Impact Evaluation of Insect-Resistant Transgenic Rice on the Feeding and Oviposition Behavior of Its Non-Target Insect, the Brown Planthopper, Nilaparvata lugens (Homptera: Delphacidae)

The feeding and oviposition behavior of the brown planthopper (BPH), Nilaparvata lugens on two transgenic indica rice homogenous genotypes (B1 and B6) with cry1Ab gene from Bacillus thuringiensis and transgenic restored line of hybrid rice (MSA) with SCK gene (a modified CpTI gene) were measured, compared with those on their corresponding non transgenic parental cultivars Jiazao935 and Minghui86 performed by BPH. Under the selection condition of host plants by BPH, loading percentage, oviposition preference and laying egg number of BPH both on transgenic cry1Ab rice and transgenic SCK rice were not significantly different from those on their controls, while their total number of probing wound caused by PBH expect for feeding on B1 plants was markedly more than that on the control. In contrast, under the non selection condition, total number of probing wound caused by BPH on either transgenic cry1Ab rice or transgenic SCK rice was pronouncedly more than those on their controls. Conversely, their honeydew amount excreted by BPH after feeding for 24 h was significantly less than those on the control. As a conclusion, three tested transgenic rice genotypes with insect resistance acted adverse effect on BHP feeding, and no marked effect on BPH oviposition.

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp （Fenneropenaeus Chinensis） were integrated into Oryza sativa L. subsp, japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in To generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

Western blot detection of PMI protein in transgenic rice

Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Modified accumulation of selected heavy metals in Bt transgenic rice

Safety assessment of genetically modified crops generally does not take into account the potential hazard of altered patterns of heavy metal accumulation in plants. A pot experiment was conducted under greenhouse conditions to evaluate the impact of heavy metal amendments on the accumulation of Cd, Cu, Pb and Zn in a Bt transgenic rice Ke-Ming-Dao （KMD） and its wild-type Xiushui 11 （Xs11）. In control soils, significant difference was only found in contents of Cu （p 〈 0.01） and Pb （p 〈 0.05） in straw between KMD and Xs11. At three levels of Cd amendments （5, 10, and 20 mg/kg）, the Cd contents in grain and straw of KMD were significantly higher than those of Xs11, and all grain Cd contents were significantly higher than the International Criteria （0.2-0.4 mg/kg） as specified by the Codex Alimentarius Commission （CAC）. These results implied that it may be unsafe for growing Bt transgenic rice in heavily Cd-polluted areas. No significant difference in Zn was found between the two varieties with the exception of roots at Zn amendment level of 600 mg/kg, while Pb contents in KMD were much higher in the straw at the lead amendment level of 1000 mg/kg and in the root at 250 mg Pb/kg. Data on the heavy metal accumulation patterns for the genetically modified rice may be used for the selection of growing areas as well as for plant residue management for Bt rice.

Response of Gas Exchange and Water Use Efficiency to Light Intensity and Temperature in Transgenic Rice Expressing PEPC and PPDK Genes

Aiming to controvert whether the photosynthetic capacity of transgenic rice expressing C4 genes is enhanced, with the C3-type untransformed rice （WT） and maize （a C4 plant） as controls, the activity of C4 photosynthetic enzymes, gas exchange parameters and water use efficiency （WUE） under different light intensities and temperatures, the stable carbon isotope ratio （8-3C） value and the metabolic index of active oxygen as well as plant yield parameters were determined in transgenic rice carrying the PEPC and PPDK genes （CK） in this study. The results showed that the light-saturated photosynthetic rate of CK was intermediate between that of WT and maize, with a slight bias towards that of maize. Under a high light intensity （1 200 μmol m^-2 s^-1） and high temperature （35℃）, CK still exhibited higher photosynthetic capacity, while the Gs decreased. The WUE of CK was only slightly increased, and was similar to that of WT. The δ13C value indicated that CK functioned as a C3 plant. In addition, the tolerance to photo-oxidation and grain yield of CK was enhanced by sprayed with NaHSO3. In conclusion, CK possesses higher photosynthetic productivity under the conditions of high photon flux density （PFD）, high temperature and spraying with NaHSO3 solution, thereby providing a new technical approach and physiological basis for constructing C4-like rice.

Characteristics of Resistance to Rice Sheath Blight of Zhongda 2,a Transgenic Rice Line as Modified by Gene “RC24”

The transgenic rice, Zhongda 2, which was genetically modified from an indica rice line Zhuxian B by rice chitinase gene (RC24), had high resistance to rice sheath blight (Rhizoctonia solan!) in laboratory and a two-year field experiment. The pathogen could invade sheath of Zhongda 2 and induce symptoms of the disease. No difference was noted in time of penetration or incubation period between Zhongda 2 and non-transgenic rice control, Zhuxian B, but the hyphae lysate could be observed earlier than control. Its resistance expressed as to inhibit the growth of mycelium in host tissue. Fis from Zhongda 2(4) crossed with other five non-transgenic rice lines showed higher resistance than donor non-transgenic parents, but the resistance was different along with the different maternal parents.

Lethal and Sub-Lethal Effects of Transgenic Rice Containing cry1Ac and CpTI Genes on the Pink Stem Borer, Sesamia inferens(Walker)

Lethal and sub-lethal effects of transgenic rice containing cry1Ac and CpTI genes on the pink stem borer, Sesamia inferens, were studied to collect information for ecological risk assessment on insect-resistance of transgenic rice. In vitro insect-feeding bioassays were conducted to evaluate the lethal and sub-lethal effects of transgenic rice lines （II YouKF6 and KF6） containing cry1Ac＋CpTI genes on S. inferens at four different growth stages, viz., seedling, tillering and elongation, booting, and milk and maturing. Transgenic rice at seedling stage showed significantly high lethal effect on S. inferens with the shortest lethal duration for 50 and 100% individuals and the highest corrected mortalities after feeding on transgenic lines at this stage for 3 and 6 d. Followed by tillering and elongation stage, 50 and 100% S. inferens were dead after feeding on transgenic lines at this stage for 4 and 10 d, respectively. Moreover, corrected mortalities for 6 d feeding on transgenic lines at this stage were significantly higher than that at booting, and milk and maturing stages. Lethal effect of KF6 on S. inferens decreased significantly at booting stage. Lethal duration for 50% S. inferens significantly extended and its corrected mortalities for 6 d feeding also declined remarkably. However, lethal effect of II YouKF6 on S. inferens did not decrease significantly at this stage. Transgenic rice at booting, and milk and maturing stages did not show significant lethal effect to S. inferens and it showed the longest lethal duration for 50% individuals and the lowest corrected mortalities for 3 and 6 d feeding. A few larvae of S. inferens could survive, pupate and emerge on these two transgenic lines at booting, and milk and maturing stages. Sub-lethal effect of two transgenic lines on S. inferens also differed significantly between different developmental stages. Continuously feeding on transgenic rice lines at seedling, and tillering and elongation stages delayed the development of larvae and pupae and decreased pupation rate, but no effect was observed on eclosion rate. Larval development was significantly inhibited after feeding on transgenic rice at booting stage, but no obvious effect was observed in pupal stage, pupation and eclosion rate. There were no significant differences for larval and pupal development, pupation, and eclosion rates between feeding on transgenic and control rice lines at milk and maturing stage. Larval and pupal weights significantly declined, but no influence was observed on fecundity when S. inferens infested on transgenic rice at any stage. These showed that there were significant differences in lethal and sub-lethal effects of transgenic rice on S. inferens among developmental stages, and the effects gradually decreased with the increase of growth stages of rice plant.

RNAi-mediated transgenic rice resistance to Rice stripe virus

Rice stripe virus（RSV） often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs（si RNAs） with transgenic virus resistance of plants using RNA interference（RNAi） is known for decades, no systematical research has been done on the profiling of si RNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein（NCP） gene. In this paper, three independent RSV-retsistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of si RNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of si RNAs were in lengths of 21, 22, and 24 nucleotides（nt）, which have validated a connection between the presence of the RSV NCP homologous si RNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines（T4-B1）, the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived si RNAs in transgenic plants, and the RSV-infected library（T4-B1-V） possessed more si RNAs than its mock inoculated libraries（T4-B1-VF）, these results indicating the inverted repeat construct and RSV could introduce abundance of si RNAs in transgenic rice. Moreover, a varied expression level of specific si RNAs was found among different segments of the NCP gene template, about 47% of NCP-derived si RNAs reads aligned with the fragment from 594 to 832 nt（239 nt in length） in NCP gene（969 nt in length） in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the si RNA profile in RSV-resistant transgenic plant using next generation sequencing（NGS） technique, we confirmed that the massive abundance of si RNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.

A mitochondrial phosphate transporter,McPht gene,confers an acclimation regulation of the transgenic rice to phosphorus deficiency

Phosphate transporters play an important role in promoting the uptake and transport of phosphate in plants. In this study, the McPht gene from the Mesembryanthemum crystallinum, a mitochondrial phosphate transporter, was isolated and constructed onto a constitutive expression vector carrying 35S：：GFP, and the recombinant constructs were transferred into Oryza sativajaponica L. cv. Kitaake to investigate the regulatory role of the McPhtgene under phosphorus deficiency. The McPhtgene encodes a protein of 357 amino acids with six transmembrane domains and is located to the mitochondria, and the mRNAtranscripts of the McPht gene are highly accumulated in the shoots of M. crystallinum in response to phosphorus deficiency. However, more mRNA transcripts of the McPht gene were accumulated in the roots of the transgenic rice under phosphorus deficiency. Measurements showed that the transgenic rice demonstrated an enhanced promotion in the root development, the root activities, and phosphate uptake under phosphorus deficiency. Transcriptome sequencing showed that the transgenic rice exhibited total of 198 differentially expressed genes. Of these, total of 154 differentially expressed genes were up-regulated and total 44 genes were down-regulated comparing to the wild type in response to phosphorus deficiency. The selective six genes of the up-regulated differentially expressed genes showed an enhanced increase in mRNA transcripts in response to phosphorus deficiency, however, the transcripts of the mitochondrial carrier protein transporter in rice, a homologous gene of the McPht, in both the transgenic line and the wild type had no obvious differences. Functional enrichment analyses revealed that the most of the up-regulated genes are involved in the cytoplasmic membrane-bounded vesicle, and most of the down-regulated genes are involved in the mitochondrion and cytoplasmic membrane-bounded vesicle. The differentially expressed genes were highly enriched in plant secondary metabolisms and plant-pathogen interaction. These results indicated that the overexpression of the McPht gene might participate in the physiological adaptive modulation of the transgenic rice to phosphorus deficiency by up- or down-regulating the differentially expressed genes.

Changes in bacterial community and abundance of functional genes in paddy soil with cry1Ab transgenic rice

A field experiment involving cry1Ab transgenic rice(GM) and its parental non-cry1Ab rice(M) has been on-going since 2014. The diversity of the bacterial communities and the abundance of the microbial functional genes which drive the conversion of nitrogen in paddy soil were analyzed during the growth period of rice in the fifth year of the experiment, using 16 S rRNAbased Illumina Mi Seq and real-time PCR on the amoA, nirS and nirK genes. The results showed no differences in the alpha diversity indexes of the bacterial communities, including Chao1, Shannon and Simpson, between the fields cultivated with line GM and cultivar M at any of the growth stages of rice. However, the bacterial communities in the paddy soil with line GM were separated from those of paddy soil with cultivar M at each of the growth stages of rice, based on the unweighted Uni Frac NMDS or PCoA. In addition, the analyses of ADONIS and ANOSIM, based on the unweighted Uni Frac distance, indicated that the above separations between line GM and cultivar M were statistically significant(P<0.05) during the growth season of rice. The increases in the relative abundances of Acidobacteria or Bacteroidetes, in the paddy soils with line GM or cultivar M, respectively, led to the differences in the bacterial communities between them. At the same time, functional gene prediction based on Illumina Mi Seq data suggested that the abundance of many functional genes increased in the paddy soil with line GM at the maturity stage of rice, such as genes related to the metabolism of starch, amino acids and nitrogen. Otherwise, the copies of bacterial amo A gene, archaeal amo A gene and denitrifying bacterial nir K gene significantly increased(P<0.05 or 0.01) in the paddy soil with line GM. In summary, the release of cry1Ab transgenic rice had effects on either the composition of bacterial communities or the abundance of microbial functional genes in the paddy soil.

Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

A total of 105 transgenic dce lines independently transformed with a pea ferritin gene （Fer） were previously obtained. After seven generations of selfing and β-glucuronidase （GUS） assisted selection, 82 transgenic lines with stable agronomic traits were got. Among the 82 transgenic lines, two high-iron transgenic rice lines Fer34 and Fer65, with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11, respectively were identified. In the two transgenic lines, the exogenous Fer gene was highly expressed, and inherited as a single locus. The transgene had no negative effect on the agronomic traits of rice plant, other mineral nutritional components, appearance quality and eating quality of the milled rice, indicating that these two lines were elite high-iron breeding lines. Furthermore, the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

Thellungiella halophila ThPIP1 gene enhances the tolerance of the transgenic rice to salt stress

Aquaporin proteins were demonstrated to play an important regulatory role in transporting water and other small molecules. To better understand physiological functions of aquaporins in extremophile plants, a novel ThPIP1 gene from the Thellungiella halophila was isolated and functionally characterized in the transgenic rice. Data showed that the ThPIP1 protein encoded 284 amino acids, and was identified to be located on the plasma membrane. The expression of ThPIP1 gene in the shoots and roots of T. halophila seedlings were induced by high salinity. The transgenic rice overexpressing ThPIP1 gene significantly increased plants tolerance to salt stress through the pathway regulating the osmotic potentials, accumulation of organic small molecules substances and the ratio of K＋/Na＋ in the plant cells. Moreover, split-ubiquitin yeast two-hybrid assay showed that Th PIP1 protein specifically interacted with ThPIP2 and a non-specific lipid-transfer protein 2, suggesting that ThPIP1 probably play a key role in responding to the reactions of multiple external stimulus and in participating in different physiological processes of plants exposed to salt stress.

Studies on the inheritance of transgenic rice with bar gene

Bar gene driven under the control of CaMV35s promotor was delivered into the immatureembroys of a japonica rice cultivar Jingying 119through biolistic approach. Two putativetransgenic plants were produced, which ex-pressed Basta-resistance. One of the Basta-re-sistance transgenic rices was completely sterile(JY 119-3), the other was self-fertile (JY119-4).

Expression of a barley ABA-responsive protein gene in transgenic rice

A cDNA clone encoding an ABA-responsiveprotein HVA1,was isolated by differentialscreening from barley aleurone layers(Hong etal.).Expression of the HVA1 gene is shownto be developmentally regulated,organ-specif-ic,and ABA-and stress-induced(Hong et

Preparative Purification and Bioassay of Bt Toxin from Cry1Ab Transgenic Rice

A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.

Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ （pinⅡ） in Transgenic Rice

The inheritance and expression of bar gene and pinII gene were studied in three transgenic rice lines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S. By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bar gene and pinII gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but a few plants in F2 have not sufficiently expressed. The wound inducible pin II gene has an expression regulated spatially and temporally, and the signal transduction pathway is not only upward, but also downward. The inducible expression of pin II in different rice transgenic lines is not completely coincident.

Assessment of Gene Flow Through Detection of SexualCompatibility Between Transgenic Rice with barand Echinochloa crusgalli var.mitis

The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. The germination and growth of rice pollen grains on barnyard grass stigmas at 30 min, and 1-4 h after crossing by hand were observed with an optical microscope. The results were compared with the germination and growth of barnyard grass pollen grains at the corresponding time after self-pollination. The results showed that germination and growth of the pollen grains of the two varieties were similar on barnyard grass stigmas, but differed significantly from self-pollination of barnyard grass. Pollen grains germinated and pollen tubes penetrated stigmas normally, and the number of pollen grains being condensing or releasing their inclusions or having released them increased with the time after self-pollination. Pollen grains of transgenic rice on the stigmas of barnyard grass couldn't germinate or grow normally after crossing, neither could they penetrate the stigmas of barnyard grass. Therefore, it could be concluded that the sexual incompatibility between transgenic rice with bar gene and barnyard grass is due to the rice pollen being unable to penetrate the stigma of barnyard grass. Further proof of incompatibility lies in the fact that the emasculated barnyard grass pollinated with the rice pollen grains could not seed.

Study of the Protective Effects in PEPC Transgenic Rice

The diurnal course of chlorophyll fluorescence parameter and active oxygen metabolism of flag leaves in PEPC transgenic and untransformed rice Kitaake were studied. The results showed that the photosynthetic rate under high light intensity has been increased by 50% and photoinhibition of photosynthesis in PEPC transgenic was alleviated after the introduction of PEPC gene from maize into rice. It was demonstrated that the increment of photosynthesis in PEPC transgenic was related to the introduction of PEPC gene using specific inhibitor of PEPC. Photoinhibition of photosynthesis in different genotypes exists at noon under natural condition. PEPC transgenic rice exhibited a less decrease in Fv/Fm, a less photoinhibition and a higher efficiency of light energy conversion to chemical energy and lower thermal energy dissipation. These results provided the physiological basis on the mechanism of tolerance to photoinhibition and rice breeding with high photosynthetic efficiency.

Improvement of foreign gene expression in transgenic rice(Oryza sativa L.)by matrix attachment regions(MARs)

Matrix attachment regions or matrix associated regions (MARs) were special DNA sequences in chromatin of eukaryotic cells that tightly associated with the nuclear matrix or scaffold in vitro after a combination of nuclease digestion and extraction. They were also called scaffold attachment regions(SARs) . It was found that MARs could improve the expression level and the stability of foreign genes in transgenic plants. The reason might be that a transgene flanked by MARs was transmitted into the plant cells, the MARs would attach the nuclear