Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

Agricultural Sciences in China

2009, 8(6): 664-670

June 2009

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis LIU Jian-xin1, 2, YU Chun-ming1, TANG Shou-wei1, ZHU Ai-guo1, WANG Yan-zhou1, ZHU Si-yuan1, MA Xiong-feng1 and XIONG He-ping1 1

Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 420105, P.R.China

2

Cotton and Fibre Research Institute of Xiaoshan, Zhejiang Academy of Agricultural Sciences, Hangzhou 311202, P.R.China

Abstract To isolate the cDNA partial sequence of key enzyme gene GalAT for pectin biosynthesis in ramie [Boehmeria nivea (L.) Gaud], and thus to understand the expression of GalAT gene in different tissues of ramie, degenerate primer was designed according to GalAT conserved sequence in other species reported, and the cDNA sequence of GalAT gene from ramie variety Zhongzhu 1 was cloned by RT-PCR method based on the degenerate primer. The cDNA revealed a 986-bp in length which encoded 328 amino acids. The cDNA sequence and putative amino acid sequence of GalAT shared high identity with previously reported Arabidopsis thaliana GAUT4 (GalAT) as 77 and 83%, respectively. Molecular evolution analysis showed that the putative amino acid sequence and Arabidopsis thaliana GAUT4 gathered to a same group. Real-time quantitative PCR analysis showed that GalAT mRNA accumulated most abundantly in root, and GalAT transcripts in all kinds of ramie tissues in turn revealed as follows: root > leaf > bast > or xylem. Key words: clone, GalAT, pectin, ramie, real-time quantitative PCR analysis

INTRODUCTION Ramie [Boehmeria nivea (L.) Gaud], belongs to Urticaceae boehmeriae, and is the perennial persistent root herbaceous plant (Lu 1992) and the industrial crops with Chinese characteristics. It is reputed as “China grass”, and its bast fiber is a kind of fine plant textile material. Pectin is one of primary colloidal matters of ramie phloem (Len et al. 2003), next to hemicellulose in colloidal substance. Pectin stuck all kinds of colloidal substance together, which makes it difficult to unfix the colloidal substance separately, so removing pectins is the key in ramie degumming (Tao 1992). Deguming, a process that get fibre from phloem

tissue, is the basal and key working procedure in ramie processing for spinning (Akin et al. 2001). The aim of ramie degumming is to remove all colloidal substance, and its key step is to remove pectin and hemicellulose (Sun 1977; Zhang and Wang 2002; Peng 1995). Degumming influences directly essential-fiber quality, yield and the further processing. At present, staple chemical degumming method in ramie industrial production have many shortcomings, such as poor bast fibre strength, lower essential fiber productivity, deteriorated spinnability of fibre, prolonged process flow; increasing deguming costs; increasing alkali content of boiling-epurate exhausted liquid, polluting surroundings badly, and consuming energy source heavily, all of which restrict severely development of bast fiber spin industry (Zeng et al. 1997).

This paper is translated from its Chinese version in Scientia Agricultura Sinica. LIU Jian-xin, Ph D, E-mail: [email protected]; Correspondence XIONG He-ping, Professor, Tel: +86-731-8998500, E-mail: [email protected]

© 2009, CAAS. All rights reserved. Published byElsevier Ltd. doi:10.1016/S1671-2927(08)60262-X

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

GalAT (α-1,4-galacturonosyltransferase) is a key enzyme for pectin biosynthesis (Kazumasa et al. 2002), which function is to catalyze 1,4-α-D-galacturonosyl to galacturonic acid polysaccaride chain’s termination to form homogalacturonan (Scheller et al. 1999), the primary pectic polysaccharide of the plant primary wall (Doong et al. 1995). Up to now, researches on pectin have focused on structural analysis, synthetic process and enzymolysis, etc. There has little study on related enzyme gene in pectin synthetic process from the molecular level, and most researches on GalAT gene focus on Arabidopsis thaliana GAUT gene family (Sterling et al. 2006). In the current study, we not only cloned the GalAT gene from ramie, and submitted it to GenBank under accession no. EU131377, but also analyzed the expression distributing of GalAT gene in ramie. The findings have practical significance to regulate and control synthetic quantity of pectin by the molecular biology method in the further research.

MATERIALS AND METHODS Materials Zhongzhu 1 came from the National Ramie Field Genebank in Changsha, the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences.

Cloning and analysis of GalAT gene Primer synthesize According to the high conserved domain of GalAT gene mRNA sequence of another plant reported such as Arabidopsis thaliana GAUT gene family (Sterling et al. 2006), by GenBank online searching, Clustalw multisequencing blast, and Primer Premier 5 software, the degenerate primer sequence was designed as: GalAT-F:5´-MTDGGDAAATACARYATHTGGAG-3´ GalAT-R:5´-CATYCCRWAWGCCCANCCACA-3´ Extraction and inverse transcription of total RNA Acquired the materials from the vigourous phloem tissue and disposed it according to the instruction of the ConcertTM Plant RNA Reagent (Invitrogen Corporation, Carlsbad, California, USA) and RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas,Vilnius, Lithuania).

66 5

RT-PCR multiplication Owing to using the degenerate primer, it was necessary to optimize the PCR reaction conditions. In the test, 12 variant annealing temperatures were set to proceed gradient PCR (Biometra T Gradient PCR, Gottingen, Germany) in order to obtain the suitable PCR parameter. PCR program was as follows: 95°C for 3 min, 95°C for 1 min, Tm 50 s (Tm was 12 temperature values acquired by PCR apparatus distributing automatically), 72°C for 90 s, 35 cycles, and finally 72°C for 15 min. PCR product were run electrophoresis in 1.1% agarose gel, and detected with the gel imaging system (Alphameger 2200TM, Alpha Innotech Corporation, San Leandro, California, USA). PCR product purification through cutting gel and reclaiming Concrete operation program was refered to TIANgel Midi Purification Kit instruction (Tiangen, Beijing, China). Ligation and transformation of PCR product Based on the character that Taq polymerase appends a “A” to PCR production tail, goal segment was linked to the Pmd 18-T vector (TaKaRa, Dalian, China), and transformed by competent cell DH5α. Screen of positive clone Prospective monoclone was screened by using four methods, i.e., Amp antibiotic screening, blue-white spotting screening, bacterial colonies PCR detection, and plastid PCR detection. DNA sequencing and analysis Prospective positive clone was send to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai) for bidirectional sequencing.

GalAT gene expression Primer design ACTIN gene primers (ACTIN gene is the housekeeping gene, as the inner reference in the experiment): ACTIN-F: 5´-GTTGAACCCTAAGGCTAACAGAG-3´ ACTIN-R: 5´-GGAATCCAGCACGATACCAG-3´ GALAT gene primers (located in high conservative region): GalAT-F: 5´-CATGGTTGAATGCGAGTTACA-3´ GalAT-R: 5´-GAAGGTGGTTGAGGATGGATA-3´ Total RNA extraction and inverse transcription were as mentioned above, and the materials came from root, leaf, xylem, and bast of the vigourous plant.

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.

66 6

Feasibility test of ACTIN gene acting as the inner reference RNA amount of four tissues was measured using extinction value method (Unicam Helios Alpha Beta, Unicam Limited, Cambridge, England), equivalent RNA was proceeded to the inverse transcription, and then equivalent cDNA was taken as templat to proceed PCR response. Finally, brightness was detected through agarose electrophoresis, while the product amount was detected accurately by measuring the extinction value. Preparation of standard curve Common PCR multiplication of GalAT gene and the housekeeping gene ACTIN was proceeded separately. The PCR product was identified by agarose gel electrophoresis first, then it was diluted in 10-fold gradient, and 103-107 concentration (5 points) was got as the standard sample to prepare standard curve. PCR program was as follows: ACTIN: 94°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles, finally 72°C for 5 min; GalAT: 94°C for 3 min, 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, 35 cycles, finally 72°C for 5 min (PE 9600 Thermal Cycler, Perkin Elmer, Inland Southern California, USA). Real-time quantitative PCR analysis In order to ensure the reaction conditions same, GalAT and ACTIN mixture (except templat and primer) were confected, then shared. The PCR reaction course was monitored by SYBR Green I fluorescence dye. Quantitative PCR cycle program was as follows: 95°C for 15 min, 95°C for 15 s, 60°C for 1 min, 40 cycles (ABI7300 Real-Time PCR System, Applied Biosystems, USA). Operation of apparatus After finishing above procedure and sampling, the 96-orificium plate with the sample was placed to ABI 7300 Real-Time PCR System to make a response, and to detect the standard curve and quality. Concentration range of the sampler of the standard curve should attach 1.00.

LIU Jian-xin et al.

cessive amino acids (Fig.1), and submited it to GenBank under the accession no. EU131377.

Bioinformatics analysis Nucleotide-nucleotide blast analysis showed GalAT cDNA sequence had very high homologue to many sequences of data bank (http://www.ncbi.nlm.nih.gov/ blast/), but those sequences which function were confirmed for coding galacturonosyltransferase in blast analysis all belong to A. thaliana galacturonosyltransferase gene family. Besides, the cDNA sequence homologue to A. thaliana GAUT4 (galacturonosyltransferase 4, NM_124152.2) was the highest to 77%. By comparing the deduced amino acid sequences (BLAST) with the protein data bank, the result showed that GalAT deduced amino acid sequence had very high homologue to protein amino acid sequences of many other species in the data bank. And the sequences with determinate function were 14 genes of A. thaliana GalAT gene family, and the homologue to A. thaliana GAUT4 was the highest to 83%, thus confirming that the translation mode was correct. By analyzing conserved domain of GalAT putative amino acid sequence, we found the putative amino acid

RESULTS cDNA cloning and analysis of GalAT gene Multiplication and clone of GalAT gene fragment We isolated a 986-bp cDNA fragment, encoding 328 suc-

Fig. 1 The mRNA sequence and putative amino acid sequence of GalAT gene.

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

sequence contained a conserved domain (Fig.2), glycosyl-transferase family 8. This family included enzymes that transfered sugar residues to donor molecules, and members were involved in lipopolysaccharide biosynthesis and glycogen synthesis. The family contained lipopolysaccharide galactosy-ltransferase, lipopolysaccharide glucosyltransferase 1, and glycogenin glucosyltransferase (Marchler-Bauer et al. 2007). After BLAST analysis on GalAT mRNA sequence and putative protein amino acid sequence, some amino acid sequences with GalAT function were chosen to produce phylogenesis tree (http://www.ncbi.nlm.nih. gov/blast/blast.cgi). As the research data on GalAT gene in data bank is limited, and all sequences which function have been determined for GalAT focus on the pattern plant A. thaliana whose genome has been sequenced completely, therefore only A. thaliana GalAT family gene coud be compared. Putative amino

1

75

66 7

acid analysis by Kimura method showed that ramie GalAT and A. thaliana GAUT4 can be gathered to the same group, homology relationship between them exceeded obviously other A. thaliana GalAT family gene (Fig.3). Through above bioinformatics analysis, it can be determined that the sequence cloned was GalAT gene sequence.

Expression of GalAT gene Total amount of every RNA after sampling was uniform in PCR experiment, but on account of different RNA extraction efficiency, inverse transcription efficiency and sample error, every cDNA amount in sampling was different. In order to proofread these mistakes, we performed proofreading by the housekeeping gene ACTIN whose expression amount is

150

225

300

328 aa

Glyco-transf-8

Fig. 2 Conserved domain of GalAT gene.

GAUT7/LGT7 (Galacturonosyltransferase 7); [Arabidopsis thaliana] GAUT5/LGT5 (Galacturonosyltransferase 5); [Arabidopsis thaliana] GAUT6 (Galacturonosyltransferase 6); [Arabidopsis thaliana] GAUT14 (Galacturonosyltransferase 14); [Arabidopsis thaliana] GAUT13 (Galacturonosyltransferase 13); [Arabidopsis thaliana] GAUT12/RX8/LGT6 (GALACTURO...; [Arabidopsis thaliana] GAUT15 (Galacturonosyltra...; [Arabidopsis thaliana] GAUT10/LGT4 (Galacturonosyltransferase 10); [Arabidopsis thaliana] QUA1 (QUASIMODO1); polygalacturonate + alpha alacturonosyltransferase; [Arabidopsis thaliana] GAUT9 (Galacturonosyltransferase 9); [Arabidopsis thaliana] GAUT1 (Galacturonosyltransferase 1); [Arabidopsis thaliana] GAUT2/LGT2 (Galacturonosyltransferase 2); [Arabidopsis thaliana] GAUT3 (Galacturonosyltransferase 3); [Arabidopsis thaliana] Boehmeria nivea Galacturonosyltransferase (GalAT) GAUT4 (Galacturonosyltransferase 4); [Arabidopsis thaliana]

Fig. 3 Molecular evolution analysis of GalAT putative amino acid sequence.

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.

66 8

LIU Jian-xin et al.

constant approximately in different samples as the inner reference. Through repeating quantitative PCR test three time, average copy number of GalAT genes in four different tissues was obtained, then divided by average copy number of ACTIN genes to obtain the relative amount of GalAT gene, finally, compared expression amount between different tissues. The ratio result of GalAT and ACTIN can be see in Table. Con-

sidering that values of bast and xylem were very closed, a significance testing was did. The result that Sig. (2tailed) value was 0.411 (less than 0.05) showed that there was not obvious difference between of them. Therefore, a conclusion was as follows: GalAT can express in all tissues tested of Zhongzhu 1, expression amount in turn was as follows: root >leaf > bast > or xylem.

Table GalAT gene copy number after proofreading Tissue

Mean of ACTIN

Mean of GalAT

GalAT/ACTIN

Root Leaf Bast Xylem

312 300 1 050 000 7 060 000 1 020 000

4 358 597 862 861 2 454 063 328 385

13.9564 0.8218 0.3476 0.3219

DISCUSSION Pectin biosynthes is believed to occur in the lumen of the Golgi endomembrane system (Zhang and Staehelin 1992). In vivo labeling and immunocytochemical analysis also have suggested that pectin is synthesized in the Golgi apparatus (Northcote and Pickett-Heaps 1966; Harris and Northcote 1971; Staehelin and Moore 1995). Immunocytochemical studies, using antibodies that recognize the pectic polysaccharides HGA and rhamnogalacturonan I suggest, that these pectic polysaccharides are synthesized within different compartments of the Golgi apparatus (Zhang and Staehelin 1992), and transported from the Golgi in vesicles that migrate to, and fuse with, the plasma membrane. The polysaccharides are then released into the extracellular space and incorporated into the wall (Northcote and PickettHeaps 1966). The pectin biosynthetic enzyme, α-1,4galacturonosyltransferase, is a key enzyme for pectin biosynthesis. The acivity of α-1,4-galacturonosyltransferase (GalAT) has been established in numerous plants which is membrane bind type and is localized in Golgi (Sterling et al. 2001). Hassid and the colleagues, using cell-free preparations and radiolabeled UDP-GalA, were able to synthesize homogalacturonan (Villemez et al. 1966, 1965; Lin et al.1966), and the enzyme responsible for this activity has been named PGAGalATransferase, that is, GalAT. GalAT’s function is to catalyze the transfer of GalA from UDP-GalA to a growing homogalacturonan chain. Homogalacturonan

Relative percent ratio (%) 90.35 5.32 2.25 2.08

is primary pectic polysaccharide of the plant primary wall (Doong et al. 1995). Despite this early attempt to identify PGA-GalATransferase activity in cell-free extracts, however, there has been little further published work concerning this enzyme (Bolwell et al. 1985; Cumming and Brett 1986). Although Ga lAT is a key enz yme of pectin biosynthesis, research on this enzyme gene is very little. According to the information obtained from several big gene retrieval data bases, all sequences whose function have been determined focus on pattern plant A. thaliana which complete genome has been sequenced. Excess pectin content influences severely on ramie deguming, moreover staple deguming methods utilized at present severely pollute environment severely and deteriorate processing quality of ramie fibre. Thus, the research on the key enzyme gene GalAT for pectin biosynthesis is important to improve ramie processing quality for spining, energy conservation, environmental protection. Moreover, although the problem on cellular localization of pectin synthesize has been solved, the report on tissue localization in plant has not yet seen. Therefore, the study on GalAT gene expression is also important to fundamental researches. Bioinformatic analysis confirmed that the sequence obtained was GalAT gene, but too little GalAT genic information in the data bank resulted in less number of sample sequences proceeding to molecular evolution analysis, suggesting that GalAT gene cloned from ramie is keep ahead to other species. The test adopted ACTIN gene as the inner reference.

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

ACTIN is one of most conservative and most abundant proteins in eukarya cell, and has extremely important physiological function as the main constituent of cytoskeletal and structure (Pollard and Cooper 1986; Coluccio and Bretscher 1989). ACTIN gene product is indispensable to maintain cellular basic structure and metabolic function, and often passes for the housekeeping gene whose expression number is constant to some extent in all kinds of cells. In order to further determine whether expression of ACTIN gene in ramie is constant approximately, semiquantitative test to ACTIN gene express efficiency in different tissues was conducted. PCR product contents were measured through agarose gel electrophoresis and extinction value method, the result showed that expression amount of ACTIN gene in ramie different tissues were uniform approximately, so ACTIN gene can be used as an inner confer of quantitative PCR analysis. Although at present there have many methods to detect gene expression in mRNA level, for instance, Northern blot, RT-PCR, etc., all of them have some limitation. Northern blot can quantify relative exactly, but its sensibility is bad, and it requires much sample; RT-PCR has good sensitivity and specificity, but it can not quantify exactly, thus only suits to semiquantitative analysis. However, real-time quantitative PCR avoids effectively the shortcomings of the above methods (Zhang et al. 2006). Fluorescence quantitative real-time PCR has many advantages, for example, wide detection range (~108), high sensibility (< 5 copies), high precision (CV < 2%), no PCR post-treatment procedure to avoid cross contamination, high rate of yield, and the ability of multiple detection (Klein 2002). SYBR Green I adopted in the test is a kind of un-probe quantitative PCR method, which major advantage is simple, convenient, operating easily, and less cost. But joining between SYBR Green I and double chain DNA is nonspecific, the nonspecific product or primer dimer generated in PCR reaction has some influence on the quantitative result (Chen et al. 2004). Through optimizing reaction conditions in the trial, analysis of peak shape of dissolve curve and the agarose gel electrophoresis detection confirmed that specificity in PCR reaction is good; correlation index analysis confirmed that fluorescence value in PCR reaction can reflect realtime change of multiplication product scale well, thus

66 9

revealing the result of quantitative analysis in the test is reliable. The 986-bp cDNA sequence of GalAT gene has been cloned in the test, expression amount of GalAT gene in various tissues in turn as follows: root > leaf > bast > or xylem. These findings have practical significance to inhibit pectin synthesize by antisense RNA or RNAi technology in our later experiments.

Acknowledgements This work was supported by the National 863 Program of China (2001AA241211).

References Bolwell G P, Dalessandro G, Northcote D H. 1985. Decrease of polygalacturonic acid synthase during xylem differentiation in sycamore. Phytochemistry, 24, 699-702. Chen Y J, Hu C J, Zhao M Q. 2004. Construction of real-time quantitative polymerase chain reaction platform with SYBR Green I. Journal of Medicine & Pharmacy, 21, 997-999. (in Chinese) Coluccio L M, Bretscher A. 1989. Reassociation of microvillar core proteins: Making a microvillar core in vitro. Journal of Cell Biology, 108, 495-502. Cumming C M, Brett C T. 1986. A galacturonyltransferase involved in pectin biosynthesis. In: Vian B, Reis D, Goldberg R, eds, Cell Walls’86. Proceedings of the 4th Cell Wall Meeting. Université Pierre et Marie Curie-Ecole Normale Supérieure, Paris. pp. 360-363. Akin D E, Foulk J A, Dodd R B, McAlister D D. 2001. Enzymeretting of flax and characterization of processed fibres. Journal of Biotechnology, 89, 193-203. Doong R L, Liljebjelke K, Fralish G, Kumar A, Mohnen D. 1995. Cell-free synthesis of pectin (identification and partial ch ar acteriz ation of polygalactu ron ate 4 -[alp h a]galacturonosyltransferase and its products from membrane preparations of tobacco cell-suspension cultures). Plant Physiology, 109, 141-152. Harris P J, Northcote D H. 1971. Polysaccharide formation in plant golgi bodies. Biochimica et Biophysica Acta, 237, 5664. Kazumasa A, Takeshi I, Tatsuya T, Toshio A, Sumihiro H. 2002. Su ccessive gycosylt ran sfer act ivity and enzymat ic characterization of pectic polygalactu ron ate 4-αgalacturonosyltransferase solubilized from pollen tubes of petunia axillaris using pyridylaminated oligogalacturonates as substrates. Plant Physiology, 130, 374-379. Klein D. 2002. Quantification using real-time PCR technology. applications and limitations. Trende in Molecular Medicine,

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.

67 0

LIU Jian-xin et al.

8, 257. Len J, Xiao A P, Nie Q L. 2003. Evaluation studies on ramie fibre quality (quality property analyse). China Fiber Inspection, 6, 31-34. (in Chinese) Lin T Y, Elbein A D, Su J C. 1966. Substrate specificity in pectin syn thesis. B ioch emical and Bio physical Research Communications, 22, 650-657. Lu H Y. 1992. Bast Fibre Plants Cultivation in China. China Agriculture Press, Beijing. (in Chinese) Marchler-Bauer A, Anderson J B, Derbyshire M K, deWeese-

galacturonosyltransferase. Proceedings of the National Academy of Sciences of the USA, 103, 5236-5241. Sterling J D, Quigley H F, Orellana A, Mohnen D. 2001. The catalytic site of the pectin biosynthetic enzyme α-1,4galacturonosyltransferase is located in the lumen of the Golgi. Plant Physiology, 127, 360-371. Sun Q X. 1977. The ramie degummed with biological method. Scientia Agricultura Sinica, (Suppl.), 130-134. (in Chinese) Tao T. 1992. The ramie degummed with biological method. Industrial Microbiology, 22, 35-36. (in Chinese)

Scott C, Gonzales N R, Gwadz M, Hao L, He S, Hurwitz D

Villemez C L, Lin T Y, Hassid W Z. 1965. Biosynthesis of the

I, Jackson J D, et al. 2007. CDD: a conserved domain database

polygalacturonic acid chain of pectin by a particulate enzyme

for interactive domain family analysis. Nucleic Acids

preparation from Phaseolus aureus seedlings. Proceedings

Research, 35, 237-240.

of the National Academy of Sciences of the USA, 54, 1626-

Northcote D H, Pickett-Heaps J D. 1966. A function of the

1632.

Golgi apparatus in polysaccharide synthesis and transport

Villemez C L, Swanson A L, Hassid W Z.1966. Properties of a

in the root-cap cells of wheat. Biochemical Journal, 98, 159-

polygalacturonic acid-synthesizing enzyme system from

167.

Phaseolus aureus seedlings. Archives of Biochemistry and

Peng Y D. 1995. Study on character of ramie deguming strain EJ3. China’s Fiber Crops, 17, 32-35. (in Chinese) Pollard T D, Cooper J A. 1986. ACTIN and ACTIN-binding proteins: A critical evaluation of mechanisms and functions. Annual Review of Biochemistry, 55, 987-1035.

Biophysics, 116, 446-452. Zeng Q F, Wang C M, Zhao X F. 1997. Enzyme processing and water pollution control on ramie cloth. Journal of Wuhan Textile College, 10, 66-70. (in Chinese) Zhang G F, Staehelin L A. 1992. Functional compartmentation

Scheller H V, Doong R L, Ridley B L, Mohnen D. 1999. Pectin

of the golgi apparatus of plant cells: immunocytochemical

biosynthesis: a solubilized galacturonosyltransferase from

analysis of high-pressure frozen-and freeze-substituted

tobacco catalyzes the transfer of galacturonic acid from UDP-

sycamore maple suspension culture cells. Plant Physiology,

gala ct u ron ic acid o n t o t h e n on -red u cin g en d of

99, 1070-1083.

homogalacturonan. Planta, 207, 512-517.

Zhang L J, Chen J F, Ke Y, Robert E M, Wen G J. 2006. Expression

Staehelin L A, Moore I. 1995. The plant Golgi apparatus:

of PEDF mRNA in non-small cell lung cancer by real-time

structure, functional organization and trafficking mechanisms.

PCR method. Chinese Journal of Lung Cancer, 9, 177-181.

Annual Review of Plant Physiology and Plant Molecular Biology, 46, 261-288. Sterling J D, Atmodjo M A, Inwood S E, Kumar Kolli V S, Quigle H F, Hahn M G, Mohnen D. 2006. Functional identification

(in Chinese) Zhang Y X, Wang Z Y. 2002. Enzymes for bio-degumming/retting and bio-pulping EJ3. China’s Fiber Crops, 24, 14-17. (in Chinese)

of an Arabidopsis pectin biosynthetic homogalaturonan (Edited by ZHANG Yi-min)

© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.