极显著提高 ,株高极显著降低 ;(3)两个纯合转基因株系的有效穗数比原品种极显著或显著提高。     

转基因(SAG12-IPT)青菜的迟衰特性

利用根癌农杆菌感染方法将融合基因SAG12 IPT导入青菜 ,转基因植株明显表现出衰老延迟的生理现象。SAG12 IPT的抗衰老作用表现为 :在衰老过程中转基因青菜叶片中叶绿素含量高于未转基因的青菜 ,PCR分析结果表明该融合基因已经转入青菜中。激素检测结果表明转基因青菜叶片中细胞分裂素含量高于未转基因植株 ,说明抗衰老与叶片内细胞分裂素含量提高有关。另外 ,转基因植株不仅表现出活体植株衰老延迟 ,而且长在植株上的与离体的叶片滞绿时间延长。这些为蔬菜的耐储存育种提供了新的思路 ,同时为该融合基因在十字花科经济作物中的应用提供了理论依据。     

Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.     

Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants

Senescence is a highly regulated process accompanied by changes in gene expression. While the mRNA levels of most genes decline, the mRNA levels of specific genes (senescence associated genes, SAGs) increase during senescence. Arabidopsis SAG12 (AtSAG12) gene codes for papain-like cysteine protease. The promoter of AtSAG12 is SA-responsive and reported to be useful to delay senescence by expressing cytokinin biosynthesis gene isopentenyltransferase specifically during senescence in several plants including Arabidopsis, lettuce and rice. The physiological role of AtSAG12 is not known; the homozygous atsag12 mutant neither fails to develop senescence-associated vacuoles nor shows any morphological phenotype. Through BLAST search using AtSAG12 amino acid sequences as query, we identified a few putative homologues from rice genome (OsSAGs; Oryza sativa SAGs). OsSAG12-1 is the closest homologue of AtSAG12 with 64% similar amino acid composition. Expression of OsSAG12-1 is induced during senescence and pathogen-induced cell death. To evaluate the possible role of OsSAG12-1 we generated RNAi transgenic lines in Japonica rice cultivar TP309. The transgenic lines developed early senescence at varying levels and showed enhanced cell death when inoculated with bacterial pathogen Xanthomonas oryzae pv.oryzae. Our results suggest that OsSAG12-1 is a negative regulator of cell death in rice.     

转基因抗早衰棉的获得

利用花粉管通道技术将含有抑制衰老嵌合基因PCSAG12-ipt的pBG121质粒转入早衰型陆地棉品种中棉所10号中,通过对T1代植株进行卡那霉素田间筛选、PCR检测及GUS组织化学染色,获得了12株转基因棉花。在棉花发育进入衰老时期,对转基因植株进行叶绿素和细胞分裂素含量的测定及形态观察,结果表明转基因植株的衰老得到延迟。     

Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean

Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.     

Metabolite fingerprinting in transgenic lettuce

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。