Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit （HMW-GS）genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of To generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20--150 mg/L PPT.Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1DylO genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1DylO. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared,but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2149.0 mL) was remarkably improved,59.6%---64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3--7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.     

转基因小麦环境释放中基因漂移研究

基因漂移供体是转基因小麦，受体有转基因小麦的非转基因分离体、受体对照品系和地方小麦品种，以外源uidA基因表达作为基因漂移的分子标记，在供体开花期间，距供体5个不同距离上收集花粉，约80％的花粉分布在距供体中心15m以内，距离35m处只检测到约6％的花粉，在11个检测样点检测受体和供体间杂交率，供体与分离体混种同一小区时，基因漂移频率较高(1．08％)；与其受体对照播种于相邻小区(相距约0．5m)时，基因漂移频率居中(0．63％)；地方品种距供体边缘0．5～2．5m处，频率较低(0．25％)；3m以外没有检测到基因漂移，认为转基因小麦基因漂移距离应在3m以内。     

Introduction of rice chitinase gene into wheat via low energy Ar+ beam implantation

A chitinase gene (RCH8) in plasmid vector pCAMBIA1308 was delivered into 3 wheat cultivars (Yangmai 158, Wan 9210, Wanmai 32) by low energy Ar⁺ beam-mediated method. Preliminary calli from treated mature embryos were first selected on hygromycin (Hm, 20 or 30 mg/L) containing medium. After the resistant calli formed, they were transferred to the regeneration medium with 10 or 20 mg/L Hm. All the three wheat varieties obtained transgenic plants. PCR and PCR-Southern assays showed that most plants regenerated from the resistant calli were positive transgenic plants. Southern blot of the positive green plants confirmed stable integration of alien DNA into wheat genome. The plant transformation frequencies varied with the variety and ion dose implanted. Wanmai 32 possessed the highest transformation frequency, reaching 3.8% at a suitable implantation dose. The transformation frequency of Yangmai 158 and Wan 9210 varied from 0.5% to 2.5% and from 0.5% to 1.4%, respectively. Progeny test for resistance to wheat scab showed that the leaf extract of R₁ generation inhibited the growth of wheat scab strain R₀ and F₁₅.     

转基因小麦T1代中外源基因的检测和分析

该研究采用Multiplex PCR和SDS-PAGE技术对转1 Dx5基因(ORF)T1代小麦进行了检测和分析.结果显示,Multiplex PCR能够扩增出转基因T1代材料中1 Dx2基因和1 Dx5基因的特征片段,表明外源基因已整合到受体基因组中;SDS-PAGE检测到一新的蛋白质亚基X,表明外源基因的插入引起了转基因T1代籽粒中HMW-GS组成的变化.该研究不仅验证了线性基因片段遗传转化策略的可行性,而且印证了普通小麦Glu-1D等位基因的多重PCR分子标记体系的有效性,为培育安全型的转基因作物新种质打下坚实的基础,加快小麦分子育种进程.     

A transgenic wheat with a stilbene synthase gene resistant to powdery mildew obtained by biolistic method

Stilbene, a kind of phytoalexin, plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spore. Stilbene synthase gene (Vst1) obtained from grapevine has been transferred into common spring wheat Jinghong 5 by using the biolistic transformation method. Five transgenic plants (T₀) were obtained from the bombarded 2014 immature embryos. One immune plantlet and 3 plantlets with mid-resistance to powdery mildew were identified from the transgenic plants of T₃ generation which came from 2 T₀ transgenic plants.     

Molecular mapping of a novel yellow rust resistance gene of wheat using microsatellite markers

Identification and genetic analysis of yellow rust resistance have suggested that wheat line R55 carries single dominant gene conferring yellow rust resistance. The bulked segregant analysis (BSA) for an F₂ population using microsatellite marker technique has indicated that the yellow rust resistance gene is located on the short arm of chromosome 1B, tightly linked to the microsatellite markers WMS11-193 bp and WMS18-184 bp, the linkage distance between the markers and the gene is 1.9 cM. This gene has been formally namedYr26. It is inferred from the pedigree, resistance and gene locus analysis that theYr26 has been transferred fromTriticum turgidum L. and is different from the other known yellow rust resistance genes.     

转基因小麦小花开放特性与基因漂移关系研究

选用3个转基因小麦、2个受体对照与170个地方品种对照，在武汉进行环境释放试验．约20％的小麦地方品种与转基因小麦花期相遇，所有地方品种开花时柱头都部分外露，且小花开放度和开放时间品种间有差异，表明存在着转基因小麦以花粉为媒介向地方品种发生基因漂移的可能性，且不同地方品种作为接受者，在不考虑其他影响因素时，因小花开放度的差异发生基因漂移的可能性也应存在差异．     

Chromosome engineering of pollen wheat

Chromosome engineering of pollen wheat is the new procedure combining anther culture and chromosome engineering techniques. It could transfer useful alien genes into wheat varieties, enhancing genetic diversity for investigation of genetics and breeding. In the present study, two new procedures, at genome level and single chromosome level, were established. Compared with the classical chromosome engineering, the chromosome engineering of pollen wheat has the following main characters: (i) diversity of products, (ii) rapid stability and (iii) high efficiency of selection. Experiments indicated that chromosome engineering of pollen wheat is an efficient way for creating alien translocation line, especially non-Robertsonian translocation line. Meanwhile, using this procedure combined with comprehensive identifying methods, the investigation of useful genes and molecular markers on rye chromosomes 1R and 6R respectively has been done. The roles and relationships between chromosome engineering of pollen wheat and gene engineering on crop improvement were discussed.     

Characterization of a PDR type ABC transporter gene from wheat （Triticum aestivum L.）

DON, as a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study, GeneChip analysis indicated that an EST encoding an ATP-binding cassette （ABC） transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers were designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance （PDR） sub-family, and hence designated as TaPDR1 （Triticum aestivum pleiotropic drug resistance）. TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCI. However, TaPDR1 expression was regulated by Al^3＋ and [Ca^2＋], indicating that [Ca^2＋]1 might mediate the signal of TaPDR1 expression.     

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

“Bainong 3217 × Mardler” BC₅F₄ wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.     

基因枪法获得可育抗除草剂转基因小麦

用基因枪法对两个春小麦品种进行了遗传转化，获得１９株独立转化的，抗除草剂Ｂａｓｔａ的小麦植株，其中１５株自交可育，ＰＣＲ和ＤＮＡ印迹检测证实了该基因在转化植株中的表达，转基因及其表达已遗传到子三代，在已检测的７个转基因后代中，有４个植株其抗除草剂性状以单位点，显性性状的孟德尔方式遗传。     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

Influence of alloplasm on resistance to wheat scab

Using alloplasmic wheat as test materials, the genetic effect of cytoplasm on wheat scab resistance was studied by three methods including the determination of resistance of explant calli toGibberella zeae toxin, the identification of inoculating single fixed position and the identification of field resistance. The results showed that among “the homonucleo-heterocytoplasmic lines”, resistance of calli toG. zeae toxin was different; the difference of cytoplasmic effect among different nucleoplasm combinations showed certain nucleoplasm interaction relation. Resistance of hybrid F₁ plants of reciprocal cross between alloplasmic line and disease-sensitive wheat variety to wheat scab was of clear difference. Hybrid F₁ of reciprocal cross betweenAe. ventricosa cytoplasmic wheat and disease-sensitive or -resistant wheat variety all exhibited fine resistance effect of alloplasm. Compared with general common wheat variety, most alloplasmic wheats tested have stronger resistance. A few selected lines havingAe. ventricosa cytoplasm have a strong and stable resistance to scab. They can be a finer new resistant source.     

白粉菌对抗、感白粉病小麦品种叶片组织侵染情况的研究

采用透明染色法对白粉菌15号生理小种侵染的小麦叶片进行处理,观察不同时间段白粉菌对京411,Brock及其杂交后代近等基因系（NIL）叶片的侵染情况。结果表明：Brock,NIL抗病耐受性均强于京411,此结果可为今后抗病基因的克隆、植物抗性信号转导机制等研究提供细胞学上的依据。     

Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP)expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the $6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.