CbPLDγ gene from Chorispora bungeana: Gene cloning, characterization, expression, and expression analysis in drought

Background: Chorispora bungeana (C. bungeana) is a typical subnival alpine species, which shows high tolerance to multiple abiotic stresses. Phospholipase D (PLD) is a crucial enzyme participated in membrane phospholipid catabolism. In this study, to explore the function of CbPLDγ in drought stress, we cloned and characterized a CbPLDγ gene, which is a part of CbPLD gene family and from C. bungeana. Methods: Use the gateway method for vector construction, using DNAstar software, PCR machine, centrifuge, pipette, electrophoresis, gel imaging system, spectrophotometer, confocal microscope, etc. Spss, Orgin software for statistical analysis. Results: The CbPLDγ gene encodes 859 amino acids containing "FIYIENQYF" domain and two HKD domains. Bioinformatics analyses showed that the CbPLDγ is highly homologous with PLDs from other plant species. Real-time quantitative PCR (qRT-PCR) and Beta-glucuronidase (GUS) assay showed that CbPLDγ was accumulated dominantly in roots and stems. Compered with the control, the expression pattern of the CbPLDγ mRNA is in response to low temperature, salt, mannitol, and exogenous ABA have up-regulated. Subcellular localisation analysis showed that the CbPLDγ was localized in the cell membrane. Compared with wild-type Arabidopsis thaliana, CbPLDγ gene overexpression plants showed higher activities of antioxidant enzymes, and lower levels of malonidiadehyde content and electrolyte leakage under drought stress. Conclusions: In this study, novel PLDγ gene was amplication from C. bungeana and was called CbPLDγ. These conrmed that CbPLDγ involved in the response to drought stress, and has the potential to improve the drought tolerance of plants. This is the rst report about cloning and characterizing the gene of CbPLDγ from C. bungeana. It laid a foundation for further research and improvement of the PLD gene family of C. bungeana. abscisic acid; Chorispora bungeana C. bungeana; CAT – catalase; Col-0 - Arabidopsis thaliana Columbia ecotype; cDNA - complementary DNA; GFP - green uorescent protein; GUS - β-glucuronidase; hiTAIL-PCR - high-eciency thermal asymmetric interlaced PCR; MDA - malondialddehyde; MS Murashige and OE - overexpressed plants; PLD - phospholipase POD - peroxidase; PA polymerase acidic protein; PCR - polymerase chain reaction; pI - isoelectric point; qRT-PCR - quantitative real time polymerase chain reaction; ROS - reactive oxygen species; RACE - rapid amplication of cDNA ends; SOD - superoxide dismutase; UTR - untranslated - wild-type; 5-bromo-4-chloro-3-indolyl-β-D-glucuronic


Introduction
Phospholipids are critical in cell membranes and also play a pivotal role in cell development, biotic stress and signal transduction. The majority of phospholipase D (PLD) can hydrolyze of phospholipids to produce phosphatidic acid (PA) and soluble head group. All of the PLD gene superfamily involves two copies of conserved HxKxxxxD sequence, which is known as the HKD motif, and it is an active site. In addition, N-terminal domain contains the binding motif lipid and C-terminal domain contains the catalytic motif. Different PLDs seem to have different but somewhat overlapping functions in cellular processes (Lein W, et al. 2001). PLD gene has been cloned from many species such as rice bran (Oryza sativa L.), Maize (Ueki J, et al. 1995), tobacco (Lein W, et al. 2001), cowpea (Vigna unguiculata L. Walp) (Ali Y B, et al. 2007), tomato (Tiwari K, et al. 2011), Arabidopsis thaliana ) and other gene subfamily which have been reported in many species. PLD genes are classi ed into six subfamilies in the showed that PLDγ are more tolerant to all stress in the Arabidopsis seedlings (Zhao J, et al. 2011). PLDγ is located in the nuclear, and this perhaps reveled that the PLDγ play a role of cell division and reproduction (Fan L, et al. 1999).
Chorispora bungeana (C. bungeana) is a cruciferae plant, which is a typical representative to subnival alpine plant. It can adapt to the environment of low oxygen partial pressure, cold and strong ultraviolet radiation. This is closely related to Arabidopsis (Zhao Z, et al. 2012). Long-term studies have shown that C. bungeana has no special morphological characteristics. The surface lint and wax layer can resist harsh environment, but it contains a lot of free fatty acid, neutral amino acid, soluble sugar, Mg 2+ -ATPase activity, and unsaturated fatty acids (Song Y, et al. 2015).
Abiotic stresses, such as salt, drought, salicylic acid (SA), abscisic acid (ABA), can also induce the PLD expression (Hong Y, et al. 2016; Guo L, et al. 2016; Kalachova T, et al. 2013; Misugi Uraji, et al. 2012). For example, PLDα1 and PLDδ in ABA signaling have a synergistic effect in Arabidopsis guard cells (Kalachova T, et al. 2013). SA can active the PLD to induce of stomatal closure in Arabidopsis (Guo L, et al. 2016). In Craterostigma plantagineum, water de ciency triggers PLD activity (Frank W, et al. 2000). In addition, PLD is more tolerant to freeze stress in C. bungeana callus (Yang N, et al. 2013). Therefore, PLDs are thought to be involved in plant responses to abiotic stresses, and have multiple functions during plant growth and development (Pinhero R G, et al. 2003). Drought is a major stress factor that limits agricultural production worldwide. In order to adapt to survive under drought stress, plants will respond through changes in morphology and physiology, biochemistry and molecular response. When stress intensi es, the plant activates the mechanism of enzyme activity and membrane structure to avoid cell death, such as increased antioxidant activity, decreased membrane permeability, and control of reactive oxygen homeostasis (Hong Y, et al. 2008;Hong Y, et al. 2010.).
To the best of our knowledge, there is no detailed report was seen on the characterization of the CbPLDγ of the C. bungeana. Thus, in this study, we cloned and analyzed the full length of CbPLDγ cDNA sequence from C. bungeana and its promoter region, examined the expression pattern of CbPLDγ in different stress conditions, subcellular localization of CbPLDγ, organizational positioning and tolerance of overexpressing plants to drought stress. It provided a better insight into the molecular mechanisms CbPLDγ, and provides a theoretical basis for further research and improvement of the gene function of the PLD gene family of C. bungeana.

Material And Methods
Plant material: The C. bungeana of plantlet was obtained and had a little of modi ed by previously. C. bungeana seedlings were cultivated on Murashige and Skoog (MS) medium with 1 mg/l 6-benzyladenine (6-BA) and 3% (w/v) sucrose.
Arbidopsis and tobacco (Nicotiana benthamiana) seeds were disinfected and germinated on MS medium. After 5 days, the germinated seeds were transferred to the soil, and all the plants were grown in a greenhouse under 25 °C light for 16 h and dark light for 8 h.
Stress treatment: The C. bungeana seedlings were grown in a growth chamber under 25 °C, photoperiods of 16 h light/8 h dark for 1 weeks. The seedlings were subjected to 150 mM NaCl, 0.3 M mannitol, 4 °C and 100 mM abscisic acid (ABA) induction, respectively, with three replicate. As controls, the plantlets were cultivated on MS. All the samples were immediately frozen in liquid nitrogen for RNA isolation and quantitative real-time PCR.
The overexpressing Arabidopsis seedlings were grown in a growth chamber under 25 °C, photoperiods of 16 h light/8 h dark for 2 weeks. The seedlings were growing without watering for 1 week, rehydrated for ve days. This sample is used for electrolyte leakage, malondialddehyde (MDA) and antioxidant activity analysis.
Cloning of CbPLDγ gene: RNAiso Plus kit (Takara, Dalian) was used to extract total RNA from the plantlets of C. bungeana according to the manufacturer's instructions, The rst strand of cDNA was synthesised using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian) according to the manufacturer's instructions.
Based on other PLDγ homologous species, Two primers (CbPLDγ1-F and CbPLDγ1-R) ( Table 1) corresponding to sequences of the conserved regions of PLD genes were used to amplify part of the CbPLDγ sequence using LA Taq DNA polymerase (Takara, Dalian). The ampli ed products were cloned as the pMD19-T vectors (Takara, Dalian) for the further sequencing. Design primers (CbPLDγ2-F and CbPLDγ2-R) ( Table 1) at the 5' end of the rst ampli ed fragment to amplify the middle fragment of CbPLDγ. The ampli ed products were cloned as the pMD19-T vectors (Takara, Dalian) for the further sequencing. The three sequences were combined to obtain the middle part of CbPLDγ.
The 5′ and 3′ rapid ampli cations of cDNA ends (RACE) were performed using the SMARTer TM RACE cDNA Ampli cation (Invitrogen, USA). Based on the instruction manual, total RNA were isolated and produced templates for 5′ and 3′ race from the C. bungeana. The perimers of 5′GSP, 5′NGSP, 3′GSP and 3′NGSP (Table 1) were designed to ampli cate of RACE products, and nest PCR products were cloned into pMD19-T vectors and sequences.
By comparing and aligning the sequences, the full length of CbPLDγ gene sequences were obtained, which were contained in middle region, the 5′ and 3′ RACE sequences. The full length of CbPLDγ gene sequences were veri ed by PCR using Primer Star HS DNA polymerase (Takara, Dalian) and sequencing.
Cloning of the promoter of CbPLDγ: Genomic DNA was isolated from the C. bungeana using the TaKaRa MiniBEST Plant Genomic DNA Extraction Kit (Takara, Dalian) to clone the CbPLDγ promoter sequence. The promoter of CbPLDγ was cloned by high-e ciency thermal asymmetric interlaced PCR (hiTAIL-PCR) using Genome Walking Kit (Takara, Dalian). Adaptors were provided by the kit and SP primers (Table 1) were designed according to the sequence of CbPLDγ. The PCR products were puri ed using TIANgel Midi Puri cation Kit (TIANGEN, Beijing), cloned into pMD19-T vector and sequenced.
Bioinformatics analysis: The similarity of nucleotide and amino acid sequence were searched in BLAST of the NCBI. The opening reading frame (ORF) was selected using the Editseq in DNAStar software. The amino acid sequences of CbPLDγ were deduced in ExPASy website (https://web.expasy.org/translate). The Megalign was used in multiple alignments of the CbPLDγ amino acid and other species by DNAstar software. The phylogenetic trees were constructed with Neighbor-Joining method by MEGA-X.
Quantitative real-time PCR: Total RNA was isolated from C. bungeana were subjected to salt, mannitol, 4 ℃, and abscisic acid (ABA) induction. The cDNAs were synthesized using the FastKing RT Kit (With gDNase) (TIANGEN, Beijin). Gene-speci c RT primer pairs designed based on the CbPLDγ sequence (Table 1) were subjected to real-time quantitative PCR using TBGreen® Premix Ex Taq™ II (Tli RNaseH Plus) (Dalian Takala). The Actin genes were used as internal controls for normalizing gene expression levels. The results were displayed using the 2 -ΔΔct method.
Histochemical location of CbPLDγ: Total RNA was isolated from C. bungeana roots, stems, and leaves using the Trizol reagent. The cDNAs were synthesized using the FastKing RT Kit, the Real-time quantitative PCR was performed. The Actin genes were used as internal controls for normalizing gene expression levels.
The promoter of CbPLDγ was cloned (Table 1)  Subcellular localization of CbPLDγ: The coding regions of CbPLDγ were ampili ed from C. bungeana by RT-PCR using primers (Table 1), and the PCR products were sequencing. The cloned gene fragment was recombined into the pMDC83 vector with recombinase. Competent DH5α cells were transformed and plasmids were veri ed by sequencing. The correct plasmids containing pMDC83-CbPLDγ-GFP vector was introduced into Agrobacterium tumefaciens GV3101. Sequencely, the recombinant plasmids were transformed into the tobacco with the epidermal cells for transient expression method (Sparkes I A, et al. 2006) and cultured in darkness at 25 °C for 48 h. The GFP uorescence was captured by confocal microscope (Leica, German).
Transformation of Arabidopsis: The CbPLDγ promoter and coding regions was ampili ed by nested PCR using primers ( Table 1). The cloned fragment was recombined into the pBIB vector with recombinase. Competent DH5α cells were transformed and plasmids were veri ed by sequencing. The correct plasmids containing pro-pBIB-CbPLDγ vector was introduced into Agrobacterium tumefaciens GV3101. Sequencely, the recombinant plasmids were transformed into the Arabidopsis Col-0 according the oral dip method and cultured in darkness at 25 °C for 24 h. Transformants were selected on MS medium containing basta. The T 4 generation of transgenic Arabidopsis plants was used for further analyses.
Determination of electrolyte leakage, malondialdehyde content, and antioxidant enzyme activities: Electrolyte leakage, malondialdehyde content, and antioxidant enzyme activities were analyzed to determine the mechanism by which overexpression of CbPLDγ conferred resistance to drought stress. The conductivity was determined E1 using a conductance bridge (DDS-11A, Yamei Electron Instrument Factory, Wuxi, China). Then, the samples were heated in boiling water for 30 min and cooled to room temperature, conductivity was read again E2.
The determination of MDA content is slightly modi ed according to the method of Lazzarino et al. (Lazzarino G, et al. 1995). Fresh leaf tissue was ground in 10% trichloroacetic acid (TCA). After su cient grinding, the grinding liquid was centrifuged at 15000 × g for 20 minutes at 4 °C. The supernatant was collected and mixed with 0.6% thiobarbituric acid (TBA) in 10% TCA. The mixture was heated in a water bath at 95 °C for 15 min, and then quickly placed on ice to cool. After the mixture was cooled, the sample was centrifuged at 10000 × g for 10 min, and the absorbance of the solution at 532, 600, and 450 nm were recorded.

Results
Cloning and sequence analysis of the CbPLDγ gene: Based on the PLDγ gene sequence in other homologous species, we have designed a pair of primers to amplify the middle of CbPLDγ gene sequence cDNA from C. bungeana. The single fragments about 480 bp were obtained. With the known cDNA sequences of the CbPLDγ genes, we obtained 712 bp after designed a pair of primers to clone the middle of CbPLDγ gene. 5′GSP and 5′NGSP were designed beside on the middle region sequence for 5′-RACE. We obtained a single fragment about 524 bp of two PCR ampli ed, which has a 160 bp 5′ untranslated region (UTR) of the ATG codon upstream. We have designed 3′GSP and 3′NGSP for 3′-RACE beside on the middle region, the 1373 bp was cloned and contained 197 bp 3′UTR in the TAA codon downstream, including a poly A tail of 12 bp. By comparing and aligning the sequences, we have deduced the fulllength cDNA from the known sequence of 2937 bp. Analysis by DNAstar, the full-length cDNA contained a 2580 bp ORF, which encoded a protein of 859 amino acids with a calculated molecular weight of 96.3 kDa and with a pI of 7.88.
The PLDζ gene in other plants was searched in NCBI, and sequence alignment was performed with DNAstar. The CbPLDγ sequence is the most similar with other species PLDγ gene at the amino acid level. This protein contained two HKD motifs: HQKTVIVD is located in the 372-379 amino acids and HSKGMVVD is located in the 710-717 amino acids. In addition, CbPLDγ sequence was contained by the "FIYIENQFF", which was the conserved sequence in the entire plant PLD gene (Fig.1).
Using MEGA-X software, the CbPLDζ gene was sequence aligned with other plant PLD genes and a phylogenetic tree was constructed. The phylogenetic tree was mainly illustrate that the CbPLDγ was classi ed to the PLDγ subfamily and more close to the Arabidopsis PLDγ gene compared with the PLD genes of other plant (Fig.2).
The expression of CbPLDγ is induced by abiotic stress: Under stress conditions, we have studied the expression changes of CbPLDγ, the gene speci c primers were used for real-time quantitative PCR. We have studied the expression pattern of the CbPLDγ mRNA in response to low temperature, salt, mannitol and exogenous ABA respectively in different times and got the transcription level of CbPLDγ. After mannitol treatment, the relative expression of CbPLDγ gene increased signi cantly and was higher than that of the control group, with the treatment time, the gene expression level was also different, it reached the peak at 12 h and the transcription levels had a 2.97 fold change. Under NaCl treatment, the relative expression of the CbPLDγ gene increased signi cantly and was higher than that of the control group. At 12 h, the relative expression of the gene reached a peak and multiplied. Under the 4 °C treatment, the transcription level of CbPLDγ was up-regulated, and the transcription level had a fold change after 24 h of treatment. After exogenous ABA treatment of C. bungeana seedlings, the relative gene expression levels increased signi cantly (Fig.3). This result showed that the CbPLDγ is in responds to low temperature, salt, mannitol and ABA.
Histochemical location of CbPLDγ: In order to explore the functions of CbPLDγ, we selected roots, stems, leaves from C. bungeana within real-time quantitative PCR, the results indicated that CbPLDγ is expressed in all tissues examined, and has a signi cant enrichment in roots (Fig.4 A).
The GUS staining experiment is further con rming this expression pattern, the plasmid pBIB-CbPLDγ-GUS was introduced into Arabidopsis Col-0 according the oral dip method by Agrobacterium GV3101. The homozygous seedlings were screened by herbicide basta and used to detect the histochemical location of GUS activity, The GUS activity results further con rmed that CbPLDγ is expressed in all the tissues, but with a signi cant enrichment in roots (Fig.4 BC). The GUS assay and qRT-PCR have an identical result.
Subcellular localization of CbPLDγ: The subcellular localization of CbPLDγ is important for understanding its function. In silico subcellular localization analysis performed using WoLF PSORT and Cell-PLoc indicated that CbPLDγ is mainly localized in the cell membrane. In order to study the subcellular localization of CbPLDγ, the CDS was linked to pMDC83 vector with GFP tag, the construct was introduced into Agrobacterium GV3101. Sequencely, the recombinant plasmids was transformed into tobacco according the epidermal cells for transient expression method. The GFP uorescence was captured by confocal microscope, and the results showed that the CbPLDγ was located in the membrane (Fig.5).
Determination of electrolyte leakage, malondialdehyde content, and antioxidant enzyme activities: The T 4 generation of transgenic Arabidopsis is adversely stimulated by the drought stress, its electrolyte leakage will increase signi cantly. In this experiment, we found that drought stress caused a signi cant increase in the conductivity of WT and OE(overexpressed plants), while the conductivity of WT plants was signi cantly higher than OE, indicating that under drought stress, the membrane damage of overexpressed plants is less than that of WT Plants (Fig.6).
MDA is a product of lipid peroxidation and is often used as an indicator of cell membrane free radical damage. Our measurement of MDA content showed that the MDA content of WT plants under drought treatment was always higher than that of transgenic lines (Fig.6). These physiological parameters indicated that transgenic lines were more resistant to drought than WT.
Under normal conditions, there was no signi cant difference in SOD, POD, and CAT relative expression between CbPLDγ transgenic lines and WT. However, the drought signi cantly increased the activities of SOD, POD, and CAT in the two plants, and the OE lines had higher antioxidant enzyme activities than the WT plants. This shows that compared with WT plants, transgenic lines are more resistant to drought stress (Fig.7).

Discussion
C. bungeana is a typical subnival alpine plant, its living environment is extremely harsh and the temperature changes greatly (Wu J M, et al. 2008.). In recent years, the research on C. bungeana has been reported from various levels, especially the anti-adversity response mechanism of C. bungeana. Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development (Distéfano Ayelen M, et al. 2015). In this study, the CbPLDγ (MF951104) gene was cloned on the basis of the cloned PLD gene family members of a C. bungeana, and its sequence was analyzed. The gene is 2937 bp in length and contains an open reading frame (ORF) of 2580 bp, 5´ and 3' untranslated regions are 160 bp and 197 bp, respectively, and also include a 12 bp poly A tail, encoding a protein of 859 amino acids. CbPLDγ includes two HKD domains, which are the active sites of PLD. In addition, CbPLDγ has the conserved motif "FIYIENQFF" in all plant PLDs, which may be related to the hydrophobic interaction between the methyl groups in the choline group, which is as important as the HKD domain (Yuan H, et al. 2005.) (Fig. 1). Phylogenetic analysis showed that C. bungeana CbPLDγ gene has high homology with other plant PLDγ genes. In addition, among the PLDγ subtypes, C. bungeana is closely related to Arabidopsis PLD gene (Fig. 2). This study explored the relative expression of CbPLDγ gene under salt, mannitol and 4 °C. It was found that 4 °C low temperature, salt and mannitol stress can induce the expression of CbPLDγ gene, indicating that CbPLDγ gene is widely involved in C. bungeana to many abiotic stress responses. Many plant growth regulators, such as ABA, participate in plant stress response and play an important role (Shaterian J, et al. 2005.). Exogenous ABA can induce the expression of CbPLDγ gene, and it is speculated that the biosynthesis of CbPLDγ may be through ABA dependent pathway under stress conditions (Fig. 3).
The different expression of PLD in various organs or different subcellular locations may be of great signi cance for further understanding the function and regulatory mechanism of each PLD. PLD exists in the plasma membrane, endoplasmic reticulum, and submicrosomes (Xu L, et al. 1996.). PLDα exists in soluble and membrane-related components, and the relative distribution between the two components depends on tissue and developmental stage (Dyer J H, et al, 1994.). Real-time uorescence quantitative PCR con rmed that the average expression level of PLDα3 in buds, owers, siliques, stems, old leaves, and roots was 1000 times lower than that of PLDα1, indicating that the expression level of PLDα1 is generally much higher than PLDα3 (Hong Y, et al. 2008). Studies have shown that GmPLDγ-GFP fusion protein is expressed in transgenic Arabidopsis roots and in tobacco leaf mitochondria (Bai Y, et al. 2020).
The expression of AtPLDδ gene in roots, owers and stems is higher than that in leaves and pods, and the expression in old leaves, stems, owers and roots is much higher than that of young leaves and pods. The subcellular localization of AtPLDδ gene in tobacco is also plasma membrane relate. PLDγ can be detected in the plasma membrane, intracellular membrane, nucleus and mitochondria, while PLDδ can only be detected on the plasma membrane (Fan L, et al. 1999). PLDε was found in microsomes, but not in the soluble fraction. When PLDε is transiently expressed in tobacco leaves, uorescence is only detected on the plasma membrane (Hong Y, et al. 2009). PLDα, PLDβ and PLDγ can exist in two states of soluble and membrane-bound state. The CbPLDγ gene is expressed in roots, stems, leaves, and is signi cantly enriched in roots and stems, and the expression level in leaves is low (Fig. 4). The difference in gene expression may be related to its function. The signi cant enrichment in roots may be because this gene is related to root growth and development. The results of subcellular localization indicate that the genes are signi cantly clustered on the cell membrane, which may imply that the CbPLDγ genes all perform their functions on the cell membrane (Fig. 5).
Drought is one of the most important limiting factors for plant growth and agricultural production (Sun X P, et al. 2013;Shahsavari N, et al. 2014). Current research further con rms the negative impact of drought on plant growth. When plants are stimulated by the external environment, they may resist the stimulation by activating the antioxidant defense system, but in the case of insu cient antioxidant defense, the resistance of the plant can be increased by exogenous application of various hormone signaling molecules (Piotrowska A, et al. 2009;Kadioglu A, et al. 2011). As we all know, PLD plays an important role in the process of cytoskeletal assembly and plasma membrane reconstruction. The permeability and stability of plant cell membranes play an important role in plant growth and development, and PLD is involved in the degradation of plant cell membrane lipids. MDA is a product of lipid peroxidation, which can reduce the level of antioxidants, leading to membrane system damage and even cell death. MDA and electrolyte leakage are usually used to analyze the degree of membrane damage under environmental stress. In this study, our measurement of MDA and electrolyte leakage content showed that wild plants under drought treatment are always higher than that of transgenic lines. This indicates that under drought stress, the membrane damage of overexpressed plants is less than that of WT plants (Fig. 6).
ROS is a substance produced by plants under various stress conditions and an important medium for plants to respond to stress. Plants have an array of antioxidant enzymes that protect cells from oxidative damage. These enzymes include SOD, POD, CAT, which work together with other enzymes to scavenge ROS (Yue Y S, et al. 2011). In our study, drought signi cantly increased the activities of SOD, POD and CAT in the two plants, and the overexpressed lines had higher antioxidant enzyme activity than WT plants. This indicates that transgenic lines are more resistant to drought than WT plants (Fig. 7).

Conclusion
In this study, novel PLDγ gene was ampli cation from C. bungeana and was called CbPLDγ. The bioinformatics analysis was completed. The hylogenetic tree was revealed that CbPLDγ is classi ed to subfamily of PLDγ, and was more close to the Arabidopsis PLDγ gene compared with the other plants.
Compered with the control, the expression pattern of the CbPLDγ mRNA is in response to low temperature, salt, mannitol, and exogenous ABA have up-regulated. Expression pattern of CbPLDγ is in roots, stems, leaves, but, it is mainly expressed in roots and stems compared with leaves. The CbPLDγ is located in cell membrane. Compared with WT Arabidopsis, CbPLDγ gene overexpression plants showed higher activities of antioxidant enzymes, and lower levels of malonidiadehyde and electrolyte leakage under drought stress. These con rmed that CbPLDγ involved in the response to drought stress, and has the potential to improve the drought tolerance of plants. This is the rst report about cloning and characterizing the gene of CbPLDγ from C. bungeana. It laid a foundation for further research and improvement of the PLD gene family of C. bungeana.

Declarations Ethical Approval and Consent to participate
This article is to study the functional identi cation of plant genes and gene functions under drought. So ethical approval and consent to participate.    Subcellular localization of CbPLDγ. Construct pMDC83-CbPLDγ-GFP was transformed into tobacco epidermal cells. All images were observed with confocal microscope.

Figure 6
Effects of drought stress on electrolyte leakage and MDA content in CbPLDγ transgenic Arabidopsis and WT. Values are the mean ± SD of at least three independent experiments. Different lowercase letters in the gure indicate signi cant differences between the same group at P <0.05. Different capital letters in the gure indicate signi cant differences at different times, P <0.05.

Figure 7
The effect of drought stress on the activity of antioxidant enzymes in CbPLDγ transgenic Arabidopsis and WT is the mean ± SD of at least three independent experiments. Different letters in the gure indicate signi cant differences between the same group (P <0.05).