Preparation And Identification Of Nitrate Reductase-deficient Mutants Of Dunaliella Salina

Dunaliella salina (D. salina) is a unicellular green alga without cell wall. It is known as probably the most halotolerant eukaryotic organism living in various marine habitats. D. salina can survival even in solutions containing 5.0 mol/L NaCl, and grow rapidly in the medium containing 1-2 mol/L NaCl. Being phototrophic, D. salina can be grown in large pools utilizing available sunlight and carbon dioxide as energy source. Furthermore, D. salina is an eukaryote and therefore it can express complex proteins that require post-translational modification. These characteristics make D. salina potentially useful as a novel bioreactor for synthesizing compounds of pharmaceutical in interest and an alternative model organism to study the molecular biology of many important cellular processes.In study of transgenic D. salina, it is critical to establish a specific selectable marker to detect whether a target gene is transformed, especially stably transformed. Antibiotics and herbicides resistance have been used as selectable markers for nuclear transformation of D. salina. However, low efficiency of transformation limits their application.Transformation systems based on complementing nitrate reductase (NR)-deficient mutants have been developed in a number of filamentous fungi and algae. The nitrate assimilation system offers several inherent advantagesfor transformation, such as high frequencies, ease of recipient isolation through resistance to chlorate, and low background growth after transformation. The transformation system based on complementing nitrate reductase-deficient mutants in D. salina has not been reported so far.Plants and algae utilize nitrate as a main source of nitrogen from the environment. They assimilate nitrate by the following pathway: after being actively transported into the cell, nitrate is reduced to nitrite by NR, and nitrite is reduced to ammonia by NiR. Ammonium ions enter the amino acid pool primarily via the action of glutamine synthetase (GS). In assimilation of nitrate, NR is considered as the key and rate-limiting enzyme. Chlorate, a nitrate analogue and substrate for both prokaryotic and eukaryotic NR, has been successfully used to select NR-deficient mutants in certain fungi, algae and higher plants, which have been shown to be resistant to chlorate toxicity in mutants but not in wild types. This fact is generally explained by assuming that chlorate itself is not toxic, but can convert into a toxic compound, chlorite, by the catalytic activity of NR.In our study, we hope to construct a selectable marker by cloning NR gene from D. salina and introducing it into NR-deficient mutants of D. salina to rescue their NR activities. Preparation and identification of NR-deficient mutants of D. salina is one of the most important steps in the process.Here, we reported that NR-deficient mutants of D. salina were prepared from mutagenized D. salina by chlorate-resistant selection and phenotype selection (growing experiments with different nitrogen cultures). And then these mutants were identified by determining their NR activities and the levels of NR mRNA using Real-time fluorescene quantitative PCR. The study may lay a solid foundation for the construction of transformation systems based on complementing nitrate reductase-deficient mutants in D. salina.Methods and Results1. Preparation of NR-deficient mutants of D. salina1.1 Mutagenizing D. salina with Ethynetrosourea (ENU)In this work, wild-type D. salina cells were collected at mid-exponential phase of the growth, thoroughly washed with nitrogen-free medium, and resuspended at approximately 2-3 x106 cells/ml in medium with nitrate (5.0 mmol/L NaNO3) as N source. In order to increase the mutagenesis rate of D. salina, mutagenic treatments were performed by adding 0.25 mmol/L and 0.50 mmol/L Ethynetrosourea (ENU) respectively, a kind of chemical mutagen.1.2 Chlorate-resistant selection of D. salina10 days after mutagenesis, the cells of D. salina were collected by centrifugation and resuspended in PKS medium with 5.0 mmol/L NaNO3 containing 200 mmol/L NaClO3 for selection, and incubated for 10 days later. Subsequently the cells were rescued in the medium with 2.5 mmol/L urea as the N source for 5-10 days, and then were streaked on solid media (10 g/L agar) containing different concentrations of NaC103 (50 mmol/L, 100 mmol/L NaClO3), respectively for further selection. After incubation for 25-30 days, mutants were isolated by selecting colonies representing chlorate resistance (showing little or no chlorate damage). The colonies of chlorate-resistant mutants picked out from solid selective media were recovered individually in the medium with 2.5 mmol/L urea as N source.1.3 Isolation of NR-deficient mutants of D. salina through phenotype selectionThe chlorate-resistant mutants were further confirmed by their ability to grow well when urea or nitrite existed in the medium, but not to grow in the medium containing nitrate as a sole nitrogen source. Those recovered cells of chlorate-resistant mutants in 2.5 mmol/L urea medium were streaked on solid media (10 g/L agar) with 5.0 mmol/L NaNO3, 5.0 mmol/L NaNO2 and 2.5 mmol/L urea respectively as nitrogen sources. After incubation for 25-30 days, the colonies on the media containing 5 mmol/L NaNO2, or 2.5 mmol/L ureawere green and similar to wild-type D. salina, while those on the medium with 5 mmol/L NaNO3 were chlorosis and grew to be pinpoint compared with wild-type D. salina. Finally through phenotype selection we isolated 30 mutants, which grew well only with urea or nitrite, by picking them out from those media, and the putative NR-deficient mutants were cultured individually for further identification. 2. Identification of NR-deficient mutants of D. salina2.1 Determining NR activities of putative NR-deficient mutants of D. salinaNR activity of D. salina was determined according to the concentration of nitrite assayed with sulfanilamide colormetry. By comparing NR activites of NR-deficient mutants with that of wild-type D. salina, the mutants were preliminarily identified. Of 30 mutants, NR activites of 6 decrease significantly (P < 0.0001) .2.2 Real-time RT-PCR quantification of NR mRNA in NR-deficient mutant of D. salinaThe total RNA isolated from wild-type D. salina and NR-deficient mutant of D. salina was reversely transcribed into cDNA respectively. The mRNA level of NR was measured using the real-time fluorescene quantitative PCR on Gene Amp 5700 Sequence Detection System with SYBR Green I chemistry. P-actin gene was used as an endogenous control. The relative ratio between NR mRNA and P-actin mRNA was used to measure the expression level of NR in NR-deficient mutant of D. salina.The real-time fluorescene quantitative PCR method was performed successfully to precisely detect the level of NR mRNA in NR-deficient mutant of D. salina. The expression level of NR are lower in 2 of 3 strains of NR-deficient mutants of D. salina than that of wild-type D. salina. The mRNA levels in mutant strain m3-4, m2-8 are respectively about 2 and 23 times less than that of wild-type D.salina.Conclusions:1. The mutants have been isolated in phenotype by mutagenizing D.salina with Ethynetrosourea (ENU), chlorate-resistant selection and growing experiments with different nitrogen sources; and finally these mutants have been identified by determining their NR activities and the level of NR mRNA using Real-time fluorescene quantitative PCR. A set of procedures of mutagenesis^ selection isolation and identification of NR-deficient mutants of D. salina is established.2. 30 putaive NR-deficient mutant strains of D. salina are isolated by chlorate-resistant selection and phenotype selection. Compared with NR activites of wild-type D. salina, of 30 mutants, NR activites of 6 mutants are decreased significantly and the levels of NR mRNA are lower in 2 strains of 3 strains of NR-deficient mutants of D. salina. Therefore the method of mutagenesis and selection of D. salina remain to be improved. The mutants acquired are also needed to furtherly select and identify. The study may lay a solid foundation for the construction of transformation systems based on complementing NR-deficient mutants in D. salina.