ANALYSIS AND EXPRESSION OF AL-TOLERANT GENES FROM SOYBEAN [Glycine max (L.) Merryl] ON FORAGE CROPS AND Escherichia coli

pastura Vol. 3 No. 2 : 70 - 74

ISSN : 2088-818X

ANALYSIS AND EXPRESSION OF AL-TOLERANT GENES FROM SOYBEAN [Glycine max (L.) Merryl] ON FORAGE CROPS AND Escherichia coli

S. Anwar, Sumarsono, Karno and F. Kusmiyati Faculty of Animal Agriculture, Diponegoro University Email: syaifulanwar06@yahoo.com, syaifulanwar2011@gmail.com

ABSTRACT

In order to analyze and to study expressions of the Al-tolerant genes, we have examined five clone genes that were isolated from soybean cv. Lumut. Soybean cv. Lumut and Slamet, Centrocema pubescens, Pennisetum purpureum and Escherichia coli were selected for futher analysis. Based on the DNA sequencing, searching enzyme restriction sites and searching DNA homology with the genebank database; the clones encoding: (1) Catalase (gmali12, that function as an antioxidant), (2) Proliferating cell nuclear antigen like protein/PCNALP (gmali15, that involved as one of transcriptional regulator in the eucaryotic cell cycle), (3) Growth hormone (gmali22, this gene may play a role on stimulation of cell growth/development), (4) Amine oxidase (gmAO, genebank accession number AF313622, a gene that function as amine oxidation and/or antioxidant), and (5) Aminoacyl peptidase (gmAP, genebank accession number AF091304, a serine protease gene). Expressions of the clone genes either on forage crops or Escherichia coli indicated that all of the clones are basic genes, but its expression increased with aluminium induction (Al-induced genes) and involved in detoxification to Al stress. From this research, we also found similar responses between oxidative stress and Al stress to gene responses.

Keywords: Analysis, Expression, Al-Tolerant Genes,

INTRODUCTION

Aluminum (Al) is regarded as one of the main toxic factors which exist in most acidic soil in Indonesia (Notohadiprawiro, 1983), even of the world, comprising 1x10⁹ hectares in the tropical and cool temperature regions (Van Wambeke, 1976) or approximately 8% by Weight (Moller et al., 1984). Most Al in soil is insoluble, associated with complex aluminosilicates and oxides. However, under acidic soil condition (pH < 5) Al is converted from insoluble forms into soluble Al ⁺³ (Marschner, 1991; Driscoll and Schecher, 1990; and Kinraide, 1991), which block growth of plant roots (Rajaram and Villegus, 1990; Kinraide and Ryan, 1991; Foy et al., 1978; Wagatsuma et al., 1987; and Taylor, 1991). Thus, Al toxicity is one of the most important soil problems that limits plant growth, particularly in the tropical regions.

Approximately 40% of the world’s arable soils are too acid, and Indonesia has over 47,6 millions hectares. A problem that is becoming increasingly severe, because of the use of nitrogenous fertilizer, industrial pollution and acid rain (Van breeman, 1985). Eventhough, normal rainfall can also cause acidification of soils by promoting the leaching of basic cations such as Ca⁺², Mg⁺², K⁺ and Na⁺ (Foy, 1984). Thus, Al toxicity is one of the most important soil problems that limits plant growth, particularly in the tropical regions (Kochian, 1995; Taylor, 1995; Matsumoto, 2000).

Identifying genetic resistance to aluminium toxicity would be a valuable contribution toward the development of tolerant crops in the tropical areas,

Soybean, Forage, E. coli

especially in Indonesia. In these low pH aluminosilicate soils, the susceptibility of field crops to aluminium toxicity leads to the inhibition of root growth into the lower soil horison. Aluminium saturates the charged sites of the soil particle and, along with the restriction of root growth, acts to impede cation exchange with subsoil elements (Ca⁺², K⁺, and Mg⁺²), which are critical for normal plant development. Determining the molecular basis of tolerance to increase levels of aluminium in certain crops (such as soybean) poses a significant challenge.

Soybean is one of important crops in Indonesia. Its specific material for Indonesian tradisional food such as tempe, tofu, sauce and soybean milk have brought the soybean to an important position in Indonesian nutrition. Demand for soybean is increasing with the increase on protein need due to improvement program on Indonesian nutrition. The development of animal husbandry in Indonesia have also increased the demand of this crop. Unfortunately, the increase in demand for soybean can not be responded by the sufficient increase in production of this crop. This research was conducted to support soybean breeding programs by molecular approach. Considering the importance of molecular information on soybean tolerance to Al, we proposed the research on molecular biology of soybean tolerance to al stress as follow-through from previous research, by two approach: (1) Analysis of the Al-tolerant and (2) Study of expression of the cloned genes. The genes also have been evaluated to forage crop by northern/slot blot hybridization (heterologous approach) and E. coli.

RESEARCH METHOD

The research consist of two programs: Research I (Analysis of Al-tolerant genes) and Research II (Study expression of Al-tolerant genes).

Research 1. There are 3 steps in this program: (a) Analysis of clone genes by nucleotide sequencing, (b) Analysis of clone genes by searching restriction enzyme sites and (c) Analysis of clone genes by searching homology with GeneBank database.

DNA synthesis for chain-termination sequencing is carried out two steps. In the first, the primed strand of DNA is extended and at the same time labelled by the incorporation of dye-nucleotide. . In the second step, dideoxynucleotides are added to the population of labelled DNA molecules (ranging in length from a few to many hundreds of nucleotides) and synthesis continues until a ddNTP is incorporated, thus terminating the chains.

Analysis of clone genes by nucleotide sequencing was started with cDNAs cloned from our previous study that is not analyzed yet (Anwar, 1999). Plasmid cDNAs cloned are prepared using the alkaline lysis method (Sambrook et al., 1989). The selected cDNA clones was sequenced by dideoxynucleotide chaintermination method (Sanger et al., 1977).

Analysis of clone genes by searching restriction enzyme sites using the amino acid and restriction enzyme sites software that have been developed by DCRG-team database, which provided information about analysis of DNA especially for searching of restriction enzyme sites, start and stop codon, amino acid sequence, including number of ATGC and amino acid.

Analysis of clone genes by searching homology with GeneBank database. The resulted cDNA sequences are then compared to the existing genes sequences in Genebank. First, we access to the NCBI (National Center for Biotechnology Information) website (http:// www.ncbi.nlm.nih.gov), and then select GeneBank database for searching similarity/homology sites for nucleotide sequence (BLAST program/BLAST web). Finally, follow instruction provided in the web electronic guide till resulted kinds of the genes.

In Research II, expression of the cloned genes have been studied by (a) using mRNA analysis by northern/slot blot hybridization method both on soybean and forage crop and (b) Escherichia coli’s exposed to Al toxic level. There are 4 steps for analysis of transcript level/mRNA analysis i.e: (a) Planting material, (b) total RNA Isolation, (c) probe preparation and (d) northern/slot blot hybridization.

Planting material was planted described by Anwar (1999). Total RNA was isolated from the root tips (± 5 mm) and/or leaf of soybean and forage crop treated and untreated with Al⁺³, using phenol/SDS method (Ausabel et al., 1987).

Northern/Slot Blot Hybridization. Total RNA (10-15 µg) samples was denatured with glyoxal and DMSO, and followed incubation in 65^oC for 15 minutes. Then,

the RNA was transferred to Hybond-N+ membranes (Amersham) by Slot-Hybridization (prior to use, the slot must be cleaned with 0.1 N NaOH and washed by steril water- DEPC treated). Probes was prepared from cDNA inserts isolated from agarose gels and labelled by non radioactive system (ECL-system). Hybridization was performed as described in Virca et al. (1990). The filter was washed twice with 2xSSC+0.4%SDS for 10 min at 42^oC, and twice with 2xSSC for 5 min at room temperature. Filter was stripped by immersion in warm (60^oC) 0.1% SDS and reprobed up to three times as described by Sambrook et al. (1989).

For Expression of clone genes by identifying tolerant-genes with its expression on Escherichia coli to Al toxic level was implemented by addition of Al toxic level on LB (Luria Bertani) culture (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl and 1% bactoagar. First of all, to set up assay for Al stress, E. coli and E. coli containing vector was cultured in LB with various Al treatment (0-500 ppm). Assay for Al-toxic level based on the reduction of E. coli’s growth on media at least 75% from control (without Al). Secondly, all of the clones was cultured at LB plate with addition of Al-toxic level based on previous study for 2 days. The clones that involved to Al tolerance was indicated by E. coli (contained the clones) growing well in the selected media.

RESULTS AND DISCUSSIONS

Analysis of Al-tolerant Genes

There are five clones that are already analyzed (cDNA isolation and sequencing, searching enzyme restriction sites and searching homology with GeneBank database) as shown on Table 1 and Figure 1-5. Based on the searching homology with the genebank database, the clones encode: (1) Catalase (gmali12, that function as an antioxidant), (2) Proliferating cell nuclear antigen like protein/PCNALP (gmali15, that involves as one of transcriptional regulator in the eucaryotic cell cycle), (3) Growth hormone (gmali22), (4) Amine oxidase (gmAO, genebank accession number AF313622), and (5) Aminoacyl peptidase (gmAP, genebank accession number AF091304).

Table 1. Characteristics of clones

No. Clones

Characteristics

• 1.    gmali12 Nucleotide length=252 bp; Amino acid=84 aa; Encode=Catalase (CAT)

• 2.    gmali15 Nucleotide length=254 bp; Amino acid=84 aa; Encode=PCNALP

  3.

  4.

  5.

  
  

gmali22 Nucleotide length=247 bp; Amino acid=65 aa; Encode= Growth Hormon (GH)

gmAO Nucleotide length=830 bp; Amino acid=250 aa; Encode= Amine Oxidase (AO)

gmAP Nucleotide length=657 bp; Amino acid=202 aa;

Encode=Aminoacyl Peptidase (AP)

Notes :

gmali12 = Glycine max aluminium induced number 12; gmali15 = Glycine max aluminium induced number 15

gmali22 = Glycine max aluminium induced number 22; gmAO = Glycine max Amine Oxidase

gmAP = Glycine max Aminoacyl Peptidas

1 tggataatgaatttccacatactgacactgagtattagggttaatatgtggaaattcatt 60

61 atccaaaaacaactcaaacttgattcccatgcaagttatatgtctttgtttgatagtttt 120

121 cttttttcttattttttttatgtgttatatttacagatgggtatatgttctttgtttcat 160

161 tttttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggcggccgctcgcgat 240

241 ctaaaactagtc 252

• Figure 1.    Nucleotide and the deduced amino acid sequence of

The gmali12 Clone. Amino acid residues are shown in the single-letter codes (coding : Catalase)

1tgattatccattgtgttgatgttcacatagtctcatacaagcataacagtttaatatggg 60

61 tgatcagttgttcccatagtctcatacaagcatatcagtttaatatgggtgatcactgtt 120

121 acgaacacaaagcaaacaatcctagatgtggacagatacacacaactgattatccattgt 180

181 gttgttgttccaacatatatttttatgacaaaaaaaaaaaaaaaagggcggccgctcgcg 240

241 atctagaactagtc 254

• Figure 2.    Nucleotide and the deduced amino acid sequence of The gmali15 Clone. Amino acid residues are shown in the single-letter codes (coding : PCNALP)

1 tgagcagaaaaggcatttatatatattacataacttatataagtgatttctatgtcttga 60 61 ttatttggtaccttacatcatccccaaatgcaggcaaaattttgaatagcctaaaaaagc 120 121 gtgatgcctatgtatgtcttaatcacatgaataatcatcatcccaaaaatgagaatcaaa 180 181 agaaggaaattagatgaaaagaaaacatgtaatgctacaaaatgaagttgttttacttta 240

241 CTGCTTC 247

• Figure 3.    Nucleotide and the deduced amino acid sequence of The gmali22 Clone. Amino acid residues are shown in the single-letter codes (coding : Growth Hormone)

1 gcaagaatggcacttcgttgaatggcagaagtggaattttcgtattggattcactcctag 60

61 ggagggtttggtaattcattcagtagcctatattgatggaagtcggggacgaagacctgt 120

121 ggcccatagattgagctttgttgagatggtagtcccgtatggagatcctaatgatcctca 180

181 ctataggaaaaatgcttttgacgctggggaagatggcctgggtaaaaatgctcattctct 240

241 caagaagggctgcgattgtttaggctatatcaaatactttgatgcgcacttcacaaactt 300

301 ctatggaggtgttgaaacaattgagaactgtgtttgtttgcatggagaagatcatggtat 360

361 tttatggaagcatcaagattggagaacaggattggctgaagttcgaaggtctagaaggct 420

421 gacagtgtctttatatgcactgtggctaactatgagtatggatttttctggcacttttat 480

481 caggatggaaaaatagaagcagagatcaagctcacaggaattctcagcttaggatcactt 540

541 caaccaggtgaactcgaaaatatggcacaaccattgcacctggactatatgcgcctgtcc 600

601 accaacattttttgttgctcgtatggacatggcagtaaattgcaagcctggtgaaacatt 660

661 taatcaggttgttgaaggtgaatgtcaaaattgaaaaaccagaaacaataatgttcctaa 720 721 caatgcattttatgctgaaaaaaaaactgcttaaatcaaaaatggaagcaatgccttgat 780

781 tgtgacctttatctgcccctccctgggattgtttggaaccctaggacttt 830

Figure 4. Nucleotide sequence of the gmAO (Glycine max Amine Oxidase) Clone.

1 atggcagctactcaggaagatgtgtactctgatcccggttctcctatgatgcggagaact 60 61 caagctgggacatacattattgccaggataaagaaggaaagtgatgaaggaagatatatt 120 121 tatactgaatggaaatggtgctacaccagaaggaaacattccattccttgatctgtttga 180 181 cataaatacaggtaaaaaaatggaacgaatctgggagagcgataaggagaagtattatga 240 241 gactgttgttgctctaatgtctgatcaagaagaaggggatttgtatttagataaactgaa 300 301 gaagatactgacttctaaagagtcaaaaactgaaaacacccaatactactttgttagctg 360 361 ggccagataaaaacatagttcaggttacaaatttccctcatccataccctcagcttgcat 420 421 ccattgcagaaagagatgatcagatatgaaagaaaagacggggttcaacttactgctaca 480 481 ttatacctaccaccaggttacaatccatcaacagatggccctttgccatgcctggtttgg 540 541 tcttacccaggagaatttaagaacaaagatgctgctggacaagttcgtggtctccaaatg 600

601 aatttgtaggctccacatcttcctgagtagctgccatcgcccgaaacttcattcgtt 657

Figure 5. Nucleotide sequence of the gmAP (Glycine max Aminoacyl Peptidase) Clone.

Expression of Al-tolerant Genes

Plants show spesific responses to many kinds of stress (biotic and abiotic) including aluminium stress. Genes response to Al stress will be reflected by increasing transcription (production mRNA) level of one or more genes. The molecular basis of these responses has not been completely worked out but there are clear examples of the expressions of many induced genes by aluminium stress.

Based on slot blot analysis (Figure 6), all of the genes are basic genes (appear at all of control media/media pH 6.0 without Al) , but its expression increased with Al stress (media pH 4.0 with Al stress). Clone gmali12 is coding catalase which involved as antioxidant. This result indicated that genes response to Al stress is similar with oxidation stress responses. This novel information is useful for genetic engineering. Similar result from the genes are expressed on Escherichia coli.

Clones (intencity)

gmali12

(%)

gmali15

(%)

gmali22

(%)

GmAO

(%)

GmAP

(%)

Glycine max cv.

Lumut a b c d

25 30 34 35

22 23 26 30

30 36 50 34

25 30 35 40

20 22 24 27

Glycine max cv.     Centrosema

Slamet          pubescens

a b c d          a b c d

25 29 30 33       25 27 31 26

25 25 31 35 I 20 28 30 34

32 30 35 48       18 20 22 25

20 20 22 35        0 0 0 0

22 20 25 27        42 42 47 48

Pennisetum purpureum a b c d

25 28 30 26

25 30 32 33

30 30 35 35

20 20 24 25

15 15 15 20

Notes : a = media pH 6.00 without Al; b = media pH 4.0 without Al

c = media pH 4.0 with 0.8 mM Al d = media pH 4.0 with 1.6 mM Al

Figure 6. Slot blot hybridization of clone genes on Glycine max, Centrocema pubescens and Pennisetum purpureum

Assay for Al-toxic level on Escherichia coli, based on the reduction of E. coli’s growth on media at least 75% from control (without Al). We found that 300 ppm Al is a critical assay for E. coli, and used it for study of expression of Al-tolerant genes on Escherichia coli. The result of research is listed on Table 2-3 and Figure 7.

CONCLUSIONS

From this research, we concluded that nucleotide and sequencing of Al-tolerant genes are coding: (1) Catalase (gmali12, that function as an antioxidant), (2)

Proliferating cell nuclear antigen like protein (gmali15, that involved as one of tanscriptional regulator in the eucaryotic cell cycle), (3) Growth hormone (gmali22), (4) Amine oxidase (gmAO, genebank accession number AF313622), and (5) Aminoacyl peptidase (gmAP, genebank accession number AF091304)

Expression of Al-tolerant genes (gmali12, gmali15, gmali22, gmAO, and gmAP) either on plant or Escherichia coli, indicated that all of the clones are genes response to Al-induction and involved in detoxification to Al-stress.

ACKNOWLEDMENT

We appreciated and thank’s to DGHE for sponsorship and for all of member’s of Forage Science Laboratory Faculty of Animal Agriculture Diponegoro University and PAU Bioteknologi IPB Bogor.

REFERENCES

Anwar, S. 1999. Cloning of Aluminium Induced Genes in Soybean [Glycine max(L.) Merryl]. Dissertation PhD Program, IPB Bogor.

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Table 3. Growth of E. coli in Luria Bertani agar media with 300 ppm Al for 24 h

Stress	Clones
	gmali12			gmali15			gmali22			gmAO			gmAP
	a	b	c	a	b	c	a	b	c	a	b	c	a	b	c
-Al:S	125	130	120	125	130	135	125	130	120	87	110	126	87	110	106
%	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
+Al:S	0	20	91	0	20	110	0	20	104	0	21	110	0	21	83
%	0	15	76	0	15	80	0	15	87	0	19	87	0	19	78

Notes : a = B+none = DH10B E.coli; b = B+VnR = DH10B E.coli withVector/plasmid insite; c = B+VR = DH10B E.coli with Vector Recombiant

Table 2. Optical density value (OD550) of growth of E. coli in Luria
Clone	Time of Stress (h)
	0	2	8	24	48
gmali12: B+none	0	0	0	0	0
B+VnR	0	0	0.115	0.247	0.225
B+VR	0	0.157	0.388	0.748	1.166
gmali15: B+none	0	0	0	0	0
B+VnR	0	0	0.115	0.247	0.225
B+VR	0	0.125	0.315	0.685	1.225
gmali22: B+none	0	0	0	0	0
B+VnR	0	0	0.115	0.247	0.225
B+VR	0	0.095	0.250	0.595	1.247
gmAO: B+none	0	0	0	0	0
B+VnR	0	0	0.088	0.282	0.200
B+VR	0	0.210	0.517	1.025	1.825
gmAP B+none	0	0	0	0	0
B+VnR	0	0	0.088	0.282	0.200
B+VR	0	0.144	0.414	0.661	0.934

Notes :

B+none = DH10B E.coli; B+VnR = DH10B E.coli withVector/plasmid insite;

B+VR = DH10B E.coli with Vector Recombiant

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