花生簇葉病菌質體 RNA 聚合酵素 Sigma factor 基因之選殖與分析(II)
Cloning and analysis of sigma factor gene of phytoplasma associated
with peanut witches' broom (II)
計畫類別：█個別型計畫 □整合型計畫
計畫編號：NSC89-2313-B002-0034
執行期間：88 年 8 月 1 日 89 年 7 月 31 日
計畫主持人：林長平 台大植病系
執行單位：台大植病系
中 華 民 國 89 年 11 月 16 日
一、中文摘要
在進行花生簇葉病病原菌質體 RNA 聚合酵素 Sigma Factor 基因選殖時，以E. coli 、 B. subtilis 、 Lactococcus lactis 、 Staphylococcus aureus 、 Clostridium acetobutylicum 及 Mycoplasma genitalium 等細菌之 rpoD、sigA 基因進行比對，根
據其高保守性區域設計出一組 PCR 引子，以花生簇葉病病原菌質體 DNA 為模板 進行 PCR 反應，得到一 600 bp 之 PCR 產物，並以此作為核酸探針，篩選花生簇 葉病病原菌質體之基因庫，得到一含 1.8 kb 嵌入片段之選殖株重組質體 pPSF3。 對 pPSF3 之嵌入片段進行核甘酸序列分析可得到一個完整的 ORF (open reading frame)，且在轉譯起始密碼 AUG 上游有互補於植物菌質體 16S rRNA 之 3’端核 酸之序列，即 Shine-Dalgarno 序列之存在，此為可能之核糖體結合位置(ribosomal binding site)。此 ORF 之核甘酸序列經轉譯為胺基酸序列後，發現其中有啟動子 -10 及-35 辨識結合區域、中心酵素結合區域、及 rpoD box，且基因大小及基因 結構均與其他細菌的 sigma factor 基因相似，故以此推斷其為 sigma factor 基因。 此基因之密碼利用性上並無以 UGA 作為 tryptophan 遺傳密碼之現象，且有與 Mollicutes 綱細菌在基因體特性上相同之"AT pressure"現象。而在南方氏雜合反 應的結果，則可發現在花生簇葉病植物菌質體中，可能有三個近似於其他真細菌 基因之 rpoD 之 sigma factor 基因。
Abstr act
In an effort to elucidate the molecular biology of phytoplasmas, a gene encoding the primary sigma factor gene of the phytoplasma associated with peanut witches’ broom (PNWB) was cloned and sequnced using PCR-based strategy. A 600-bp
rpoD gene fragment was PCR-amplified and used as a nucleic acid probe for
screening λ ZAP II genomic library of PNWB phytoplasma. The complete nucleotide sequence of the 1.8 kb insert DNA of pPSF3, one of the in vivo excised
recombinants carrying a complete open reading frame (ORF), was determined. The ORF encodes a polypeptide contains -10 recognition and binding region, -35 recognition and binding region, core binding region and rpoD box. The gene organization and the nucleotide sequence in conserved region of the ORF are similar to other rpoD homologous sigma factor genes. According to the nucleic acid and
amino acid sequence analyses and the results of Northern hybridization analyses, this gene was identified as a putative sigma factor gene. Based on the results of Southern hybridization analyses with the 600-bp fragment as a probe, it is suggested that three
rpoD homologous sigma factor genes may exist in PNWB-phytoplasma.
二、緣由與目的
Introduction
Phytoplasmas, which are a group of cell-wall-free prokaryotes, are associated with a variety of plant diseases in several hundreds of plant species (McCoy et. al., 1989; Seemuller et. al., 1998). Understanding the physiology, biochemistry and molecular biology of phytoplasmas is limited because of their resistance to culturing under axenic conditions (Lee and Davis, 1986), while procedures available for
purification of phytoplasmas from disease plants are tedious and unsatisfactory (Clark et. al., 1989; Jiang and Chen, 1987) due to the inevitable contamination of plant component. However, molecular methods had been applied to improve the
methodology of the studies of phytoplasmas since late 1980s, especially phytoplasma DNA can be purified and isolated from plant DNA by CsCl equilibrium buoyant density centrifugation based on its extreme AT bias of codon usage (Kirkpatrick et. al., 1987; Sears et. al., 1989). Consequently, the molecular biology of phytoplasmas can now be studied, and it seems to be the best way to approach and characterize these mysterious prokaryotes. Several kinds of genes are now identified and sequenced. Among these, the sequences of 16S rRNA gene and some ribosomal protein genes of several different phytoplasmas had accumulated, so that a phylogeny-based
classification system of phytoplasmas has established on the sequences of 16S rRNA genes (Gundersen et. al., 1994; Seemuller et. al., 1998). Few other genes have also identified, including elongation factor G and Tu (Berg and Seemuller, 1999) and an antigenic protein gene (Yu et. al., 1998).
Sigma factor is a key subunit of prokaryotic RNA polymerase, which is an essential enzyme in prokaryotic genes transcription. To initiate transcription, DNA dependent RNA polymerase has to associate with sigma factor to form a holoenzyme. Sigma factors could recognize and bind to two consensus sequences, -10 and -35 sequences, in the promoter region to facilitate the initiation of gene transcription. Most bacterial species synthesize several different sigma factors which direct the RNA polymerase holoenzyme to distinct classes of promoters with a different consensus sequence (Wosten, 1998). Based on sequence similarity, bacterial sigma factors could be roughly divided into two broad classes: primary sigma factors ó70 and other ó70-related sigma factors, which initiate transcription of the housekeeping genes; and ó54 and other alternative sigma factors, which recognize promoters with different sequences that are responsible for growth under different physiological conditions Helmann and Chamberlin, 1988). Four regions with high conservation are observed
after aligning the amino acid sequences of the known ó70 genes (sigA and rpoD)
(Lonetto et al., 1992). Each of the four regions can be further divided into several subregions, some are suggested to be functional domains, including core binding, recognition and binding of -10 and –35 consensus sequences and rpoD box (Daniels et al., 1990; Waldburger et al., 1990; Gardella et al., 1989). The initiation of transcription is the most prominent step in gene regulation. Thus the study of sigma factor could increase our understandings to how phytoplasmas transcribe their gene and how the expression of the genes be regulated. In this study, we describe a gene encoding the primary sigma factor gene of the phytoplasma associated with peanut witches’ broom (PNWB) was cloned and sequnced using PCR-based strategy.
三、結果與討論
Results and Discussion
Amplification of a rpoD gene fr agment of PNWB phytoplasma
A specific 0.6 kb DNA fragment was amplified using DNA templates from PNWB-phytoplasma-infected periwinkle plants and phytoplasma DNA purified with CsCl-bisbenzimide density-gradient centrifugation method (lanes 2 and 3, Fig. 1), but no PCR product was obtained using DNA templates prepared from healthy plants. It reveals that the DNA fragment was amplified from PNWB phytoplasma. The amplified DNA fragment was then sequenced and aligned to other prokaryotic rpoD
genes. This DNA fragment contains nucleotide sequences identical to highly conserved regions , including rpoD box and –10 and –35 sequences recognition and binding region, of the rpoD genes compared. More than 80% of nucleotide
sequence identity with other rpoD genes also shows that it should be a gene fragment
of phytoplasma rpoD gene.
Cloning of phytoplasma rpoD gene
After screening recombinant phages of the PNWB phytoplasma genomic library, several positive clones PSF1, PSF2, PSF3, PSF6, PSF7 and PSF8 were obtained. Each was confirmed by Southern hybridization analyses using the same nucleic acid probe for library screening. All of these 6 clones shared the same positive signal and all contained a 1.8kp insert. Clones PSF1, PSF2 and PSF3 containing plasmids pPSF1, pPSF2 and pPSF3 were selected for further sequence analyses. Both end of the inserts were first partially sequenced. There were no differences among the
nucleotide sequences of these three clones. Consequently, only pPSF3 was selected for entire nucleotide sequences determination.
Sequence analyses of the rpoD gene
The complete nucleotide sequence of the 1768 bp insert DNA of pPSF3 was determined (Fig. 2). Based on the universal codon usage, one putative ORF was identified. The ORF contains 1377 bp (nt 138-1514), Starting from the ATG initiation codon and stopping at translation termination codon TAA. The coding region of the gene encodes a polypeptide of 458 a. a. with a calculated MW of 54.8 kD. A putative Shine-Dalgano sequence AGGAGG, which is complementary to the 3’ end sequence of 16S rRNA of phytoplasmas, was found, but no consensus –10 and –35 sequences were identified.
The base composition of the gene is 41.06 mol% of A, 12.12 mol% of C, 16.65 mol% of G, and 30.17 mol% of T (Table 1). A low G+C content (28.77%) of the gene, a preferential use of A- and T- rich codons, and the high frequency of the use of A or T residues at the 5’-end (1st base) (64.85%) and 3’-end (3rd base) (72.05 %) of codons were observed, which are similar to the features of other phytoplasmas (Kirkpatrick et al., 1987; Lim and Sears, 1991b; Sears et al., 1989). Fifty AAA lysine codons appeared in the gene, which made it the most frequent codon. In the
rpoD gene, 12 tryptophan residues are encoded by UGG. The result agrees with that
of a previous study on the ribosomal protein genes for Oenothera phytoplasma (Lim
and Sears, 1991b). In the class Mollicutes, mycoplasmas and spiroplasmas both
utilize UGG and UGA triplets are tryptophan codons (Inamine et al., 1990; Lim and Sears, 1992), but Acholeplasma laidlawii utilizes UGG only (Tanka et al., 1989; Lim
and Sears, 1992). Sequence similar ities
The nucleotide sequence and the deduced amino acid sequence of the ORF were compared with other rpoD genes. This ORF shares 51% of nucleotide sequence
identity and 62% of amino acid sequence identity with sigA gene of Mycoplasma genitalium. The result of multiple alignments of amino acid sequences was shown
as Fig. 3. It reveals the gene organization and the nucleotide sequence in conserved region of this ORF is similar to other rpoD sigma factor genes. The ORF also
contains -10 recognition and binding region, -35 recognition and binding region, core binding region and rpoD box. All of those regions are functional domain and responsible to most biological function of sigma factor, thus it suggests that this ORF encodes primary sigma factor of PNWB-phytoplasma.
deduced amino acid sequence using the method developed by Garnier et al. (Garniel et al., 1978) (Fig. 4). Most of the rpoD genes could be divided into four regions.
Region 1 is mainly composed of â sheet, while other regions are mainly composed of á helix. The putative secondary structure of phytoplasma rpoD gene is similar to
other rpoD genes, except á helical region 1.
Souther n hybr idization analysis
Hybridization patterns were shown as Fig. 5. Total DNA of PNWB
phytoplasma– infected periwinkle was digested with XbaI (lane 1), BamHI (lane 2)
and EcoRI (lane 3) and hybridized with the 600 bp-amplified rpoD gene fragment.
Three bands of hybridization signals were detected in each lane. No positive signal was observed for the DNA prepared from healthy plants in the Southern hybridization under both low and high stringencies. In many eubacteria, more than one sigma factor responsible for the transcription of house keeping genes were identified. These results suggest that three rpoD homologous sigma factor genes may exist in
PNWB-phytoplasma.
Nor ther n hybr idization analysis
The result of Northern hybridization is shown in Fig. 6. Total RNA purified from healthy periwinkle (lane 1) and from diseased periwinkle affected with PNWB-phytoplasma (lane 2) were applied in the study. One 3.7 kb positive signal was detected for the RNA prepared from infected periwinkle, while no signal was observed for the prepared from healthy plant. It reveals that this rpoD gene does
expressed in PNWB phytoplasma.
Acknowledgements
This work was supported in part by a grant NSC-89-2313-B002-034 from the National Science Council, Republic of China.
四、計畫成果自評 本計畫於已於本年度將可順利完成此一計畫。 本計畫可充分利用貴會補助之重要儀器設備，發揮其研究效益，而對基礎科 學之研究上，則可因研究之成果而能對植物菌質體提供重要之分子生物學上之資 訊，尤以在植物菌質體基因之研究上有所貢獻，而且或能因對其基因之瞭解，進 而對植物菌質體與寄主植物或媒介昆蟲細胞間之辨識關係能有進一步之認知，如
此對整體防治策略之擬定當有極大助益。
參與之工作人員能熟悉植物病原菌質體之抗原及核酸分離技術，亦能熟 悉基因選殖及各種核酸分析之技術 (如 hybridization, sequencing, PCR ...等)，此 外亦能參與實驗之設計，瓶頸之克服，並學習將基礎研究之成果延用於實際植物 病理研究之相關工作上。
五、參考文獻
References
Berg, M., Seemuller, E., 1999. Chromosome organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma. Gene 266, 103-109.
Chen, M.F., Lin, C.P., 1997. DNA probes and PCR primers for the detection of a phytoplasma associated with peanut witches’-broom. Europ. J. Plant Pathol. 103, 137-145.
Clark, M.F., Morton, A., Buss, S.L., 1989. Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies against primula yellows MLO-associated antigens. Ann. Appl. Biol. 114, 111-124. Daniels, D., Zuber, P., Losick, R., 1990. Two amino acids in an RNA polymerase
sigma factor involved in the recognition of adjacent base pairs in the -10 region of a cognate promoter. Proc. Natl. Acad. Sci. USA 87, 8075-8079.
Garnier, J., Osguthorpe, D.J., Robson, R., 1978. Analysis of the accuracy and implications of a simple method for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97-120.
Gundersen, D.E., Lee, I.-M., Rehner, S.A., Davis, R.E., Kingsbury, D.T., 1994. Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification. J. Bacteriol. 176, 5244-5254.
Helmann, J.D., Chamberlin, M.J., 1988. Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57, 839-872.
Higgins, D.G., Sharp, P.M., 1988. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237-244.
Inamine, J.M., Ho, K.C., Loechel, S., Hu, P.J., 1990. Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae,
Mycoplasma genitalium, and Mycoplasma gallisepticum. J. Bacteriol. 172,
504-506.
Jiang, Y.P., Chen, T.A., 1987. Purification of mycoplasma-like organisms from lettuce with aster yellows disease. Phytopathology 77, 949-953.
Kirkpatrick, B.C., Stenger, D.C., Morris, T. J., Purcell, A. H., 1987. Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organisms. Science 238, 197-200.
Ko, H.C., Lin, C.P., 1994. Development and Application of cloned DNA probe for a mycoplasmalike organism associated with sweetpotato witches’ broom.
Phytopathology 84, 468-473.
Lee, I.M., Davis, R.E., 1986. Prospects for in vitro culture of plant pathogenic mycoplasmalike organisms. Ann. Rev. Phytopathol. 24, 339-354.
Lim, P.O., Sears, B.B., 1991. DNA sequence of the ribosomal protein genes rpl2
and rpsl9 from a plant-pathogenic mycoplasma-like organism. FEMS Microbiol.
Lett. 84, 71-74.
Lim, P.O., Sears, B.B., 1992. Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two
ribosomal protein gene sequences. J. Bacteriol. 174, 2606-2611. Lonetto, M., Gribskov, M., Gross, C.A., 1992. The σ70 family: sequence
conservation and evolutionary relationships. J. Bacteriol. 174, 3843-3849. McCoy, R.E., Caudwell, A., Chang, C.J., Chen, T.A., Chiyowski, L.N., Cousin, M.T.,
Dale, J.L., de Leeuw, G.T.N., Golino, D.A., Hackett, K.J., Kirkpatrick, B.C., Marwitz, R., Petzold, H., Sinha, R.C. Sugiura, M., Whitcomb, R.F., Yang, I.L., Zhu, B.M., Seemuller, E., 1989. Plant diseases associated with mycoplasma-like organisms, and Mycoplasmas of plants and Arthropods. R. F. Whitcomb and J. G. Tully, eds. Academic Press, San Diego, CA.
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.
Sears, S.E., Lim, P.O., Holland, N., Kirkpatrick, B.C., Klomparens, K.L., 1989. Isolation and characterization of DNA from a mycoplasmalike organism. Mol. Plant-Microbe Interact. 2, 175-180.
Seemuller, E., Marcone, C., Lauer, U., Goschl, M., 1998. Current status of molecular classification of the phytoplasmas. J. Plant Pathol. 80, 2-26. Shen, W.C., Lin, C.P., 1993. Production of monoclonal antibodies against a
mycoplasmaloke organism associated with sweetpotato witches’ broom. Phytopathology 83, 671-675.
Shine, J., Dalgarno, L., 1974. The 3‘-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites . Proc. Natl. Acad. Sci. USA 71, 1342-1346.
Tanaka, R., Andachi, Y., Muto, A., 1989. Nucleotide sequence of truptophan tRNA gene onAcholeplasma laidlawii. Nucleic Acids Res. 17, 5842.
Waldburger, C., Gardella, T., Wong, R., Susskind, M.M., 1990. Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition. J. Mol. Biol. 215, 267-276.
Wosten, M.M.S.M., 1998. Eubacterial sigma-factor. FEMS Microbiol. Rev. 22, 127-150.
Yeh, K.W., Juang, R.H., Su, J.C., 1991. A rapid and efficient method for RNA isolation from plants with high carbonhydrate content. Focus 13, 102-103. Yu, Y.L., Yeh, K.W., Lin, C.P., 1998. An antigenic protein gene of a phytoplasma
