Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3).

Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol™ extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 µg/mL after the production and purification of different dsRNAs, reducing production costs by 72%.

Revealing the differential protein profiles behind the nitrogen use efficiency in popcorn (Zea mays var. everta).

We investigated the proteomic profiles of two popcorn inbred lines, P2 (N-efficient and N-responsive) and L80 (N-inefficient and nonresponsive to N), under low (10% of N supply) and high (100% of N supply) nitrogen environments, associated with agronomic- and physiological-related traits to NUE. The comparative proteomic analysis allowed the identification of 79 differentially accumulated proteins (DAPs) in the comparison of high/low N for P2 and 96 DAPs in the comparison of high/low N for L80. The NUE and N uptake efficiency (NUpE) presented high means in P2 in comparison to L80 at both N levels, but the NUE, NUpE, and N utilization efficiency (NUtE) rates decreased in P2 under a high N supply. DAPs involved in energy and carbohydrate metabolism suggested that N regulates enzymes of alternative pathways to adapt to energy shortages and that fructose-bisphosphate aldolase may act as one of the key primary nitrate responsive proteins in P2. Proteins related to ascorbate biosynthesis and nitrogen metabolism increased their regulation in P2, and the interaction of L-ascorbate peroxidase and Fd-NiR may play an important role in the NUE trait. Taken together, our results provide new insights into the proteomic changes taking place in contrasting inbred lines, providing useful information on the genetic improvement of NUE in popcorn.

Limited Nitrogen and Plant Growth Stages Discriminate Well Nitrogen Use, Uptake and Utilization Efficiency in Popcorn.

The extensive use of nitrogen (N) in agriculture has caused negative impacts on the environment and costs. In this context, two pot experiments were performed under different N levels and harvested at different vegetative stages to assess two popcorn inbred lines (P2 and L80) and their hybrid (F1 = P2 × L80) for the N use, uptake and utilization efficiency (with the inclusion and exclusion of root N content); to find the contrasting N levels and vegetative stages that effect nitrogen use efficiency (NUE) and to understand the relationship between the traits related to NUE. The hybrid and P2 were confirmed better than L80 for all the studied traits. NUE is mainly affected by the shoot dry weight, uptake and utilization efficiency. Extremely low and high N levels were found to be more discriminating for N use and dry weight, respectively. At the V6 (six fully expanded leaf) stage, root N content (RNC) should be considered; in contrast, at the VT (tasseling stage) stage, RNC should not be considered for the uptake and utilization efficiency. The genetic parameter performance for N use, uptake, shoot dry weight and N content could favor the achievement of the genetic gain in advanced segregating generations.

Relative importance of gene effects for nitrogen-use efficiency in popcorn.

The objective of this study was to evaluate the effects of additive and non-additive genes on the efficiency of nitrogen (N) use and N responsiveness in inbred popcorn lines. The parents, hybrids and reciprocal crosses were evaluated in a 10x10 triple lattice design at two sites and two levels of N availability. To establish different N levels in the two experiments, fertilization was carried out at sowing, according to soil analysis reports. However, for the experiments with ideal nitrogen availability, N was sidedressed according to the crop requirement, whereas for the N-poor experiments sidedressing consisted of 30% of that applied in the N-rich environment. Two indices were evaluated, the Harmonic Mean of the Relative Performance (HMRP) and Agronomic Efficiency under Low Nitrogen Availability (AELN), both based on grain yield at both N levels. Both additive and non-additive gene effects were important for selection for N-use efficiency. Moreover, there was allelic complementarity between the lines and a reciprocal effect for N-use efficiency, indicating the importance of the choice of the parents used as male or female. The best hybrids were obtained from inbred popcorn lines with contrasting N-use efficiency and N responsiveness.

Comparação de testadores em famílias S2 obtidas do híbrido simples de milho-pipoca IAC-112 / Comparison of testers in S2 families obtained from the popcorn single hybrid 'IAC-112'

Este trabalho teve como objetivo avaliar três testadores (o híbrido simples modificado IAC-112, a variedade BRS-Angela e a linhagem L.8.2, extraída de um material genético norte americano), para discriminar 64 famílias S2 derivadas do híbrido simples de milho-pipoca IAC-112. As famílias foram avaliadas por seu desempenho per se e em cruzamentos com os testadores. As características avaliadas foram rendimento de grãos (RG) e capacidade de expansão (CE). Foram obtidas estimativas das capacidades geral e específica de combinação, heterose dos topcrosses e a discriminação de cada testador por meio do índice de diferenciação proposto por FASOULAS (1983). Com base nesses parâmetros, o melhor testador para as famílias S2 foi a linhagem L8.2, tanto para RG quanto para CE

This work aimed to analyze three testers (the single hybrid IAC-112, the open pollinated variety BRS-Angela and the S9 inbred line L.8.2) for the evaluating of 64 S2 families derived from the single popcorn hybrid IAC112. The families per se and their respective crosses with testers were evaluated for grain yield (GY) and popping expansion (PE). The general and specific combining ability were estimated according to the partial diallel model, topcross heterosis and the discrimination capacity of each tester through the differentiation index proposed by Fasoulas were also evaluated. The inbred line L.8.2 was the best tester for both grain yield and popping expansion, discriminating reliably the families.