Sequence analysis of cereal sucrose synthase genes and isolation of sorghum sucrose synthase gene fragment

Sorghum (Sorghum bicolor (L.) Moench) is an important staple food for about 500 million people in semi arid regions of the world. Recently, sorghum has been identified as a main plant species for the comparative analysis of grass genomes and as a source of beneficial genes for agriculture. Recent studies have shown that there is conservation of gene order at the chromosome level in rice, sorghum and maize. Therefore, a high-resolution alignment between these genomes will be needed to utilize them constructively for sorghum gene discovery. Sorghum sucrose synthase gene fragment was amplified by primers designed at conserved exon position of cereal sucrose synthases. Sorghum sucrose synthase gene fragment I shared homology with other cereal sucrose synthase at the exon positions 6, 7, 8, 9 and 10. Sorghum sucrose synthase fragment II shared homology from exon 2 to 6.


INTRODUCTION
Sorghum, a C 4 grass that diverged from maize just 15 million years ago, is the fifth most important cereal grown worldwide (Doggett, 1988).Recently, sorghum has been identified as a key plant species for the comparative analysis of grass genomes and as a source of beneficial genes for agriculture.Sorghum is relatively small genome (750 Mbp) (Arumuganathan and Earle, 1991)) and incremental divergence from maize and rice (Doebley et al., 1990), make it ideally suited to aid the discovery and analysis of grass genes through comparative genomics.Sucrose synthase genes have been isolated from many of plants, mostly from starch storing plants such as maize (Werr et al., 1985;Hung et al., 1994), rice (Wang et al., 1992;Yu et al., 1992), wheat (Marana et al., 1990), potato (Salanoubat and Belliard, 1987), pea (Barratt et al., 2001) as well as Arabidopsis thaliana (Chopra et al., 1992;Martin et al., 1993).

Multiple sequence alignment
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.

Isolation of plant DNA
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 dis-
tilled water two times and then placed in tissue culture bottle containing ½ MS medium.Sorghum seeds were kept at 28 o C (BOD incubator) for germination.Sorghum genomic DNA was isolated by CTAB method (Dellaporta et al., 1983).The isolated genomic DNA was treated with RNase to make it RNA free.DNA quantitation was carried out by agarose gel analysis.2 -5 µl of sorghum genomic DNA was loaded in 0.7% agarose gel.Known amount of I Kb ladder was loaded as control in adjacent well.The quantity of DNA in the sample was estimated by comparison with the intensity of 3 kb band by eye judgment.

PCR
Primer sequences were designed at the conserved coding region to amplify a sucrose synthase gene fragment from sorghum (Table 1).Purification of amplified DNA from PCR reactions was performed using Qiaquick PCR purification kit (QIAGEN, Germany).pBluescript and UA cloning vector (QIAGEN PCR Cloning Kit) was used to clone the PCR amplified product

Transformation of E. coli cells
One vial of competent cells was taken out from -70 o C and thawed by keeping on the ice. 2 ml of plasmid DNA or 15 µl of ligation mixture was added and kept on ice for 30 min.The cells were subjected to heat shock at 42 p C for 2 min, immediately plunged on ice for 5 min.To this 900 µl of Luria broth was added and kept at 37 o C with shaking speed of 220 rpm for 1 h.The Cells were pelleted at 10,000 rpm for 1 min and resuspended in 100 µl of Luria broth, plated and incubated at 37 o C for growth of transformed cells.

Cloning and sequencing
The recombinant clone was sequenced using T7 and SP6 reverse primers.Sequencing was carried out by Sanger dideoxy DNA sequencing method.

Multiple sequence alignment
Sucrose synthase is found in all plant tissues and is found at high levels particularly in sink tissues

Primer design and PCR amplification
As the sucrose synthase gene sequence is highly conserved among cereals especially at the coding region, from multiple sequence alignment, highly conserved exon positions were chosen to design primers and using those to amplify sucrose synthase gene fragment form sorghum. Schematic diagram showing the exon position where the primers were designed and sorghum sucrose synthase gene fragments were cloned is shown in Figure 1.The forward primer GP1F 5' GC GTC GAC CCA AGA GCT TGG TTT GGA GAA GG 3' was designed based on the region of homology at the exon position 6 and the reverse primer GP1R 5' GC TCT AGA CTG TGA ACT GGC ATG AGA AGT GG3' was designed based on the region of homology at the exon position 10.The purified sorghum genomic DNA was used for amplification.Various annealing temperatures ranging from 53 -63 o C were tested for amplification of the specific sequence.PCR reaction was run for 25 cycles and the reaction was electrophoresed on the 0.8% agarose gel.Among various annealing temperature tested, amplification was obtained at 56 o C, which showed a single band of expected size of about 1 kb.No nonspecific amplification was observed (Figure 2).The amplified product was eluted from gel cloned in Sal I and Xba I pBluescript.To confirm the size of the insert, plasmid isolated from the transformed colony was digested with same two restriction enzymes.The expected fragment of ~1kb was obtained.

Cloning of sorghum sucrose synthase gene fragment
Sorghum sucrose synthase gene fragment cloned in pBluescript was sequenced and the sequence was aligned with other cereal sucrose synthase gene sequences, which were already taken for multiple sequence    vulgare and S. officinanum, so the PCR amplified and cloned fragment in pBluescript vector was confirmed as sucrose synthase gene fragment of sorghum (Figure 3).
To clone an upstream sequence of sucrose synthase gene, one more forward primer GP 2 F was designed at the exon position 2 and reverse primer GP2F was designed at exon position 6.PCR amplification was done at various annealing temperatures ranging from 50 to 60 o C using sorghum genomic as template.The electrophoresis of PCR products was done on 0.8% agarose gel.The sharp single band of expected size of about 1.5 kb was obtained at 55 o C annealing temperature.Thermostable polymerase enzyme will add extra adenine nucleotide at the end of the amplifying strand at each cycle.This property was utilized to clone a Taq amplified PCR product in TA cloning vector.The 1.5 kb PCR amplified product was cloned in pDrive TA cloning vector.Sequencing was done by Sanger dideoxy DNA sequencing method and the nucleotide sequence was given in Figure 4.The sequence of 1.5 kb gene fragment was compared with other cereal sucrose synthase.It shared homology at exon No. 2, 3, 4, 5, and 6 of RSS 1 , RSS 2 , RSS3 of rice, Suc 2, Suc1C of maize, SS2 of H. vulgare and S. officinanum.So the 1.5 kb fragment was confirmed as sorghum synthase gene fragment.Highlighted portions are exon sequences sharing homology with cereal sucrose synthases 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.Sucrose synthase gene sequences of cereals such as rice, maize, sugarcane and barley were taken from NCBI Genbank database and multiple sequence alignment was carried out using MegAlign programme of DNA star software.Genes Suc S 2 , RSs 1 and SUS 2 are highly conserved with respect to their exon and intron position and number.All the three genes have 16 exons.All the gene sequences compared have conserved gene sequences at their coding part and not in the non-coding region.13 th exon of rice RSS 2 is split in two.14 th exon of RS 2 is sharing homology with 16 th exon of maize SuS 2 and rice RSS 1 .In case of rice RSs 3 and Ss 2 of H. vulgare the 3 rd exon position is replaced with a new exon.This new exon of RSS 3 of rice and Ss 2 of H. vulgare does not share homology with 3 rd exons of RSS 1 , RSS 2 of rice, SucS 2 , SuS 1 of maize, SS 2 of S. officinarum.5 th exon of RSS 3 of rice and Ss 2 of H. vulgare is missing in both.A long leader intron is characteristic of sucrose synthase genes (Werr et al., 1985, Wang et al., 1992;Yu et al., 1992).It is present in all the sucrose synthase genes isolated so far, except for ASuS I from Arabidopsis (Martin et al., 1993).The Cis elements present in the leader intron may be involved in gene re-gulation.
Sorghum sucrose synthase gene fragment cloned in pBluescript was sequenced and the sequence was aligned with other cereal sucrose synthase gene sequences, which were already taken for multiple sequence alignment.The cloned fragment shared homology in the region of exon 6, exon 7, exon 9 and exon 10 with Rss 1 , Rss 2 and Rss 3 of rice, SUC 2 , SUCI of maize, Ss 2 of H. vulgare and S. officinanum.Therefore the PCR amplified and cloned fragment in pBluescript vector was confirmed as sucrose synthase gene fragment of sorghum.
It was decided to amplify the gene portion between exon 2 and exon 6. Primers were designed at exon 2 and exon 6 and 1.5 kb sucrose synthase gene fragment II was PCR amplified and cloned in pDrive cloning vector.The nucleotide sequence, which shared sequence similarity at their exon positions 2 to 6 with other cereal sucrose synthases.The position and the length of sequence homology were highlighted in the Figure 4. Exonic sequences not encoding protein (exon 1 and 16) of all the sucrose synthases taken for comparison do not exhibit conservation as that of coding region of the gene.
High level sequence divergence of the introns relative to that of exons was observed.This indicates that selection pressure against mutation abolishing or reducing the function of the gene.It was observed that sequences at exon/intron boundaries are highly conserved.These findings suggest that mutations in such a manner do not alter their ability to be recognized as introns.A very small percentage of sequence variations in exonic sequences encoding protein observed is an indication of the signifycance of evolution.

Figure 1 .Figure 2 .
Figure 1.Schematic diagram showing the exon position where primers were designed and sorghum sucrose synthase gene fragments were cloned.GP-Gene specific primer; F -forward primer; R -reverse primer.

Table 1 .
Nucleotide sequences of primers.

Table 2 .
Gene name, accession number, exon numbers, exon position and region of homology different cereal sucrose synthases

Table 2 .
Contd.∆ = 3 rd exon of RSs3 (Oryza sativa) and SS2 (Horgeum vulgare) share homology with each other but does not share a region of homology with 3 rd exon of other sucrose synthase gene sequences compared; * = 4 th exon of RSs3 and SS2 share region of homology with 3 rd exon of other sucrose synthase gene sequences compared; ∇ = 5 th exon of RSs3 and SS2 share region of homology with 5 th exon of other sucrose synthase gene sequences compared.;α= RSs3 and SS2 does not have an exon to share homology with 5 th exon of other sucrose synthase gene sequences compared.!= 6 th exon is highly conserved and shares highest region of homology in all the sucrose synthase gene sequences.= 13 th exon of RSs2 shares similarity with 13 th exon of RSs3 and Ss2 and not with the RSs1, SucS2 and SVS2.= 14 th exon of RSs2 shares similarity with 14 th exon of RSs1 and Sus2 and not with the RSs3, Ss2 and SucS2.0 = 15 th exon of RSs2 shares region of homology only with Sus2 and not with other sucrose synthase gene sequences compared.