Transgenic Petunia hybrida with Silicon Transporter Protein OsLsi1 and OsLsi2 Genes and Its Drought Resistance Analysis

Yang Tao,Zhao Ran,Zhao Jing,Wan Liang,and Fan Jin-ping*

1 College of Horticulture,Northeast Agricultural University,Harbin 150030,China

2 College of Chengdong,Northeast Agricultural University,Harbin 150030,China

3 District Commissioner's Office of Aihui,Heihe 164300,Heilongjiang,China

Introduction

Petunia hybrida is a solanaceae petunias perennial herbaceous plant.It is often cultivated annually and used for flower bed,planter and natural decoration (Liang et al.,2006).Petunia's growth requires high environment requirements,drought and other abiotic stress can cause the death of plants (Wang et al.,2004).Drought is not only causing serious limitations to Petunia's growth,but bringing great burdens for artificial irrigation,and causing the waste of resources.The genetic engineering research of Petunia mainly concentrated on changing flower color and color formation mechanism,etc.,while researches rarely in drought resistance.

Silicon is the second most abundant element in soil and the earthcrust,which is regarded as the essential element for the plant growth and development.Soluble silica can trigger multiple defense reaction mechanism in plants.Increasing the silicon content in plants can enhance drought and lodging resistances,improve the abilities of light absorption and reduce transpiration loss,etc (Fauteux et al.,2005;Ma et al.,2006).We found that there existed multiple genes coding transporter silicon in rice,some of which improved the utilization of silicon,including input transport protein gene Lsi1 (low silicon rice 1) and release of silicon transfer protein gene Lsi2 (low silicon rice 2).

Compared with the wild type rice,the rice mutants with silicon absorption loss (Lsi1) and the low silicon rice (Lsi2),Ma et al.(2006) found the whole protein family Lsi1 gene and assumpted anion transporter Lsi2 gene.Lsi1 gene had an open reading frame of 897 bp,containing four exons and five introns,and encoding 298 amino acids,the encoding products through six transmembrane regions contained membrane proteins similar with water channel protein and two Asn-Pro-Ala (NPA) sequence motives.Lsi1 gene was mainly expressed in root,and the function of encoding protein was mainly to transfer silicon from external soil to plant root cells,the transportation had the specificity of silicon (Li et al.,2010).

Lsi2 gene,with an open reading frame of 1 419 bp,contained two exons and one intron,encoding the protein composed of 472 amino acid residues.Lsi2 gene encoded membrane proteins through 11 transmembrane regions;the transport process of silicon was an active process driven by proton gradient energy (Ma et al.,2007).Silicon was transported to cortical cells and released first by Lsi1 gene,then acrossed aerenchyma into the apoplast with radial structure through Lsi2 gene,finally by Lsi1 gene translocated to the endothelial cells and was released into the column by Lsi2.Therefore,the silicon efficiently transporting into the stele across cells in casparian strip needed the coupling of Lsi1 and Lsi2 genes (Lily et al.,2010).Lsi1 and Lsi2 genes provided a new prospect for researching system of the absorbing silicon in higher plants.

Hu (2012) transformed silicon transporter protein OsLsi1 and OsLsi2 genes into Verbena,improving the cold resisitance of transgenic plants.The objectives of this study were to transform silicon transporter protein OsLsi1 and OsLsi2 genes cloned from rice into Petunia by Agrobacterium mediated method then tried to improve the drought resistance ability by effective absorption of silicon in soil,which had important practical significance in fostering new Petunia varieties with resistance.

Materials and Methods

Materials

Petunia hybrida was provided by the Garden Station in Northeast Agricultural University,Harbin,China.Agrobacterium tumefaciens GV3101 and plant expression vectors pBIL1 and pBIL2 containing Lsi1 and Lsi2 genes were saved in the Garden Plant Breeding Laboratory in Northeast Agricultural University (Fig.1).

Mediums

This test required five kinds of culture media.One of them which was used in Agrobacterium culture YEB liquid containted beef extract 5 g · L-1,yeast extract 5 g · L-1,peptone 5 g · L-1and sucrose 5 g · L-1.All the medium components were mixed and adjusted to pH 7.0 prior.The other four types of the culture medium ingredients in addition to shown in Table 1 also needed to add 0.7% agar and 3% sucrose then adjusted pH to 5.8 prior.

Petunia hybrid transformation

The plant expression vector plasmids pBIL1 and pBIL2 were transformed into Agrobacterium GV3101 by freezing and thawing methods.The transformants grew on YEB liquid culture medium containing Km 100 mg · L-1and Rif 50 mg · L-1.No vaccine blade was punched and made into semals with a diameter of 7 mm punch,inoculated on M1culture medium with the adaxial surface down,precultured for 2-4 days,then infected for 10-15 min with the above Agrobacterium.The infected explants were inoculated on M1medium,cocultured for 2-3 days at 28℃ in darkness,then transferred to M2medium for adventitious bud differentiation and screening at 25℃ in light.When the adventitious buds grew to 3-4 cm,they were inoculated into M3medium for rooting culture until roots completed.

Fig.1 Structure of plant expression vector pBIL1 and pBIL2

Table1 Evaluation index of liver function (UI · L-1)

PCR and RT-PCR identif ication of transgenic Petunia hybrid plants

Extracted the total DNA of transgenic Petunia leaves by CTAB method and total RNA by Trizol method,and PCR amplification was performed with 25 μL reaction volume.The amplification conditions were at 94℃ for 7 min,followed by 30 cycles of 94℃for 30 s,55℃ for 30 s,72℃ for 2 min,then at 72℃for 10 min.PCR products were electrophoresed on a 1.2% (w/v) agarose gel containing ethidium bromide and photographed under UV light.

Table2 Primers used in PCR and RT-PCR idetification

Drought stress of transgenic Petunia hybrid plants

Selected no-transgenic and transgenic T0generation plants 20 strains planted in the pots and watered with water and 1/8 MS nutrient solution added NaSiO3,alternately.The drought treatments of 0,4,7,10 and 14 days were performed with four strains in each group when the seedlings grown 4-6 true leaves,and observed the form of each strain under drought stress,then rewatered to observe plant recovery.

The non-transgenic and transgenic T0generation plants were taken by the drought treatments for 0,4,7,10 and 14 days,then determinated the physiological indexes.

(1) The malondialdehyde (MDA) content determination: glucosinolates barbituric acid method (Zou,2000);

(2) The determination of the free proline (Pro) content: acidic triketohydrindene hydrate method (Zou,2000);

(3) The super oxide dismutase (SOD) activity determination: NBT method (Li,2003);

(4) The peroxidase (POD) activity determination: the guaiacol method (Li,2003).

We repeated the experiment three times,recorded the experiment data,and used the software of Excel and SPSS to compare and multiple comparisons.

Results

Transgenic petunias plant

The silicon transporter protein OsLsi1 and OsLsi2 genes cloned from rice were transformed into Petunia by agrobacterium mediated method,getting 54 and 62 transgenic plants,respectively.The growth of genetic transformation plants at various stages are shown in Fig.2.

Fig.2 Regeneration and resistant transformations of Petunia hybrid

Identification of transgenic Petunia hybrid plants of OsLsi1 gene

Taking pBIL1 plasmid as the positive control,DNA of the no-transgenic Petunia leaves and water were as negative controls respectively,54 strains of OsLsi1 gene transgenic lines were identified by regular PCR (Fig.3).Electrophoresis showed that 29 lines were positive,and PCR detection positive rate was 53.7%,preliminary confirming that OsLsi1 gene had been integrated into Petunia.

To further detect the expression of OsLsi1 gene in transgenic Petunia,RT-PCR was performed on leaves of 26 transgenic and wild-type Petunia plants.The results showed that all the 26 lines were RTPCR positive plants,proving that OsLsi1 gene was successfully transformed into Petunia.Parts of the electrophoresis are shown in Fig.4.

Fig.3 PCR analysis of regeneration plant with OsLsi1 gene

Fig.4 RT-PCR analysis of regeneration plant with OsLsi1 gene

Identification of transgenic Petunia hybrid plants of OsLsi2 gene

Taking pBIL2 plasmid as the positive control,DNA of the no-transgenic Petunia leaves and water as the negative controls respectively,the 62 strains of OsLsi2 gene transgenic plants were identified by regular PCR (Fig.5).The results showed that the 46 plants were positive,and PCR detection positive rate was 74.2%,preliminary confirming that OsLsi2 gene had been integrated into Petunia.For the purpose of detecting the expression of OsLsi2 gene in Petunia,RT-PCR was performed on leaves of the 46 transgenic and wild-type Petunia plants.The results showed that there were 32 positive lines,proving that OsLsi2 gene was successfully transformed into Petunia.Parts of the electrophoresis are shown in Fig.6.

Determination of related physiological indexes under drought stress

Determination of MDA content

From Fig.7,we found that with the increase of the stress days,the malondialdehyde content of the three strains was increasing,OsLsi2 gene strain was the most significant,then CK,while OsLsi1 gene strain was not significant.No significant differences among the three strains in non stress;but with the prolonging of drought stress time,the three strains of MDA content had different degrees of difference.After the stress of 4 days,OsLsi1 gene strain and other two strains had a significant difference (P＜0.05),and the difference was extremely after 7 days (P＜0.01);OsLsi2 gene strain showed significant difference in CK strain after 7 days (P＜0.01).The comparison proved that the drought-resistant ability of OsLsi1 gene strain was the strongest,and OsLsi2 gene was the weakest.

Fig.5 PCR analysis of regeneration plant with OsLsi2 gene

Fig.6 RT-PCR analysis of regeneration plant with OsLsi2 gene

Fig.7 Multiple alignment of MDA content under drought stress at different time of strains

Determination of free proline content (Pro) content

From Fig.8,we found that after 0-7 days drought stress,proline content of the three strains increased,and the content of the differences were not significant,but OsLsi1 gene strain had been rising and rising greatly;strain OsLsi2 reached the maximum content in stress for 7 days,then decreased;CK strain in stress for 10 days reached the maximum content.

Multiple comparison analyses indicated that under drought stress for 0 day and 7 days proline content of the three plants was no significant difference in stress,the difference of OsLsi2 gene strain was significant (P＜0.05);while for 14 days,the three strains had extremely significant differences (P＜0.05).

Determination of SOD activity

As shown in Fig.9,after 0-7 days drought stress,SOD activity of the three trains increased,OsLsi1 gene strain rose most,then CK;since after treated for 7 days,SOD activity of OsLsi1 gene strain was significantly higher than that of the other two strains,resulting that the drought-resistant ability of OsLsi1 gene strain was the strongest.

Fig.8 Multiple alignment of free proline content between strains at different time under drought stress

Fig.9 Multiple alignment of SOD activity under drought stress at different time of strains

From multiple comparison analysis,stress from 0 to 4 days,there was no significant difference with CK,OsLsi1 gene and OsLsi2 gene strains;after 7 days,CK,OsLsi1 gene and OsLsi2 gene strains began to present a significant difference (P＜0.05),when 10-14 days,three strains reached extremely significant differences (P＜0.01),proving that OsLsi1 gene strain had the strongest drought resistance,secondly,CK and OsLsi2 gene strains were the worst.

Determination of POD activity

As shown in Fig.10,POD activity of the three strains had been rendered on a trip of the difference.CK strain had a maximum after 10 days'treatment;OsLsi1 gene strain was steadily rising,and ultimately did not reach a maximum activity;and OsLsi2 gene strain reached the highest POD activity after 7 days'treatment of the drought,then declined dramatically.Multiple comparison analyses showed that as the extension of the drought treatment time,the differences were extremely significant among the three strains (P＜0.01).

Fig.10 Multiple alignment of POD activity under drought stress at different time of strains

Though comprehensive analysis and judgment,the drought resistance of OsLsi1 gene strain was significantly better than that of CK and OsLsi2 gene strains,after the drought stress for 14 days the limit of defense system had not broken;the indicators of OsLsi2 gene strain began to decline by a large margin after 7 days of drought stress,showing that after drought stress of 7 days,tissues and organs inside the plants had damaged and began to affect the normal growth of plants,reaching the limit of the mechanism system under stress.In comparison,under the stress of 10 days,CK strain reached the limit of the defense system.Therefore,the drought-resistant ability of the three strains from strong to weak was OsLsi1＞CK＞OsLsi2.

Discussion

To verify the drought-resistant ability of the transgenic plants,as indexs of resistance,MAD content,Pro content,SOD activity and POD activity were measured after drought stress.With different drought treatments,the physiological indexes changed.The droughtresistant ability of the three strains was,from strong to weak,OsLsi1＞CK＞OsLsi2.Therefore,OsLsi1 gene may absorb silicon from the soil environment,making plants enhance the drought-resistant ability;while OsLsi2 gene may release its own silicon to the outside,making the resistance of OsLsi2 gene plants weak.

As one of the most widely used annual flowers in garden,Petunia has rich color and wide varieties,which is used in flower beds and flower grooves.In 1995,Netherlanders firstly transformed DFR gene into Petunia hybrida by agrobacterium,getting the bright orange genetic transgenic plants (Mol et al,1995).Since then,genetic engineering breeding of Petunia was extensively developed.The main development was to change the color and the formation mechanism of design and color,greatly enriching the varieties of Petunia,improving ornamental and application.In 1996,Shao et al.transformed CHS gene into Petunia,the flowers'color changed from purple into white or purple and white phenotype.In 1996,Jorgensen et al.found that 139 strains significantly changed shapes in 189 strains of transgenic plants with CHS gene.

And the resistances were mainly in the aspects of disease resistance and insect pests resistance.In 2006,Liu et al.transformed the aphid-resistance gene (snow lotus lectin gene,GNA) into Petunia,and obtained the aphid-resistance plants.In 2007,Feng et al.successfully transformed antiviral protein gene (PAP) into Petunia,obtained new Petunia varieties with certain disease-resistance.But the genetic transformation of Petunia about drought-resistance was still rare.In our study,we transformed OsLsi1 and

OsLsi2 genes into Petunia by Agrobacterium method,developed the drought-resistant Petunia varieties,and made the drought-resistance been improved,which greatly improved the ornamental value of Petunia,reduced the limits of drought on growth and the burdens of maintenance management,laid a foundation for the further study of Petunia's drought resistance,as well as provided a new train of thought for improving the efficiency of water saving in landscaping.

Conclusions

The results showed that MDA content,Pro content,SOD activity and POD activity were almost rising,except SOD activity had a little declined during 10-14 days water stress,which suggested that OsLsi1 gene against drought resistance was the strongest with 14 days drought stress and reached the limits of its own defense system;while resistance to limit OsLsi2 gene plants was 7 days,and CK plants were 10 days as a contrast.All the results proved that the silicon transporter protein OsLsi1 and OsLsi2 genes were normally transcripted and expressed in transgenic Petunia hybrida;OsLsi1 gene could improve the abilities of plant resistance against drought and recover after drought stress,while OsLsi2 gene could reduce the abilities above.The order of the drought resistance ability of the three strains from strong to weak was OsLsi1＞CK＞OsLsi2;silicon indeed improved the ability of drought resistance as well.The whole experiment offered a new way to improve the drought resistance of the Petunias,and laid a foundation for improving the ability of the garden plant drought resistance and water saving.

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