巴西橡胶Pto类抗病同源序列的克隆与系统发育重建（英文）

张影波 庞玉新 莫廷辉 曾立星

Abstract：The tomato Pto gene encodes a serine/threonine kinase （STK） whose molecular characterization provides valuable insights into the disease resistance mechanism of tomato and it is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study， a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein were used to amplify similar sequences in Hevea brasiliensis. A fragment of -550 bp was amplified， cloned and sequenced. The sequence analysis of several clones revealed twelve distinct sequences were highly similar to STKs. Based on their significant similarity with the tomato Pto protein （BLASTX E value <3e-53）， seven of them were classified as Pto resistance gene candidates （Pto-RGCs）. Multiple sequence alignment of the hevea Pto-RGC products revealed that these sequences contained several conserved subdomains in most STKs and also several conserved residues that are crucial for Pto function. Moreover， the phylogenetic analysis showed that the hevea Pto-RGCs were clustered with Pto suggesting a common evolutionary origin with this R gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in hevea.

Key words：disease resistance gene， Pto， serine/threonine kinase， Hevea brasiliensis

CLC number：Q943.2， S667.7

Document code：A

Article ID：1000-3142（2017）04-0485-12

摘 要：番茄Pto基因是一類可以编码丝氨酸/苏氨酸激酶（STK）序列的广谱抗性候选基因，其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础。在该研究中，一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列，扩增得到了一个约550 bp的基因片段，其随后被克隆并测序。序列分析发现，其中的7个抗病同源序列与Pto基因高度同源（BLASTX E value <3e-53），所以其被认为是Pto基因抗病同源序列（Pto-RGCs）。通过巴西橡胶的Pto-RGCs多序列比对表明，这些序列包含了多个STKs保守的次级结构域。此外，系统发育分析也表明，巴西橡胶的Pto-RGCs属于Pto基因同源的R基因。该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源。

关键词：抗病基因， Pto， 苏氨酸/丝氨酸激酶， 巴西橡胶

Natural rubber is not only an important industrial material but also an important strategic resource， the Para rubber tree （Hevea brasiliensis）， is the only commercial source at present， due to its high rubber content and quality （Backhaus， 1985）. Natural rubber production is currently threatened by a wide spectrum of pathogens including viruses， bacteria， fungi and nematodes. They reduce yield， affect the quality， debilitate or kill the host plant （Jacob et al，1989； Lespinasse et al， 2000）. Currently， the ascomycete fungus Microcyclus ulei （South American leaf blight， SALB） which was originated from South America， have become the most important disease of there， even destruction some rubber plantation there， more worse there is no evidence that there is an absolute effective chemical control exist（Alencar et al， 1975； Albuquerque et al， 1987）. Genetic resistance is the most suitable strategy to control these pathogens in the field and there are sources of resistance to both of them in wild clones germplasm. However， breeding for pathogen resistance in hevea is limi-ted by the long life cycle， trip-loidy and sterility in most commercial cultivars. Molecular biotechnology has the potential to overcome these constraints by transferring single or even multiple disease resistance （R） genes into the genome of susceptible hevea cultivars using either biolistics or agrobacterium-mediated transformation. Although transformation technologies have been developed for hevea in various laboratories around the world， no hevea R gene has been isolated to date （Huang et al， 2010）.

The tomato Pto gene is one of the best-characterized and most intensively studied R genes （Pedley & Martin， 2003）. Pto confers hypersensitive response-mediated resistance against strains of Pseudomonas syringae pv. tomato that express the avirulence proteins AvrPto or Avr-PtoB （Martin et al， 1993； Kim et al， 2002）. Overexpression of Pto in tomato under the control of the strong cauliflower mosaic virus （CaMV） 35S promoter has been shown to activate defense responses in the absence of pathogen inoculation. Pto-overexpressing plants show resistance not only to P. syringae pv tomato but also to Xanthomonas campestris pv. vesicatoria and to the fungal pathogen Cladosporium fulvum （Tang et al， 1999）. With the Bioinformatics analysis software， the Pto encodes a cytoplasmic serine/threonine protein kinase （STK） and also with several conserved subdo-mains， southern hybridization using the tomato Pto gene as probe revealed the presence of Pto-RGCs in many plant species such as Arabidopsis， bean， soybean， pea， rice， maize， barley， wheat and sugarcane （Martin et al， 1993）. Pto-type disease resistance gene analogues had successfully amplified from banana， potato， bean， grapevine etc. （Vleeshouwers et al， 2001； Vallad et al， 2001； Digaspero & Cipriani， 2003）， but since no molecular characterization of hevea Pto-RGCs has been published， the objectives of this study were to obtain Pto-RGCs from hevea using degenerate PCR and to determine the structure and phylogenetic relationships of the hevea Pto-RGCs.

1 Materials and Methods

1.1 Plant material and DNA extraction

The hevea wild germplasm‘XJ000072 was chosen for PCR amplification of Pto-RGC sequences because it shows resistance to a range of hevea pathogens， including the most destructive such as Microcyclus ulei and Oidium heveae . The genomic DNA was isolated using QIAGEN DNeasy Plant Minikit（QIAGEN Inc.， Valencia， CA）according to manufacturers instructions.

1.2 Degenerate PCR

A pair of degenerate primers designed by Vallad et al， （2001）， forward 5′-TNGGNSANGGNGKNTTYGG-3′and reverse 5′-ACNCCRAANGARTANACRTC-3′， was used to amplify the region between the subdomains I and IX of STKs. The degenerate PCR reaction was performed in a 50 μL reaction volume containing 300 μmol·L-1 of dNTPs， 4 μmol·L-1 of each degenerate primer forward and reverses， 1 U of Taq DNA polymerase （InvitrogenTM）， 1 × PCR buffer， 1.5 mmol·L-1 MgCl2 and approximately 200 ng of genomic DNA. PCR conditions were 95 ℃ for 3 min， followed by 35 cycles of 95 ℃ for 30 s， 45 ℃ for 30 s and 72 ℃ for 1 min； and an additional 10 min extension at 72 ℃ was included.

1.3 Cloning and sequencing

PCR products were visualized on a 1% agarose gel stained with ethidium bromide. A band of the expected size was excised from the gel and purified using the QIAquick Gel Extraction Kit（QIAGEN Inc.， Valencia， CA）according to manufacturers instructions. Purified PCR products were cloned into the pGEM-T easy plasmid vector （Invitrogen Corp.， Carlsbad， CA）. Plasmids were transferred by electroporation into Escherichia coli DH 5α competent cells. Bacteria were plated onto LB medium containing ampicillin， X-Gal and IPTG， and recombinant plasmids were chosen by blue/white selection （Sambrook & Russell， 2001）. Plasmid DNA was purified by the alkaline lysis method （Sambrook & Russell， 2001） and sequenced using the BigDye terminator sequencing kit version 3.1 （Applied Biosystems） according to manufacturers instructions. The sequencing products were separated with an ABI 3 730 automatic sequencer （Applied Biosystems） through the capillary separation service of the Chinese academy of agricultural science. Selected clones were sequenced in both orientations.

1.4 Sequence edition， similarity searches and multiple-sequence alignment

All sequences were assembled and edited using the programs SEQMAN and EDIT， respectively of the Lasergene software package version 4.03 （DNASTAR， Madison， WI， USA）. The degenerate primer sequences were removed from each sequenced clone so only the region between the end of subdomain I and the start of subdomain IX of STKs was considered for further analysis. Predicted amino acid sequences were generated using the translate tool of the EDIT program （Lasergene software）. Similarity searches were conducted with the BLASTX program （Altschul et al， 1997） through the National Center for Biotechnology Information （NCBI） GenBank database （http：//www.ncbi.nlm.nih.gov） using the default settings. Percent amino acid identity between predicted protein sequences was determined with the MEGALIGN program of the Lasergene software using the default settings. Determination of conserved amino acids in hevea Pto-RGC sequences was carried out with the programs Clustal X version 2.1 （Larkin et al， 2007） and WebLogo version 3.0 （Crooks et al， 2004） （http：//weblogo.berkeley.edu/） using the default settings.

1.5 Phylogenetic analysis

Phylogenetic trees were constructed by the neighbor-joining （NJ） method using the NJ algorithm implemented in the Molecular Evolutionary Genetics Analysis （MEGA） software version 5.10 with the Poisson correction. Bootstrapping （1 000 replicates） was used to evaluate the degree of support for a particular grouping pattern in the phylogenetic tree. Protein sequences belonging to twelve groups of characterized STKs from Arabidopsis thaliana （Hardie，1999）， a phosphoenolpyruvate carboxylase kinase （PEPck） （GenBank accession No. AF162660） from A. thaliana （Hartwell et al， 1999）， the tomato Pto protein （GenBank accession No. A49332） and Pto-RGCs from different plant species were retrieved from the GenBank for the phylogenetic tree constructions. The tomato Pto disease resistant protein was used as query in BLASTP （Altschul et al， 1997） searches to retrieve amino acid sequences of Pto-RGCs from the GenBank. Only the region between the end of subdomain I and the start of subdomain IX was considered for the phylogenetic tree constructions.

2 Results and Analysis

2.1 Identification of Pto resistance gene candidates in hevea

PCR amplification of hevea genomic DNA with a pair of degenerate primers previously used by Vallad et al （2001） generated an expected band of -550 bp. This band was cloned and a total of 50 clones were sequenced. The primer sequences were removed from each sequenced clone for further analysis. Of the 50 sequenced clones （STK-1 to STK-50）， 32 presented uninterrupted open reading frames （ORFs）， while the other eighteen sequences presented multiple stop codons in all reading frames， and as a result they were not further investigated. Similarity searches of the 32 hevea sequences using the BLASTX algorithm （Altschul et al， 1997） against the NCBI non-redundant database revealed significant similarity to known STKs （E value < 3e-53）， including the disease resistance protein Pto from tomato. A threshold value of 85% amino acid identity previously used by Vallad et al （2001） to classify Pto-RGC clones from bean into classes or groups was used in the present study， therefore hevea clones with greater than 85% amino acid identity were considered to be part of the same group.

A total of twelve distinct groups of STK-like sequences were identified， most of which contained redundant or highly similar clones （ > 97% amino acid identity）. Seven groups were designated as Pto resistance gene candidates （Pto-RGCs） based on their significant similarity with the tomato Pto disease resistance protein （E value < 3e-53）. The other five groups showed significant similarity to other types of STKs， which are described later in the text. Each group was designated by the name of a single clone representative of the group and used for further analysis. Percent amino acid identity between the predicted amino acid sequence of Pto-RGCs and the corresponding region of the Pto protein ranged from 64.3% （STK40） to 70.9% （STK25） （Table 1）， whereas amino acid identity among the Pto-RGCs ranged from 70.3% （STK 33 vs. STK 40） to 99.5% （STK3 vs. STK 12）（Table1）. BLASTX searches also revealed that no Pto-RGCs were highly similar （> 92% amino acid identity） to hevea sequences present in the GenBank database.

2.2 Isolation of other hevea serine/threonine kinase-like sequences

The degenerate primers used in this study were designed from the conserved subdomains I and IX of the STKs Pto， Fen and Pti1 of tomato， and MHK and APK1 of Arabidopsis （Vallad et al， 2001）. Therefore， these primers have the potential to isolate not only Pto-RGCs but also other types of plant STKs. In agreement with this observation five additional STK-like sequences from hevea were identified in BLASTX searches （Table 2）. Four of them （STK8， STK15， STK17 and STK 48） showed significant similarity to the receptor-like kinase （RLK） subfamily （E value < 2e-64）， whereas the remaining sequence STK43 showed a significant similarity to a receptor-like kinase （E value=3e-123） from Platanusx acerifolia.

2.3 Multiple sequence alignment and phylogenetic analysis

A multiple sequence alignment using the Clustal X program was performed with the predicted amino acid sequences of the seven hevea Pto-RGCs and the corresponding region of the tomato Pto protein （Fig. 1）. The alignment revealed that several features of the Pto protein are highly conserved in the hevea Pto-RGCs such as the STK subdomains internal to the degenerate primer sequences， the presence of the activation domain between subdomains Ⅶ and Ⅷ， and its internal P+1 loop site， which is responsible for the specific binding of AvrPto （Frederick et al， 1998）， and also several invariant amino acids distributed throughout the sequences. In addition， three of the four autophosphorylation sites （serine or threonine） in the activation domain of Pto （Sessa et al， 2000） are conserved in the corresponding region of all hevea Pto-RGCs. The alignment also showed that the entire hevea Pto-RGCs presented a two amino acid deletion （subdomain V） and a three amino acid insertion （subdomain VIa） with regard to the Pto protein. We found that the two amino acid deletion was also present in Pto-RGCs from other monocot species such as Solanum habrochaites （GenBank accession No. AAK11567）， S. berthaultii（GenBank accession No. AAK82689）， Phaseolus vulgaris（GenBank accession No. AAK52079）， Oryza sativa （GenBank accession No. XP476621） and Tritium aestivum （GenBank accession No. AAL51075）. This deletion was also present in Pto-RGCs from other dicot species such as Arabidopsis thaliana （GenBank accession No. NP197789） and Cucumis sativus （GenBank accession No. AAP57674） but absent in Phaseolus vulgaris （GenBank accession No. AF363819）. The extent and significance of this polymorphism in both monocot and dicot Pto-RGCs awaits further research. In the case of the three amino acid insertion， it is present in Pto-RGCs from other monocot and dicot species but absent in Pto-RGCs from the Solanaceae family （Vleeshouwers et al， 2001）.

In order to highlight the Pto autophosphorylation sites that are conserved in the hevea Pto-RGCs and other critical residues for Pto function located in the activation domain， a sequence Logo was generated with the hevea Pto-RGC products and it is shown in Fig 2. Of the three Pto autophosphorylation sites （Thr195， Ser198 and Thr199） conserved in the hevea Pto-RGCs， Ser198 is required for the AvrPto-Ptomediated hypersensitive response （Sessa et al， 2000） and it is present in the majority of hevea Pto-RGCs （Fig. 2）.

The phylogenetic analysis of Fig. 3 shows that the seven hevea STK-like sequences identified as Pto-RGCs formed a cluster with the tomato Pto protein， which is supported by a high bootstrap value （99%）. This result supported the designation of the seven hevea STK-like sequences as Pto-RGCs. Regarding the other hevea STK like sequences， four of them were related to receptor-like kinases as previously observed in the BLASTX results and the remaining sequence STK43 formed a highly supported （with bootstrap value 69%） cluster with a receptor-like kinasea（Fig. 3）. This phylogenetic tree also showed that the protein kinase region used for its construction contains sufficient sequence information to represent clusters defined by analysis with full sequence data of the kinase catalytic domain （Hardie，1999）. Furthermore， phylogenetic analysis of the hevea Pto-RGCs with Pto-RGCs from different plant species （Fig. 4） revealed that the hevea Pto-RGCs were more closely related to Pto-RGCs from other plant species than each other. Another interesting finding was that the clades where the hevea Pto-RGCs were grouped （Ⅰ， Ⅱ and Ⅲ） also contain Pto-RGCs from different species， suggesting that the origin of this type of sequence may have preceded the divergence of monocot and different plants.

3 Discussion

There is evidence that Pto-RGCs are highly conserved in many plant species. Southern hybridization using the tomato Pto gene as probe revealed the presence of Pto-RGCs in many plant species such as Arabidopsis， bean， soybean， pea， rice， maize， barley， wheat and sugarcane （Martin et al， 1993）. Recent studies report the cloning and characterization of Pto-RGCs from potato， bean and grapevine （Vleeshouwers et al， 2001； Vallad et al， 2001； DiGaspero & Cipriani， 2003）. Furthermore， other Pto-RGC sequences from different plant families have been deposited in the GenBank （http：// www.ncbi.nlm.nih.gov）， however these sequences have not been characterized. In this study， a set of Pto-RGC sequences and other STK-like sequences were identified from the hevea cultivar ‘XJ000072. These sequences were isolated by PCR using a pair of degenerate primers previously designed and used by Vallad et al （2001） to isolate Pto-RGCs from bean. In total， seven distinct Pto-RGC sequences and five other STK-like sequences were identified in the hevea genome. These sequences were isolated by PCR using a pair of degenerate primers previously designed and used by Vallad et al （2001） reported the identification of a lower number of Pto-RGCs （five distinct sequences sharing from 56.9% to 63.9% amino acid identity with Pto） and no further cloning of other STK-like sequences. In this study， the PCR annealing temperature was lower （45 ℃） in comparison to the previous study （60 ℃）， which may explain the broader diversity of STK-like sequences isolated in hevea. This low PCR annealing temperature could explain the isolation of the receptor-like sequence or the protein-like sequence， which was quite divergent from the rest of the hevea STK-like sequences isolated （Pto-RGCs and RLKs） （Fig. 3）. Overall， our data demonstrate that the degenerate primers used are capable of amplifying Pto-RGCs and other types of STK-like sequences from a monocot species.

The complete genome sequence of Arabidopsis （genomesize of 130 Mbp） revealed the presence of 15 Pto-RGCs （Arabidopsis Genome Initiative 2000）， while a draft of the rice genome sequence （genome size of 420 Mbp） revealed a similar number of Pto-RGCs with fourteen. These data indicate that the number of Pto-RGCs in these two plant genomes is conserved even though the rice genome is 290 Mbp larger than Arabidopsis， and also indicate that the number of Pto-RGCs in a plant genome is small in comparison to the NBS-LRR class of R genes， which has a large number of divergent genes in the Arabidopsis and rice genomes， with 149 and 480 genes， respectively （Meyers et al， 2003； Zhou et al， 2004）. The genome size of hevea is estimated to be -2 100 Mbp （Leitch et al， 1998）， assuming that the number of Pto-RGCs in a plant genome do not increase significantly according to the genome size， then it is possible that in hevea the number of Pto-RGCs could be similar to Arabidopsis or rice. Hence， it is tempting to speculate that the number of Pto-RGC sequences identified in this study represents a significant proportion of the total number of Pto-RGC sequences in the hevea genome.

All hevea Pto-RGC products displayed conserved serine/threonine kinase sub-domains （Hanks & Quinn 1991）， suggesting that the uncovered genes are likely to encode active kinases. Moreover， most residues of the Pto activation domain involved in pathogen recognition and HR induction （Pedley & Martin， 2003） are highly conserved in hevea Pto-RGCs suggesting that these residues might play a similar role in hevea. Indeed， the cloning of the full cDNA sequence and protein expression of these hevea Pto-RGCs will allow the possibility to answer some fundamental questions regarding for example， whether the Pto-RGCs encoded proteins are auto-phosphorylated in vitro and also whether substitution of tyrosine by aspartate in the corresponding site of Pto （Tyr207） will lead to a HR-like induction. Regarding the other STK-like sequences reported in this study，some of them are similar to receptor like kinases that are known to be involved in the response to pathogens， for example hevea STK8， STK15， STK17 and STK48 were related to the Constitutive triple response 1 （CTR1）group. CTR1， a member of this kinase group in Arabidopsis， also CTR1 is the immediate downstream target of ethylene receptors in A. thaliana， a putative Raf-like MAPK kinase kinase. Other interesting example is STK43， which was related to the leucine-rich repeat kinase1 （LRK1） group. LRK1 is an LRR-RLK isolated from Arabidopsis thaliana， and expression of the gene is induced by ABA， dehydration， high salt， and low temperature （Hong et al， 1997）， but the function of RPK1 was still unclear. The role of other hevea STK-like sequences in disease resistance remains to be determined.

Phylogenetic analyses of Pto and Pto-RGC sequences have suggested that these sequences form a unique group of kinases in plants （Vallad et al， 2001； Vleeshouwers et al，2001）. In agreement with this finding the hevea Pto-RGCs formed a highly supported group with the Pto disease resistance protein （Fig. 3） suggesting that these sequences share a common evolutionary origin with the tomato Pto protein and possibly a similar function in disease resistance. Furthermore， phylogenetic analysis of Pto-RGCs from different Solanum species has revealed that Pto orthologue genes are more similar than paralogues suggesting that the origin of Pto could predate the radiation of Solanum species （Vleeshouwers et al， 2001）. This ancient origin of Pto is further supported by the fact that both Pto and a Pto orthologue （LhirPto） are functional in Nicotiana benthamiana （Riely & Martin， 2001）. Additional evidence of this ancient origin is the presence of Pto-RGCs in other dicot species and also monocots that have been recently deposited in the GenBank （http：//www.ncbi.nlm. nih.gov）. The phylogenetic analysis of Fig. 4 supports and extends these previous observations since all hevea Pto-RGCs were grouped in clades that contained Pto-RGCs from both monocot and dicot species suggesting that the origin of this type of sequence might have predated the divergence of monocot and dicot plants which took place about （200 ± 40） million years ago （Wolfe et al， 1989）.

The tomato Pto protein is capable of recognizing at least two Avr proteins （AvrPto and AvrPtoB） from P. syringae （Kim et al， 2002）. Surprisingly， these two Avr proteins share limited sequence similarity. This dual recognition specificity has also been reported in R proteins of the NBS-LRR class， for example the Rpm1 protein from Arabidopsis confers resistance to P. syringae and recognizes two different avirulence proteins， AvrB and AvrRpm1 （Bisgrove et al， 1994）. Another interesting example is the Mi-1 gene from tomato， which confers resistance to a nematode and an aphid pest （Vos et al， 1998）. This dual （and perhaps even multiple） pathogen recognition specificity for a single R protein may prove to be common in R genes （Martin et al， 2003） and raises the possibility that Pto may confer resistance to pathogens other than bacteria. Whether the hevea Pto-RGCs are involved in conferring bacterial resistance as in tomato or are involved in conferring resistance to other types of pathogens will require functional analysis， which could be carried out with genetic complementation or loss of-function experiments. In the case of genetic complementation， the hevea Pto-RGCs could be used as probes to screen a hevea BIBAC library for the isolation of BIBAC clones containing Pto-RGCs. These Pto-RGC-BIBAC clones could be used to transform a hevea disease susceptible cultivar using Agrobacterium tumefaciens （Huang et al， 2010）. These experiments would lead to a collection of Pto-RGC-BIBAC transgenic lines ready to be used for disease resistance tests. The BIBAC technology coupled with Agrobacterium- mediated transformation not only promises to unravel the function of hevea RGCs but also the development of disease resistance in this crop. In the case of the loss-of-function strategy， the hevea Pto-RGC sequences could be used in RNA interference （RNAi） constructs （Waterhouse & Helliwell， 2003） in order to silence their corresponding targets in a resistant genotype. Those resistant plants that show disease symptoms after the infection with a particular type of pathogen would allow the identification of an R gene. The RNAi technology has been recently used to determine the function of genes involved in disease resistance in barley （Douchkov et al， 2005）. The hevea Pto-RGCs could also be used to produce molecular markers tightly linked to R-genes for genomic mapping and positional cloning. In this respect， several RGCs of the NBS-LRR class have been shown to be quite useful as molecular markers to assist the isolation of functional R genes through map-based positional cloning （McDowell et al， 1998； Zhao et al， 2005）.

The Pto gene is considered as a promising candidate for engineering broad-spectrum pathogen resistance in tomato since plants over-expressing this gene display resistance to both bacterial and fungal pathogens （Tang et al， 1999）. Moreover， expression of Pto mutants such as ptoThr204Asp or ptoTyr207Asp can constitutively activate a HR-like response in the absence of P. syringae （Rathjen et al， 1999）. Expression of these engineered Pto genes under the control of a defined inducible promoter has been considered as another promising strategy to protect crops against pathogens through the hypersensitive response （Rathjen et al， 1999）. The cloning of the full cDNA sequences of the hevea Pto-RGCs will permit assessing their potential to confer disease resistance using the strategies mentioned above.

In summary， this study has uncovered a set of hevea Pto-RGC sequences and provided the first insights about their amino acid sequence structure and evolution. The presence of several conserved amino acids in the hevea Pto-RGCs that are crucial for Pto function， and the fact that these sequences were phylogenetically closely related to Pto， make of them a valuable sequence resource for plant-pathogen interaction studies in hevea. The hevea Pto-RGCs could be used to generate not only a collection of BIBAC clones or RNAi constructs for functional analysis but also they might be useful as molecular markers for genetic mapping. The availability of these sequences will facilitate the cloning of their corresponding full gene sequences， which in turn will allow further genetic and biochemical characterization that may lead to the development of specific or even broad-spectrum pathogen resistance in hevea. Moreover， the other hevea STK-like sequences identified in this study may be used as a research platform for further studies in this crop.

In addition to their potential use for genetic improvement， RGCs also provide opportunities and tools to answer some fundamental questions about disease resistance genes， such as structure， R gene organization， distribution and evolution （Michelmore & Meyers 1998； Meyers et al， 2003）. The use of PCR with degenerate primers targeting the highly conserved subdomains of STK proteins has also proven to be an efficient method for isolating Pto resistance gene candidates （Pto-RGCs） in bean and grapevine （Vallad et al， 2001； DiGaspero & Cipriani， 2003）， indicating that this approach could be used to retrieve this type of gene from other plant species.

REFERENCES：

ALBUQRERQUE PEP， PEREIRA JCR， SANTOS AFD， 1987. Efficiency of impaction of fungicides by thermal fogging in leaves of Hevea spp. [J]. Revista Theobroma， 17：189-199.

ALENCAR H， PEIXOTO E， FERREIRA HIS， 1975. Controle do mal das-folhas （Microcyclus ulei） da seringueira na Bahia [C]. II. Relacao custo/beneficio da aplicacao aerea de fungicida， regiao.

ALTSCHUL SF， MADDEN TL， SCHAVER AA， et al， 1997. Gapped BLAST and PSI-BLAST：a new generation of protein database search programs [J]. Nucl Acids Re， 25：3389-3402.

BACKHAUS RA， 1985. Rubber formation in plants-a mini-review [J]. Israel J Bot， 34：283-293.

BISGROVE SR， SIMONICH MT， SMITH NM， et al， 1994. A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes [J]. Plant Cell， 6：927-933.

CROOKS GE， HON G， CHANDONIA JM， et al， 2004. WebLogo：A sequence logo generator [J]. Genome Res， 14：1188-1190.

DE ITUBERA， 1972-1973. Revista Theobroma， 5：12-20.

DIGASPERO G， CIPRIANI G， 2003. Nucleotide binding site/leucine-rich repeats， Pto-like and receptor-like kinases related to disease resistance in grapevine [J]. Mol Genet Genom， 269：612-623.

DOUCHKOV D， NOWARA D， ZIEROLD U， et al， 2005. A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells [J]. Mol Plant Micr Inter， 18：755-761

FREDERICK RD， THILMONY RL， SESSA G， et al， 1998. Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase [J]. Mol Cell， 2：241-245.

HANKS SK， QUINN AM， 1991. Protein kinase catalytic domain sequence database：identification of conserved features of primary structure and classification of family members [J]. Method Enzymol， 200：38-62.

HARDIE DG， 1999. Plant protein serine/threonine kinases：classifications and functions [J]. Ann Rev Plant Physiol Plant Mol Biol， 50：97-131.

HARTWELL J， GILL A， NIMMO GA， et al， 1999. Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression [J]. Plant J， 20：333-342.

HONGONG SW， JON JH， KWAK JM， et al， 1997. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid， dehydration， high salt and cold treatments in Arabidopsis thaliana [J]. Plant Physiol， 113， 1203-1212.

HUANG TD， LI Z， SUN AH， et al， 2010. Establishment of Agrobacterium tumefaciens -mediated anther calli transformation system in Hevea brasiliensis [J]. Acta Agron Sin 36（10）：1691-1697.

LARKIN MA， BLACKSHIELDS G， BROWN NP， et al， 2007. Clustal W and Clustal X version 2.0. Bioinformatics， 23：2947-2948.

LE SPINASSE D， RODIER-GOUD M， GRIVET L， et al， 2000. A saturated genetic linkage map of rubber tree （Hevea spp.） based on RFLP， AFLP， microsatellite， and isozyme markers [J]. TheoR Appl Genet 100：127-138.

LEITCH AR， LIM KY， LEITCH I J， et al， 1998. Molecular cytogenetic studies in rubber， Hevea brasiliensis Muell Arg. （Euphorbiaceae） [J]. Genome， 41：464-467.

JACOB JL， PREVOT JC， ROUSSEL D， et al， 1989. Hevea brasiliensis yield-limiting factors， latex physiological parameters， latex diagnosis and clonal typology [M]//DAUZAC J， JACOB JL， CHRESTIN H. Physiology of rubber tree latex. Boca Raton， FL：CRC Press Inc.：345-382.

KIM YJ， LIN NC， MARTIN GB， 2002. Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity [J]. Cell， 109：589-598.

KOICHIRO TAMURA， DANIEL PETERSON， NICHOLAS PETERSON， 2011. MEGA5：Molecular evolutionary genetics analysis using maximum likelihood， evolutionary distance， and maximum parsimony methods [J]. Mol Biol Evol， 28（10）：2731-2739.

MARTIN GB， BROMMONSCHENKEL SH， CHUNWONGSE J， et al， 1993. Map-based cloning of a protein kinase gene conferring disease resistance in tomato [J]. Science， 262：1432-1435

MCDOWELL JM， DHANDAYDHAM M， LONG TA， et al， 1998. Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis [J]. Plant Cell， 10：1861-1874.

MEYERS BC， KOZIK A， GRIEGO A， et al， 2003. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis [J]. Plant Cell， 15：809-834.

MICHELMORE RW， MEYERS BC， 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process [J]. Genome Res， 8：1113-1130.

NARAYANAN C， MYDIN KK， 2011. Breeding for disease resistance in Hevea spp. -status， potential threats， and possible strategies [C]//Proceedings of the 4th International Workshop on Genetics of Host-Parasite Interactions in Forestry.

PEDLEY KF， MARTIN GB， 2003. Molecular basis of Pto-mediated resistance to bacterial speck disease in tomato [J]. Ann Rev Phytopathol， 41：215-243.

RATHJEN JP， CHANG JH， STASKAWICZ BJ， et al， 1999. Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto [J]. EMBO J， 18：3232-3240.

RIELY BK， MARTIN GB， 2001. Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto [J]. Proc Natl Acad Sci USA， 98：2059-2064.

SANTY PE， ANDREW JK， BLONDY CC， et al， 2007. Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in banana [J]. Mol Genet Genom， 278：443-453.

SAMBROOK J， RUSSELL D， 2001. Molecular cloning：a laboratory manual [M]. 3rd ed. Cold Spring Harbor：Cold Spring Harbor Laboratory Press.

SESSA G， D ASCENEO M， MARTIN GB， 2000. Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto-Pto-mediated hypersensitive response [J]. EMBO J， 19：2257-2269.

TANG X， XIE M， KIM YJ， et al， 1999. Overexpression of Pto activates defense responses and confers broad resistance [J]. Plant Cell， 11：15-29.

VALLAD G， RICKIN M， VALLEJOS C， et al， 2001. Cloning and homology modelling of a Pto-like protein kinase family of common bean （Phaseolus vulgaris L.） [J]. Theor Appl Genet， 103：1046-1058.

VLEESHOUWERS VGAA， MARTENS A， VAN DOOIJEWEERT W， et al， 2001. Ancient diversification of the Pto kinase family preceded speciation in Solanum [J]. Mol Plant Micr Inter， 14：996-1005.

VOS P， SIMONS G， JESSE T， et al， 1998. The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids [J]. Nat Biotechnol， 16：1365-1369.

WATERHOUSE PM， HELLIWELL CA， 2003. Exploring plant genomes by RNA-induced gene silencing [J]. Nat Rev， 4：29-38.

WOLFE KH， GOUY M， YANG YW， et al， 1989. Date of the monocot-dicot divergence estimated from chloroplast DNA sequence data [J]. Proc Natl Acad Sci USA， 86：6201-6205.

ZHAO B， LIN X， POLAND J， et al， 2005. A maize resistance gene functions against bacterial streak disease in rice [J]. Proc Natl Acad Sci USA， 102：15383-15388.

ZHOU T， WANG T， CHEN JQ， et al， 2004. Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes [J]. Mol Gen Genom， 271：402-415.

uncertainties of the temperature sensitivity of soil respiration [J]. Acta Ecol Sin， 31（8）：2301-2311. [楊庆朋， 徐明， 刘洪升， 等， 2011. 土壤呼吸温度敏感性的影响因素和不确定性 [J]. 生态学报， 31（8）：2301-2311.]

ZHAI XY， CHEN ST， LIU YF， et al， 2014. Effects of warming and simulated acid rain on soil respiration in a winter wheat-soybean rotation field [J]. J Agro-Environ Sci， 33（12）：2381-2388. [翟晓燕， 陈书涛， 刘义凡， 等， 2014. 增温及模拟酸雨对冬小麦-大豆轮作农田土壤呼吸的影响 [J]. 农业环境科学学报， 33（12）：2381-2388.]

ZHANG DQ， SHI PL， ZHANG XZ， 2005. Some advance in the main factors controlling soil respiration [J]. Adv Earth Sci， 20（7）：778-785. [张东秋， 石培礼， 张宪洲. 2005. 土壤呼吸主要影响因素的研究进展 [J]. 地球科学进展， 20（7）：778-785.]

ZHANG JE， OUYANG Y， LING DJ， 2007. Impacts of simulated acid rain on cation leaching from the Latosol in south China [J]. Chemosphere， 67（11）：2131-2137.

ZHANG PH， SHEN XY， XU XL， et al， 2005. Effects of simulated acid rain on the microbes and enzyme activity in soil of Atractylodes macrocephala [J]. Chin J Soil Sci， 36（2）：227-229. [张萍华， 申秀英， 许晓路， 等， 2005. 酸雨对白术土壤微生物及酶活性的影响 [J]. 土壤通报， 36（2）：227-229.]

ZHANG Y， WANG LX， CHEN ST， et al， 2011. Effects of simulated acid rain on soil respiration in a northern subtropical secondary forest [J]. China Environ Sci， 31（9）：1541-1547. [张勇， 王连喜， 陈书涛， 等， 2011. 模拟酸雨对北亚热带天然次生林土壤呼吸的影响 [J]. 中国环境科学， 31（9）：1541-1547.]

ZHOU LY， ZHOU XH， ZHANG BC， et al， 2014. Different responses of soil respiration and its components to nitrogen addition among biomes：a meta-analysis [J]. Glob Change Biol， 20（7）：2332-2343.

ZHOU MK，LI ZW，LI ZG， et al， 2012. Effects of simulated acid rain on photosynthesis of leaves and organic acid secreted by roots of Cycas debaoensis [J]. J Southern Agr， 43（5）：587-591. [周明昆， 李正文， 李志刚， 等， 2012. 模拟酸雨对德保苏铁叶片光合作用及根系分泌有机酸的影响 [J]. 南方农业学报， 43（5）：587-591.]

ZHOU P， LIU GB， XUE S， 2009. Review of soil respiration and the impact factors on grassland ecosystem [J]. Acta Prat Turae Sin， 18（2）：184-193. [周萍， 劉国彬， 薛萐， 2009. 草地生态系统土壤呼吸及其影响因素研究进展 [J]. 草业学报， 18（2）：184-193.]

ZHOU YP， 2013. Effects of simulated experimental warming and acid rain on CO2 and CH4 fluxes from winter wheat and soybean farmland [D]. Nanjing：Nanjing University of Information Science and Technology：1-68. [周迎平， 2013. 模拟增温和酸雨对冬小麦-大豆旱作农田CO2和CH4通量的影响 [D]. 南京：南京信息工程大学：1-68.]