Analysis on Tissue-specific Expression of DpXTH1 and DpXTH2 Genes in Dahlia

Pingping ZHANG,Lei WANG,Chi CHEN,Lifen WANG,Biping ZHENG,Lixin QIAN,Jianzhong TAN

Department of Horticulture,College of Architecture,Soochow University/Key Laboratory of Architecture and Urban Environment of Suzhou City,Suzhou 215123,China

Xyloglucan endotransglycosylase/hydrolase(XTH)is a kind of cell wall remodeling enzyme,and it can change the structure of cellulose in cell wall.XTHs are encoded by a variety ofXTHgenes.They are involved in the expansion and degradation of cell walls[1],hydrolysis of xyloglucans for energy supply[2]and changes in fluidity of cell plates[3].In addition,they are closely related to the elongation of plant tissues,softening processoffruits,programmed death of cells,etc.At present,XTH family has been found in a variety of plants,and in various tissues of the plants,the activities of XTHs have been detected[4].Total 25 XTHs have been found in tomato.Among them,SlXTH5 is highly expressed in mature fruits[5].It is predicted that there are 29 open reading frames in rice[6].Among them,OsXTH8 is found to be expressed in sheaths[7],andOsXTH5,OsXTH19,OsXTH20,OsXTH24 andOsXTH28 are involved in the extension of rice pedicels[8].Among the cloned 33 XTH genes fromArabidopsis thaliana,most are concentrated to be expressed in root elongation zone;butAtXTH2isonlyexpressedin pollen,andAtXTH3 isexpressed abundantly in inflorescences,flowers and stamens[9].DifferentXTHgenes show significant differences in expression and regulation model among different organs and tissues of plants,and they constitute the entireXTHgene family.

In this study,theDahlia pinnataCav. ‘Danbanhuang’ was used as tested material,and the expression levels ofDpXTH1 andDpXTH2 in different tissues and organs were detected by real-time PCR.The expression characters ofXTHgene in dahlia were analyzed so as to provide certain experimental basis for revealing the biologicalfunctionsofDpXTH1andDpXTH2 genes.

Materials and Methods

Plant material

The tested material wasDahlia pinnataCav. ‘Danbanhuang’.At the full blooming stage,the roots,stems,leaves and petals of dahlia were collected.They were cleaned with distilled water,and dried for the extraction of total RNA.

Extraction of total RNA and synthesis of first-strand cDNA

The total RNA in the four tissues of dahlia was extracted with RNAprep pure Plant Kit(Tiangen).The integrity of extracted RNA was examined by 1%agarose gel electrophoresis.TheODvalues of RNA at the wavelengths of 260 and 280 nm were measured,and according to theOD260/OD280,the concentrations and purities of total RNA were assessed.The first-strand cDNA synthesis was performed by transcription with First Strand cDNA Synthesis Kit(Thermo Scientific).

Primer design and synthesis

According to the design principles forquantitative PCR primers,the primers were designed using Primer 5.0 based on the registered gene sequence of dahlia.The primer sequences ofβ-actin(ID:AB621922.1),with target fragment size of 117 bp,were as follows:F,5’-CCCGACTGTCCCTGTT-3’;R,5’-CGGCGTTGTTACTTT G-3’.The primer sequences ofDpXTH1(ID:HM053613.1),with target fragment size of 239 bp,were as follows:F,5’-GGTCGGATTCTCACAT-3’;R,5’-TTGCCCAC TTCGGTTT-3’.The primer sequences ofDpXTH2(ID:JQ948092.1),with target fragment size of 132 bp,were as follows:F,5’-AGACAACTTGAGGGTGGTAG-3’;R,5’-CGCAGAGTCTCCAGGAATAA-3’.The primers were all synthesized by the Sangon Biological Engineering Technology&Services Co.,Ltd(Shanghai,China).

Standard preparation and real-time PCR

The cDNA,obtained by reverse transcription,was used as template for verifying the specificities of synthesized primers.The PCR system(25 μl)was as follows:10×TaqBuffer 2.5 μl,dNTP Mix (2 mmol/L for each)2 μl,forward primer(10 μmol/μl)0.4 μl,reverse primer(10 μmol/μl)0.4 μl,MgCl2(25 mmol/L)2 μl,TaqDNA Polymerase 0.125 μl,template cDNA 1.5 μl,sterile distilled water 16.075 μl.The reaction program was as follows:predenaturation at 94℃for 3 min;denaturation at 94℃for 30 s,annealing at 50℃for 30 s,extension at 72℃for 45 s.35 cycles;extension at 72℃for 7 min.The specificities of amplification products were examined by 1.6%agarose gel electrophoresis.

Using the standards of the 3 genes and cDNA from 4 tissues as the templates,real-time quantitative PCR was performed with QuantiNova SYBR Green PCR Kit in a PCR instrument(Prism 7300,ABI).The reaction system (20 μl)was as follows:SYBR Premix ExTaqTM(2×,TaKaRa)10μl,forward primer(10 μmol/μl)0.4 μl,reverse primer(10 μmol/μl)0.4 μl,ROX Reference Dye(50 ×)0.4 μl,cDNA 2.0 μl,dH2O 6.8 μl.The PCR program was as follows:95℃1 min;95℃15 s,60℃31 s,45 cycles.After the amplification,the melting curves were analyzed.There were 3 replicates for each sample.

Preparation of standard curve and data analysis

The amplified cDNA product was diluted by 10,102,103,104and 105times,respectively for the preparation of standard curve.Taking the Ct values ofβ-actin,DpXTH1 andDpXTH2 as the abscissa and the logarithms of their concentrations as the ordinate,the standard curves were drawn.The copies ofβ-actin,DpXTH1 andDpXTH2 in different tissues of dahlia were calculated using Microsoft Excel 2007,and the expression differences inDpXTH1 andDpXTH2 among different tissues of dahlia were analyzed.

Results and Analysis

Quality analysis of total RNA from different tissues of dahlia

A certain amount(3 μl)of extracted RNA from each tissue was examined by 1%agarose gel electrophoresis.Apparent 18 S and 28 S rRNA bands were obtained.TheOD260/OD280values of total RNA from roots,stems,leaves and petals were 1.734,1.800,1.813 and 1.900,respectively.It indicated that the extracted RNA had mild degradation,high purity and high quality,and it had not been contaminated by genomic DNA.Thus the extracted RNA could be used for reverse transcription.

RT-PCR results

Using the cDNA from roots,stems,leaves and petals of dahlia as the templates,the real-time PCR was performed with the corresponding primers forβ-actin,DpXTH1 andDpXTH2.The results showed that the bands ofDpXTH1 andDpXTH2 of roots were relatively dim,and the expected target fragments were detected in all the stems,leaves and petals of dahlia (Fig.1).It indicated that the designed primers were suitable for SYBR Green real-time quantitative PCR.

Amplification results of the samples and standard curves

The different-concentration standards ofβ-actin,DpXTH1 andDpXTH2 were amplified by PCR.As shown in Fig.2,the amplification curves were all smooth,and only single peaks were shown in the melting curves.So it was further illustrated that the designed 3 pairs of primers all had good specificities.

Based on the obtainedCtvalues and calculated copies ofβ-actin,DpXTH1 andDpXTH2,the regression equations were deduced:β-actin,y=-2.807x+31.24(R2=0.998);DpXTH1,y=-3.185x+33.14(R2=0.996);DpXTH2,y=-2.889x+29.94(R2=0.995).According to the following formula:

The amplification efficiencies were calculated as 1.27,1.06 and 1.22,respectively.There were no significant differences in amplification efficiency between the reference gene and target genes,indicating that the prepared standard curves met the requirements by real-time quantitative PCR and they could be used for relatively quantitative analysis.

Expression differences of DpXTH1 and DpXTH2 genes among different tissues

In order to determine the expression differences betweenDpXTH1 andDpXTH2 among different tissues and organs of dahlia,the total RNA from roots,stems,leaves and petals of dahlia was extracted,and the real-time quantitative PCR was performed using β-actin as the reference gene.The Ct values ofDpXTH1 andDpXTH2 from roots were all higher than 35,so it could be concluded that theDpXTH1 andDpXTH2 genes were not expressed in dahlia roots.The relative expressionlevelsofDpXTH1andDpXTH2 in stems,leaves and petals of dahlia were analyzed.The results showed that there were significant differences in relative expression levels ofDpXTH1 andDpXTH2 among different tissues of dahlia (Fig.3).BothDpXTH1 andDpXTH2 genes were highly expressed in petals of dahlia,and their expression levels were significantly higher than those in the same-growth period stems and leaves.It suggested that at the full blooming stage,the transcription ofDpXTH1 andDpXTH2 was mainly concentrated in petal cells;at that time,the expression level ofDpXTH1 was moderate in leaves of dahlia,but the expression level ofDpXTH2 was relatively low in stems and leaves of dahlia.In addition,the relative expression level ofDpXTH1washigherthanthatofDpXTH2,indicating that the roles ofDpXTH1 andDpXTH2 might be different in the physiological metabolism of dahlia atthe fullblooming stage.Therefore,it could be speculated that the expression and regulation ofDpXTH2 were dominant during the growth and development of dahlia at the full blooming stage.

Discussion

Xyloglucan endotransglycosylase/hydrolases (XTHs)are a typical family of proteins encoded by multiple genes.They are closely associated with the growth and development of plants,and participate in the elongation of tissues[10-12],growth and senescence[13],fruit ripening[14]and other physiological processes.The expression ofXTHgenes is tissue-and organ-specific[15].Moreover,the expression peaks of differentXTHgenes may be different in the same tissue among different growth periods.For example,inArabidopsis thaliana,AtXTH1 gene is expressed in siliques;AtXTH9 is expressed in shoot apical meristems,flower buds and flowering branches,and it is involved in the elongation of stems and flowering branches;AtXTH27 is expressed in vein channels and expanded leaves,and it takes part in the development of vessels[12,16].In the ripening persimmon fruits,the expression peak ofDkXTH1 appears before the softening of fruits,while the expression peak ofDkXTH2 appears in the mid of ripening period[14].In this study,theDpXTH1andDpXTH2 genes are not expressed in roots of dahlia,but they are highly expressed in petals.So it can be concluded that theDpXTH1 andDpXTH2 genes are petal-specificgenes,and theyare closely associated with the growth and development of petal cells.

On the other hand,inArabidopsis thaliana,mostXTHgenes are abundantly expressed in floral organs,as well as in other tissues and organs.However,AtXTH3 is only highly expressed in floral organs,and its transcription level is relatively low in other tissues and organs.Therefore,it can be speculated thatAtXTH3 has a flower-specific promoter[17].In this study,compared with roots,stems and leaves of dahlia,DpXTH1 andDpXTH2 show higher transcription levels in petals.So it is speculated that there may be also a flower-specific promoter in the gene structure,and it regulates the expression levels ofDpXTH1 andDpXTH2 genes during the development of dahlia petals.

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