Expression of Aquaporin BnPIP-like Gene from Rapeseed (Brassica napus) Enhances Salt Resistance in Yeast (Pichia pastoris).
A new aquaporin (AQP) BnPIP-like gene was isolated and sequenced from rapeseed (Brassica napus L.). It encoded a putative protein with 281 amino acids, sharing 95.3% identity with Arabidopsis plasma membrane intrinsic proteins (PIP, Genbank: NP_195236.1). Prediction of transmembrane structure showed that BnPIP-like gene contained five loops and six transmembrane helices. Online analysis also indicate that BnPIP-like protein existed as homo-tetramers. In order to research its functions in eucaryon, BnPIP-like gene was fused into the pPIC3.5K and then the recombinant vector, as well as the pPIC3.5K, were induced into methylotrophic yeast (Pichia pastoris Strain GS115), respectively. In BMMY media, the BnPIP- like protein was sufficiently expressed after methanol (MeOH) induction for 24 h. The salt stress (300 mM NaCl) treatment was operated after 18 h of normal growth conditions.
The OD600 values of BnPIP-like-transgenic P. pastoris and the control were determined every 12 h. The growth curves were obtained and it showed that the growth of both P. pastoris with or without BnPIP-like protein was obviously inhibited. However, the concentrations of BnPIP-like-transgenic P. pastoris was always higher than the control, indicating that the inhibition of growth in BnPIP-like-transgenic P. pastoris was slighter because of the over-expression of BnPIP-like gene. Thus the resistance to salt explained the eukaryotic functions of BnPIP- like protein. Moreover, it provided a theoretical possibility for a more comprehensively industrial utilization of fermenting yeast.
Keywords: Brassica napus; Aquaporin; Salt resistance; Eukaryotic expression
Salt stress usually interrupts the endocellular ionic or osmotic equilibrium, inhibits the growth and metabolisms in higher plants; secondarily it also results in consequent oxidative stress, or even death (Niu et al., 1995; Zhu, 2001), among which the inhibition of growth was the most significant. Salt stress is one of the most crucial abiotic stresses in agriculture. There are about 20% of the world's cultivated lands and nearly 50% of all irrigated lands are affected by it (Zhu, 2000; 2001). It lead to the reduction of plant growth and crop production (Munns et al., 2006). As to the rapeseed (Brassica napus L.), one of the most important oil-crops in the world, soil salinity affects its yield and quality characters significantly (Zadeh and Naeini, 2007; Zamani et al., 2010; Jian et al., 2014). There were some methods to solve the salinity problems.
Besides irrigation with fresh water and improving soil drainage, studying salt tolerance in plant with a view to identify and eventually to manipulate the genes involved in salt resistance is another promising approach (Zhu, 2000).
Higher plants had different regulating metabolism pathways in response to salt stress, such as eliminating the reactive oxygen species (ROS), synthetizing the osmotic regulators, utilizing ATPase to maintain the Na+ or Cl- ion concentrations, expressing late embriogenesis abundant protein (LEA) and transporting H2O molecules by aquaporin (AQP) etc. Aquaporin is a kind of integral membrane proteins, which transport water molecules across membrane selectively and efficiently. It is a member of major intrinsic protein (MIP) super-family (Zardoya, 2005). The first AQP protein, named CHIP28 or AQP1, was isolated from human red blood cell in 1988 (Denker et al., 1988); its function as membrane water channel in Xenopus laevis oocytes was confirmed in 1992 (Preston et al., 1992). One year later, the first plant AQP protein g-TIP was isolated from Arabidopsis (Maurel et al., 1993).
AQPs from higher plants such as tobacco, spinach, maize, rice, wheat etc. were cloned (Maurel et al., 2008; Ludewig and Dynowski, 2009), as well as some non-vascular plant (Danielson and Johanson, 2008). Besides plants, AQPs were also found in creatures: archaebacteria, bacteria, fungus, animals etc. Discovery and researches on AQPs overturned the long-standing theory that free diffusion driven by osmotic potential was the only way for water molecules to transport across the membrane. According to their sequence homology and sub-cellular location, plant AQPs could be subdivided in four subgroups: plasma membrane intrinsic proteins (PIPs): PIP1, PIP2 and PIP; tonoplast intrinsic proteins (TIPs), which further subdivided into 5 subgroups: a, b, g, d and e-TIP; nodulin 26-like intrinsic proteins (NIPs); small and basic intrinsic proteins (SIPs, further subdivided into 2 subgroups: SIP1 and SIP2); GlpF-like intrinsic proteins (GIPs).
Besides the water molecular transporting and consequent drought or salt tolerance, AQPs also took part in some other biological processes such as photosynthesis (Uehlein et al., 2003), flowering (Bots et al., 2005), seed germination (Schuurmans et al., 2003), seed maturing and so on; it also transports other small molecules like glycerol (Schuurmans et al., 2003), H2O2 (Bienert et al., 2007) and CO2 (Uehlein et al., 2003).
In the present study, a new aquaporin (AQP) BnPIP-like gene was first isolated and sequenced from rapeseed. Prediction of transmembrane structure showed that BnPIP- like protein contains five loops and six transmembrane helices and online analysis also indicate that it existed as homo-tetramers. In order to study its functions in eucaryon, BnPIP-like gene was fused into the pPIC3.5K. After the recombinational plasmid was linearized, BnPIP-like gene was then induced into methylotrophic yeast (Pichia pastoris Strain GS115). After induced by methanol for 18 h in BMMY media, the BnPIP-like protein was sufficiently expressed. The salt stress (300 mM NaCl) treatment was operated. The OD600 values of BnPIP-like-transgenic P. pastoris and the control showed that both kinds of P. pastoris cells (with or without BnPIP-like protein) were obviously inhibited.
However, the concentrations of BnPIP-like-transgenic P. pastoris was always higher than the control, indicating that inhibition of growth in BnPIP-like-transgenic P. pastoris was slighter because of the over-expression of BnPIP-like gene. Thus the resistance to salt explained the eukaryotic functions of BnPIP-like protein. Unlike previous research, here the BnPIP-like gene was induced into and expressed in yeast, rather than Xenopus laevis oocytes. Thus it also provide a possibility for industrial yeast to enhance their survive rate in tough conditions.
Materials and Methods
Materials and Vectors
Seeds of Brassica napus L. (cultivar JR9), eukaryotic expression vector pPIC3.5K and yeast cells (Pichia pastoris Strain GS115) were kept in Crop Research Institute, Sichuan Academy of Agricultural Sciences (SAAS). Intermediate cloning vector pEASY-T Simple, competent cell TOP 10 and high fidelity Trans Taq-T DNA polymerase were purchased from Transgen Biotech Company. RNA extraction kit and reverse transcription kit were purchased from TianGen Company. Seeds of B. napus were sterilized by 75% ethanol (EtOH) and 0.1% Hg2Cl2 successively, and then plated on MS solid medium (Murashige and Skoog, 1962). The aseptic seedlings were grown in climatic chamber at 24degC with 16/8 h of light/dark cycle and 60% humidity.
Cloning and Sequencing of BnPIP-like Gene
20 days after germination, the whole aseptic seedlings were used to extract the total RNA. Total RNA was prepared from these 20-days old seedlings using TianGen RNA extraction kit according to the instructions. Reverse transcription was performed with oligo-T primer, by Reverse Transcriptase kit in accordance with the instructions. Thus, the total cDNA was obtained. Then PCR was performed with specific primers PIP-1 and PIP-2 (PIP-1: 5'- ATGTCGAAAGAAGTGAGCGAAGA-3'; PIP-2: 5'- TCAGTTTGTTGCGTTGCTTCGGA-3'). The primers were designed by Primer Premier 5.0 with homologous cloning method, according to the sequence of Arabidopsis plasma membrane intrinsic proteins gene (PIP, Genbank: NP_195236.1). The PCR cycling procedure consisted of 2 min at 94degC, 33 cycles for 30 s at 94degC, 30 s at 52degC and 55 s at 72degC, and a final 15-min extension at 72degC. The PCR products were then purified from agarose gel using gel extraction and purification kit.
After the fragment was ligated into the pEASY-T simple vetor, recombinant plasmid was induced into Escherichia coli DH5a. The positive colonies were then selected and sequenced.
Structural Analysis and Prediction of BnPIP-like Protein
After the fragment was sequenced, physical and chemical properties of nucleic acids and amino acids were analyzed by ProtParam (http://web.expasy.org/protparam/); while relative molecular weights (Mw) and theoretical isoelectric point (pI) were calculated by pI/Mw tool (http://web.expasy.org/compute_pi/); protein secondary structures were analyzed by NPS (http://npsa-pbil.ibcp.fr/cgi- bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html); prediction of transmembrane helices in protein was operated online by bioinformatics tool TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Simulative three-dimensional (3-D) model was made by comparative modeling online (http://swissmodel.expasy.org/). Prediction of subcellular location was analyzed online (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).
Expression of BnPIP-like Gene in Rapeseed under Salt Stress
Plump and full seeds of rapeseed were selected to determine the transcriptional expression of BnPIP-like gene. After 3 days jarovization at 4degC, the seeds were germinated in soils-mixture of vermiculite: peat (1:1) in a climatic chamber at 25degC with 16/8 h of light/dark cycle and 60% humidity. 18 days after germination, the salt stress treatment was started: 300 mmol/L NaCl solution was sprayed to the seedlings and added into the soil sufficiently. The seedlings at the same size were picked at 0, 2, 4, 6, 8 and 20 h, respectively in order to extract the total RNA of the whole plant. The reverse transcription was performed as described above and then the qPCR was operated.
Specific primers to detect the transcriptional expression of BnPIP-like gene was designed (Q-P1: 5'-CCCATTACCGGAACTGGAAT-3'; Q-P2: 5'- AACGGACCAACCCAGAAGAT-3') and the ACTIN gene (actin-F: 5'-TGGTGAAGGCTGGTTTTGCT-3'; actin-R: 5'-TTCTGACCCATCCCAACCAT-3') was used as an internal control and also amplified simultaneously from each sample. The qPCR cycling procedure consisted of 3 min at 95degC, 27 cycles for 30 s at 95degC, 30 s at 55degC and 30 s at 72degC and a final 5-min extension at 72degC. The qPCR cycling consisted of 1 min at 95degC, 40 cycles of 10 s at 95degC, 40 s at 56degC, 45 s at 72degC (data collection), followed by a melting curve procedure of 1 min at 95degC, 1 min at 56degC, 78 cycles for 56degC ramping to 95degC at the rate of 0.5degC/10 s. The qPCR was performed on a Bio-Rad iCycler fluorescence thermocycler (Bio-Rad, Hercules, CA). The fluorescence master mix reagent for the reaction was Sybr Green (Toyobo).
All of the cycle threshold (Ct) values of BnPIP-like amplification were normalized by the corresponding ACTIN Ct values. Three parallel repeats were done and the results were summarized as averages and the standard deviation (SD). The data were analyzed and plotted using Microsoft Office software.
Expression of BnPIP-like gene Induced by Methanol in Yeast
Full-length BnPIP-like gene fragment was fused into eukaryotic expression vector pPIC3.5K with restriction sites BamH I and EcoR I. The recombinant plasmid pPIC3.5K- BnPIP-like gene was then induce into E. coli (TOP 10) competent cells. Colonies were picked up and cultured in LB media with ampicillin, followed by plasmid extraction and restriction enzyme digestion with BamH I and EcoR I in order to screen out the positive ones. Then the recombinant plasmid pPIC3.5K-BnPIP-like gene containing fusion genes was then linearized by restriction enzyme Sal 1, followed by electroporation transformation (1500 V, 5 ms) of competent yeast (GS115) cells; as well as the original pPIC3.5K plasmid.
The cells were plated on RDB+G418 media at 30degC for 4~5 days and single colonies were respectively picked up and cultured in YPD liquid media with 5 antibiotics (ampicillin, kanamycin, streptomycin, chloramphenicol and tetracycline), followed by extraction of total DNA and consequently PCR in order to screen out the positive colonies. The PCR was performed with specific primers (AOX1-1:5'-GACTGGTTCCAATTGACAAGC-3'; AOX1-2: 5'-GCAAATGGCATTCTGACATCC-3'). The PCR cycling procedure consisted of 5 min at 94degC, 30 cycles for 30 s at 94degC, 30 s at 52degC and 1 min at 72degC, and a final 15-min extension at 72degC. Then the positive colonies were picked up and cultured successively in YPD, BMGY and BMMY (all with 5 antibiotics) media, in order to induce the protein expression with methanol. The protocol was according to Promdonkoy et al. (2014).
Determination of the Salt Resistance in BnPIP-like-Transgenic Yeast
Positive yeast cells, including BnPIP-like-transgenic yeast and control (transformed with original pPIC3.5K fragment integrated into the genome) was respectively cultured in YPD liquid media with 5 antibiotics (described above) at 30degC for 24 h. One mL germ solutions were then added into transitional BMGY media 5 antibiotics at 30degC for 24 h. One mL germ solutions were then added into inducing BMGY media 5 antibiotics at 30degC for 18~24 h, respectively. The OD600 values were determined and aseptic NaCl powder was added to 300 mM. The OD600 values were then determined every 12 h and the growth curves were obtained. Methanol was replenished every 12 h.
The Sequence of BnPIP-like Gene
An 846 bp-length fragment was successfully obtained by RT-PCR and verified by the agarose gel electrophoresis (Fig. 1a) and then followed by extracting and sequencing. Online analysis showed that nucleic acid sequence of this fragment, named BnPIP-like gene, shared the highest identity (91.7%) with Arabidopsis plasma membrane intrinsic proteins (PIP, Genbank:NP_195236.1), which belongs to the aquaporin (AQP) family. BnPIP-like gene encoded a putative protein with 281 amino acids and their sequence of BnPIP-like protein showed 95.3% identity with Arabidopsis PIP (Fig. 1b). The sequences had the conserved domain SGXHXNPAVT of MIP super-family and the conserved domain GGGANXXXXGY and TGINPARSLGAA of PIP family, as well as the conserved domain NPA (Fig. 1b).
Further alignment with known AQPs from rapeseed indicated that BnPIP-like gene had 64.97%, 65.31%, 31.93% and 74.56% identities with EU487188.1, EU487187.1, AF118381.1 and AF118383.1, respectively (data not shown). Thus, BnPIP-like protein was considered a new member of AQPs family.
The formula was C1382H2111N351O366S10 with predicted theoretical pI of 8.99 and molecular weight of 29.8197 kDa. The instability index (II) was computed to be 30.39 and it classified the protein as stable. Prediction of transmembrane helices in protein was operated online by bioinformatics tool TMHMM Server v. 2.0, finding BnPIP-like gene contained five loops and six transmembrane helices (Fig. 2a). N-terminal and C-terminal were both intracellular, as well as loop B and loop D. Loop A, loop C and loop E were extracellular. Moreover, high conserved sequences "Asn- Pro-Ala" were found in both loop B and loop E (data not shown). Three-dimensional (3-D) model was made by comparative modeling online. It showed that BnPIP-like protein, like other PIPs in higher plant, existed as homo- tetramers (Fig. 2b).
Expression of BnPIP-like Gene in Rapeseed Seedlings Induced by Salt Stress
Salt stress inhibited the growth of plants and resulted in withering. 2 h after the 300 mMol/L NaCl solution was sprayed to the seedlings and added into the soil, the leaves and stems started to wither. Then whole seedlings were picked successively and total RNA was extracted and reverse transcription was operated. Relative expression on transcriptional level was determined by qPCR and it showed that the expression significantly suppressed (to about 15% of the control) 2 h after the salt stress started (Fig. 3). But after 4th h, the transcriptional level recovered to normal level as control. As the salt stress continued, the expression of BnPIP-like gene decreased again (Fig. 3).
Expression Induced by Methanol of BnPIP-like gene in Yeast
Restriction sites BamH I and EcoR I were used to fuse BnPIP-like gene fragment into vector pPIC3.5K with mictic tag at N-terminal and His tag at C-terminal (Fig. 4a). The recombinant plasmid pPIC3.5K-BnPIP was then induced into E. coli (TOP10) and consequently was extracted from positive clones and checked by double-restriction enzyme (BamH I and EcoR I) digestion. Agarose gel electrophoresis (Fig. 4b) showed that a big fragment about 9 kb and a small fragment about 1 kb were obtained as expected, and sequencing result also indicated it correct (data now shown). Different to E. coli, the electroporation transformation of competent yeast cells needed to linearize the expression vector first. So restriction enzyme Sal I was utilized and the cells were treated at 1500 V for 5 ms. Four days after growing on RDB+G418 media at 30degC, clones were picked and cultured in YPD liquid media with 5 antibiotics.
Then the PCR was operated and the clones obtaining 1 kb band on agarose gel were considered positive ones (Fig. 4c). The positive yeast cells were cultured in YPD, BMGY and BMMY successively and the target protein was induced by 0.5 methanol after 24 h extracted and then checked on PAGE. The target protein was found successfully expressed (Fig. 4d).
Expression of BnPIP-like Gene in Yeast Improved the Cell Survival Ratio under Salt Stress
The transgenic yeast and control shared the same OD600 values at the 18th h and then the salt stress treatment started. Under 300 mM NaCl salt stress, both transgenic yeast and control suffered growth inhibition. But from the 30th h on, the BnPIP-like-transgenic yeast became stronger than the control. In other words, the transgenic yeast (OD600=2.525) suffered less inhibition than control (OD600= 2.465, Fig. 5). As the salt stress continued, this difference got more significant: on the 42th h, it showed that the OD600 value of transgenic yeast got 3.659, while the OD600 value of control got only 3.265 (Fig. 5). After that, the difference gradually got smaller; however, the transgenic yeast cells always grew slightly stronger than the control (data not shown).
Environment affects higher plant growth greatly. Salinity decreases the germination rate of seeds of plants, depresses the photosynthesis efficiency, inhibits respiration and protein synthesis and disturbs the reactive oxygen species (ROS) or even nucleic acid metabolism (Niu et al., 1995; Zhu, 2000; 2001). As to crops, it inhibits the growth and decreases the yield. Besides irrigation and improving soil drainage, studying salt tolerance genes in plant was another promising approach.
There are several genes in different pathways enhancing salt tolerance studies: ROS-eliminating Mn- containing superoxide dismutase ThMSD from salt cress (Eutrema halophilum) enhanced salt tolerance in transgenic Arabidopsis (Xu et al., 2014); by regulating the osmotic regulator betaine, a BADH gene from Atriplex micrantha could enhance salinity tolerance in transgenic maize plants (Di et al., 2015); besides, late embryogenesis abundant (LEA) gene AtEm6 from Arabidopsis was also found to enhance salt tolerance in transgenic rice cell lines by regulating expression of Ca2+-dependent protein kinase genes (Tang and Page, 2013). Aquaporin (AQP) as an integral membrane protein, transporting water molecules across membrane efficiently (Denker et al., 1988; Zardoya, 2005), was investigated in this study.
A new AQP BnPIP-like gene was first isolated from rapeseed and analyzed. Its full-length cDNA had 846 bp (Fig. 1a) and it encoded a putative protein with 281 amino acids. Online sequence alignment showed that in NCBI database, the one which shared highest identity (95.3%, Fig. 1b) with BnPIP-like gene was PIP (Genbank: NP_195236.1) from Arabidopsis. The theoretical pI of BnPIP-like protein was 8.99 and molecular weight was 29.8 kDa. It had the conserved domain SGXHXNPAVT of MIP super-family and the conserved domain GGGANXXXXGY and TGINPARSLGAA of PIP family, as well as the conserved domain NPA; prediction of transmembrane structure showed that BnPIP-like protein contained five loops and six transmembrane helices (Fig. 2a), existing as homo-tetramers (Fig. 2b), which was in accord with the classical structures for PIP (Chaumont et al., 2001; Li et al., 2013).
Further analysis found that protein EU487188.1, EU487187.1 and AF118383.1 had higher similarity in structure with BnPIP-like protein than AF118381.1 had: AF118381.1 had 7 transmembrane domains and its C- terminal was extracellular (data not shown). This was probably attributed to that they belonged to different subfamilies: protein AF118381.1 belonged to g-TIP subfamily (located in tonoplast).
To study BnPIP-like gene expression model in plants, rapeseed cultivar JR9 was planted and then treated by 300 mmol/L NaCl. It was showed that 2 h after the NaCl solution was sprayed to the seedlings and added into the soil, the plants started to wither, and at this time, the expression was significantly suppressed (to about 15% of the control, Fig. 3). But from the 4th h on, the transcriptional level recovered nearly to level as control. As the salt stress treatment continued, the expression of BnPIP-like gene decline again (Fig. 3).
In order to further investigate functions of BnPIP-like protein, yeast rather than E. coli was utilized as an expression system. Because BnPIP-like protein had 6 transmembrance domains in such a small fragment described as above, it was impossible to be expressed correctly in prokaryotic E. coli. It was found inclusion bodies formed in E. coli BL21 cells. In our previous experiments, the BnPIP-like gene fragment was fused into prokaryotic expression vector pET30a and then induced into Escherichia coli BL21 (DE), in order to operate the prokaryotic expression (Xu et al., 2014). But the products were expressed mainly as inclusion bodies rather than transmembrane proteins (data not shown). Then yeast was considered, since its eukaryotic expression could complete the posttranscriptional modifications, which were better for eukaryotic gene expression.
Moreover, the theoretical pI of BnPIP-like protein was 8.99, far from the pH value of BMMY media (pH 6.0), so it could solve the inclusion body problem efficiently.
This was exactly the reason why we then turned to eukaryotic expression system yeast. Methylotrophic Pichia pastoris (Strain GS115) could avoid that problem and determine the protein functions sooner than in model plants. BnPIP-like gene was fused into vector pPIC3.5K (Fig. 4a). Then the recombinant plasmid pPIC3.5K-BnPIP and the original pPIC3.5K were linearized and induced into yeast cells, respectively obtaining the transgenic yeast and the control stains. Positive transgenic yeast was screened out by PCR (Fig. 4c), then the induction conditions were explored and the target protein was successfully expressed (Fig. 4d). After cultured in YPD and BMGY media successively, the yeast cells were induced into BMMY media. The salt stress treatment started on the 18th h in BMMY media with methanol.
The OD600 values of BnPIP-like-transgenic P. pastoris and control were determined every 12 h. Although both yeast were obviously inhibited, the concentrations of BnPIP-like-transgenic P. pastoris was always higher than control (Fig. 5) slightly, indicating that the inhibition of growth in BnPIP-like-transgenic P. pastoris was slighter because of the over-expression of BnPIP-like gene. Thus the resistance to salt explained the eukaryotic functions of BnPIP-like protein. Moreover, it provided a theoretical possibility for a more comprehensively industrial utilization of fermenting yeast. It also established the base for its further functional research in plants.
In this study, BnPIP-like gene a new aquaporin gene from rapeseed was isolated and sequenced. Theoretical biochemical characters, especially its transmembrane structure, were analyzed. When induced by methanol, BnPIP-like could enhance the salt resistance in yeast (GS115).
We thank the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-13), the National Key Research and Development Plan (JFYS2016ZY03002156), the Ministry of Agriculture Experimental Observation of the Upper Reaches of the Yangtze River Oil Crop Science Station (09203020), Sichuan Crop Breeding Community, Innovation Ability Promotion Project of Sichuan Provincial Finance (2016zypz-013), Sichuan Province Innovation Team Funding.
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|Author:||Chai, Liang; Li, Hao-Jie; Zhang, Jin-Fang; Tan, Hao; Cui, Cheng; Jiang, Jun; Zheng, Ben-Chuan; Zhang|
|Publication:||International Journal of Agriculture and Biology|
|Date:||Dec 31, 2016|
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