THE EGYPTIAN SOCIETY OF GENETICS Volume 46 July 2017 No . 2 ISOLATION AND SEQUENCE ANALYSIS OF A NOVEL PARTIAL VACUOLAR Na + / H + ANTIPORTER cDNA FROM Capparis orientalis , Lycium shawii AND Zygophyllum album

Egyptian native (nondomesticated) plant species from different families such as Capparis orientalis, Lycium shawii and Zygophyllum album were collected from North Western coast of Marsa Matrouh Governorate, Egypt. Plant vacuolar Na+/H+ antiporter candidate gene from tolerant plant species; several are localized on the tonoplast, plays an important role in several plant species (Halophytes and xerophytes) under abiotic stress. Then, once the genes will be identified from tolerant plant species, the overall goal of this study is to identify partial the vacuolar antiporter NHX1 candidate gene. According to NHX1 family homologous sequence conservative region; one degenerate oligonucleotide primer pair was used to amplify core (partial middle) fragment of cDNAs vacuolar Na+/H+ antiporter with about size of 600 bp, approximately. Touchdown PCR program (TD-PCR) of cDNAs were success to increase specificity, sensitivity and yield to amplify core cDNA of vacuolar Na+/H+ antiporter gene. Sequence analysis provided us with a novel partial fragment length of cDNAs about 548 bp, 557 bp and 557 bp of a novel CoNHX1, LsNHX and ZaNHX was deposited in GenBank database with NCBI, GenBank accession no. KJ452345.1, KJ452346.1 and KJ452347.1 and amino acid sequences about 182 a.a, 185 a.a and 185 a.a with GenBank accession no. AHY19036.1, AHY19037.1 and AHY19038.1, respectively. BLASTN of sequences result and phylogenetic relationship analysis indicated that all were clustered into the vacuolar Na+/H+ antiporter group. The deduced amino acid sequences showed high identities with other plant vacuolar-type Na+/H+ antiporters. Taken together, these results suggest that CoNHX, LsNHX and ZaNHX are new members of the vacuolar Na+/H+ antiporter family. The ultimate goal of this study provided a basic foundation information about a Novel partial vacuolar Na+/H+ antiporter gene to develop 5` and 3` RACE technique (Rapid amplification cDNA Ends).

n Egypt, as in the majority of the arid and semi arid regions, drought and salinity are responsible of substantial losses of culture yield, deterioration of plant cover and erosion of soils.Water is one of the most important constraints of agriculture production with faces salinity and drought stress.
In the North coast of Marsa Matruh Governorate -Egypt, there are many desert plant species that tolerant drought and salinity stresses.Among these species, Capparis orientalis (LASAF/KABBAR): a leaf succulent obligate halophyte and a species of plant belong to family Capparaceae.Ahmed et al. (1972) reported that some Egyptian Capparis species contain of glucosinolates, glucoiberin, glucocapparin, sinigrin, glucocleomin, glucobrassicin and glucocapangulin were isolated, characterized and identified.In the Arabian folk medicine, several Capparis species have many uses (Shahina, 1994).

Lycium shawii (AWSAJ) belongs
to family Solanaceae, semi-succulent and a thorny perennial shrub.Fukuda et al. (2001) reported that Lycium species most-ly occur in arid and semi-arid climates, and a few are known from coastal zones in somewhat saline habitat types.Solanaceae are known for having a diverse range of alkaloids.As far as humans are concerned, these alkaloids can be desirable, toxic, or both.It grows along sandy stone ridges.It has purple, sometimes white, trumpet-like flowers and sharp thorns.The leaves are elliptical and congested in closed clusters (Omar et al., 2007).Cherouana et al. (2013)  Zygophyllum album is a succulent cushion-like under shrub frequently reaching 1 m in height.The leaves and branches are blue-green, mealy pubescent, and present in oases, eastern Egyptian desert, Red sea coastal region and Sinai (Täckholm, 1974).Hassanean et al. (1993) characterized besides the two known saponins, quinovic two new glycosides were isolated from the aerial parts of Zygophyllum album growing in Egypt.Moustafa et al. (2007) detected the chemical constituents of Zygophyllum album (L.) (family Zygophyllaceae) isolated three flavonoids via Kaemferol, Isorhmnetin and Quercetin-3-O-gluoside.
In this study, two modified protocols for RNA isolation are described here as a simple, fast, convenient and does not require DEPC (diethylene pyrocarbonate) treatment.Reverse transcription of the RNA followed by PCR amplification was used to confirm that the RNA produced is able to generate cDNA.One degenerate primer pair with touchdown RT-PCR program is used to amplify a partial middle fragment cDNAs of a novel vacuolar Na + /H + antiporters (NHX) from Capparis orientalis, Lycium shawii and Zygophyllum album which were isolated, sequenced and analyzed.

Plant materials
One hundred milligram of collected frozen tissue samples from three plant

Total RNA extraction and purification
For establishing the suitable method for isolating total RNA from plants under this study, two different methods were evaluated TRIzol® Reagent and RNeasy Plant Mini Kit.For TRIzol® method, total RNA was extracted using 100 mg tissue/1 mL TRIzol® Reagent according to the manufacturer's instruction (Invitrogen, Cat no. 15596-026).Isolated RNA was cleaned via RNeasy Plant Mini Kit.For RNeasy Plant Mini Kit method, 100 mg of plant tissue was used for each reaction.Polyethylene Glycol (PEG-MW 6000) was add to RLC or RLT buffer provided with kits breach of a concentration of 30 mg/1 ml and was incubated at 60C for 3 hours and keep worm before use.
Fine powder of samples was subjected to RNA extraction following the manufacturer's procedure according (RNasy mini plant Kit Cat No: 74904).RNA was suspended in 30-50 µl in RNase free water and stored in -80C for further analysis.
Purified RNA samples were measured using NanoDrop spectrophotometer (NanoDrop, Technologies Inc.).The integrity of total RNA was verified using 1.2% non-denaturing agarose gel electrophoresis.

RNA analysis by one step RT-PCR
Positive RNA (2X 105 copies/ µL), provided by TaKaRa one step RNA PCR Kit was applied as control according to the manufacturer's instruction (TaKaRa Bio INC.Cat.No. RR024A).Two micro liter of total RNA extraction from plant species were used as a template.Primers ubiquitous 18s rRNA universal primer sequences were used as positive control for one step RT-PCR to amplify 1 kb of the 18S rRNA, (18S-F: 5`-CAG TAG TCA TAT GCT TGT CTC AAA-3`/ 18S-R: 5`-GAC TAG GAC GGT ATC TGA TCG T-3`), (Brunner et al., 2004;Ashoub et al., 2006).Sample amplified products were analyzed using 1.2% agarose gel electrophoreses staining with ethidium bromide.

Amplification of partial cDNA Na + /H + antiporter gene by RT-PCR two steps
Two steps were performed to amplify the cDNA for the NHX gene.For preparing the first strand cDNA synthesis of total RNA 10 pg -500 ng, oligo (dT) 20 and M-MLV RT (SuperScript III Reverse Transcriptase) were mixed and used according to the manufacturer's instructions (Invitrogen, Cat No. 18080-085).The cDNA synthesis reaction was stored at -20C to be used for second step PCR.

Oligonucleotide primers designing
One degenerate primer pair was designed from Na + /H + antiporter genes at the conserved nucleotide sequences region which were determined based on the multiple sequence alignment of other Na + /H + antiporter gene families sequences from selected plant species in the universal database.Alignments between related species to plants under this study showed a homology at some regions of the gene (ORF) were species specific.
A volume of 10 µl of each sample was analyzed using 1.2% agarose gel electrophoreses and stained with ethidium bromide (Eth-Br).The PCR fragments of each sample were excised and purified from the agarose gel with a clean, sharp scalpel.The gel slice was weighed in a colorless tube and the QIAquick ® Gel Extraction Kit (Qiagen, cat. no. 28706) was used to elute the DNA from the gel.For PCR product, the sample was centrifuged according to the manufacturer's procedure in the QIAquick PCR Purification Kit (Qiagen, Cat.No. 28106).uk/clustalw).Evolutional distances were calculated using the neighbor-joining method (Saitou and Nei, 1987).Sequence analysis at BLAST search and ExPASYtranslate tool data analysis were conducted on the NCBI platform.
Partial middle cDNA of vacuolar Isolating RNA using both the commercial reagent TRIzol ® Reagent (Invitrogen) followed by purification step with a Qiagen spin-column and RNeasy plant mini Kit (Qiagen) purified with PEG for RNA extraction showed high efficiency for isolating high-quality and quantity RNA suitable for using in sensitive downstream applications.In RNeasy Plant mini Kits (Qiagen), RLC or RLT extraction buffer contain β-mercaptoethanol to prevent sample oxidation and to inhibit RNase release from tissue prior to chloroform extraction.Polyethylene glycol (PEG, MW: 6000) probably played an important role in separating polyphenols and polysaccharides from RNA.Results showed good quality of the isolated RNA provided by NanoDrop spectrophotometric measurements, as it gave A 260 /A 280 absorbance ratio between 1.9-2.0;indicating that RNA was relatively free of DNA and protein contamination.Isolated total RNA was run on 1.2% agarose gel electrophoresis.Clear photo of isolated RNA was obtained and both 28S rRNA with molecular size (3.7 Kb) and 18S rRNA with molecular size (1.9 Kb) were separated clearly from mRNA, discrete ribosomal RNA with no apparent RNA degradation, indicating that RNA is also relatively free of RNase (Fig. 2).Similar results were also reported by Kiefer et al. (2000), Tattersall et al. (2005) and Portillo et al. (2006).
The RNA quality was tested by one step RT-PCR for TaKaRa kit using universal oligonucleotide primers, which were designed based on the conserved 18S rRNA.Reaction products and 1 Kbp DNA leader plus, were separated on 1.2% agarose TAE of gel electrophoresis and visualized under UV light following Et-Br staining (Fig. 3).
Results showed that the expected 1 kb DNA fragment of the 18S rRNA was amplified as a positive control from plant species.This result is in agreement with those observed by Brunner et al. (2004) and Ashoub et al. (2006).This protocol allowed RNA isolation with high purity from plant species under this study.The method may be suitable for other plant species from different families rich in polyphenols and polysaccharides.

Amplification of NHX1 partial cDNA by RT-PCR two steps
One degenerate primer pair (NHX F-dp and NHX R-dp) was used to amplify a partial middle fragment of cDNA Na + /H + antiporter gene in plant species (Capparis orientalis, Lycium shawii and Zygophyllum album) which gave one fragment with size about 600 base pair (Fig. 4).Using touchdown PCR program (TD-PCR) of cDNA was able to increase specificity and sensitivity to amplify partial a according to Hecker and Roux (1996) and Korbie and Mattik (2008).

Phylogenetic relationship based on sequence analyses
All purified PCR products were sequenced.The partial fragment sequence showed sequence identity with Na BLASTN of result and phylogenetic relationship analysis indicated that all obtained fragments were clustered into the vacuolar Na + /H + antiporter group.The deduced amino acid sequences showed high identities with other plant vacuolartype Na + /H + antiporters (Figs 8 and 9).Taken together, these results suggest that CoNHX, LsNHX and ZaNHX are new members of the vacuolar Na + /H + antiporter families.Multiple alignments of vacuolar Na + /H + antiporters (the deduced amino acid sequence) showed that CoNHX, LsNHX and ZaNHX share high identity with other plant vacuolar Na + /H + antiporters as shown in Table (1) and in Figs ( 8 and 9).Some putative membrane spanning domains (M4, M5, M6 and M7) were recognized by SOSUI software program (http://harrier.nagahama-ibio.ac.jp/sosui/ cgi-bin/adv_sosui.cgi;Hirokawa et al., 1998).

SUMMARY
species, Capparis orientalis, Lycium shawii and Zygophyllum album were placed in sterile 2 ml microfuge and immediately dipped in liquid nitrogen, and crush into fine powder using Tissue Lyser II machine (Qiagen) for homogenization to avoid browning and degradation during RNA extraction.All reagents and materials including UltraPure™ DNase/RNase-Free distilled water were autoclaved, without treating with DEPC (Diethyl pyrocarbonate).

Fig. ( 6
Fig. (6): Partial fragment (557 bp) of Na + /H + antiporter gene of LsNHX and deduced amino acid sequence of LsNHX from Lycium shawii, GenBank accession number is KJ452346.1/AHY19037.1.Nucleotide sequence and deduced sequence of amino acid residues of Na + /H + antiporter gene are indicated by a single letter code.

Fig. ( 7
Fig. (7): Partial fragment (557 bp) of Na + /H + antiporter gene of ZaNHX and deduced amino acid sequence of ZaNHX from Zygophyllum album, GenBank accession number is KJ452347.1/AHY19038.1.Nucleotide sequence and deduced sequence of amino acid residues of Na + /H + antiporter gene are indicated by a single letter code.
isolated two known flavonoid glycosides from arial parts of Lycium arabicum, the compounds were identified by spectral analysis.
*Homology values of nucleotide are bold.Homology values of amino acid are in brackets.