通过根癌农杆菌介导的共转化获得具有蜡质基因反义片段的转基因水稻

在构建剔除潮霉素抗性基因的反义蜡质基因重组质粒p13W8的基础上,用分别携带p13W8质粒和有潮霉素抗性基因的pCAMBIA1300质粒的根癌农杆菌,在不同处理条件下对粳稻3个品种进行共转化.试验结果显示,共感染混合液中pCAMBIA1300菌液比例高,抗性愈伤组织的转化频率也较高;抗性愈伤组织的PCR检测结果表明,潮霉素抗性基因的阳性检测率为98%,反义蜡质基因的阳性检测率为68%.在105株转基因植株中,反义蜡质基因阳性株为30株,占转基因植株的28.6%.乙酰丁香酮处理组的平均转化频率略高于未处理组;在乙酰丁香酮的各种处理中,直接浸泡愈伤组织的转化频率稍高于其它处理方式.     

通过共转化和花药培养快速获得直链淀粉含量降低且无抗性标记的转基因水稻

用根癌农杆菌共转化的方法将p13W8质粒(含反义蜡质基因)和pCAMBIA1300质粒(含潮霉素抗性基因)导入水稻.经PCR检测,发现在29个T1群体中发生抗潮霉素抗性基因和反义蜡质基因的分离;从1 264个T1单株中筛选到183个只带反义蜡质基因片段的转基因植株.选择了4个直链淀粉含量降低的T1单株进行花药培养,获得正常结实的花培苗34株;经PCR检测,有23株为只含反义蜡质基因而无潮霉素抗性基因的植株.从转化到获得直链淀粉含量降低、遗传稳定的转基因植株仅用了一年半时间.     

杂交水稻直链淀粉含量遗传分析

杂交水稻稻谷是从Ｆ１代杂合体收获的Ｆ２代种子,由于Ｆ２代属分离群体,直链淀粉含量等稻米品质会发生分离现象．本研究选择直链淀粉含量低、中、高的５个亲本配组的５个杂交组合,按单粒分析法分析双亲亲本、Ｆ１和Ｆ２代的种子的直链淀粉含量．结果表明,Ｆ２代呈现１（低）：３（中＋高）孟德尔分离规律．在５个组合的单粒分析中发现分离的Ｆ２代群体呈双峰的连续分布,该现象表明高直链淀粉含量对低直链淀粉含量为不完全显性,除单一基因控制外,还受微效多基因修饰．因此杂交水稻育种工作中,尤其是优质杂交稻新组合选育时,必须注意选择双亲的直链淀粉含量基本一致,以防分离现象影响米质．     

Field Performance of Transgenic indica Hybrid Rice with Improved Cooking and Eating Quality by Down-regulation of Wx Gene Expression

High amylose content (AC) in rice endosperm is correlated with poor grain quality, particularly in indica hybrid rice. We have generated several homozygous transgenic parent lines of indica hybrid rice carrying an antisense Waxy (Wx) gene and demonstrated that the AC in seeds of these lines decreased dramatically. Two transgenic maintainer lines (L25B and L18B), derived from one of the key maintainer parents of an indica hybrid rice in China, Long-te-fu B (LTF-B), were selected and the antisense Wx gene was subsequently introgressed into the male-sterile counterpart, LTF-A, with the aim to generate improved indica hybrids. The indica hybrids derived from the selected transgenic male-sterile lines and restorer lines were tested for quality and agronomic performance under normal field conditions. Our results demonstrated that the reduction of AC in the homozygous transgenic maintainer lines stably passed down in five successive generations and the improved quality was also found in their relevant transgenic hybrids produced. The other two key characters of rice cooking and eating quality, the gel consistence (GC) and gelatinization temperature (GT), were also improved in the grains of both the transgenic maintainer lines and their relevant hybrids. In addition, no change was observed for most of the agronomic characters of the transgenic maintainer lines and the relevant transgenic hybrids. Although the grain weight of the transgenic line was reduced, the grain yield of the homozygous transgenic parent lines and the transgenic hybrids was similar when compared with that of the wild-type controls. These results suggest that transgenic approaches are an effective way to obtain rice lines with both improved qualities and high yield, especially for indica hybrid rice.     

基因枪法转基因水稻中hpt基因稳定遗传

基因枪转化将潮霉素磷酸转移酶基因（ｈｐｔ）导入粳稻品种７７１７０,获得可育的转基因植株,研究外源基因遗传的稳定性。自交后代（Ｔ１和Ｔ２）经潮霉素筛选获得抗性植株和敏感植株,分子鉴定结果表明抗性植株带有ｈｐｔ基因,而敏感植株中没有ｈｐｔ基因存在。Ｔ１和Ｔ２代中潮霉素抗性表现为显性单基因位点的遗传方式,符合孟德尔分离规律,并得到分子鉴定结果的证实。Ｓｏｕｔｈｅｒｎ杂交结果显示,ｈｐｔ基因多拷贝整合在水     

玉米pepc基因在杂交转育的转基因水稻后代中的传递和表达特征

在合肥、海南两地以高光效转玉米pepc基因水稻为父本,与培矮64S、2302S、2304S、2306S、5129、02428、皖粳97和双九A等8个受体杂交,转育转pepc基因水稻新种质材料。至2002年已转育成一批转pepc基因水稻品系,对这些材料世代跟踪研究显示：(1)玉米pepc基因以一个显性基因稳定传递给后代,符合孟德尔分离规律,自交F2呈3：1分离,BC1为1：1分离;(2)玉米pepc基因在杂交转育的转基因水稻中高水平表达,与受体亲本相比,PEPC活性提高3.7～17．4倍,表达水平与受体亲本和转基因拷贝数有关,来源相同的世代间有相似的表达水平,但同一世代不同个体间表达水平有差异,这可能与其位置效应、拷贝数和环境条件有关;(3)杂交后代结实率不高,容易产生异交结实导致转基因材料混杂分离。采取抗生素催芽初筛、PCR分析抽检、PEPC活性测定和田间表型观测的转玉米pepc基因水稻选育筛选体系,辅之以受体为轮回亲本适当回交可有效地控制。利用该途径已育成3个稳定的高表达的转玉米pepc基因水稻品系H1596、H1597和Y1470,说明通过常规杂交转育的方法,可以培育实用的稳定高表达的转玉米pepc基因水稻品种。     

水稻蜡质基因引导区的两个SSR序列与直链淀粉含量的相关性(英文)

分析了蜡质基因引导区的两个简单重复序列 (SSR) (CT) n 和 (AATT) n 在 74份水稻材料中的多态性及其与直链淀粉含量 (AC)的关系。这些材料包括了籼稻 (OryzasativaL .ssp .indica)、粳稻 (O .sativassp .japonica)和普通野生稻 (O .rufipogon) ,其AC值覆盖了栽培稻AC分布的整个范围。以 (CT) n 作标记检测到 8个等位基因 ,粳稻品种趋于含有重复数目较多 (n≥ 16 )的等位基因 ,重复次数较少 (n≤ 14)的等位基因只出现在籼稻中。 (AATT)n检测到 2个等位基因 ,野生稻中少数植株表现出杂合性。分析表明AC与这两个SSR序列基因型高度相关 ,高AC (>2 2 .0 % )品种具有 (CT)重复次数较少 (n≤ 14)的等位基因 ;相反 ,除了糯米外 ,所有低或者中等AC的品种都有 (CT)重复数较多 (n≥ 16 )的等位基因。具有重复次数较多的 (AATT) 6等位基因的品种多为高AC ,具有重复次数较少的(AATT) 5等位基因的品种多为低或中等AC。不同SSR基因型品种间AC差异极显著。虽然目前还不能确定这两个SSR序列在直链淀粉合成中的直接功能 ,SSR变异与AC间近乎完全的相关性可作为分子标记直接用于水稻的品质改良。     

导入反义蜡质基因改良水稻稻米的食味品质和营养品质

经PCR、Southern blot和稻米GUS检测,蜡质基因反义片段与gus构成的融合基因已整合到8株水稻基因组中,并能正确表达。通过稻米GUS染色追踪分析获得转反义蜡质基因纯合后代。将转基因植株的稻米送农业部稻米及制品质量监督检验测试中心进行糙米蛋白质含量和直链淀粉含量测定。结果显示：（1）在转反义蜡质基因纯合的超2-10粳稻后代中,大多数单株糙米在直链淀粉含量降低的同时,蛋白质含量有不同程度提高,糙米蛋白质含量和直链淀粉含量呈显著负相关关系;（2）对照糙米直链淀粉平均含量和糙米蛋白质平均含量分别为13.4%和9.5%。在转基因植株中,稻米直链淀粉含量最低的为11.4%,而蛋白质含量也相对最高,为13.5%。本试验结果表明在水稻中导入反义蜡质基因不仅能够降低水稻稻米的直链淀粉含量,还有可能提高水稻稻米的蛋白质含量。     

水稻bicoid反义基因转基因植株胚胎发育的形态变化

Bicoid是调控果蝇胚胎极性及以后分节发生过程的重要基因,这一基因编码的蛋白是一种转录调控因子,对一组早期胚胎发育过程中的多齿基因的表达具有调控作用,并被称为形态发生剂。在以前的工作中,从水稻cDNA文库中分离了一个同果蝇bicoid有相当同源性的克隆-Rb24基因（EMBL登录号：AJ2771380）。为进一步了解它在水稻中功能,将Rb的反义基因片段通过农杆菌的介导转入水稻中。通过Gus活性检测和PCR鉴定所获的抗性植株为转基因植株,转基因植株一个很明显的变化就是大约有50%的种子是败育的。通过石蜡切片观察了转基因水稻幼胚的发育情况,结果表明细胞的组织的分化在胚的发育过程中被阻断了。因此Rb基因同控制水稻胚的发育有关。     

高赖氨酸蛋白基因导入水稻及可育转基因植株的获得

构建了一个植物高效表达质粒,使来源于四棱豆（Psophocarpus tetragonolobus(L.)DC)的高赖氨酸蛋白基因（lys）受控于单子叶植物ubiqutin强启动子下表达。用基因枪法将其导入水稻(Oryza sativa L.)幼胚诱导的愈伤组织,经潮霉素抗性筛选,得到可育的再生植株。经PCR和Southem blotting检测,表明该基因已整合到水稻的基因组织。GUS组织化学染色表明转基因水稻植株的叶、茎和根中均有gus基因的表达。测定112株转基因水稻叶片中赖氨酸叶量,大部分植株有不同程度的提高,最高幅度为16．04％。     

cry1A基因在转基因玉米中的遗传与表达

通过Southern杂交、ELISA分析等方法, 研究了cry1A基因在转基因玉米中的遗传与表达.结果表明,cry1A基因在转基因玉米中呈单位点显性基因遗传.cry1A杀虫蛋白在转基因玉米不同株系中的表达量存在显著差异;在转基因玉米同一植株不同组织中的表达量也明显不同,在叶片、苞叶等绿色组织中的表达量显著高于在髓、花丝等非绿色组织中的表达量;在玉米叶片中的表达量随着发育期的推进有上升的趋势;在研究的3个转基因玉米株系中,cry1A杀虫蛋白的表达量在R2、R3、R4代之间无显著差异.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

cry1A基因在转基因玉米中的遗传与表达(英文)

通过Southern杂交、ELISA分析等方法 ,研究了cry1A基因在转基因玉米中的遗传与表达。结果表明 ,cry1A基因在转基因玉米中呈单位点显性基因遗传。cry1A杀虫蛋白在转基因玉米不同株系中的表达量存在显著差异 ;在转基因玉米同一植株不同组织中的表达量也明显不同 ,在叶片、苞叶等绿色组织中的表达量显著高于在髓、花丝等非绿色组织中的表达量 ;在玉米叶片中的表达量随着发育期的推进有上升的趋势 ;在研究的 3个转基因玉米株系中 ,cry1A杀虫蛋白的表达量在R2 、R3 、R4代之间无显著差异。     

Inheritance and segregation of exogenous genes in transgenic cotton

Three transgenic cotton varieties (lines) were chosen for the study of inheritance and segregation of foreign Bt (Bacillus thuringiensis toxin) andtfdA genes in cotton. The transformed cotton varieties CCRI 30 and NewCott 33B expressing the BtcryIA gene, and cotton line TFD expressing thetfdA gene were crossed with CCRI 19, CCRI 12 and Lumian 6. The results confirm inheritance and segregation of (i) the exogenous Bt gene in transgenic CCRI 30 and NewCott 33B, governing resistance to bollworm, and (ii) the exogenoustfdA gene in transgenic TFD, governing resistance to the herbicide 2,4-D. Both resistance characters were governed by a single dominant nuclear gene, and were not affected by cytoplasm. Our data support the conclusion that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. Our results also indicate that a practical backcross breeding program could be used to develop cotton cultivars combining one or more resistance traits from foreign and native gene sources.     

Transgenic Rice Plants Expressing Bacillus subtilis Protoporphyrinogen Oxidase Gene Show Low Herbicide Oxyfluorfen Resistance

Transgenic rice plants harbouring Bacillus subtilis protoporphyrinogen oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T₁ generation by Southern and mRNA expression in T₂ generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improved Performance of Transgenic Glycinebetaine-Accumulating Rice Plants under Drought Stress

Plasmid DNA (pChlCOD), containing the selectable hygromycin phosphotransferase hpt gene for hygromycin B resistance and the Arthrobacter globiformis codA gene for choline oxidase which catalyzes the direct conversion of choline to glycinebetaine, was delivered into rice plants using Agrobacterium-mediated gene transfer via scutellum-derived calli. Southern, Northern and Western blot analyses demonstrated that the foreign gene had been transferred, integrated into rice chromosomal DNA and expressed. Drought test indicated that glycinebetaine acts as an osmoprotectant and its production in transgenic rice plant helped the cells to maintain osmotic potential and increased root growth, and thus enhanced the ability of the plants to tolerate water deficit This revised version was published online in July 2006 with corrections to the Cover Date.     

脆茎基因在水稻亚种间杂种后代中的遗传

脆茎基因在水稻粳型品种间杂种后代中按孟德尔遗传比例传递,而在亚种间杂种后代中呈脆茎(bcl bcl)频率显著增加和显著降低两种偏离孟德尔比例的异常分离型。Kamairazu／／Ketan Nangka／Kamairazu F1呈脆茎频率增加型,F2群体则呈正常分离,但F3和F4仍出现少数增加型和降低型的株系。IR36／Kamairazu的F2呈降低型分离,F3和F4株系中有正常型和降低型两种,F4还出现个别增加型株系。Ketan Nangka／IR36／／Kamairazu的F2中除了有增加型和降低型外,出现部分正常型。水稻bcl基因的异常分离与花粉育性无关,而是由雄配子选择性授精引起,与位于第3染色体的配子体基因ga2、ga3和ga14有关。     

Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)

We investigated the evolutionary dynamics of duplicated copies of the granule-bound starch synthase I gene (GBSSI or Waxy) within polyploid Spartina species. Molecular cloning, sequencing, and phylogenetic analyses revealed incongruences between the expected species phylogeny and the inferred gene trees. Some genes within species were more divergent than expected from ploidy level alone, suggesting the existence of paralogous sets of Waxy loci in Spartina. Phylogenetic analyses indicate that this paralogy originated from a duplication that occurred prior to the divergence of Spartina from other Chloridoideae. Gene tree topologies revealed three divergent homoeologous sequences in the hexaploid S. alterniflora that are consistent with the proposal of an allopolyploid origin of the hexaploid clade. Waxy sequences differ in insertion–deletion events in introns, which may be used to diagnose gene copies. Both paralogous and homoeologous coding regions appear to evolving under selective constraints.     

水稻直链淀粉和蛋白质突变系筛选及其与主要农艺性状的关联分析

研究利用重离子辐照杂交籼稻9311创建农艺性状突变体库,使用化学方法筛选直链淀粉、蛋白质突变材料,并分析籽粒品质性状与农艺性状之间的相关性,为后续筛选直链淀粉、蛋白质突变体工作奠定基础。结果显示:在169份直链淀粉、蛋白质突变体中,直链淀粉含量变幅范围是7.64%~32.37%,其中高含量直链淀粉突变体材料有11份,低含量直链淀粉突变体材料有5份;蛋白质含量变幅范围是7.05%~13.79%,其中高蛋白突变体有34份,低蛋白突变体有3份。从169份突变体中筛选出直链淀粉、蛋白质含量都有梯度差异的突变体材料,筛选出材料的农艺性状分析结果表明:在直链淀粉、蛋白质突变体材料的农艺性状中,每穗实粒数、有效穗数、结实率和株高这四个农艺性状与籽粒品质性状间有关联,变异系数分别为37.53%、30.72%、24.70%、15.38%。相关性结果表明:直链淀粉含量和蛋白质含量呈负相关,直链淀粉含量与农艺性状呈正相关,蛋白质与农艺性状呈负相关,其中蛋白质含量和每穗实粒数、结实率呈极显著负相关,相关系数分别为-0.504、-0.592。     

水稻rbcS启动子控制的外源基因在转基因水稻中的特异性表达

为将不同启动子用于转基因水稻的研究,从武运粳8号水稻中克隆了Rubisco小亚基基因(rbcS)的5'上游调控区,构建了由rbcS启动子引导的GUS融合基因,并经农杆菌介导导入到水稻中.对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS启动子可驱动GUS报告基因在转基因水稻植株叶片和叶鞘内的叶肉细胞中特异性高效表达,而在茎、根和种子等器官中不表达或表达活性极弱,表现出明显的组织与细胞特异性.结果还表明,光诱导处理可明显提高rbcS启动子启动的外源基因的表达量.