Cloning and Expression Analysis of FaSRP Gene in Festuca arundinacea under Abiotic Stresses

Erru YU, Xiaodong LI, Jianhong SHU, Jiahai WU, Xiaoli WANG*

1. Guizhou Institute of Oil Crops, Guiyang 550006, China;

2. Guizhou Institute of Prataculture, Guiyang 550006, China

Festuca arundinacea (Poaceae:Festuca) is a leaf-broad herb.Due to fast growth, broad adaptability, strong regeneration ability,strong resistance and long green period, Festuca arundinacea has become an important grass cultivar for establishing artificial grassland and reseeding natural grass pastures in temperate and subtropical regions[1-2].Festuca arundinacea can be used as both turfgrass and forage.Its growth is affected by drought, temperature, nutritional level, salinity and other abiotic stresses. The researches on molecular response mechanism of Festuca arundinacea under abiotic stresses and breeding of stress-tolerant cultivars are of great significance for cultivation and promotion of Festuca arundinacea.

In previous researches on model plants and crops, a number of functional resistant genes have been successfully identified, including genes directly protecting plants against environmental damage, genes indirectly protecting plants from drought and high temperature damage by regulating expression of other genes or other metabolic pathways, and genes encoding enzymes for synthesis of various osmotic-adjusting substances and for membrane lipid modification. The encoded proteins mainly belong to the functional enzymes[3-6]and important transcription factors[7-9].

Current studies have shown that SRPs (Stress Related Protein) are a kind of function-conserved protein making responses to stresses. In Arabidopsis, the expression of SRP gene is significantly induced by drought, high temperature and other stresses[10]. In rubber, the expression of SRP gene is not only associatedwith the production of rubber, but also highly correlated with resistance of rubber trees[11].The homologous genes of SRP in dandelion regulate the expression of genes making responses to bZIP class abiotic stresses by the ABA pathway to regulate the adaptability of dandelion to environmental stresses. The homologous gene CaSRP1 (Capsicum annuum stress-related protein 1) is over-expressed in Arabidopsis to promote the growth of roots and stems and to bring forward blotting. The over-expression of CaSRP1 can improve drought tolerance of plants, indicating CaSRP1’s involving in dual regulation of development and drought resistance in plants. It has been also revealed that SRP related genes are associated with resistance of non-rubber producing plants[13].

In this study,on the basis of transcriptome data,a functional gene making response to low nitrogen stress was isolated from the cDNA of Festuca arundinacea, and the full-length cDNA sequence of SRP gene was obtained by RACE technology. The secondary structure and molecular functions of SRP protein were predicted using bioinformatics software. To further verify whether SRP has the same function in Festuca arundinacea, the changes in expression of SRP gene in Festuca arundinacea were detected under low nitrogen,drought,high temperature, high salt and other abiotic stresses. The results showed that the expression of SRP in Festuca arundinacea was induced by low nitrogen,drought and high temperature stresses,but was insensitive to high slat stress. This study will provide certain basis for screening of resistance genes in pasture and theoretical foundation for stress tolerance molecular breeding of Festuca arundinacea.

Materials and Methods

Materials

Plant material Qiancao 1 is new national forage cultivar bred by the Guizhou Institute of Prataculture in 2005.Its registration number is 299.

Main reagents The E. coli DH5α competent cells were purchased from the Tiangen Biotech (Beijing) Co., Ltd.The cloning vector pMD 19-T, RNA extraction kit (TaKaRa RNAiso Reagent), RNA reverse transcription kit (RevertAid First cDNA Synthesis Kit) were purchased from the Thermo Scientific Company. The 5’RACE kit(5’RACE System for Rapid Amplification of cDNA Ends, Version 2.0) was purchased from the Shanghai Invitrogen Biotechnology Co., Ltd. The 3’RACE kit (SMARTer®RACE cDNA Amplification Kit) and Taq enzyme(Advantage 2 PCR kit) were purchased from the Clontech Laboratories Inc. The DNA Marker DL2000,100 bp DNA Ladder Marker, LA Taq enzyme, EASY Dilution, DNaseI and In Vitro Transcription T7 kit were purchased from the TaKaRa Biotechnology(Dalian)Co.,Ltd.The agarose DNA extraction kit was purchased from the Omega BioTek Inc. The yeast extract,peptone and agarose were purchased from the Oxoid Inc. The X-gal, IPTG and Ampicillin were purchased from the Beijing Soledad Technology Co.,Ltd.

Methods

Preparation and low-nitrogen treatment of plant material The Festuca arundinacea plants with similar genetic background were selected as tested material. Their full seeds were selected and soaked in water at 50 ℃overnight. Subsequently, the seeds were soaked and stirred in 75%ethanol for 30 s. And then, they were rinsed three times with sterile water.After soaked in 0.1% HgCl for 4 min,the seeds were rinsed 5-6 times with sterile water. The filter paper was laid in petri dishes,and then sterile distilled water was added into the dishes with a pipette.The seeds were placed on the filter paper, which was maintained moist with sterile distilled water. The seeds were inoculated in a light incubator (L∶D=16∶8, 22 ℃)[14]. After the germination, the seeds were transferred to self-made hydroponic culture cups containing Hoagland solution.After a 30-d culture, the nitrogen-free treatment (nitrogen stress treatment,and the NO3-in the solution was replaced by Cl-) and normal nitrogen treatment (control treatment, and the NO3-concentration in the Hoagland solution was as usual)were performed for the seeds. The nutrient solution was replaced once every three days.At the same time, in order to reduce the effect of microenvironment in the greenhouse,the planting pots were rotated regularly. The leaves of Festuca arundinacea in both of the two treatment groups were sampled every day.They were frozen immediately with liquid nitrogen, and then preserved at-80 ℃for use. In the high salt treatment group, the Festuca arundinacea plants were soaked in the Hoagland solution containing NaCl (400 mmol/L)for 24 h 7 d after their germination. In the high temperature stress treatment group, the 30-d-old Festuca arundinacea plants were treated by high temperature (42 ℃) for 24 h. In the drought treatment group, the Festuca arundinacea plants were soaked in 30% PEG solution for 24 h 7 d after their germination. The sampling was performed on hour 0,0.5,1.0,2.0,6.0,12.0 and 24.0,respectively.

Extraction of total RNA from Festuca arundinacea The total RNA in leaves of Festuca arundinacea was extracted using extraction kit (TaKaRa RNAiso Reagent)[15]. The absorbances of the extracted RNA at 260 and 280 nm were determined with a UV spectrophotometer. The A260/A280ratio was calculated to determine the purity of extract RNA. The integrity of extract RNA was examined by 1.0% agarose gel electrophoresis. The extracted RNA was reverse transcribed into cDNA using ReverseAid First Strand cDNA Synthesis Kit, and finally, the cDNA was preserved at-80 ℃for use.

Cloning of full-length cDNA of SRP gene in Festuca arundinacea The genetic sequence of SRP in Festuca arundinacea, obtained by transcriptome sequencing was used as the templates. Using the Primer Premier 5.00,one pair of primers was designed to verify the accuracy of sequencing and splicing. They were named as SRP-fwd1 (5’-GCTGAGGCGGCTGGAGTT-3’) and SRP-rev1 (5’-CACCTTGGCGATGCGCTC-3’). According to the instructions of 3’RACE and 5’RACE kits, three specific downstream primers were designed to clone the 5’ end of the target fragment, including SRP-rev2 (5’-AGTCGACGGCGTCGAC-3’), SRP-rev3 (5’-CGTGGCCCTTGGCGAGCG-3’)and SRPrev4(5’-TAGAGCCCCGCGATGCAC-3’).At the same time,two specific up-stream primers were designed to clone the 3’ end of the target fragment, including SRP-fwd2 (5’-TGTTCCCGCAGGTGGCGCAGAT-3’), and SRPfwd3 (5’-GCCGCCTACTGGTGCGAGAAG-3’). The PCR product was examined using 0.1% agarose gel electrophoresis. And then, the PCR product was recovered, purified and inserted into the T vector and cloned. The sequencing was completed by the Shanghai Sangon Biological Engineering Technology & Services Co.,Ltd. The sequencing results were checked and spliced by DNAMAN.Thus the full-length cDNA sequence of SRP gene in Festuca arundinacea was obtained.

Bioinformatic analysis of SRP sequence in Festuca arundinacea

The cDNA sequence of SRP gene in Festuca arundinacea was aligned with those of SRPs by BlastX in NCBI.The sequences of SRPs among which the homologies were higher than 30%were selected for phylogenetic analysis.The relative molecular weight,isoelectric point and hydrophobicity of SRP were analyzed using ExPASy(http://web.expasy.org/protparam/;http://web.expasy.org/protscale/). The secondary structure of SRP was analyzed using SOPMA (http:// npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl page=npsa_sopm.html?).The subcellular localization analysis was performed at http://psort.hgc.jp/form.html.

Fluorescence quantitative PCR analysis of SRP gene expression in Festuca arundinacea under different abiotic stresses The expression levels of SRP gene in Festuca arundinacea under low nitrogen,drought, high temperature and high salt stresses were detected using qRT-PCR. Using the cDNA sequence of SRP gene in Festuca arundinacea,obtained by transcriptome sequencing,as the template, the primers were designed (SRP-F: 5’-AGCACCTCGCCATCTCCACC-3’; SRP-R: 5’-ACCCTGTTGTACTTCTCGCACC-3’). The reference gene was UBI (UBI-F:5’-CACCTCGATCACCCACCTCT-3’;UBI-R:5’-AGGGTCTCCGATAACCTCCA-3’).The final expression levels of SRP gene in Festuca arundinacea were calculated using the 2-ΔΔctmethod[15].There were three biological replicates for each sample, and there were three technical replicates for each biological replicate.

Results and Analysis

Cloning of SRP gene in Festuca arundinacea

Using the cDNA sequence of Festuca arundinacea as the template,the 3’ and 5’ ends of the SRP gene were amplified using the 3’RACE and 5’RACE methods. The primers were designed by spicing sequence fragments from transcriptome sequencing(Fig.1a, Fig.1b). Based on the RACE results, the primers were designed to amplify the full-length cDNA sequence SRP gene. The accuracy of amplified cDNA sequence of SRP gene was examined by aligning with spliced sequence fragments (Fig.1c). The fulllength cDNA sequence of SRP gene in Festuca arundinacea was obtained.The full length of SRP gene sequence is 1 165 bp, composed of 5’ untranslated region (47 bp), 3’ untranslated region (263 bp) and an open reading frame(855 bp).The SRP gene in Festuca arundinacea encodes a protein composed of 284 amino acids(Fig.2).

Bioinformatic analysis of SRP gene in Festuca arundinacea

Using the ExPASy software, the relative molecular weight and isoelectric point of SRP protein were speculated as 30.323 kDa and 5.24,respectively. The subcellular localization analysis showed that SRP protein is located in cytoplasm.The hydrophobicity analysis showed that the maximum hydrophobic value of SRP protein is 2.300, and minimum hydrophobic value is -1.989. SRP protein has total 9 hydrophilic peaks (E＜-1.5).The secondary structure of SRP protein was predicted by SOPMA. The results showed that SRP protein contains about 54.23% of the α-helix, 5.99%of β-corner and 30.28% of irregular curling.

Conserved domain analysis of SRP protein and homology analysis of SRP genes

The structure prediction of SRP protein showed that FaSRP has a REF domain in the amino acid range of 12-234. To determine the genetic rela-tionships between SRP protein of Festuca arundinacea and SRPs of other plants, the amino acid sequences of SRPs were aligned by DNAMAN and MEGA. The results showed that the amino acid sequences of SRPs were mainly classified into two groups, dicot group and monocot group(Fig.3).In the monocot category, the homologies of SRP amino acid sequences between Festuca arundinacea and Brachypodium distachyon (XP010234141.1), Oryza brachyantha(XP006658100.1),Setaria italica (XP004958627.1), Sorghum bicolor (XP002461188.1), Zea mays(ACG39345.1) and Hordeum vulgare(BAJ95174.1)range from 64%to 78%.However, the genetic distances between SRP of Festuca arundinacea and those of dicots are farer(Fig.4).In FaSRP, the conserved area is mainly concentrated in the ERF domain in the middle of the sequence.

Expression characteristics of SRP gene in Festuca arundinacea under abiotic stresses

In order to explore the responses of SRP gene in Festuca arundinacea to various abiotic stresses, the changes in expression level of FaSRP were studies by qRT-PCR under low nitrogen, drought, high temperature and high salt stresses. Under low nitrogen stress, the expression level of SRP in Festuca arundinacea was increased significantly 30 min after the starting of stress, and it reached the peak 1 h after the starting of stress.However, the expression of SRP was inhibited after 2 hours. During the stress interval of 6-24 h, the expression level of SRP was basically the same with that under normal conditions(Fig.5a).Under drought stress,in the first period(0-6 h), the expression level of FaSRP showed no significant difference between stress and normal environments; in the late period (12-24 h), the expression of FaSRP was significantly induced (Fig.5b). Under high temperature stress, the expression level of FaSRP was significantly increased 1 h after the starting; in the stress interval of 2-12 h,no significant difference was found in FaSRP expression level between the treatment and control groups; the expression of FaSRP was significantly induced even 24 h after the starting (Fig.5c). Under high salt stress, the expression of FaSRP was slightly inhibited, but no significant difference was found between the treatment and control groups(Fig.5d).

Conclusions and Discussion

Forage is mainly planted in the arid and semi-arid areas in North China and remote mountainous areas in South China, and it is more susceptible to environmental stress. In South China, drought, heat stress, nutritional deficiency and other hazards cause greater harms to crops[16],and they are major factors limiting crop yield. However, traditional breeding of highly outcrossing forage crops often requires more resources and time. Therefore,screening and verification of functional genes are of great significance for germplasm innovation by genetic modification.

In previous studies, a number of low nitrogen-resistant genes have been screened from Festuca arundinacea by transcriptomics and proteomics technologies. At both transcription and protein levels, the expression of SRP gene is significantly induced. The bioinformatic analysis shows that the SRP gene of Festuca arundinacea has high homologies with those of other crops, especially of Oryza brachyantha, Setaria italica,Sorghum bicolor, Zea mays and other monocots (homology ＞60% ). The REF domain shows higher conservatism, indicating functional conservatism of SRP gene in evolution.

Plant growth and development is often affected by many adverse environmental factors. There have been some reports on simultaneous responses of tobacco and Arabidopsis to multiple environmental stresses[17].Under multiple or single environmental stresses, differentially expressed genes are sometimes overlapped in plants.The overlapped genes can regulate the resistance of plants to multiple stresses. They are also candidate genes for breeding of multiple stresses-resistant germplasms[18-20]. SRPsare found to be capable of making responses to drought, high temperature and other environmental stresses[10,17].In this study, the changes in expression level of FaSRP under low nitrogen, high temperature, drought and high slat stresses were studied. The results showed that the expression level of FaSRP differs with the proceeding of low nitrogen stress, as well as drought and high temperature stresses. However, the expression level of FaSRP is not significantly changed under high salt stress. The variations in FaSRP expression level are not exactly the same under different abiotic stresses. It suggests that SRP gene is involved in adaptability regulation of plants to several stresses, but the regulatory pathways are different.This study will provide certain candidate gene and technical reserve for breeding of drought- and high temperature-tolerant, nutritious and highly efficient Festuca arundinacea cultivars.

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