Conglin Huang

Over expression of Zmda1-1 gene increases seed mass of corn

Genetic engineering of seed size and increasing biomass in crop plants has an important significant contribution to the world. Arabidopsis DA1 is one of the key factors that negatively control seed and organ size by restricting the period of cell proliferation, and the mutant of Arabidopsis DA1, da1-1 (DA1R358K) can dramatically increase the size of seed. However, it is not clear whether overexpression of Zmda1-1, the mutant of ZmDA1 which is homology of DA1 in Arabidopsis, has the same biological effect as da1-1 in Arabidopsis. Therefore, in this study, the plant expression vector harboring both Zmda1-1 driven by the corn ubiquitin promoter and a PAT selectable marker gene driven by 35S CAMV promoter was constructed and introduced into maize inbred line ‘ji444’ using pollen-tube-pathway method. Screened with herbicide phosphinothricin (PPT), 22 seedlings of 2563 transformed samples survived, and 21 independence lines of which were positive in polymerase chain reaction (PCR) analysis, and the transformation rate of T0 generation was about 0.82%. Further PCR-southern blotting results proved that the Zmda1-1 had integrated into maize genome, and the Zmda1-1 had expression in low level by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The seed mass of transgenic maize increased at an average of 33.6% of empty vector control lines, and the harvest yield was increased by 23.6 to 114.1% in different lines than empty vector control lines. The result suggests that Zmda1-1 can be used to engineer higher harvest yield in crops plant, thus providing the first successful example of increasing the harvest yield of maize by transgenic technology. Key words : Transgenic maize, pollen-tube pathway, Zmda1-1 , seed mass.

Nucleotide Variation in the NCED3 Region of Arabidopsis thaliana and its Association Study with Abscisic Acid Content under Drought Stress

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with anova analysis suggested that the 274th site (P<-->S) and the 327th site (P<-->R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

The systematic or long-distance signal transmission plays crucial roles in animal lives. Compared with animals, however, much less is known about the roles of long-distance signal communication in plant lives. Using the model plant Commelina communis L., we have probed the root to shoot communication mediated by heat-shock signals. The results showed that a heat shock of 5 min at 40°C in partial roots, i.e. half or even 1/4 root system, could lead to a significant decrease in stomatal conductance. The regulation capability depends on both heat shock temperature and the amount of root system, i.e. with higher temperature and more roots stressed, the leaf conductance would decrease more significantly. Interestingly, the stomatal regulation by heat shock signal is in a manner of oscillation: when stomata conductance decreased to the lowest level within about 30 min, it would increase rapidly and sometimes even exceed the initial level, and after several cycles the stomata conductance would be finally stabilized at a lower level. Feeding xylem sap collected from heat-shocked plants could lead to a decrease in stomata conductance, suggesting that the heat shock-initiated signal is basically a positive signal. Further studies showed that heat shock was not able to affect ABA content in xylem sap, and also, not able to lead to a decrease in leaf water status, which suggested that the stomatal regulation was neither mediated by ABA nor by a hydraulic signal. Heat shock could lead to an increase in xylem sap H2O2 content, and moreover, the removal of H2O2 by catalase could partially recover the stomatal inhibition by xylem sap collected from heat-shocked plants, suggesting that H2O2 might be able to act as one of the root signals to control the stomatal movement. Due to the fact that heat-shock and drought are usually two concomitant stresses, the stomatal regulation by heat-shock signal should be of significance for plant response to stresses. The observation for the stomatal regulation in an oscillation manner by presently identified new signals should contribute to further understanding of the mystery for the pant systematic signaling in response to stresses.

Synthesis of medium-chain-length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering

Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3′-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line S4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mcl-PHAs in tobacco via chloroplast genetic engineering.

Detection of herbicide-resistant maize by using loop- mediated isothermal amplification of the pat selectable marker gene

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that reagents react to under isothermal conditions with high specificity, efficiency and rapidity. We used LAMP for detection of herbicide-resistant maize, which is a widespread genetically modified crop. Pat gene, a widely used selectable marker, was amplified by a set of four special primers that recognized six distinct sequences of the target. We studied the optimized conditions for LAMP using different Mg 2+ , dNTPs, betaine and primers concentrations. Furthermore, we tested the sensitivity and specificity of LAMP as compared to PCR. This work shows that the LAMP method can detect a specific pat gene from herbicide-resistant maize and their test results are consistent with the results of the conventional PCR methods. However, LAMP assay results were found to be more sensitive than the conventional PCR. Key words: Herbicide-resistant maize, loop-mediated isothermal amplification (LAMP), transgenic maize detection, pat gene.

Purification and Characterization of ZmRIP1, a Novel Reductant-Inhibited Protein Tyrosine Phosphatase from Maize

Protein tyrosine phosphatases (PTPases) have long been thought to be activated by reductants and deactivated by oxidants, owing to the presence of a crucial sulfhydryl group in their catalytic centers. In this article, we report the purification and characterization of Reductant-Inhibited PTPase1 (ZmRIP1) from maize (Zea mays) coleoptiles, and show that this PTPase has a unique mode of redox regulation and signaling. Surprisingly, ZmRIP1 was found to be deactivated by a reductant. A cysteine (Cys) residue (Cys-181) near the active center was found to regulate this unique mode of redox regulation, as mutation of Cys-181 to arginine-181 allowed ZmRIP1 to be activated by a reductant. In response to oxidant treatment, ZmRIP1 was translocated from the chloroplast to the nucleus. Expression of ZmRIP1 in Arabidopsis (Arabidopsis thaliana) plants and maize protoplasts altered the expression of genes encoding enzymes involved in antioxidant catabolism, such as At1g02950, which encodes a glutathione transferase. Thus, the novel PTPase identified in this study is predicted to function in redox signaling in maize.

Over-expression of ZmPti1, a homologue to Pti1, increases salt tolerance of Arabidopsis thaliana

Studies have shown that Pti1 plays an important role in plant disease resistance pathway. However, Pti1 have not been studied for its roles under salt stress condition. Previous study has shown that maize ZmPti1 is induced by salicylic acid (SA), low-temperature, mannitol and salt. In order to analyze the further biological functions of ZmPti1, ZmPti1 was over-expressed in Arabidopsis. Under salt stress, compared to wild type, transgenic plants grew better, had higher seedling fresh and dry weight (FW, DW), seed yields and the superoxide dismutase (SOD) activity; Seedling FW and DW of S1-1, S2-1 transgenic plants increased significantly than that of WT; Seed weight of S1-1, S2-1 transgenic plants increased 71 and 106% respectively; However malondialdehyde (MDA) content and relative electric conductivity (ion leakage) of transgenic plants were kept to a relative lower level. Based on the present knowledge, this is the first report that showed that the over-expression of Pti1-like gene enhances the salt resistance in plants.

Polyploidy levels of Chinese large-flower chrysanthemum determined by flow cytometry

Flow cytometry was used to determine the ploidy level of 405 Chinese large-flower chrysanthemum ( Chrysanthemum morifolium Ramat.) cultivars. Sixty-three cultivars are triploid, 175 cultivars tetraploid, 32 cultivars pentaploid, 46 cultivars hexaploid and 1 cultivar heptaploid. Forty-eight cultivars were then randomly selected for confirmation by chromosome-counting; the results are in agreement with the classification of ploidy level by flow cytometry. Most cultivars are aneuploid. The high percentage of tetraploid and triploid, instead of hexaploid in previous studies, represents the first evidence of low ploidy in large-flower chrysanthemum, which indicated a wider range of ploidy variation in this population. The results also offer further insights to the possible evolution and the regulation of flower size of this large-flower population. Additionally, the combination of flow cytometry and chromosome-counting is proved to be efficient and necessary for large-scale ploidy screening of chrysanthemum. Keywords: Chrysanthemum, ploidy level, flow cytometry

Cloning and Characterization of Maize ZmASK1, a Homologue to shaggy/GSK-3-Like Gene, Involved in Plant Responses to Abiotic Stresses

Abstract Shaggy/GSK-3 -like genes play important roles in plant development and stress responses. One full-length cDNA encoding a shaggy/GSK-3 homologue, known as ZmASK1 (for abiotic stress-induced kinase), was isolated from maize ( Zea mays L.) by RT-PCR. The predicted ZmASK1 protein has 426 amino acids with an estimated molecular mass of 48.5 kD and an isoelectric point of 8.7. RT-PCR analysis showed that the ZmASK1 expression was induced by mannitol, salt, and abscisic acid. Furthermore, ZmASK1 showed different expression patterns in different tissues and was the most abundant in ovaries. These results suggest that ZmASK1 may play multiple roles in abiotic stress responses as well as in plant reproductive development. This is the first isolation of shaggy/GSK-3 -like gene associated with stress and development reported in maize.

First Report of Chrysanthemum Stem Blight and Dieback Caused by Tomato spotted wilt virus in China

Cultivated chrysanthemum (Chrysanthemum morifolium) is a popular flower species from China, where it is widely planted for use as tea and in medicines, as well as for ornamental purposes. In July 2016, a serious stem necrotic disease was observed on chrysanthemum in a commercial greenhouse in Beijing, China. Most chrysanthemum varieties were infected, with a total disease incidence above 50%, rising to over 90% in some sensitive cultivars. Infected chrysanthemum plants exhibited two main symptoms: (i) irregular chlorotic spots on the upper leaves with shortened internodes (cultivar Silvia), or (ii) discontiguous black streaks on stems near the base of the upper petioles, followed by eventual dieback (cultivars Dante, Radost, Stellini, and Olive). These symptoms are very similar to those of chrysanthemum infected with the tospoviruses Tomato spotted wilt virus (TSWV) or Chrysanthemum stem necrotic virus (CSNV). To identify the pathogen, infected chrysanthemum leaves were analyzed using reverse-transcription polymerase chain reaction (RT-PCR) with tospovirus universal primers (Hassani-Mehraban et al. 2016). After extracting the total RNA with a Plant RNA kit (Tiangen, Beijing, China), RT-PCR was performed using an RT-PCR kit (Vazyme, Nanjing, China), with infected chrysanthemum showing black streaks on stems as positive controls and virus-free plantlets as negative controls. RT-PCR products were bidirectionally sequenced by Sangon Biotech (Shanghai, China) and aligned in DNAstar. Three clones of 10 infected leaf samples (two of each cultivar) were collected from different sections of the greenhouse and sequenced, revealing high sequence similarity with fewer than 2-bp difference between them. The most conserved sequence, isolated from sample BLZ01, was deposited in GenBank under accession number MF196915. This sequence was 99% identical to TSWV S RNA segments in GenBank and distinctly different from CSNV RNAs, indicating that the devastating disease was caused by TSWV, not CSNV. This diagnosis was further confirmed using commercial ELISA kits (Luyuan Bode Biological, Beijing, China), which revealed TSWV but not CSNV in all symptomatic tissue. Additional RT-PCRs were performed using TSWV-specific (Chung et al. 2006; Qiu et al. 1998) and CSNV-specific (Takeshita et al. 2011) primers complementing the RNA-dependence RNA polymerase gene (L RNA), Gn/Gc gene (M RNA), and nucleocapsid protein gene (NP, S RNA), respectively, and the results confirmed those of the ELISA. Finally, the virus isolated from BLZ01 was mechanically transmitted from infected cultivar Olive collected from the original greenhouse to 1-month-old virus-free chrysanthemum seedlings using chilled 0.01 M phosphate buffer (pH 7.2) containing 0.1% sodium sulfite. After 2 weeks, most inoculated plants (13 of 18) exhibited localized necrotic spots and black streaks on their stems. RT-PCR and sequencing revealed that the partial sequence of the TSWV NP gene from the inoculated chrysanthemum was identical to the virus from the greenhouse materials. TSWV has spread and is causing epidemics on chrysanthemum in many countries. To our knowledge, this is the first report of TSWV detected on chrysanthemum in China, and this article will act as a warning to prevent the spread of TSWV in Beijing.