The Inheritance and Expression of cry1A Gene in Transgenic Maize

We investigated the inheritance and expression of cry1A gene in transgenic maize ( Zea mays L.) by Southern blotting analysis and enzyme_linked immunosorbent assays (ELISA). The results showed that cry1A had been transmitted to progeny of transgenic maize as a single gene. Contents of cry1A insecticidal protein were significantly different among transgenic maize lines and various tissues of the same transgenic lines. High expression of cry1A protein occurred in green tissues, such as leaf and husk leaf, and low expression occurred in pith, tassel, ear pith, pollen and silk. The results also showed that the contents of cry1A insecticidal protein in leaves of transgenic maize increased with the advance of development and there was no significant difference in cry1A expression level among various generations of transgenic maize.

The Influence of Bt-Transgenic Maize Pollen on the Bacterial Diversity in the Midgut of Chinese Honeybees, Apis cerana cerana

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase（rbcS） small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize（Zea mays） immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Screening Method for Transgenic Maize with Kanamycin Resistance

[Objective]The paper was to establish simple and effective method to screen marker gene in maize with kanamycin resistance.[Method]Using inbred line "Chang 7-2" and hybrid "Zhengdan 958" of maize as test materials,their seeds were soaked with different concentrations and volumes of kanamycin for3 and 4 d,respectively,the rate of albino seedlings and average seedling height after sowing for10 d were investigated.[Result]The rate of albino seedlings not only was related to kanamycin concentration,but also had relationship with solution volume during soaking process.The difference between inbred line and hybrid was no significant.When 100 ml of kanamycin solution with concentration of 200 mg/L was used to soak seeds for 3 d,the rate of albino seedlings basically could reach 100%.When 100 ml of kanamycin solution with concentration of 100 mg/L was used to soak 20 seeds for 3 d to carry out resistance screening,the accuracy was up to 84.8% after verifying the screening test of T1 transgenic maize plants.[Conclusion]The method was feasible,which could be used as a simple method for screening transgenic gene maize with kanamycin resistance.

Maize ABP2 enhances tolerance to drought and salt stress in transgenic Arabidopsis

Abiotic stresses, especially drought and salt, severely affect maize production, which is one of the most important cereal crops in the world. Breeding stress-tolerant maize through biotechnology is urgently needed to maintain maize production. Therefore, it is important to identify new genes that can enhance both drought and salt stress tolerance for molecular breeding. In this study, we identified a maize ABA （abscisic acid）-responsive element （ABRE） binding protein from a 17-day post-pollination （dpp） maize embryo cDNA library by yeast one-hybrid screen using the ABRE2 sequence of the maize Cat1 gene as bait. This protein, designated, ABRE binding protein 2 （ABP2）, belongs to the bZIP transcription factor family. Endogenous expression of ABP2 in maize can be detected in different tissues at various development stages, and can be induced by drought, salt, reactive oxygen species （ROS）-generating agents, and ABA treatment. Constitutive expression of ABP2 in transgenic Arabidopsis plants enhanced tolerance to drought and salt stress, and increased sensitivity to ABA. In exploring the mechanism by which ABP2 can stimulate abiotic stress tolerance, we found that ROS levels were reduced and expression of stress-responsive and carbon metabolism-related genes was enhanced by constitutive ABP2 expression in transgenic plants. In Short, we identified a maize bZIP transcription factor which can enhance both drought and salt tolerance of plants.

Construction of Maize Universal Expression Vector PGM-35Sbar and Maize Transformation

[Objective] The paper was to construct maize universal expression vector, in order to provide basis for using transgenic methods to improve abiotic stress tolerance of maize. [Method] Based on the transformation of existing pGreen0229 plant expression vector, phosphinothricin-resistant selectable marker-bar gene driving by CaMv35S promoter was constructed, which could be used to connect target gene of maize expression vector PGM-35Sbar, and transform Ji444 maize inbred lines by pollen tube pathway. [Result] The universal expression vector for PGM-35Sbar maize had been successfully constructed. When the maize plants were transformed, 14 resistant plants were obtained, and 12 plants were identified to be positive plants by PCR detection. [Conclusion] The study provided basis for rapid construction of maize expression vector containing specific target gene.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Establishment of multiple shoot clumps from maize(Zea mays L.)and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene a/s (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene a/s has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R1 plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

Aldehyde dehydrogenases（ALDHs） represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of the ALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maize ALDH7 subfamily member（ZmALDH7B6） was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance of ZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-specific and stress-induced expression patterns, the 1.5-kb 5′-flanking ZmALDH7B6 promoter region was fused to the β-glucuronidase（GUS） reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was significant induction of GUS activity in response to ammonium supply, confirming ammonium-dependent expression of ZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by the ZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested that ZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue.

Breeding for Parents of Hybrid Rice with Maize pepc Gene

The results of the investigation on transgenic rice with maize C4-specific phosphoenolpyruvate carboxylase (pepc) gene showed that the transgenic rice plants with the maize pepc gene expressed at high level and the maize PEPC expression was inherited in the progenies in a Mendelian manner. The transgenic plants had PEPC activity of more than 10-fold higher than untransformed plants. As compared with untransformed plants, the panicle per plant, spikelet per panicle, 1000-grain weight and grain-weight per plant for transgenic plants increased by 14.9 % , 5.7%, 1.3 % and 13.9 %, respectively. By crossing the maize pepc gene was incorporated into the parents of hybrid rice, which were the photo-sensitive genie male sterile (PGMS) lines of two-line hybrid rice such as Peiai64s, 7001s, 2302s, 2304s and 2306s-1, and the BT type of cytoplas-mic male sterile (CMS) line of three-line hybrid rice such as Shuangjiu A, and restorer lines 5129, Wanjing97 in the spring of 1998. The following progresses were made: (1) The inheritance of the high-level expression of the maize PEPC was stable in different genetic background of rice; (2) PEPC activity of hybrid was the mean of the two parents. Its saturated photosynthetic rate (Pn) rose to 50 % higher than that of the receptor parent. These results demonstrated that it is possible to increase the vigor of the rice plant by transgenic approach with maize pepc gene; (3) The activity of PEPC in leaf could be considered as the major physiological index because the correlation coefficient between PEPC activity and Pn was 0.6470* * ; (4) We have developed three rice lines with maize pepc gene; (5) The selection method of high photosynthetic efficiency rice has been established, which includes soaking seeds into solution of hygromycin phosphotransferase to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities and Pn of rice plants with maize pepc gene.

Effect of three insect-resistant maizes expressing Cry1le,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker)

Three transgenic maize events(IE09S034,Shuangkang 12--5 and C0030.3.5)produced Cry1le,Cry1Ab/Cry2Aj and G10-EPSPS,Cry1Ab and EPSPS,respectively,all of which target the Asian corn borer.The oriental armyworm Mythimna separata(Walker)is the secondary target.In this study,the effects of the three Bt maizes on the development and survival of armyworm were studied.The results showed that IE09S034 had insecticidal activity against 1st instar larvae,and the survival rate of armyworm fed with Bt maize for 10 days was 462%,significantly lower than that of the control.The larvae at 3rd--6th instar were more tolerant of the Bt toxin than the early instar larvae.However,Shuangkang 12-5 had good insecticidal activity against 1st-5th instar larvae.The mortality was nearly 100%when the larvae were fed with Shuangkang 12-5 before 3rd instar,and the toxin had quick acting eficacy.This event significantly ihibited the development of armyworm;that is,the larval duration of the 3rd and 4th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days,respectively.The pupal weight and egg number were also significantly lower than those of the control.For C0030.3.5,it could control 1st--5th instar larvae effectively.The mortality rates were all over 50%if 1st-3rd larvae were fed with this event.The pupal weight of 4th-6th instar larvae fed with Bt maize were only 53.9,56.8 and 54.6%,respectively,compared to that of the control.The number of eggs laid was significantly less than the control.The results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of armyworm.which can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm.

Transfer and Detection of barstar Gene to Maize Inbred Line 18-599 (White) by Particle Bombardment

In China, the purity of maize hybrid strain is discomforting to the development of seed industrialization. Finding a new method for reproduction of maize hybrid strain is necessary. In this study, using particle bombardment, barstar gene was transferred into maize inbred line 18-599 （White）, which is an antiviral and high quality maize inbred line. By molecular detection of the anther of transgenic maize, two plants transferred with barstar gene were gained in this study, which are two restorer lines. The two plants showed normal male spike, and lively microspores. But the capacity of the two restorer lines should be studied in the future. The aim of this study is to find a new method of reproduction of maize hybrid strain using engineering restorer lines and engineering sterility lines by gene engineering technology.

Effect of Low Temperature Treatment on Agrobacterium-mediated Transformation of Maize Seed Buds

[Objectives]This study was conducted to improve the genetic transformation rate of maize. [Methods]The seeds of maize ＂ Zheng 58＂ as the experimental material were germinated and treated under freezing temperature of-18 ℃ for 30 and 35 min. The EGFP gene was transformed into the growth points of these seed buds by Agrobacterium tumefaciens EHA105. The transformation effect was determined by fluorescence protein detection on transformed buds. [Results]After a certain period of treatment at-18 ℃ following germination and transformation of maize bud growth points with A. tumefaciens,although the survival rate of maize buds was reduced,the percentage of transformed cells significantly increased. [Conclusions]Low temperature treatment can improve the transformation efficiency of A. tumefaciens to the growth point of maize bud.

The antioxidant protein ZmPrx5 contributes resistance to maize stalk rot

Maize(Zea mays L.)stalk rot is a devastating disease worldwide,causing severe yield losses.Although previous studies have focused on the genetic dissection of maize resistance to stalk rot,the mechanisms of resistance remain largely unknown.We used a comparative proteomics approach to identify candidate proteins associated with stalk rot resistance.Statistical analyses revealed 763 proteins differentially accumulated between Fusarium graminearum and mock-inoculated plants.Among them,the antioxidant protein ZmPrx5,which was up-accumulated in diseased plants,was selected for further study.ZmPrx5 transcripts were present in root,stalk,leaf,ear,and reproductive tissues.The expression of ZmPrx5 in three inbred lines increased significantly upon F.graminearum infection.ZmPrx5 was localized in the cytoplasm.Compared to control plants,maize plants overexpressing ZmPrx5 showed increased resistance to F.graminearum infection,and ZmPrx5 mutant plants were more susceptible than wild-type plants.Defense-associated pathways including plant–pathogen interactions,phenylalanine metabolism,and benzoxazinoid and flavonoid biosynthesis were suppressed in ZmPrx5 homozygous mutant plants compared with wild-type plants.We suggest that ZmPrx5 positively regulates resistance against stalk rot in maize,likely through defense-oriented transcriptome reprogramming.These results lay a foundation for further research on the roles of Prx5 subfamily proteins in resistance to plant fungal diseases,and provide a potential genetic resource for breeding disease-resistance maize lines.

Uncertainty in Detecting Specific Fragment of Genetically Modified Maize Event NK603 by Real-time Fluorescence Quantitative PCR

In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment （specific fragment） in samples containing 2% genetically modified maize event NK603. The uncertainty of detection results was evaluated based on various uncertainty sources, such as PCR amplification system, data analysis and micropipette. The results showed that A-type uncertainty （ uA ）, B-type uncertainty （ uB ）, combined standard uncertainty （ uC ） and expanded uncertainty （ U95 ） were 0. 000 8,0.1301,0. 001 and 0. 002, respectively; the final detection result was 1.9% ±0.002. Thus, the main uncertainty in detecting flanking fragment of genetically modified maize event NK603 with realtime fluorescence quantitative PCR method was derived from the random effect in the experimental process.

Application of Genetically Modified Technology in Maize Breeding

The achievements of genetical y modified （GM） maize in herbicide toler-ance, insect resistance, disease resistance and alkali resistance were reviewed in this paper so as to provide some reference for the researches on molecular breed-ing of maize in China.

Effects of Acetylacetone Solution Soaking on Agrobacterium - transformed Maize Seed Buds

[Objectives]The effect of acetosyringone seed soaking on the transformation of maize seed buds was analyzed,so as to improve the genetic transformation efficiency of maize and to provide technical support for transgenic breeding of maize.[Methods]The seeds of the"Zheng 58"maize inbred line were used as experimental materials.When the seeds were germinated,AS was added to the water at concentrations of 70,140,210,and 280μmol/L,respectively,and the seeds germinated without the addition of AS served as the CK.The Agrobacterium-mediated method was used to transform bud growth points of maize seeds,and green fluorescent protein detection was performed on the young shoots transformed with EGFP(enhanced green fluorescent protein)gene.The effect of soaking seeds with acetosyringone solution on the transformation of maize bud growth points by Agrobacterium was studied according to the detection results.[Results]Soaking seeds in acetosyringone solutions for germination had the effect of inhibiting the germination of maize seeds and inhibiting sprout elongation,and the higher the concentration of acetosyringone,the stronger the inhibition.When the concentration of acetosyringone solution was 280μmol/L,the germination rate of seeds was only 36.2%of the CK,while soaking seeds with 70-140μmol/L acetosyringone solution for germination could not only ensure a higher germination rate of maize seeds,but also significantly increased the transformation efficiency of maize bud growth points.When the seeds were soaked with 70μmol/L acetosyringone solution for germination,the positive rate of transformed maize buds was the highest,reaching 32.1%.[Conclusions]When maize bud growth points were used as the receptor of Agrobacterium transformation,soaking seeds with 70-140μmol/L acetosyringone for germination basically did not affect the germination of seeds,and was beneficial to the activation of Agrobacterium,thereby promoting the transformation.

Quantification of CrylAb Toxin in Bt Maize for Ostrinia nubilalis （Lep.： Crambidae） Bioassay

Two variants of diet composition were prepared to evaluate the susceptibility of ECBs to CrylAb toxin as follows： 1） 38-0600 Stonefly Heliothis Diet mixed with purified CrylAb protein and 2） 38-0600 Stonefly Heliothis Diet mixed with lyophylized leaves of Bt maize MON 810-YieldGard. A method of sample preparation and extraction of Bt toxin for reproducible ELISA quantification were optimized. The qualitative DAS-ELISA kit from Agdia was optimized for use in quantitative analysis of Cry lAb toxin. The mortality of ECB larvae from the laboratory strain on the diet with CrylAb toxin did not differ significantly from the mortality on the diet with Bt maize leaves with the same rate of Cry lAb toxin. Similarly, the mortality of the ECB larvae from the field population on the diet with Bt maize leaves did not differ significantly from the mortality of ECB larvae from the laboratory strain on the same type of diet. Therefore, the incorporation of Bt maize leaves into the diet did not influence the efficacy of CrylAb toxin against ECBs. Using this method, a susceptibility of one field population of ECBs from the Czech Republic to CrylAb toxin was determined （LC50 of 2.16 μg of Cry 1Ab g^-1 of diet）.

Recombinant protein detection and its content in total protein, lipids and toxic antinutritional substances in Mexican maize

The engineering genetic technology has developed Bt maize events which contain recombinant protein that will be safe for the consumer. The aflatoxins are contaminants present in maize capable of producing cancer and decreasing the immune response in human, additionally contained polyphenols compounds considered non nutritive. The objective of this study was to identify the presence of recombinant protein in hybrid and local varieties of corn and evaluate the content of aflatoxins and tannins. 25 samples of white grain maize for human consumption were collected, 12 were for hybrid maize and 13 local varieties, from the states of Hidalgo, Mexico and Morelos. Samples were analyzed for Cry1Ab/Cry1Ac, using lateral flow strip method, crude protein and lipids by standard methods. Aflatoxins were assessed by comercial Elisa kit and tannins by spectroscopy method. The data were grouped in a completely random model and an analysis of variance was performed. The results indicated that 44.5% of hybrid corn was positive by Bt-Cry1Ab/1Ac proteins, containing 9.02% ± 2.5 lipids and 11.33% ± 2.2 crude protein, 189 ± 0.92 mg/g of tannins and 6.36 ± 3.3 μg·g-1 aflatoxins. The local maize samples (55.5%) were negative to Bt-Cry1Ab/ Cry1Ac, which protein content was of 8.68% ± 0.90, 6.14% lipids ± 2.3, 273 ± 0.40 mg/100g tannin and 7.15 ± 3.3 μg·g-1 of aflatoxins. In conclusion, we observed an improvement of nutrient composition in hybrid maize with Bt proteins, and decrease in tannins content comparing with some local varieties without Bt proteins. The effectiveness of Bt maize expressing the Cry1Ab/ Cry1Ac in reducing aflatoxin contamination was not observed, therefore, additive affects of aflatoxins contamination in maize Bt-Cry need to be further investigated in cancer disease development.