籼稻转反义蜡质基因后代的直链淀粉含量测定和纯系选育

通过根癌农杆菌介导将反义蜡质基因导入籼型雄性不育保持系龙特甫B中,获得30个PCR检测为阳性的转基因植株,其中,28个为Southern检测阳性.T1种子直链淀粉含量测定结果表明,有21个转基因植株比龙特甫B明显下降,下降幅度为3%-13%,并在部分转基因植株的种子中观察到蜡质状籽粒;对6个转基因植株进行了不同世代的直链淀粉含量测定,在L3和L5的T4代中,选择到直链淀粉含量分别为15.9%和8.4%的纯合株系,经凝胶电泳测定,Wx蛋白量明显降低,并与直链淀粉含量下降表现一致.以L3-1-1-1(15,9%)和L5-8-2-1(8.4%)纯合株系为亲本,分别与龙特甫A进行成对杂交和回交,并测定了F1和B1F1种子直链淀粉含量,以L3-1-1-1作亲本的F1为21.4%,B1F1为17.1%;以L5-8-2-1作亲本的F1为13.6%,B1F1为9.3%,结果表明在不育系的转育过程中,以中低直链淀粉含量的转基因纯合株系为亲本,能有效降低F1和B1F1的种子直链淀粉含量.     

转反义蜡质基因水稻亲本后代性状分析

以单质粒转化的粳稻广粳一号和双质粒共转化的籼稻01早5202所获得的转反义蜡质基因水稻后代为供试材料, 通过潮霉素抗性、PCR检测等手段分析了外源基因的遗传分离特性, 同时, 还分析了转基因材料的直链淀粉含量、waxy蛋白含量的变化特性。结果表明, 无论是采用标记基因(hpt)与目的基因(Anti-sense waxy)连锁的单质粒转化, 还是采用双质粒共转化, 其供试的后代植株材料都发生了外源基因分离现象, 且转基因植株材料的直链淀粉含量都有所下降, 有些单株的直链淀粉含量已降至10%(质量百分比)以下, 远低于对照(其直链淀粉含量为22.04%); SDS-PAGE检测结果显示, 供试的转基因材料的waxy蛋白的含量与对应的直链淀粉含量呈正相关性。     

籼稻和粳稻中蜡质基因座位上微卫星标记的多态性及其与直链淀粉含量的关系

稻米直链淀粉是在由蜡质基因Wx编码的颗粒结合淀粉合成酶（GBSS）的催化下合成的。最近,在Wx基因的区段内发现了一段多态性微卫星序列（CT）n。对74个非糯籼稻和粳稻材料的（CT）n多态性进行了分析,并探讨了其与直链淀粉含量之间的关系。在74个品种（系）中共发现7种（CT）n片段（Wx等位基因）,即（CT）8,（CF）10,（CT）11,（CT）16,（CT）17,（CT）18,（Ch）19。在籼粳亚种间,不同（CT）n的分布存在差异较大：在籼稻中,以（CT）11和（CT）18为主,占92．6％,另有（CT）10和（CT）8各2份,（CT）17型1份;在粳稻中,以（CT）16、（CT）17为主,共占20份材料中的90．0％。在上述74个品种（系）中,以（CT）n表示的Wx基因型对稻米直链淀粉含量的决定系数R2达0．912,也即Wx基因型差异可解释这些材料直链淀粉含量变异的91．2％。还发现6份籼稻材料Wx座位上为杂合的（CT）18／（CT）11,其中2份为推广早籼优质品种浙9248和舟优903,并对其在遗传和育种研究中的意义作了探讨。     

籼稻和粳稻中蜡质基因座位上微卫星标记的多态性及其与直链淀粉含 …

稻米直链淀粉是在由蜡质基因Ｗｘ编码的颗粒结合淀粉合成酶（ＧＢＳＳ）的催化下合成的,最近,在Ｗｘ基因的区段内发现了一段多态性微卫星序列（ＣＴ）ｎ,对７４个非糯籼稻和粳稻材料的（ＣＴ）ｎ多态性进行了分析,并探讨了其与直链淀粉含量之间的关系,在７４个品种（系）中共发现７种（ＣＴ）ｎ片段（Ｗｘ等位基因）邓（ＣＴ）８,（ＣＴ）１０,（ＣＴ）１１,（ＣＴ）１６,（ＣＴ）１７,（ＣＴ）１８,（ＣＴ）１９,在籼粳     

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。     

水稻蜡质基因引导区的两个ＳＳＲ序列与直链淀粉含量的相关性

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不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（Oryza sativa L.ssp.indica)、粳稻（Ｏ.sativa ssp.japonica)和普通野生稻（O.rufipogon),其ＡＣ值 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（＞２２．０％）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,所有低或中等ＡＣ的品种都有（ＣＴ）重复较多（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。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。     

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.     

转反义蜡质基因‘湘晴’及其杂交稻米的直链淀粉含量研究

利用根癌农杆菌介导将反义蜡质基因(anti-Waxy)导入三系恢复系湘晴水稻获得转基因植株.从T1代外源基因呈单拷贝整合的转基因湘晴水稻中选取3个稻米直链淀粉含量降低较明显的单株继续种植得到纯合后代.以纯合转基因植株及湘晴水稻(对照)为恢复系分别与寒丰不育系杂交,获得4组杂交稻后代(F2).3个T3代纯合转基因湘晴水稻糙米(T4)直链淀粉含量分别为14.42%、13.96%和14.72%,对照为16.04%;3组由转基因湘晴水稻为父本杂交制种后代(F2)糙米直链淀粉含量分别为14.53%、13.77%和14.64%,对照杂交稻(F2)为16.22%.研究表明,导入湘晴水稻中的反义蜡质基因不仅能够降低湘晴稻米直链淀粉含量,还能够进一步抑制杂交后代的稻米直链淀粉合成.     

小麦蜡质基因座位上SSR的多态性及其与直链淀粉含量的关系

分析了蜡质基因(Wx)3′端的一个简单重复序列(SSR)(AT(AT)n AT)在32份小麦中的多态性及其与直链淀粉含量(AC)的关系。这些材料包括了山东省内的不同小麦品种,其AC含量差别较大。在扩增的小麦品种中均出现两条带,一条204bp(Wx-Dla)位于7DS染色体上,另一条225—346bp(Wx-Ala)位于7AS染色体上。也就是说在扩增的小麦品种中均存在Wx-Ala和Wx- Dla基因,并且较长的片段显示了长度多态性。分析表明AC与7D上这个SSR序列基因的多态性呈正相关,高AC小麦品种扩增的片段较长,电泳迁移率较小;相反,低AC小麦品种扩增的片段较短,迁移率较大,不同长度SSR品种间AC差异显著。虽然目前还不能确定这个SSR序列在直链淀粉合成中的直接功能,但SSR变异与AC之间的相关性可作为分子标记直接用于小麦品种改良。     

云南软米直链淀粉含量的遗传分析与基因定位

以低直链淀粉( 软米)品种毫木细与高直链淀粉品种桂朝2号杂交F2代分离群体为试验材料,研究水稻低直链淀粉含量的遗传规律和基因定位。结果表明,软米品种毫木细直链淀粉含量受一个隐性主效基因/QTL控制,该基因位点（QTL）与糯性基因(wx)为非等位。除主效QTL外,可能还有微效基因的作用。用SSR标记检测到该主效基因/QTL位于11号染色体上RM224附近,LOD值为15.4,可解释的表型变异率为32%。     

Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch

The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the rice waxy gene, which includes the region between exon 4 and exon 9, was inserted in an antisense orientation between the 35 S promoter and the GUS gene of pBI221. The resultant plasmid, pWXA23, was introduced into rice protoplasts by electroporation. GUS activity was clearly detected in derived callus lines, suggesting that the antisense component of the fusion gene was also expressed. Transgenic rice plants were regenerated from these callus lines and their GUS activity was confirmed. Some of the rice seeds from these transformants showed a significant reduction in the amylose content of grain starch, even though they had become polyploid. These results suggest that even when intron sequences are included, antisense constructs can bring about a reduced level of expression of a target gene. The utility of GUS, included as a reporter gene, for the simple detection of expression of an antisense gene, was apparent from these results.     

Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.     

抗虫基因转化优良籼型保持系D297B的研究

本文报道了用基因枪法将雪花莲外源凝集素基因(gna)转移到优良籼型杂交稻保持系D297B中。通过PCR、Southern blotting和Western blotting等分子检测方法证明,外源基因已整合到受体材料的基因组中并得到表达。蛋白活性测定结果显示转基因在受体中表达的蛋白具有生物活性。潮霉素抗性分析表明外源基因多数是以单位点形式整合并以3：1方式遗传。另外,PCR证据也说明抗虫基因与选择标记基因基本上是共整合和共遗传的。     

Activity of starch synthase and the amylose content in rice endosperm

The content of amylose in endosperm of non-waxy japonica rice (Oryza sativa cv Akitakomachi) was increased by lowering the growth temperature from 25° to 15° during the ripening period. The activities of sucrose synthase, ADPglucose pyrophosphorylase, starch branching enzyme (Q-enzyme) and soluble starch synthase in endosperm developed at 15° were lower than or similar to those at 25°, when compared on a endosperm basis at the similar ripening stage. In contrast, the activity of starch granule-bound starch synthase, which is considered to be indispensable for amylose synthesis, was higher by 3–3.5-fold in the endosperm developed at the low temperature than that at the high ambient temperature. The results suggest that the low temperature specifically accelerates the expression of the bound starch synthase gene (waxy gene) in rice endosperm, which resulted in elevated amylose biosynthesis in the endosperm when developed at lower temperatures.     

Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene

Stemborers and leaffolders are two groups of lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, a cry1C* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin was synthesized by codon optimization as the first step towards gene stacking in our resistance management strategy of transgenic rice. Agrobacterium-mediated transformation of this gene into Minghui 63 (Oryza sativa L.), an elite indica CMS restorer line, produced 120 independently transformed plants, 19 of which had a single-copy transgene. Preliminary screening of T₁ families of these 19 transformants in the field identified five lines showing a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Hybrids were produced by crossing these five lines with Zhenshan 97A, the male-sterile line for Shanyou 63, the most widely cultivated hybrid in China. These five lines and their hybrids were highly resistant to yellow stemborer (Tryporyza incertulas) as revealed by an insect bioassay. The content of Cry1C* protein varied considerably among the five lines as well as among the corresponding hybrids. T1c-19, a line showing the highest content of Cry1C* protein, and its hybrid were tested in the field for insect resistance and agronomic performance and found to be highly resistant to stemborers and leaffolders throughout the growth period, resulting in a significantly increased grain yield compared with the respective controls. These results indicate that T1c-19 can be used for production of insect-resistant hybrid rice and as a germplasm for gene stacking to produce rice plants with two toxins.     

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2^nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.