水稻和拟南芥中几丁质酶的分析

Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) February 2007, 34(2): 138-150 Received: 2006-02-17; Accepted: 2006-05-31 This work was supported by the 863 Program (No. 2002AA245041), the National Natural Science Foundation of China (No. 30260006), and the R rice; Arabidopsis; secreted characteristics; phylogenetics Chitinases (EC4), which are present in various organisms, catalyze the hydrolytic cleavage of the -1, 4-glycosidic bond in biopolymers N- acetyl- glucosamine and are largely found in chitin of ar- thropods 1 . However, their endogenous substrate has not been found in plants 2 . Chitinases use two differ- ent hydrolytic mechanisms: Substrate-assisted cataly- sis 3and acid catalysis 4 . Chitinases have some domains based on their amino acid sequences. Typical plant chitinases have an N-terminal signal region, a main structural domain (or a catalytic domain), and a C-terminal region that exists only in vacuolar chitinases. Some chitinases have one or more chitin-binding domains (CBD) fol- lowing the N-terminal signal region. In addition, there is a variable linking region between CBD and the catalytic domain. Each domain has distinct func- tions 5 . The classification system of plant chitinases has been revised several times 5 . Chitinases can generally be divided into two categories, endochitinases and exochitinases, with respect to their hydrolytic sites. Plant chitinases, however, are divided into seven classes, , on the basis of their structure, sub- Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana 139 strate specificity 6 , mechanisms of catalysis, and sen- sitivity to inhibitors. Class chitinase is further di- vided into two subclasses, Class a and Class b. Class a chitinases are acidic and have a C-terminal region with approximately six amino acids located in vacuole; Class b chitinases are basic and secreted in apoplast 5,7,8 and do not have a C-terminal region. Chitinases of Classes a, b, , , and be- long to Class PR (pathogenesis-related proteins)-3, Class belongs to PR-8, and Class to PR-11 9 . In healthy plants, some forms of chitinases, both vacuolar and apoplastic, are produced continu- ously 10,11 . Production of chitinases is regulated by a variety of stress factors, both biotic and abiotic, in- cluding infection, wound, drought, cold, ozone, heavy metals, excessive salinity, and UV light 1, 10, 12-14 . In addition, phytohormones, such as ethylene, jasmonic acid, salicylic acid, auxin, and cytokinin, induce chitinase expression 2 . It is well known that chitinases are usually in- volved in active or passive defense against patho- gens 11,12,15 . However, chitinases are also known to regulate growth and development by generating or degrading signal molecules 16-18and through pro- grammed cell death (PCD) 19,20 . Interestingly, apoplastic chitinases show antifreeze activity in monocotyledonous plants 21but not in dicotyledo- nous plants. Therefore, these enzymes have been studied by several researchers. Rice (Oryza sativa ssp. japonica) and Arabidopsis thaliana are two model plants representing monocotyledons and dicotyledons, respectively, whose genomes have been sequenced completely, but the structure and function of their genes and proteins are still not clear. It will be useful to first predict the structure and function of proteins using bio-software before confirming them through experiments. For example, the analysis of chitinases in rice and Arabidopsis has not been reported so far. In this article, an attempt was made to predict the structure, function, and classification of chitinases in both the plants using bio-software. 1 Materials and Methods 1. 1 Materials The sequences were downloaded from the web sites /pub/data/Eukaryotic_Projects/ o_sativa/ annotation_dbs/pseudomolecules/version_ 2.0/all_chrs/all.pep for rice and http:/www.arabi- / for Arabidopsis. On the basis of de- scription of the rice variety, Nipponbare and Arabidopsis thaliana genomes, the number of chitinases in the two plants was found to be 37 and 24, respectively. The chitinases studied in this article are putative chitinases, chitinases-like, and proteins with chitinase activity. To exactly predict the cluster chiti- nases from rice and Arabidopsis, the sequences of some known chitinases from other plants were downloaded from the NCBI protein database as stan- dards. They include: Class : AAA62421 (acidic) (Zea mays), AAB23692 (acidic) (Dioscorea japon- ica), AAC24807 (Solanum tuberosum), AAD04295 (extracellular) (Vitis vinifera); Class : AAB96340 (S. tuberosum), CAB99486 (Hordeum vulgare subsp. vulgare), AAX83262 (Triticum aestivum), AAC36359 (Capsicum annuum); Class : CAA77657 (basic) (Nicotiana tabacum), CAA77656 (acidic) (N. ta- bacum), CAA76203 (Lupinus albus), BAC65326 (V. vinifera), AAO47731 (acidic) (Rehmannia glutinosa), AAM08773 (Oryza sativa), AAN37391 (C. annuum); Class : CAA74930 (Arabidopsis thaliana), BAD77932 (Cryptomeria japonica), AAQ10093 (V. vinifera), ABA39179 (Linum usitatissimum), AAM95447 (V. vinifera), AAB01665 (Brassica napus ); Class : AAM18075 (Momordica charan- tia), CAA54373 (N. tabacum); Class : P11218 (Ur- tica dioica); and Class : AAP80801 (Gossypium hirsutum). 1. 2 Methods 1.2.1 Overall analysis of the chitinases 22-24Four bio-software were used for the analysis of 140 Journal of Genetics and Genomics 遗传学报 Vol.34 No.2 2007 secreted chitinases: SignalP3.0 (http:/www.cbs.dtu. dk/services/SignalP) for prediction of signal peptides in proteins, TMHMM2.0 (http:/www.cbs.dtu. dk/ services/TMHMM/) for prediction of transmembrane helices in proteins, TargetP1.1 (http:/www.cbs. dtu.dk/ services/TargetP) for identification of the subcellular location, and big-Pi Predictor (http:/ mendel.imp. univie.ac.at/sat/gpi/gpi_server.html) for analysis of GPI-anchor site. The alignment search was carried out using BLAST (basic local alignment search) from the web site, /BLAST/. The pro- gram used was blastp (Protein-protein BLAST), and the database nr was selected. Typical domains were analyzed using the on-line software from the web site, http:/smart.embl.de/smart/set_mode.cgi?GENOMIC =1. 1.2.2 Phylogenic tree of chitinases. The multiple protein sequences alignments were carried out using the program Clustal X from the web site, http:/bips.u-strasbg.fr/fr/Documentation/Clustal X/, and the parameters were auto-generated. The mo- lecular evolutionary genetic tree through the Neighbor-joining method was constructed using MEGA3.1 from the web site, http:/www.megasof /index.html. The phylogenic tree was tested using Bootstrap (1,000 replicates; seed = 64,238). Pairwise deletion was selected for gaps/missing data. 2 Results 2. 1 Characteristics of the chitinases On the basis of genome annotation, chitinases genes were found on all 11 chromosomes except on chromosome 7 of rice, whereas in Arabidopsis, the chitinases genes were found on all of the five chro- mosomes (Table 1). The length of the open reading frames (ORFs) ranged from 200 aa to 400 aa except for 9631.m02567, which had 127 aa. The average ORF length is 301 aa. The BLAST alignment search showed that most of the top matched proteins were chitinases, chitinase precursors, and chitinase-like proteins. In addition, the top-ho- mologous species of each ORF are different. 2.1.1 Signal peptides (SPs) and GPI-anchor in the chitinases SPs are responsible for targeting proteins to the endoplasmic reticulum (ER) for subsequent transport through the secretory pathway 40, 41 . However, not all chitinases had SPs at their N-termini. In rice and Arabidopsis genomes, two thirds or 25 and 16 chiti- nases, respectively, had SPs at the N-termini (Table 2). ORFs with SPs are listed in Table 1. The genes for secreted chitinases were dispersed on different chro- mosomes. The SP sequence had an average of 24.8 aa. The GPI anchor is usually found at the C-termini of the protein. The results obtained using Big-Pi Pre- dictor, however, indicated that all chitinases of rice or Arabidopsis do not have this structure. 2.1.2 Transmembrane domains in the chitinases When 37 rice and 24 Arabidopsis chitinases were analyzed with TMHMM2.0, it was found that 15 rice chitinases had one transmembrane domain, which was located within the first 40 N-terminal amino acids, and one, 9638.m03593 had two transmembrane heli- ces at 1234 and 139161. Only 4 Arabidopsis pro- teins had transmembrane helices within the N-terminal amino acid region. Of all the 61 proteins in both rice and Arabidopsis, only 9638.m03593 had a real transmembrane domain at 139161 site, because TMHMM, however, may not distinguish signal pep- tide from transmembrane domains in the region. 2.1.3 Subcellular location of the chitinases Target P1.1 analysis of 61 chitinases showed that these enzymes could be divided into five categories according to their subcellular locations, i.e., chloro- plast, mitochondrion, secretory pathway, other loca- tions, and “unknown”. Most of the enzymes exist in the secretory pathway. In rice, only three chitinases are located inside mitochondrion; one inside chloro- Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana 141 Table 1 Overall analysis of ORFs of rice and Arabidopsis ORF L Top-matched clone Top-matched protein S E-value Top-homologous species R 9629.m01787 290 AAC95376 Chitinase 399 3.00E-130 Cynodon dactylon - 9629.m04512 301 AAB47176 PRm 3 411 1.00E-113 Zea mays 25 9629.m06349 296 AAQ21404 Chitinase III 376 5.00E-103 Medicago truncatula 26 9630.m03783 271 NP_191010 ATEP3; chitinase 350 5.00E-103 Arabidopsis thaliana - 9631.m00319 256 CAA90970 Chitinase 323 3.00E-87 Vitis vinifera 27 9631.m02567 127 No No No No No No 9631.m02983 326 ABD47583 Chitinase 537 3.00E-151 Musa paradisiaca - 9632.m02919 479 CAA54374 Chitinase V 158 6.00E-37 Nicotiana tabacum 28 9632.m03968 229 AAT40051 Chitinase 325 1.00E-87 Zea diploperennis 29 9632.m03974 288 AAT40036 Chitinase 379 1.00E-103 Tripsacum dactyloides 29 9633.m00400 295 AAD54935 Chitinase precursor 335 2.00E-90 Petroselinum crispum - 9633.m01399 297 CAC87260 Putative xylanase inhibitor protein 298 2.00E-79 Triticum turgidum subsp. durum 30 9633.m01410 293 CAC87260 Putative xylanase inhibitor protein 268 1.00E-70 Triticum turgidum subsp. durum 30 9633.m01415 297 CAC87260 Putative xylanase inhibitor protein 248 2.00E-64 Triticum turgidum subsp. durum 30 9633.m03044 340 AAG53609 EndochitinaseI-antifreeze protein precursor 411 2.00E-113 Secale cereale 30 9633.m03045 299 AAR18735 Chitinase; BoCHI1 428 1.00E-118 Bambusa oldhamii - 9633.m03046 340 AAF04454 Chitinase 330 4.00E-89 Poa pratensis - 9634.m04963 320 ABD47583 Chitinase 481 2.00E-134 Musa paradisiaca - 9634.m04964 323 ABD47583 Chitinase 514 2.00E-144 Musa paradisiaca - 9636.m04120 315 CAC87260 Putative xylanase inhibitor protein 277 5.00E-73 Triticum turgidum subsp. durum 30 9636.m04169 316 NP_172076 ELP; chitinase 431 3.00E-119 Arabidopsis thaliana - 9637.m02807 326 NP_172076 ELP; chitinase 446 6.00E-124 Arabidopsis thaliana - 9638.m02367 307 ABD32310 Glycoside hydrolase, family 18 311 3.00E-83 Medicago truncatula - 9638.m02374 288 Q9SLP4 Chitinase 1 precursor 306 8.00E-82 Tulipa bakeri 31,32 9638.m03591 261 CAA55345 Chitinase 412 9.00E-114 Hordeum vulgare subsp. vulgare - 9638.m03593 296 CAA55345 Chitinase 349 7.00E-95 Hordeum vulgare subsp. vulgare - 9639.m04440 304 CAC87260 Putative xylanase inhibitor protein 279 1.00E-73 Triticum turgidum subsp. durum 30 9639.m04442 290 CAC87260 Putative xylanase inhibitor protein 321 2.00E-86 Triticum turgidum subsp. durum 30 9639.m04443 292 CAC87260 Putative xylanase inhibitor protein 282 1.00E-74 Triticum turgidum subsp. durum 30 9639.m04445 289 CAC87260 Putative xylanase inhibitor protein 299 8.00E-80 Triticum turgidum subsp. durum 30 9639.m04446 284 CAC87260 Putative xylanase inhibitor protein 299 1.00E-79 Triticum turgidum subsp. durum 30 142 Journal of Genetics and Genomics 遗传学报 Vol.34 No.2 2007 (Table 1 continued) ORF L Top-matched clone Top-matched protein S E-value Top-homologous species R 9639.m04447 280 CAC87260 Putative xylanase inhibitor protein 305 2.00E-81 Triticum turgidum subsp. durum 30 9639.m04448 304 CAC87260 Putative xylanase inhibitor protein 394 2.00E-108 Triticum turgidum subsp. durum 30 9639.m04449 300 CAC87260 Putative xylanase inhibitor protein 287 3.00E-76 Triticum turgidum subsp. durum 30 9639.m04450 301 CAC87260 Putative xylanase inhibitor protein 280 5.00E-74 Triticum turgidum subsp. durum 30 9639.m04451 302 CAC87260 Putative xylanase inhibitor protein 276 7.00E-73 Triticum turgidum subsp. durum 30 9640.m01849 336 CAC87260 Putative xylanase inhibitor protein 134 5.00E-30 Triticum turgidum subsp. durum 30 At1g02360.1 272 AAD54936 Chitinase precursor 416 5.00E-115 Petroselinum crispum - At1g05850.1 321 AAP80801 Chitinase precursor 507 3.00E-142 Gossypium hirsutum - At1g56680.1 280 CAA43708 Chitinase 226 6.00E-58 Brassica napus 33 At2g43570.1 277 AAK62047 Chitinase 4-like protein 436 4.00E-121 Brassica napus - At2g43580.1 265 CAA43708 Chitinase 437 2.00E-121 Brassica napus 33 At2g43590.1 264 CAA43708 Chitinase 468 1.00E-130 Brassica napus 33 At2g43600.1 273 CAA43708 Chitinase 210 4.00E-53 Brassica napus 33 At2g43610.1 281 CAA43708 Chitinase 291 2.00E-77 Brassica napus 33 At2g43620.1 283 CAA43708 Chitinase 287 3.00E-76 Brassica napus 33 At3g12500.1 322 AAF69777 Chitinase 616 4.00E-175 Arabis fecunda 34 At3g16920.1 333 AAQ56599 Chitinase-like protein 541 1.00E-152 Gossypium hirsutum 35 At3g47540.1 214 CAA43708 Chitinase 291 9.00E-78 Brassica napus 33 At3g54420.1 273 CAA40474 Chitinase 408 1.00E-112 Phaseolus vulgaris 36 At4g01700.1 280 CAA57773 Chitinase 428 1.00E-118 Arachis hypogaea 37 At4g19720.1 363 CAA55128 Chitinase/lysozyme 298 2.00E-79 Nicotiana tabacum 38 At4g19730.1 332 CAA54374 Chitinase 253 1.00E-65 Nicotiana tabacum 39 At4g19740.1 289 CAA55128 Chitinase/lysozyme 185 2.00E-45 Nicotiana tabacum 38 At4g19750.1 362 CAA55128 Chitinase/lysozyme 318 2.00E-85 Nicotiana tabacum 38 At4g19760.1 365 CAA54374 Chitinase 322 1.00E-86 Nicotiana tabacum 39 At4g19770.1 261 CAA54374 Chitinase 223 6.00E-57 Nicotiana tabacum 39 At4g19800.1 398 CAA55128 Chitinase/lysozyme 356 9.00E-97 Nicotiana tabacum 38 At4g19810.1 379 CAA55128 Chitinase/lysozyme 443 5.00E-123 Nicotiana tabacum 38 At4g19820.1 366 CAA55128 Chitinase/lysozyme 344 4.00E-93 Nicotiana tabacum 38 At5g24090.1 302 BAC11879 Acidic endochitinase 603 3.00E-171 Arabidopsis lyrata subsp. petraea - 9629.m01787 to 9640.m01849 are ORFs on Chromosome 1 to Chromosome 12 except for Chromosome 7 of rice in turn. At1g ?.1 to At5g ?.1 are ORFs on Chr.1 to Chr.5 of Arabidopsis in turn. L: length; S: score; R: reference; -: unpublished and direct submission. Fenghua Xu et al.: Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana 143 Table 2 Sequences of signal peptides in the ORFs of rice and Arabidopsis genomes ORF SPs length C-domain and H-domain N-domain Cluster 9629.m01787 31 MAKPTPAPRATPFLLAAVLSIVVVAASG AEA 9629.m04512 26 MAANKLKFSPLLALFLLAGIAVT SRA 9629.m06349* 20 MQMLIMVVVALAGLAAG ARA 9630.m03783 25 MARRLSLLAVVLAMVAAVSAST AAA 9631.m02983 21 MRALAVVAMVATAFLAAA VHA 9632.m02919 24 MADKNGLLLLSTIAAVTLSSL SLA 9632.m03974* 29 MANSPTPTMLAFLALGLALLLSATGQ ASA 9633.m01399 21 MASRRLAPLLVLLLSSSL AAG 9633.m01415 24 MALRRHAALLSLAVVLLFAGL AAA 9633.m03044 32 MSTPRAAASLAKKAALVALAVLAAALATA ARA 9633.m03046 32 MIAARAANLQVAMKALALAVLALAYAAAT ARA 9634.m04963 18 MRALALAVVAMAVVA VRG 9634.m04964 20 MRALAVVVVATAFAVVA VRG 9636.m04169 27 MRTSRAAAAAASLPLLLLVALLVA AEG 9637.m02807 28 MKRKTRNKIILTTLL