Analysis on Drought Tolerance of TaPIMP1 Transgenic Wheat

[Objective] This study aimed to evaluate the application potential of wheat MYB protein-encoding gene TaPIMP1 in the breeding of drought-tolerant wheat germplasm. [Method] The homozygous transgenic lines with TaPIMP1 gene which is driven by ubiquitin promoter and their recipient Yangmai 158 were cultivated under simulated drought condition. Then, seed germination, seedling growth, and some biochemical parameters associated with abiotic stress of the three cultivars were an- alyzed. [Result] In the medium containing 20% PEG6000, the expression level of TaPIMP1 gene in all the three wheat cultivars changed greatly within the first 24 h; the seed germination rate, the lengths of coleoptile and radical of two transgenic lines B64 and B208 were significantly higher than those of control Yangmai 158. In the medium containing 10%-20% PEG6000, the leaf relative water content and sol- uble sugar contents of two TaPIMP1 transgenic lines were significantly higher than those in control. In the medium containing 15%-20% PEG6000, the MDA content of two transgenic lines was significantly lower than that of the control. [Conclusion] The drought tolerance of the two transgenic lines has been improved obviously com- pared with Yangmai 158, so the TaPIMP1 gene can be introduced to develop new drought-tolerant wheat cultivars.

Overexpression of GmDREB1 improves salt tolerance in transgenic wheat and leaf protein response to high salinity

The transcription factor dehydration-responsive element binding protein(DREB)is able to improve tolerance to abiotic stress in plants by regulating the expression of downstream genes involved in environmental stress resistance.The objectives of this study were to evaluate the salt tolerance of GmDREB1 transgenic wheat(Triticum aestivum L.)and to evaluate its physiological and protein responses to salt stress.Compared with the wild type,the transgenic lines overexpressing GmDREB1 showed longer coleoptiles and radicles and a greater radicle number at the germination stage,as well as greater root length,fresh weight,and tiller number per plant at the seedling stage.The yield-related traits of transgenic lines were also improved compared with the wild type,indicating enhanced salt tolerance in transgenic lines overexpressing GmDREB1.Proteomics analysis revealed that osmotic-and oxidative-stressrelated proteins were up-regulated in transgenic wheat leaves under salt stress conditions.Transgenic wheat had higher levels of proline and betaine and lower levels of malondialdehyde and relative electrolyte leakage than the wild type.These results suggest that GmDREB1 regulates the expression of osmotic-and oxidative-stress-related proteins that reduce the occurrence of cell injury caused by high salinity,thus improving the salt tolerance of transgenic wheat.

RNA-seq analysis of unintended effects in transgenic wheat overexpressing the transcription factor GmDREB1

The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than the genetic modification for simple monogenic traits. For this reason, it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. We selected drought-and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, to investigate unintended pleiotropic effects using RNA-seq analysis. We compared the transcriptome alteration of transgenic plants with that of wild-type plants subjected to salt stress as a control. We found that GmDREB1 overexpression had a minimal impact on gene expression under normal conditions.GmDREB1 overexpression resulted in transcriptional reprogramming of the salt response,but many of the genes with differential expression are known to mitigate salt stress and contribute incrementally to the enhanced stress tolerance of transgenic wheat. GmDREB1 overexpression did not activate unintended gene networks with respect to gene expression in the roots of transgenic wheat. This work is important for establishing a method of detecting unintended effects of genetic engineering and the safety of such traits with the development of marketable transgenic crops in the near future.

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0 - 1.5mm in size, C58cl of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production, shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptll ELISA (nptII enzyme assay), PCR, Southern blot and leaf bleach, 29 positive plants were identified from two genotypes of Bobwhite and Yangmai 10, with an average transformation efficiency of 0.82%. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.

Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Metabolic profiling of DREB-overexpressing transgenic wheat seeds by liquid chromatographymass spectrometry

DREBs are transcription factors that regulate abiotic stress tolerance in plants.Previously,we reported that wheat transgenic lines overexpressing Gm DREB1 showed increased tolerance to drought and salt stress.However,the molecular basis of increased tolerance is still poorly understood,and whether the overexpression of DREB will cause unexpected effects is also of concern.We performed seed metabolic profiling of the genetically modified(GM)wheat T349 and three non-GM cultivars with LC-MS to identify the metabolic basis of stress tolerance and to assess the unexpected effects of exogenous gene insertion.Although we did not note the appearance of novel metabolites or the disappearance of existing metabolites,overexpression of the transcription factor Gm DREB1 in T349 wheat influenced metabolite levels in seeds.Increased levels of stress tolerance-associated metabolites were found in the stress-sensitive non-transgenic acceptor counterpart J19,while metabolites associated with cell membrane structure and stability accumulated in T349.Among these metabolites in T349,most showed levels similar to those in the non-GM wheats.Overexpression of Gm DREB1 in T349 may cause a shift in its metabolic profile leading to down-regulation of several energy-consuming processes to favor increased yield under stress conditions,which is a reasonable expectation of breeders while creating the GM wheat and Gm DREB1 overexpression did not cause unexpected effects in T349 seeds.These results may be helpful for GM crop research and risk assessment.

Transgenic expression of TaTLP1, a thaumatin-like protein gene, reduces susceptibility to common root rot and leaf rust in wheat

Thaumatin-like protein (TLP) plays an important role in combating plant pathogen infection.Common root rot caused by Bipolaris sorokiniana and leaf rust caused by Puccinia triticina (Pt) are major fungal diseases in wheat.The disease responses of TaTLP1-overexpressing transgenic lines (TaTLP1-OE) were evaluated after inoculation with each pathogen.The TaTLP1-OE lines had no apparent differences in tiller number and 1000-kernel weight from the wild type Jinan Wheat No.1 (JW1),whereas resistance to leaf rust and common root rot was improved,resulting from activated peroxidase and b-1,3-glucanase after B.sorokiniana infection,and reactive oxygen species-related genes were upregulated in Ta TLP1-OE lines after Pt infection.These results indicated that stable expression of TaTLP1 increased resistance against both diseases.

Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10-42 residues （Hpa110-42） in the Hpa1 sequence is critical as the isolated Hpa110-42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110-42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110-42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes （EIN2, PP2-A, and GSL10） and consistent with induced expression of defense response genes （Hel, PDF1.2, PR-1b, and PR-2b）. As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110-42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110-42 can be integrated into breeding germplasm of the agriculturally signiifcant crop with a great potential of the agricultural application.

Study on the Obtaining of Transgenic Wheat with GNA Alien Gene by Biolistic Particle

The immature embryos of wheat plants, cv. Jing 411, 12 - 14 days after pollination, were cultured on SD2 medium for callus induction. After 10 days culture, 800 wheat calli were bombarded by biolistic particle coated with the DNA of plasmid pBI121-2 harboring both Galanthus nivalis agglutinin gene and bar gene. 67 green plants were finally regenerated from the bombardment calli on selection medium containing 4mg/L Basta. The results of bioassay by both inoculating wheat aphids onto the plants and applying Basta solution of 50 mg/L and 75 mg/L onto the wheat leaves in the field, and the molecular analysis, such as PCR and Southern blotting, indicated that 8 T2 plants contaning the target genes were obtained.

Rabbit Defensin(NP-1) Gene Transformation of Wheat and In Vitro Microbicidal Activity Assay of Transgenic Plants

Monocot high expression vector pBARUNP1, harboring rabbit defensin(NP 1) gene and selective bar gene for resistance to the herbicide Basta, were constructed and then transferred into immature embryos of wheat (“Bobwhite” and “Zhong 60634”)via particle bombardment. Southern and RNA dot blots showed the stable integration and transcription of foreign NP 1 gene in the wheat genome. Furthermore, in vitro microbicidal activity assay indicated the proper translation of defensin. Crude protein extraction of transgenic plants exhibited to some extent cytotoxic to several pathogens including G. saubinetii, B. subtilis, E.coli, and A. tumefaciens.

Characterization of transgenic wheat lines expressing maize ABP7 involved in kernel development

Wheat is one of the major food crops in the world.Functional validation of the genes in increasing the grain yield of wheat by genetic engineering is essential for feeding the ever-growing global population.This study investigated the role of ABP7,a bHLH transcription factor from maize involved in kernel development,in regulating grain yield-related traits in transgenic wheat.Molecular characterization showed that transgenic lines HB123 and HB287 contained multicopy integration of ABP7 in the genome with higher transgene expression.At the same time,QB205 was a transgenic event of single copy insertion with no significant difference in ABP7 expression compared to wild-type(WT) plants.Phenotyping under field conditions showed that ABP7 over-expressing transgenic lines HB123 and HB287 exhibited improved grain yield-related traits(e.g.,grain number per spike,grain weight per spike,thousand-grain weight,grain length,and grain width) and increased grain yield per plot,compared to WT plants,whereas line QB205 did not.In addition,total chlorophyll,chlorophyll a,chlorophyll b,and total soluble sugars were largely increased in the flag leaves of both HB123and HB287 transgenic lines compared to the WT.These results strongly suggest that ABP7 positively regulates yieldrelated traits and plot grain yield in transgenic wheat.Consequently,ABP7 can be utilized in wheat breeding for grain yield improvement.

Resistance to Virus Gene Expression in Transgenic Wheat Plants

This paper presents a definitive evidence for using BYDV CP gene to build resistancegene and obtain resistance to BYDV transgenic wheat plants by transformation via a pollentube pathway.

Molecular Detection and Disease Resistance Identification of Transgenic Wheat with pti5-vp16 Gene

The immature embryos of wheat cultivars Liaochun10, Tiechun1 and Fengqiang3 were bombarded with gold particles coated with pti5 vp16 by gene gun and disease resistant regenerated plants were attained. In order to confirm that the plants are genuine transformed ones, a series of molecular tests were conducted as follows. Firstly, transient GUS expression test on embryos two days after bombardment was done. There were many obvious blue spots produced on the surface of bombarded embryos after GUS staining, in which the maximum reached to 85 blue spots per embryo. Secondly, PCR test was performed with DNA from the regenerated plants obtained after double selection with ppt. 6 plants were found PCR test positive. At last, further verification analysis using dot hybridization and southern blotting was carried out on those PCR positive plants and the strong hybridization signals appeared as expected. All the above tests were uniformly indicated that the disease resistant regenerated plants were true transgenic plants. When inoculated with Blumeria graminis, transgenic wheat plants of PCR positive results were mostly resistant(R) after 7 days, and resis tant, moderate resistant(MR), moderate susceptible(MS) at 14 days respectively. The disease severity of them was distinctively lighter than that of control.

Transgeni根癌农杆菌介导的小麦转基因植株再生(英文)

根癌农杆菌菌株Ａｇｌ Ⅰ的Ｔｉ 质粒ｐＵＮＮ－２ 带有Ｕｂｉ１ 启动子驱动的ｎｐｔ Ⅱ基因。７ 种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同浓度巴龙霉素的筛选，３ 种基因型小麦产生抗性愈伤组织并再生植株。再生植株经ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交鉴定为转基因植株，转化频率（ 再生转基因植株的小麦愈伤组织数／ 用于转化实验的愈伤组织数） 为３．７％ ～５ ．９％ 。小麦基因型及转化材料的起始生理状态是影响ＴＤＮＡ转移的重要因素。

Overaccumulation of glycine betaine makes the function of the thylakoid membrane better in wheat under salt stress

Wheat(Triticum aestivum L.) lines T1, T4, and T6 were genetically modified to increase glycine betaine(GB) synthesis by introduction of the BADH(betaine aldehyde dehydrogenase, BADH)gene from mountain spinach(Atriplex hortensis L.). These transgenic lines and WT of wheat(T. aestivum L.) were used to study the effect of increased GB synthesis on wheat tolerance to salt stress. Salt stress due to 200 mmol L-1Na Cl impaired the photosynthesis of the four wheat lines, as indicated by declines in net photosynthetic rate(Pn), stomatal conductance(Gs),maximum photochemical efficiency of PSII(Fv/Fm), and actual photochemical efficiency of PSII(ФPSII) and an increase in intercellular CO2concentration(Ci). In comparison with WT, the effect of salinity on the three transgenic lines was mild. Salt stress caused disadvantageous changes in lipids and their fatty acid compositions in the thylakoid membrane of the transgenic lines and WT. Under salt stress, the three transgenic lines showed slightly higher chlorophyll and carotenoid contents and higher Hill reaction activities and Ca2+-ATPase activity than WT. All the results suggest that overaccumulation of GB resulting from introduction of the BADH gene can enhance the salt tolerance of transgenic plants, especially in the protection of the components and function of thylakoid membranes, thereby making photosynthesis better. Changes in lipids and fatty acid compositions in the thylakoid membrane may be involved in the increased salt stress tolerance of the transgenic lines.

Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.

TaNAC48 positively regulates drought tolerance and ABA responses in wheat(Triticum aestivum L.)

NAC family transcription factors(TFs) are important regulators in plant development and stress responses. However, the biological functions of NAC TFs in wheat are rarely studied. In this study, 43 putative drought-induced NAC genes were identified from de novo transcriptome sequencing data of wheat following drought treatment. Twelve wheat NACs along with ten known stress-related NACs from Arabidopsis and rice were clustered into Group II based on a phylogenetic analysis. Ta NAC48, which showed a higher and constitutive expression level in Group Ⅱ, was selected for further investigation.Ta NAC48 transcript was up-regulated by drought, PEG, H_(2)O_(2) and abscisic acid(ABA) treatment and encoded a nuclear localized protein. Overexpression of Ta NAC48 significantly promoted drought tolerance with increased proline content, and decreased rates of water loss, malondialdehyde(MDA), H_(2)O_(2) and O_(2)^(-) content. Root length and a stomatal aperture assay confirmed that Ta NAC48-overexpression plants increased sensitivity to ABA. Electrophoretic mobility shift assay(EMSA) and luciferase reporter analysis indicated that Ta AREB3 could bind to a cis-acting ABA-responsive element(ABRE) on Ta NAC48 promoter and activate the expression of Ta NAC48. These results suggest that Ta NAC48 is essential in mediating crosstalk between the ABA signaling pathway and drought stress responses in wheat.

Transformation of Coat Protein Encoding Gene from Soil-Borne Mosaic Virus into Wheat

CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat variety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 -5 mg L-1 Bialaphos were obtained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformation frequency had 0.99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstrated that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ to CP1- in T1 generation was 1.0 to 1. 3, and didn't agree with Mendelian rule. RT-PCR result from T2 plants showed that the alien gene CWMV-CP1 had stable expression in wheat genetic background.

Wheat Genetic Transformation as Efficient Tools to Fight against Fungal Diseases

Wheat ranks first among cereal crops cultivated in the world. In its production, diseases like powdery mildew, fusarium head blight and rusts caused by fungal pathogens represent a major problem. They produce different symptoms that cause severe crop damage by infecting the spikes, leaves, roots, stems and grains. They are causing losses both by reducing the quantity of the harvested crop and the quality of the product. Quality problems of the harvested product can be due to shrivelled seed, which are frequently found as a consequence of the infection by leaf pathogens, such as mildews, rusts and Septoria. Fusarium head blight is the major culprit for mycotoxin contamination from the harvested grain, causing economic losses and in the worst casing human and animal health problems. In severe epidemics, all these fungal diseases can significantly reduce yield. Resistance to fungi is beneficial not only from a commercial point of view （yield）, but also because of the reduced levels of mycotoxins. The integration of transgenic approaches offers a potential chemical-free and environment-friendly solution for controlling fungal pathogens. This is an essential asset for wheat world food security.

小麦TaLEA_4-1D基因的克隆及功能验证

干旱胁迫是影响小麦生长和产量的主要因素之一。胚胎发育晚期丰富蛋白(LEA)能够响应多种非生物胁迫,在提高植物抗逆性方面发挥重要功能。为了探索LEA蛋白在小麦抵御干旱胁迫中的作用,本研究通过对小麦耐旱材料H611干旱胁迫前后的RNA-seq数据进行分析,筛选出10个TaLEA蛋白基因;克隆了TaLEA_4-1D基因,并对其进行生物信息学分析、表达分析和亚细胞定位,同时构建TaLEA_4-1D过表达拟南芥株系进行功能验证。结果表明,TaLEA_4-1D编码214个氨基酸,含有一个LEA_4保守结构域,蛋白分子量为21.97 kD,等电点为8.89,是亲水的稳定蛋白;二级结构和三级结构预测表明,此蛋白主要由α-螺旋(85.98%)和无规则卷曲(11.21%)组成。顺式作用元件分析发现,TaLEA_4-1D含有多个与植物激素诱导、生长发育和逆境胁迫响应相关的顺式调控元件。系统进化树分析显示,TaLEA_4-1D基因与节节麦、大麦、二穗短柄草、水稻等的LEA亲缘关系较近;亚细胞定位显示该蛋白位于细胞核和细胞质中。干旱胁迫下,过表达TaLEA_4-1D转基因拟南芥根长显著增加,其对干旱胁迫的耐受性增强,过表达植株表型优于野生型。