Abstract: Regulation of expression of the transgene by use of appropriate promoters is most important for durability and specificity of resistance. Sucrose synthase promoter of cereals like Rice and maize was reported for their phloem specificity. Promoters are more efficient in their expression in the organism from which it has been isolated rather than in heterologous system. Taking these points into consideration, to control the sap sucking pests of Sorghum, Sucrose synthase promoter was isolated by Adapter PCR technique and in silico analysis of promoter sequence reveals the presence of phloem specific cis regulatory elements.
INTRODUCTION
Cereals are the most important food crops among which sorghum is the fifth most important crop in the world. Sorghum is mainly cultivated in semi arid tropics and is extensively grown in Africa, Asia and North America. Stem borers cause major yield losses in cereals. Sorghum yield and yield stability are hampered by several insect species. The sorghum stem borer (Chilo partellus) is one of the serious pests of sorghum which infects the crop usually a month after sowing and causes dead hearts. Shoot fly is an important insect pest of seedling sorghum primarily in Asia and Africa. Depending on the plant stage during insect attack, feeding may lead to loss of leaf area, dead hearts, or stem and peduncle tunneling. Although each kind of damage may result in serious economic loss, not all are significant in a given location.
During development and differentiation, plants need to integrate a wide range of tissue, developmental and environmental signals to regulate complex patterns of gene expression (Karam and Singh, 1998). Manipulating the genetic makeup of plants to improve valuable traits such as seed oil composition, to make them insect pest resistant, pathogenic disease resistant, herbicide resistant or to introduce novel characteristics like production of vaccines or plastics is a challenging task. Appropriate control of genes that are introduced into plants is critical to success in this undertaking and the need for precision is dictated by the fundamental organization of plants as complex living systems (Datta and Selvaraj, 1995). For efficient pest control, it is important that effective levels of insecticidal proteins are expressed at the site where the insects feed. Regulation of expression of the transgene by use of appropriate promoters is most important for durability and specificity of resistance.
Sucrose synthase in source tissues is involved in phloem loading and phloem transport (Martin et al., 1993; Fu and Park, 1995; Hanggi and Fleming, 2001). Sucrose synthase genes have been isolated from many of plants, mostly from starch storing plants such as maize (Werr et al., 1985), rice (Wang et al., 1992; Yu et al., 1992), wheat (Marana et al., 1990), potato (Salanoubat and Belliard, 1987; Fu et al., 1995), pea (Barratt et al., 2001), bean (Silvente et al., 2003) as well as Arabidopsis thaliana (Chopra et al., 1992; Martin et al., 1993). Many plants have multiple, distinct isoforms of sucrose synthase genes, which have different patterns of expression in different organs of plant (Marana et al., 1990; Zeng et al., 1998; Barratt et al., 2001; Klotz et al., 2003; Kateri et al., 2006; Haagenson et al., 2006). For example, the two genes encoding sucrose synthase in maize Sh1 and Sus1 showed opposite responses to changes in tissue carbohydrate status (Koch et al., 1992).
Rice (Shi et al., 1994) and maize (Yang and Russell, 1990) sucrose synthase gene promoters have been reported for phloem specific expression. The present study to isolate Sucrose synthase gene promoter from sorghum by adapter PCR technique with the objective of expressing insecticidal genes in a tissue specific manner under the control of homologous promoter to control pests which targets phloem.
MATERIALS AND METHODS
Isolation of Plant DNA
Sorghum seedlings were grown in tissue culture lab of National Research
Centre on Plant Biotechnology, New Delhi during June, 2003. Two weeks old sorghum
seedlings grown in ½ MS medium (Murashige and Skoog, 1962) were taken
for total genomic DNA isolation. Sorghum (var. CSV 15) seeds were kept in 0.1%
HgCl2 for 10 min with intermittent shaking. After decanting 0.1%
HgCl2 solution, the sorghum seeds were thoroughly washed in sterile
distilled water two times and then placed in tissue culture bottle containing
½ MS medium. Sorghum seeds were kept at 28°C (BOD incubator) for
germination. Sorghum genomic DNA was isolated by CTAB method (Dellaporta et
al., 1983).
PCR Amplification of Sorghum Sucrose Synthase Gene Fragment
Multiple sequence alignment of sucrose synthase gene sequences of various
cereals like rice, maize and barley were accessed from NCBI Genbank database
and multiple sequence alignment was done using MegAlign programme of DNA star
software. Primer sequences were designed at the conserved coding region to amplify
a sucrose synthase gene fragment from sorghum. Gp1F 5’ CTCCTCTCATCCCAATG
3’ forward primer was designed at second exon position two and Gp2R 5’
CCCCTTCTCCAAACCAAG 3’ reverse primer was designed at sixth exon position.
Sorghum genomic DNA was used as template. 1.5 kb amplicon obtained was purified
using Qiaquick PCR purification kit (QIAGEN, Germany). UA cloning vector (QIAGEN
PCR Cloning Kit) was used to clone the PCR amplified product. The recombinant
clone was sequenced using T7 and SP6 reverse primers.
Sequencing was carried out by Sanger dideoxy DNA sequencing method.
Southern Hybridization
Localization of particular sequences within genomic DNA is usually accomplished
by the transfer techniques described by Southern (1975).
DNA Restriction and Electrophoresis
Ten to fifteen microgram of plant DNA was taken and restricted by restriction
enzymes such as BamHI, EcoRI, BglII and SalI separately.
Five unit of each enzyme were taken per μg of DNA in 1X reaction buffer
and incubated overnight at 37°C. After incubation the reaction was stopped
by addition of 1 mM EDTA. The digested DNA fragments were resolved by gel electrophoresis.
For separation of DNA, 0.8% agarose gel was prepared in 1X TAE. The voltage
applied across the gel was 40 V for 8-10 h. HindIII digested DNA was
used as marker. Ethidium bromide at the rate of 0.5 μg mL-1
was added to the gel before casting the gel.
Transfer to Membranes
After the completion of run, the position of marker bands was recorded on
a cellophane sheet and the gel was soaked in 0.25 N HCl for 10-15 min with gentle
shaking for depurination. The gel was then washed twice with double distilled
sterile water and 0.4 N NaOH for 45 min to 1 h. The gel was carefully placed
on a Whatman No. 3 blotting paper whole sides dip in a tank of solution containing
0.4 N NaOH and 1.5 M NaCl over a clean glass plate. A nylon N+ membrane
(Hybond-N+, Amersham) was cut to same size as the gel and placed
over the gel after soaking it in 0.4 N NaOH for 30 sec.
Three to five pieces of Whatman No. 3 blotting paper were cut to same size as the gel, soaked in 0.4 N NaOH and placed on the membrane. Air bubbled trapped between the gel and membrane was removed by rotating a clean glass rod over the Whatman paper. Then 5-7 cm thick stack of blotting sheet (of the same size as the gel), a glass plate and weight of approximately 500 g were placed over it. The capillary transfer was allowed continue for 14 to 15 h, after which the assembly was dismantled. The membrane was cut at the side of HindIII digested marker loaded position and the membrane was cross-linked over UV transilluminator for 30 sec, air-dried and kept in a desiccator till further use.
Pre-Hybridization
The membrane was placed in a clean dry hybridization bottle (Amersham) and
10-20 mL of pre-hybridization buffer was allowed to continue for 2-4 h at 65°C
at a speed of 10-15 rpm in a hybridization oven.
Preparation of Probe
Deca LabelTM DNA labeling kit (MBI Fermentas) was used for the
preparation of probe.
Washing of Filter and Autoradiography
High stringency i.e., low buffer and high temperature was employed during
washing of filters. After decanting the hybridization solutions to safe disposal,
the filter was washed with 2X SSPE, 0.1% w/v SDS at 55°C for 10 min and
subsequent wash with 0.5X SSPE and 0.2X SSPE with 0.1% w/v SDS. The washed membrane
was then kept on a support (usual X-ray film) covered with saran wrap after
removing trapped air bubbles, if any and then exposed to X-ray film (Hyperfilm,
Amersham) in a cassette with an intensifying screen.
The cassette was kept at -70°C for appropriate exposure time based on the CPM of 32P on the membrane and the stringency of washing the film was developed in Kodak developer for 2-3 min, rinsed in distilled water and fixed in Kodak fixer for 10 min. The film was washed thoroughly under running tap water and air-dried.
Adapter PCR
Adapter PCR technique was followed to get the upstream sequence of sorghum
sucrose synthase gene. It is kind of PCR technique in which one gene specific
primer and one random primer is used. Adapter PCR technique was done to get
the upstream sequence of sorghum sucrose synthase gene.
Restriction Digestion of Genomic DNA
Sorghum genomic DNA was taken (15-20 μg) and digested overnight with
the BamHI, restriction enzyme. This enzyme will produce the same compatible
overhang and finally the digested DNA was purified using PCR purification kit
(QIAGEN).
End Filling Reaction
The digested DNA was partially end filled with dGTP and dATP (2 mM each)
in the presence of 10x klenow buffer and klenow enzyme (10 U μL-1)
in a reaction volume of 50 μL and was incubated at 37°C for 30 min
followed by purification using PCR purification kit (QIAGEN).
Adapter Preparation
Two partially complementary primers 33 and 11 mer in a equimolar concentration
was taken and kept at 95°C for 3 min and allowed to come down gradually
to the room temperature for the purpose of annealing.
Ligation with Adapters
The partially end filled DNA was ligated to the adapter in a ligation reaction
at 16°C for 24 h in a 20 μL reaction volume containing 8-12 mg DNA,
1.5 μg adapter and 1 μL of T4 DNA ligase (2 U μL-1)
excess adapters were removed again through Qiaquick columns (QIAGEN).
PCR Amplification
The adapter ligated genomic DNA was suitably diluted and used for amplification
in a 50 μL reaction mixture under the following amplification conditions.
| • | 94°C3 minInitial denaturation |
| • | 92°C1 min |
| • | 55°C1 min |
| • | 72°C2 minStep 2-4 cycled for 30 times |
| • | 72°C5 minFinal extension |
T7 and gene specific primers were used for amplification. Genomic DNA amplified with T7 primer alone was used as negative control. The purified products of first round of amplification were re-amplified with the second gene-specific primer.
Cloning and Sequencing
The DNA bands obtained after re-amplification were cloned in P drive TA
cloning vector. The recombinant clone was sequenced using T7 and
SP6 reverse primers. Sequencing was carried out by Sanger dideoxy
DNA sequencing method.
Adapter PCR (Flow Chart)
Cloning and Sequencing
The DNA band obtained in the third round PCR with Gp5R gene specific primer
was cloned in pDrive TA cloning vector. The recombinant clone was sequenced
using T7 and SP6 reverse primers. Sequencing was carried
out by Sanger’s dideoxy DNA sequencing method.
RESULTS
Primer Design and PCR Amplification
Multiple sequence alignment of sucrose synthase gene sequence from different
cereals revealed that it is highly conserved at their coding regions. Highly
conserved exon positions were chosen to design primers and using those to amplify
sucrose synthase gene fragment form sorghum.
| Fig. 1: | Schematic diagram showing the exon position where primers were designed. Gp: Gene specific primer, F: Forward primer, R: Reverse primer |
| Fig. 2: | PCR amplification of Sorghum sucrose synthase gene fragment using second set of gene specific primers. Lane M: 1 kb Ladder and Lane 1: PCR product of Sorghum sucrose synthase gene fragment II |
Schematic diagram showing the exon position where the primers were designed (Fig. 1) and the DNA fragment size of 1.5 kb was amplified and cloned in UA cloning vector is shown in Fig. 2 and 3. The amplicon was sequenced and multiple sequence alignment was done with Rss1, Rss2 and Rss3, of rice, SUC 2, SUCI of maize, Ss2 of Hordeum vulgare and Saccharum officinanum. The amplicon shared homology at the exon position second and six as expected and confirmed as Sorghum sucrose synthase gene fragment (Fig. 4).
Southern Hybridization of Sorghum Genomic DNA
Genomic DNA was isolated from two weeks old sorghum seedlings and RNase
treatment was given to make the genomic DNA free of RNA. Southern hybridization
was carried out to find out suitable restriction enzyme and design adapter with
complementary overhang.
| Fig. 3: | Sorghum sucrose synthase gene fragment II of ~ 1.5 kb size. Lane M: 1 kb Ladder and Lane 1: P drive UA cloning vector having sorghum sucrose synthase gene fragment II restricted with EcoRI |
| Fig. 4: | Nucleotide sequence of sorghum sucrose synthase gene fragment II Gp1F-Gp2R. Note: Highlighted portions are exon sequences sharing homology with cereal sucrose synthases |
Sorghum genomic DNA was restricted independently with various kinds of enzymes such as BamHI, EcoRI, BglII and PstI. 1.5 kb Sorghum sucrose synthase gene fragment obtained by PCR amplification using primers Gp1F and Gp2R was used as hybridization probe. Lane loaded with BamHI restriction enzyme digested genomic DNA gave two hybridization signals of sizes about 6 and 4 kb. EcoRI digested sample gave a hybridization signal size of around 10 kb. PstI and BglII digested sample gave multiple hybridization signals of various size ranges (Fig. 5).
| Table 1: | Nucleotide sequences of primers |
| Fig. 5: | Southern hybridization of sorghum genomic DNA using sucrose synthase gene fragment as probe. Lane 1: Sorghum genomic DNA restricted with BamHI restriction enzyme showing two hybridization signals with the approximate size band of 6 and 4 kb, Lane 2: Sorghum genomic DNA restricted with EcoRI restriction enzyme, Lane 3: Sorghum genomic DNA restricted with BglII restriction enzyme and Lane 4: Sorghum genomic DNA restricted with PstI restriction enzyme |
Adapter PCR
Adapter PCR was carried out to amplify the upstream region of sorghum sucrose
synthase gene. Three nested sets of primers with the interval of 200 bp were
designed near the second exon position (Fig. 1, Table
1). In the Southern hybridization, BamHI restricted sorghum genomic
DNA sample gave two hybridization signals of size 4 and 6 kb. To carry out adapter
PCR, Sorghum genomic DNA of same concentration (5-7 μg which was taken
for doing Southern hybridization) was taken and over nightly restricted with
BamHI, restriction enzyme and agarose gel electrophoresis was carried
out at
low voltage. In the UV illuminator, the restricted genomic DNA was observed as smear. The smear corresponding to ~6 and 4 kb marker band was cut and gel purified. Both 6 and 4 kb fragments which gave hybridization signals with BamHI restriction enzymes were taken for Adapter ligation as any one of fragment might carry the upstream sequence. Adapter primers were designed to include T7 promoter primer sequence. Adapter primers were end filled with d ATP and d GTP using klenow enzyme in order to have a complementary cohesive sequence, which can be ligated to BamHI, restricted fragment. Adapter primers were ligated to gel purified BamHI digested 4 and 6 kb fragments independently. PCR was carried out by T7 as forward primer and gene specific primer as reverse primer using adapter ligated to 4 and 6 kb fragment as template. To avoid non-specific amplification, three nested sets of gene specific primers were designed. First PCR was carried out with T7 primer and extreme outward gene specific primer (Gp3R). The size of the DNA band obtained was ~1.6 kb. The PCR product obtained in the first set of reaction was used as template in the second PCR reaction where forward and reverse primers were T7 and 2nd outward gene specific primer GP4R, respectively. In the second PCR 1.4 kb DNA band was obtained when the reaction sample was electrophoresed at 0.8% agarose gel.
| Fig. 6: | Adapter PCR amplification of upstream sequence of Sorghum. Sucrose synthase gene using nested set of gene specific primers. Lane M: 1 kb Ladder, Lane 1: ~ 1.6 kb PCR product obtained using gene specific primer (Gp3R) and T7 Primer, Lane: 2 ~1.4 kb PCR product obtained using gene specific primer (Gp4R) and T7 Primer and Lane: 3 ~1.2 kb PCR product obtained using gene specific primer (Gp5R) and T7 Primer |
| Fig. 7: | Sorghum sucrose synthase promoter cloned in P drive UA cloning vector. Lane M: 1 kb Ladder and Lane 1: Pdrive UA cloning vector having sorghum sucrose synthase promoter of ~1.2 kb size restricted with EcoRI |
In 3rd PCR reaction, 2nd PCR reaction product was used as template. The forward and reverse primers were T7 primer and Gp5R, respectively. The size of PCR product was 1.2 kb (Fig. 6). The amplicon size of ~1.2 kb was cloned in UA cloning vector (Fig. 7) and sequencing was carried out by Sanger dideoxy DNA sequencing method (Fig. 8).
In silico Analysis of Promoter
Plant CARE is a database of plant cis-acting regulatory elements for in
silico analysis of promoter sequences. The 1.2 kb sorghum sucrose synthase
promoter sequence was analysed in Plant CARE database.
| Table 2: | Sorghum sucrose synthase promoter sharing homology with cis-regulatory elements of other Cereals (Ref: Plant CARE software) |
| Fig. 8: | Nucleotide sequence of sucrose synthase promoter of sorghum |
The conserved box and the cis-elements which were sharing similarity with other plant promoter cis-elements were given in Table 2. The transcription start site was found to be at 578 bp. TATA box is reported to be important for RNA polymerase II recognition. There are TATA less promoters, which were reported to be found in nuclear encoded photosynthetic genes. The consensus sequence for TATA box was TACAaac found at 541th nucleotide position. This sequence is homologous to the consensus sequence for TATA box for rice. The CAAT box was found upstream of TATA box at nucleotide position 439. This CAAT box was found to be homologous with CAAT box found in Barley. This is a common cis-acting element in promoter and enhancer region. A-box motif, cis-acting element conserved in alpha-amylase promoters was found at the nucleotide position 176 bp. The GC-motif with the consensus sequence at CGCGCa was found at the nucleotide position 636 down stream of TATA box, which shared homology with rice in which it is said to be enhancer like element involved in endosperm expression. Similarly GCN4 motif was found at 442 bp, which is 99 bp upstream of TATA box, which is a cis- regulatory element, involved in endosperm expression. I box was found at two positions 248 and 381 bp and G box was found at 170 and 593 bp. Both are cis-acting element involved in light responsiveness. P box is gibberellin responsive element (ABRE) a cis acting element involved in the abcisic acid responsiveness was found at nucleotide position 317 bp. Two cis acting elements BoxII at nucleotide position 600 and GATA motif at three positions 250, 278 and 381 bp in the negative strand were found. It was reported that both act combinatorily in phloem specific expression of RTBV promoter (Yin et al., 1997). As2 box was found in the negative strand at position 284 bp responsible for shoot specific expression.
DISCUSSION
Sucrose synthase is found in all plant tissues and is found at high levels particularly in sink tissues. In monocotyledonous plants sucrose synthase is encoded by a small gene family. Phloem specific expression of sucrose synthase gene promoters have been reported in monocots such as Rss1 of Rice (Shi et al., 1994) and Sh1 of maize (Yang and Russell, 1990) and dicots such as Sus3 of Potato (Fu et al., 1995) and Asus1 of Arabidopsis (Martin et al., 1993).
Southern hybridization of Sorghum genomic DNA restricted with different restriction enzymes such as, EcoRI, BglII and PstI was done using Sorghum Sucrose synthase gene fragment as a probe. There were two hybridization signals corresponding to the size of 6 and 4 kb found with restricted DNA sample (Lane 1). Lane 2 loaded with EcoRI digested DNA sample (Fig. 5) was observed with three bands of size 8, 5 and 1 kb. Lane 3 with BglII digested DNA sample was observed with two hybridization signals above 10 kb and around 7 kb. Lane 4 with PstI restricted DNA sample was with four hybridization signals of different size i.e., 9, 7, 4 and 3 kb. Sorghum genomic DNA restricted with four different restriction enzymes (BamHI, EcoRI, BglII and PstI) all four of them was observed with minimum of two hybridization signals. This suggests that there should be at least two sucrose synthase isoforms independently coded by two genes in Sorghum. Lane 2 and Lane 4 was observed more than two hybridization signals indicates the possibility of presence of two restriction sites within the gene makes them get detected twice.
Sucrose synthase promoter of sorghum was amplified by Adapter PCR technique, which shared sequence conservation with neither Rss1 promoter nor with Maize Sh1 promoter. But it has similar cis regulatory elements as that of Maize Sh1 and Rice Rss1 promoter. It has cis elements for phloem specific expression such as Box II and GATA motif as that of RTBV promoter. RTBV promoter has three cis elements viz., Box II, the ASL Box and the GATA motif, which were shown to confer phloem specific gene expression (Chen et al., 1997).
The GATA family of transcriptional factors is zinc finger proteins that specifically recognize the GATA consensus sequences and are proven to be important for tissue specific gene expression, differentiation and development in mammalian systems (Simon, 1995). GATA motif has been found in most of the light regulated promoters and has been shown to be important for the activity of these promoters (Terzaghi and Cashmore, 1995). A GATA motif found in 17 bp element involved in binding of the protein factor Gs3A-F1 to the minimal-132 Gs3A gene promoter, which retained phloem specific gene expression (Brears et al., 1991). Likewise, the phloem specific AHA3 promoter has a GATA motif, although its function has not been characterized, a similar GATA motif was found at the nucleotide position 250 and 278 of sorghum sucrose synthase promoter which suggests that it may be involved in phloem-specific expression.
Sucrose synthesis plays an important role in seed development. GCN4 motif was found at the nucleotide position 442 and 568, which shares sequence similarity with rice GCN4 motif that is involved in endosperm expression. As2 box which was found at the nucleotide position 284 of minus stand, shares sequence similarity with N. tabacum, which is responsible for shoot specific expression.
Combinatorial interactions in transcriptional regulation have been well documented in plants (Benfey and Chua, 1990). For example, the combinatorial interactions of different tissue specific sub domains (Cis elements) results in constitutive expression from the CaMV 35S promoter (Benfey and Chua, 1990). Regulatory mechanism may reflect the evolution and differentiation of phloem and xylem. It has been proposed that phloem appeared much earlier during evolution of higher plants than did xylem and phloem always differentiates before xylem during plants development (Aloni, 1987). So, different combinations of different cis elements seem to serve as a code to direct spatial and temporal expression of different genes. To further understand the nature of these combinatorial interactions and their regulation by environmental signals, it may well be necessary to clone and characterize the factors binding to these elements.
The constitutive expression of the insecticidal gene in transgenic plants is likely to increase the risk of pests developing resistance and may also result in yield penalties as the plant directs more resources than necessary to the defense. To avoid this, tissue specific promoter can be used, thereby limiting exposure to the toxin, which could be expressed in those parts of the plant affected by insect. To manage the insects belonging to the suborder Homoptera, such as plant hoppers and aphids, which feed by sucking phloem sap of the transgenic plant, an insecticidal gene can be expressed in phloem tissues to achieve better results. The sorghum sucrose synthase promoter isolated in the present study can be used to direct phloem specific expression of such insecticidal genes.