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Abstract With reported pathogenic genes of Botrytis cinerea Pers. and unknown proteins related to pathogencity as experimental materials， the pathogenic genes were studied by bioinformatics methods， and these unknown proteins were analyzed with bioinformatics tools and softwares， so as to lay a theoretical basis for the control of tomato grey mould at molecular level in future. The results showed that through the alignment analysis by DNAMAN software， three pairs of unknown proteins with higher comprehensive matching degrees were obtained， and these proteins were predicted to be coded by hrp gene cluster of type III secretion system； through Bioedit and ProtParam， the amino acid composition， size， molecular weight， isoelectric point and hydrophobicity of the each of the six unknown proteins were obtained； and corresponding secondary structures were obtained using DNAMAN. The results of this study could guide deep research of such genes.
Key words Tomato； Grey mould； Pathogenic gene； Bioinformatics
Received： October 8， 2017 Accepted： December 13， 2017
Supported by Science and Technology Pilot Project of Xinjiang Agricultural Reclamation Academy of Science （50YYD201610）.
Qiang ZHANG （1977-）， male， P. R. China， associate researcher， master， devoted to research about botany.
*Corresponding author. Email： [email protected]
Tomato （Lycopersicon esculentum Mill.） is an important economic crop in Xinjiang， which is native to tropical zone， widely planted in various areas of China. There are various tomato varieties in China， and as one of good fruits in summer， it is popular due to its fragrant and sweet taste. However， with the expansion of the planting area of tomato and the continuous improvement of commercialization and production scale， tomatorelated diseases also increase. Among the various diseases， tomato grey mould commonly occurs at home and abroad， and becomes one of the three diseases harming tomato production in the world， thereby seriously affecting tomato yield and quality. Tomato grey mould is caused by Botrytis cinerea Pers. in Deuteromycotina[1-2]， which not only jeopardizes tomato， but also other Solanaceae crops[3].
Before 2009， studies at home and abroad mainly focused on detection of Aac， biological characteristics， law of disease development， chemical control and screening of diseaseresistant varieties， and few studies had been done on the pathogenicity of Botrytis cinerea Pers. at molecular level. Later， studies on pathogensis appeared， including the construction of genomic library and mutant library， pathogenic mechanism and pathogenic genes. In 2009， Ren et al.[4] constructed an Aac mutant libray by triparental mating， and indicated that T3SS is a key factor for pathogenicity. Wei et al.[5] found and cloned a congener of tvrR gene in the whole genome of Aac， and the results showed that the change in pathogenicity of the mutant is caused by the change of its growing ability. In 2010， Wang et al.[6] demonstrated that the product coded by hrp gene of Aac affects the pathogenicity of pathogenic bacteria on host plant. In the same year， Zhao et al.[7] reported that the mutation of luxR genes would cause the decrease of pathogenicity of the pathogen and the ability of producing signal molecules is weakened. In 2011， WMJ et al.[8] verified that the deficit of His is related to the pathogenicity of the pathogen. Li et al.[9] studied the survival time of Aac at different places. Gao et al.[10] constructed a mutant of hpaP gene， and demonstrated that the gene plays an important role in motility of the pathogen， formation of biological membrane and expression of hrcV. In 2012， Yan et al.[11] predicted Aac pathogenic gene and target gene by comparative genomics， which provides guidance for such research in future. In 2016， Wang et al.[12] constructed a luxR4229deleted mutant by homologous recombination method， and found that luxR4229 plays a role of regulating the pathogenicity， motility and biomembrane formation of Aac. The research on the pathogenesis of Aac mainly includes the research on type III secretion system， quorum sensing system and flagellum and fimbria[13]. The research method is generally to construct a mutant library by randomly inserting the chromosome of Aac strain using transposon MiniTn5， and then screen the mutants losing pathogenicity by pathogenicty examination. This method for the research on the pathogenic genes of Aac has the disadvantages of randomness and blindness. The rapid development of bioinformatics provides a new strategy for the research of molecular pathogenisis of Aac. In this study， predicted unknown proteins were analyzed applying bioinformatics method. This method saves time for searching information and avoids the blindness when searching pathogenic genes by the construction of a mutant library through the insertion of the chromosome of Aac strain using transposon MiniTn5， laying a foundation for further research of such pathnogenic genes.
Materials and Methods
Experimental materials
The resistant parent material ‘TS107’ and susceptible tomato parent material ‘TS100’ of tomato grey mold were both provided by Xinjiang Agricultural Reclamation Academy of Science. Based on consulting a great deal of literatures， there were few studies on bioinformatics of Aac proteins， yp_968837， yp_968846， yp_968848， yp_966269， yp_970484 and yp_970742. This study selected these proteins， and analyzed the products of these proteins by bioinformatics methods. The serial numbers of these proteins were input and searched on NCBI， and it was found that the serial numbers of these proteins has been revised. The revised accession numbers （AC） were input， and the amino acid sequences of related unknown proteins were downloaded. The main network resources included NCBI and protParam. The used software included DNAMAN， BIOEDIT and Excel.
Experimental methods
Acquasion of pathogenic genes of B. cinerea Pers.
Based on consulting a great deal of literatures， the genes verified to be related with the pathogenicity of Aac were screened， and the pathogenic genes in references were inducted and reorganized， so as to analyze their pathogenic genes and pathogenesis.
Bioinformatics analysis
The amino acid sequences of Aac proteins， yp_968837， yp_968846， yp_968848， yp_969269， yp_970484 and yp_970742， and corresponding amino acid sequences of the pathogenic genes of other plant bacterial pathogens， AAZ34500， AAW73346， AAF61285， AAZ35693， CEL40510 and CAO95583， were obtained from NCBI. The searched sequences were input into Bioedit and DNAMAN， and converted to fasta form and output. The output amino acids， yp_968837 and AAZ34500， yp_968846 and AAW73346， yp_968848 and AAF61285， yp_969269 and AAZ35693、yp_970484 and CEL40510， and yp_970742 and CAO95583 were aligned in pairs by DNAMAN software， the identity， similar residues and gap of every two sequences were obtained， and the biological significances of these values were predicted. A local resource library was constructed with Bioedit software， and combined with ProtParam tool， the amino acid size， composition， molecular weight， isoelectric point and typical hydrophobic region of each protein from yp_968837， yp_968846， yp_968846， yp_969269， yp_970484 and yp_970742 were analyzed. Then， their secondary structures were analyzed with DNAMAN software， and above data were organized with Excel and used for prediction analysis. Results and Analysis
Analysis of Aac pathogenic genes
As shown in Table 1， there have been studies reporting that most Aac genes are related to T3SS， for instance， hrcV， hrcR， hrp and hpaP are all genes in hrp gene cluster. These genes are all conserved genes of T3SS. Among them， the mutation of hrcV， hrcR and hrp would cause the condition that Aac loses its pathogenicity and ability to induce allergic necrosis on tobacco， while hrpP gene is regulated by hrpX， the protein coded by it has chaperon function， and after mutation， the motility， formation of biomembrane and expression of hrcV gene of this strain are affected to reduce the pathogenicity of the pathogen. Furthermore， tvrR gene， luxR gene and his synthetase gene reduce the pathogenicity of Aac by influencing its growth system， quorum sensing system and his synthetase process， respectively. However， the difference between tvrR gene and his synthetase gene is that tvrR gene mutant still could activate tobacco tobacco allergic reactions， while his synthetase gene could not.
Table1 Analysis of pathogenic genes in Botrytis cinerea Pers.
Pathogenic geneCategoryFunctionYearAuthor
HrcVT3SSThe conserved gene in T3SS， mutated， abolishes the loss of pathogenicity and the ability to induce allergic necrosis on tobacco2006COPELAND
hrcRT3SSConserved genes in T3SS suggest that the protein encoded by the hcR gene is located in the bacterial endometrium and is a major component of the secretory pathway First， the ability to mutate Aac to lose pathogenicity and induce allergic necrosis in tobacco2009Ren
tvrRGrowthrelated genesPathogenesis of tvrR mutations is associated with changes in their ability to grow， and mutants can also trigger tobacco hypersensitivity2009Wei
luxRGroup sensing systemPathogenicity of l uxR gene after mutation decreased， the ability to produce signal molecules weakened2010ZHAO
hrpT3SSAs a key component of T3SS， it affects the pathogenicity of pathogenic bacteria to host plants and the allergic reaction to nonhost and diseaseresistant plants2011WANG
hisA， hisBhis a synthase geneGene mutation， not only lost the ability to stimulate tobacco allergic reactions， and its pathogenicity significantly decreased2011WANG
hisC， hisD， hpaPT3SSThe conserved gene in T3SS was regulated by hrpX and the encoded protein had chaperone function， which affected the motility， biofilm formation ability and expression of hrcV gene2011GAO DNAMAN alignment analysis
The criterion of protein sequence homology alignment is that in the protein sequence regions comprising more than 80 amino acids， if the homology of the amino acid sequences at the two ends is not lower than 25%， it is generally acknowledged that they have similar biological property. However， it is found that under this criterion， the two sequences might have similar functions， but also might be proteins with totally different properties. Therefore， the protein judgment only according to sequence homology has an accuracy which is not high， and other comparisons are also needed[15]. Similar residues represent the matching degree of two proteins， and the higher the similar residues， the higher the similarity of their biological functions. Gap represents the situation that sequence interruption occurs during sequence alignment. Generally， the lower the gap value， the higher the similarity.
The protein sequences of Aac were compared with corresponding amino acid sequences of pathogenic genes of other plant pathogens. It could be seen from Table 2 that yp_968837 and AAZ34500， yp_968846 and AAW73346， yp_968848 and AAF61285， yp_96929 and AAZ356963， yp_970484 and CEL40510， and yp_970742 and CAO95583 were aligned. The homology values were not high， but the similar residues all reached one half or more， and except that protein yp_970484 had a higher value， the gap values were all lower than one half. Comprehensively， the three pairs of proteins with higher matching degrees were yp_968848 and AAF61285， yp_969269 and AAZ35693， and yp_968837 and AAZ34500， indicating that the similarity between the functions of each pair was higher. Because these pathogenic genes of other plant bacterial pathogens are all from the hrp gene cluster of type III secretion system， these proteins were also predicted to be coded by hrp gene cluster.
Analysis of Aac pathogenic proteins
According to amino acid composition and physicochemical properties of proteins， their basic characteristics were analyzed， including amino acid size， composition， molecular weight， isoelectric point， hydrophobicity and secondary structure. At present， the prediction analysis of the physicochemical properties of unknown proteins is an important means for the understanding of unknown proteins， which is of certain guiding significance on related research[16].
Analysis of physicochemical properties
A local resource library was constructed with Bioedit software， and combined with ProtParam， the amino acid size， molecular weight， isoelectric point， and amino acid composition of each of yp_968837， yp_968846， yp_968848， yp_969269， yp_970484 and yp_970742 were analyzed. It could be seen from Table 3 that the proteins had 126-557 amino acids， their molecular weights were in the range of 13 819.14-53 506.96Da， and the isoelectric point ranged from 5.42 to 10.92. It is indicated that these proteins have wide spans of amino acid size， molecular weight and isoelectric point， and though they all belong to predicted gene proteins of hrp gene cluster， they differ greatly in physicochemical properties. Table 3 The size， molecular weight， theoretical isoelectric point and hydrophobicity of the six unknown proteins
Accession numberProtein numberThe number of amino acids/oneMolecular weight/DaTheoretical isoelectric point
WP_011793639yp_96883755753 506.966.94
WP_011793648yp_96884621122 825.0310.92
WP_011793650yp_96884812613 819.146.03
WP_011794053yp_96926936137 362.7010.43
WP_011795249yp_97048433235 163.929.33
WP_011795504yp_97074245441 612.715.42
Analysis of hydrophobicity
The hydrophobility of the amino acid side chain of protein is represented by the value obtained by subtracting the hydrophobicity of Gly from the hydrophobicity of various amino acids. The analysis of hydrophobicity is shown in Fig. 1-Fig. 6. The vertical coordinate Mean Hydrophobicity represents the mean value of hydrophobicity； the horizontal ordinate Position represents the position of amino acid； Kyte & Doolittle Scale Mean Hydrophobicity Profile refers to the mean of hydrophobicity profile determined by KD method； and Scanwindowsize=13 indicates that the size of the scan window is equal to 13， and by setting the window size as n， the figure would show the mean value of hydrophobicity from position n-4 to position n+4. Typical hydrophobic regions could obtained by analyzing Fig. 7-Fig. 12， and the results are shown in Table 4. It could be seen from Table 4 that the hydrophobic regions of most proteins were located within 10-25 amino acids， the hydrophobic regions of larger proteins， such as yp_970484， included about 50 amino acids， and hydrophobic regions of smaller proteins， such as yp_968848， comprised 8 amino acids.
Analysis of secondary structures
The prediction of the secondary structure of protein could provide a great deal of information for tertiary structure and function， and facilitates the determination of the relationship between the spatial structure and function of protein. The secondary structures predicted by DNAMAN software are shown in Fig. 7-Fig. 12. The labels of the secondary structures are Helix （αhelix）， Strands and Coils （random coil）， and the vertical coordinate Probability represents the probability of the three structures. It could be seen from the prediction results that the secondary structures of proteins yp_968837 and yp_968848 were mostly alternated folding of Helix and Coils， rarely Strands； the secondary structures of proteins yp_968848 and yp_970484 comprised a few Strands； and the three structures might all exist in the secondary structure of yp_969269； and the secondary structure of yp_970742 differed greatly from the secondary structures of other proteins， as Strands and Coils had larger proportions， and there was a longer segment of Strands. Fig.1 The Hydrophobicity profile of proteinyp_968837
Fig. 2 The Hydrophobicity profile of protein yp_968846
Fig. 3 The Hydrophobicity profile of protein yp_968848
Fig. 4 The Hydrophobicity profile of protein yp_969269
Fig. 5 The Hydrophobicity profile of protein yp_970484
Fig. 4 The Hydrophobicity profile of protein yp_969269
Fig. 7 The secondary structure of protein yp _ 968837
Fig. 8 The secondary structure of protein yp _ 968846
Discussion and Conclusion
Grey mould is a kind of serious disease on melons in production， which mainly occurs on leaves and fruit of tomato[17]， and after the occurrence of many scabs， the fruit rots， resulting in serious economic loss. Due to the lack of ideal control agents and diseaseresistant varieties， the diseased scope is enlarged year by year， and the potential hazard becomes higher and higher. Therefore， the research of the pathogenic genes of the pathogen has important guiding significance.
Fig. 9 The secondary structure of protein yp _ 968848
Fig. 10 The secondary structure of protein yp _ 969269
Fig. 11 The secondary structure of protein yp _970484
Fig. 12 The secondary structure of protein yp _ 970742
In this study， predicted unknown proteins were analyzed by bioinformatics method. At first， through alignment analysis using DNAMAN， the optimal matched protein pairs were yp_968848 and AAF61285， yp_969269 and AAZ35693， and yp_968837 and AAZ34500， each of which was predicted to have higher function similarity. The analysis results of physiochemical properties showed that these proteins comprised 126-557 amino acids， their molecular weight were in the range of 13819.14-53506.96Da， and the isoelectric point ranged from 5.42 to 10.92. It is indicated that these proteins have wide spans of amino acid size， molecular weight and isoelectric point， and though they all belong to predicted gene proteins of hrp gene cluster， they differ greatly in physicochemical properties.
It was predicted through the amino acid composition results of proteins that protein yp_968848 might be located in the function region at the inner side of T3SS system； protein yp_969269 had complicated amino acid composition， and its function might be more important； protein yp_970484 might have two regions， the hydrophilic region and the hydrophobic region； and protein yp_970742 might be a hydrophilic protein. The composition of amino acids is closely related to their functions， the 20 amino acids constituting proteins carry sidechain groups with different polarities， respectively， and their functions could thus be predicted through their composition. It could be known from the hydrophobic region analysis of the proteins that the hydrophobic regions of most proteins were located within 10-25 amino acids， the hydrophobic regions of larger proteins， such as yp_970484， included about 50 amino acids， and hydrophobic regions of smaller proteins， such as yp_968848， comprised 8 amino acids.
It could be seen from the prediction of the secondary structures of proteins that the secondary structures of proteins yp_968837 and yp_968848 were mostly alternated folding of Helix and Coils， rarely Strands； the secondary structures of proteins yp_968848 and yp_970484 comprised a few Strands； the three structures might all exist in the secondary structure of yp_969269； and the secondary structure of yp_970742 differed greatly from the secondary structures of other proteins， as Strands and Coils had larger proportions， and there was a longer segment of Strands.
References
[1] SCHAAD NW， SOWELLG， GOTH RW， et al. Pseudomonas pseudoalcaligens subsp citrulli subsp nov[J].Int J Syst Bacteriol，1978， 28（1）： 117-125.
[2] WILLEMSA， GOOR M， THIELEMANS S， et al. Transfer of several phytopathogenic Pseudomonas species to Acidovorax avenae subsp. Avenae subsp. nov， Combnov.， Acidovorax avenae subsp. cilrulli， Acidovorax avenae subsp. catteyae， and Acidovorax avenae konjaci [J]. Int I Syst bacteriol， 1992， 42： 107-119.
[3] QIU JW， LIAO XL. Research progress on prevention and control of melon seeds bacterial fruit spot pathogens [J]. Science of Biological Hazards， 2014， 37（1）： 70-73.
[4] REN ZG， HOU L， SONG ZG， et al. Meloidogyne baccatus pathogenic mutants screening and cloning of hrcR gene [J]. Journal of Plant Pathology， 2 009， 39 （5）：501-506.
[5] WEI N， QIAN GL， LI XX， et al. Gramnegative bacterial fruit germ tvrR gene gLong and its impact on the pathogenicity[J].Journal of Nanjing Agricultural University， 2010， 33（6）：68-74.
[6] WANG X， WANG W， QIAN GL， et al. Gourd bacterial fruit leaf spot bacteria allergic reactions and induced cloning and functional analysis of partial genes of pathogenic （hrp） gene cluster [J]. Agricultural Biotechnology Reported， 2011， 19（1）： 36-44.
[7] ZHAO YQ， HAN ZH， HIGH AZALEA， et al. Melon Bacterial fruit spot bacteria luxR gene Functional Analysis [J]. Journal of Agricultural Biotechnology， 2011， 19（3）： 542-548.
[8] WANG MJ， HAN ZH， WU Z， et al. Lack of histidine nutrition in melon bacterium depression and pathogenicity related [J]. Acta Microbiologica Sinica， 2011， 51（9）：1185-1193.
（Continued on page 86）
Key words Tomato； Grey mould； Pathogenic gene； Bioinformatics
Received： October 8， 2017 Accepted： December 13， 2017
Supported by Science and Technology Pilot Project of Xinjiang Agricultural Reclamation Academy of Science （50YYD201610）.
Qiang ZHANG （1977-）， male， P. R. China， associate researcher， master， devoted to research about botany.
*Corresponding author. Email： [email protected]
Tomato （Lycopersicon esculentum Mill.） is an important economic crop in Xinjiang， which is native to tropical zone， widely planted in various areas of China. There are various tomato varieties in China， and as one of good fruits in summer， it is popular due to its fragrant and sweet taste. However， with the expansion of the planting area of tomato and the continuous improvement of commercialization and production scale， tomatorelated diseases also increase. Among the various diseases， tomato grey mould commonly occurs at home and abroad， and becomes one of the three diseases harming tomato production in the world， thereby seriously affecting tomato yield and quality. Tomato grey mould is caused by Botrytis cinerea Pers. in Deuteromycotina[1-2]， which not only jeopardizes tomato， but also other Solanaceae crops[3].
Before 2009， studies at home and abroad mainly focused on detection of Aac， biological characteristics， law of disease development， chemical control and screening of diseaseresistant varieties， and few studies had been done on the pathogenicity of Botrytis cinerea Pers. at molecular level. Later， studies on pathogensis appeared， including the construction of genomic library and mutant library， pathogenic mechanism and pathogenic genes. In 2009， Ren et al.[4] constructed an Aac mutant libray by triparental mating， and indicated that T3SS is a key factor for pathogenicity. Wei et al.[5] found and cloned a congener of tvrR gene in the whole genome of Aac， and the results showed that the change in pathogenicity of the mutant is caused by the change of its growing ability. In 2010， Wang et al.[6] demonstrated that the product coded by hrp gene of Aac affects the pathogenicity of pathogenic bacteria on host plant. In the same year， Zhao et al.[7] reported that the mutation of luxR genes would cause the decrease of pathogenicity of the pathogen and the ability of producing signal molecules is weakened. In 2011， WMJ et al.[8] verified that the deficit of His is related to the pathogenicity of the pathogen. Li et al.[9] studied the survival time of Aac at different places. Gao et al.[10] constructed a mutant of hpaP gene， and demonstrated that the gene plays an important role in motility of the pathogen， formation of biological membrane and expression of hrcV. In 2012， Yan et al.[11] predicted Aac pathogenic gene and target gene by comparative genomics， which provides guidance for such research in future. In 2016， Wang et al.[12] constructed a luxR4229deleted mutant by homologous recombination method， and found that luxR4229 plays a role of regulating the pathogenicity， motility and biomembrane formation of Aac. The research on the pathogenesis of Aac mainly includes the research on type III secretion system， quorum sensing system and flagellum and fimbria[13]. The research method is generally to construct a mutant library by randomly inserting the chromosome of Aac strain using transposon MiniTn5， and then screen the mutants losing pathogenicity by pathogenicty examination. This method for the research on the pathogenic genes of Aac has the disadvantages of randomness and blindness. The rapid development of bioinformatics provides a new strategy for the research of molecular pathogenisis of Aac. In this study， predicted unknown proteins were analyzed applying bioinformatics method. This method saves time for searching information and avoids the blindness when searching pathogenic genes by the construction of a mutant library through the insertion of the chromosome of Aac strain using transposon MiniTn5， laying a foundation for further research of such pathnogenic genes.
Materials and Methods
Experimental materials
The resistant parent material ‘TS107’ and susceptible tomato parent material ‘TS100’ of tomato grey mold were both provided by Xinjiang Agricultural Reclamation Academy of Science. Based on consulting a great deal of literatures， there were few studies on bioinformatics of Aac proteins， yp_968837， yp_968846， yp_968848， yp_966269， yp_970484 and yp_970742. This study selected these proteins， and analyzed the products of these proteins by bioinformatics methods. The serial numbers of these proteins were input and searched on NCBI， and it was found that the serial numbers of these proteins has been revised. The revised accession numbers （AC） were input， and the amino acid sequences of related unknown proteins were downloaded. The main network resources included NCBI and protParam. The used software included DNAMAN， BIOEDIT and Excel.
Experimental methods
Acquasion of pathogenic genes of B. cinerea Pers.
Based on consulting a great deal of literatures， the genes verified to be related with the pathogenicity of Aac were screened， and the pathogenic genes in references were inducted and reorganized， so as to analyze their pathogenic genes and pathogenesis.
Bioinformatics analysis
The amino acid sequences of Aac proteins， yp_968837， yp_968846， yp_968848， yp_969269， yp_970484 and yp_970742， and corresponding amino acid sequences of the pathogenic genes of other plant bacterial pathogens， AAZ34500， AAW73346， AAF61285， AAZ35693， CEL40510 and CAO95583， were obtained from NCBI. The searched sequences were input into Bioedit and DNAMAN， and converted to fasta form and output. The output amino acids， yp_968837 and AAZ34500， yp_968846 and AAW73346， yp_968848 and AAF61285， yp_969269 and AAZ35693、yp_970484 and CEL40510， and yp_970742 and CAO95583 were aligned in pairs by DNAMAN software， the identity， similar residues and gap of every two sequences were obtained， and the biological significances of these values were predicted. A local resource library was constructed with Bioedit software， and combined with ProtParam tool， the amino acid size， composition， molecular weight， isoelectric point and typical hydrophobic region of each protein from yp_968837， yp_968846， yp_968846， yp_969269， yp_970484 and yp_970742 were analyzed. Then， their secondary structures were analyzed with DNAMAN software， and above data were organized with Excel and used for prediction analysis. Results and Analysis
Analysis of Aac pathogenic genes
As shown in Table 1， there have been studies reporting that most Aac genes are related to T3SS， for instance， hrcV， hrcR， hrp and hpaP are all genes in hrp gene cluster. These genes are all conserved genes of T3SS. Among them， the mutation of hrcV， hrcR and hrp would cause the condition that Aac loses its pathogenicity and ability to induce allergic necrosis on tobacco， while hrpP gene is regulated by hrpX， the protein coded by it has chaperon function， and after mutation， the motility， formation of biomembrane and expression of hrcV gene of this strain are affected to reduce the pathogenicity of the pathogen. Furthermore， tvrR gene， luxR gene and his synthetase gene reduce the pathogenicity of Aac by influencing its growth system， quorum sensing system and his synthetase process， respectively. However， the difference between tvrR gene and his synthetase gene is that tvrR gene mutant still could activate tobacco tobacco allergic reactions， while his synthetase gene could not.
Table1 Analysis of pathogenic genes in Botrytis cinerea Pers.
Pathogenic geneCategoryFunctionYearAuthor
HrcVT3SSThe conserved gene in T3SS， mutated， abolishes the loss of pathogenicity and the ability to induce allergic necrosis on tobacco2006COPELAND
hrcRT3SSConserved genes in T3SS suggest that the protein encoded by the hcR gene is located in the bacterial endometrium and is a major component of the secretory pathway First， the ability to mutate Aac to lose pathogenicity and induce allergic necrosis in tobacco2009Ren
tvrRGrowthrelated genesPathogenesis of tvrR mutations is associated with changes in their ability to grow， and mutants can also trigger tobacco hypersensitivity2009Wei
luxRGroup sensing systemPathogenicity of l uxR gene after mutation decreased， the ability to produce signal molecules weakened2010ZHAO
hrpT3SSAs a key component of T3SS， it affects the pathogenicity of pathogenic bacteria to host plants and the allergic reaction to nonhost and diseaseresistant plants2011WANG
hisA， hisBhis a synthase geneGene mutation， not only lost the ability to stimulate tobacco allergic reactions， and its pathogenicity significantly decreased2011WANG
hisC， hisD， hpaPT3SSThe conserved gene in T3SS was regulated by hrpX and the encoded protein had chaperone function， which affected the motility， biofilm formation ability and expression of hrcV gene2011GAO DNAMAN alignment analysis
The criterion of protein sequence homology alignment is that in the protein sequence regions comprising more than 80 amino acids， if the homology of the amino acid sequences at the two ends is not lower than 25%， it is generally acknowledged that they have similar biological property. However， it is found that under this criterion， the two sequences might have similar functions， but also might be proteins with totally different properties. Therefore， the protein judgment only according to sequence homology has an accuracy which is not high， and other comparisons are also needed[15]. Similar residues represent the matching degree of two proteins， and the higher the similar residues， the higher the similarity of their biological functions. Gap represents the situation that sequence interruption occurs during sequence alignment. Generally， the lower the gap value， the higher the similarity.
The protein sequences of Aac were compared with corresponding amino acid sequences of pathogenic genes of other plant pathogens. It could be seen from Table 2 that yp_968837 and AAZ34500， yp_968846 and AAW73346， yp_968848 and AAF61285， yp_96929 and AAZ356963， yp_970484 and CEL40510， and yp_970742 and CAO95583 were aligned. The homology values were not high， but the similar residues all reached one half or more， and except that protein yp_970484 had a higher value， the gap values were all lower than one half. Comprehensively， the three pairs of proteins with higher matching degrees were yp_968848 and AAF61285， yp_969269 and AAZ35693， and yp_968837 and AAZ34500， indicating that the similarity between the functions of each pair was higher. Because these pathogenic genes of other plant bacterial pathogens are all from the hrp gene cluster of type III secretion system， these proteins were also predicted to be coded by hrp gene cluster.
Analysis of Aac pathogenic proteins
According to amino acid composition and physicochemical properties of proteins， their basic characteristics were analyzed， including amino acid size， composition， molecular weight， isoelectric point， hydrophobicity and secondary structure. At present， the prediction analysis of the physicochemical properties of unknown proteins is an important means for the understanding of unknown proteins， which is of certain guiding significance on related research[16].
Analysis of physicochemical properties
A local resource library was constructed with Bioedit software， and combined with ProtParam， the amino acid size， molecular weight， isoelectric point， and amino acid composition of each of yp_968837， yp_968846， yp_968848， yp_969269， yp_970484 and yp_970742 were analyzed. It could be seen from Table 3 that the proteins had 126-557 amino acids， their molecular weights were in the range of 13 819.14-53 506.96Da， and the isoelectric point ranged from 5.42 to 10.92. It is indicated that these proteins have wide spans of amino acid size， molecular weight and isoelectric point， and though they all belong to predicted gene proteins of hrp gene cluster， they differ greatly in physicochemical properties. Table 3 The size， molecular weight， theoretical isoelectric point and hydrophobicity of the six unknown proteins
Accession numberProtein numberThe number of amino acids/oneMolecular weight/DaTheoretical isoelectric point
WP_011793639yp_96883755753 506.966.94
WP_011793648yp_96884621122 825.0310.92
WP_011793650yp_96884812613 819.146.03
WP_011794053yp_96926936137 362.7010.43
WP_011795249yp_97048433235 163.929.33
WP_011795504yp_97074245441 612.715.42
Analysis of hydrophobicity
The hydrophobility of the amino acid side chain of protein is represented by the value obtained by subtracting the hydrophobicity of Gly from the hydrophobicity of various amino acids. The analysis of hydrophobicity is shown in Fig. 1-Fig. 6. The vertical coordinate Mean Hydrophobicity represents the mean value of hydrophobicity； the horizontal ordinate Position represents the position of amino acid； Kyte & Doolittle Scale Mean Hydrophobicity Profile refers to the mean of hydrophobicity profile determined by KD method； and Scanwindowsize=13 indicates that the size of the scan window is equal to 13， and by setting the window size as n， the figure would show the mean value of hydrophobicity from position n-4 to position n+4. Typical hydrophobic regions could obtained by analyzing Fig. 7-Fig. 12， and the results are shown in Table 4. It could be seen from Table 4 that the hydrophobic regions of most proteins were located within 10-25 amino acids， the hydrophobic regions of larger proteins， such as yp_970484， included about 50 amino acids， and hydrophobic regions of smaller proteins， such as yp_968848， comprised 8 amino acids.
Analysis of secondary structures
The prediction of the secondary structure of protein could provide a great deal of information for tertiary structure and function， and facilitates the determination of the relationship between the spatial structure and function of protein. The secondary structures predicted by DNAMAN software are shown in Fig. 7-Fig. 12. The labels of the secondary structures are Helix （αhelix）， Strands and Coils （random coil）， and the vertical coordinate Probability represents the probability of the three structures. It could be seen from the prediction results that the secondary structures of proteins yp_968837 and yp_968848 were mostly alternated folding of Helix and Coils， rarely Strands； the secondary structures of proteins yp_968848 and yp_970484 comprised a few Strands； and the three structures might all exist in the secondary structure of yp_969269； and the secondary structure of yp_970742 differed greatly from the secondary structures of other proteins， as Strands and Coils had larger proportions， and there was a longer segment of Strands. Fig.1 The Hydrophobicity profile of proteinyp_968837
Fig. 2 The Hydrophobicity profile of protein yp_968846
Fig. 3 The Hydrophobicity profile of protein yp_968848
Fig. 4 The Hydrophobicity profile of protein yp_969269
Fig. 5 The Hydrophobicity profile of protein yp_970484
Fig. 4 The Hydrophobicity profile of protein yp_969269
Fig. 7 The secondary structure of protein yp _ 968837
Fig. 8 The secondary structure of protein yp _ 968846
Discussion and Conclusion
Grey mould is a kind of serious disease on melons in production， which mainly occurs on leaves and fruit of tomato[17]， and after the occurrence of many scabs， the fruit rots， resulting in serious economic loss. Due to the lack of ideal control agents and diseaseresistant varieties， the diseased scope is enlarged year by year， and the potential hazard becomes higher and higher. Therefore， the research of the pathogenic genes of the pathogen has important guiding significance.
Fig. 9 The secondary structure of protein yp _ 968848
Fig. 10 The secondary structure of protein yp _ 969269
Fig. 11 The secondary structure of protein yp _970484
Fig. 12 The secondary structure of protein yp _ 970742
In this study， predicted unknown proteins were analyzed by bioinformatics method. At first， through alignment analysis using DNAMAN， the optimal matched protein pairs were yp_968848 and AAF61285， yp_969269 and AAZ35693， and yp_968837 and AAZ34500， each of which was predicted to have higher function similarity. The analysis results of physiochemical properties showed that these proteins comprised 126-557 amino acids， their molecular weight were in the range of 13819.14-53506.96Da， and the isoelectric point ranged from 5.42 to 10.92. It is indicated that these proteins have wide spans of amino acid size， molecular weight and isoelectric point， and though they all belong to predicted gene proteins of hrp gene cluster， they differ greatly in physicochemical properties.
It was predicted through the amino acid composition results of proteins that protein yp_968848 might be located in the function region at the inner side of T3SS system； protein yp_969269 had complicated amino acid composition， and its function might be more important； protein yp_970484 might have two regions， the hydrophilic region and the hydrophobic region； and protein yp_970742 might be a hydrophilic protein. The composition of amino acids is closely related to their functions， the 20 amino acids constituting proteins carry sidechain groups with different polarities， respectively， and their functions could thus be predicted through their composition. It could be known from the hydrophobic region analysis of the proteins that the hydrophobic regions of most proteins were located within 10-25 amino acids， the hydrophobic regions of larger proteins， such as yp_970484， included about 50 amino acids， and hydrophobic regions of smaller proteins， such as yp_968848， comprised 8 amino acids.
It could be seen from the prediction of the secondary structures of proteins that the secondary structures of proteins yp_968837 and yp_968848 were mostly alternated folding of Helix and Coils， rarely Strands； the secondary structures of proteins yp_968848 and yp_970484 comprised a few Strands； the three structures might all exist in the secondary structure of yp_969269； and the secondary structure of yp_970742 differed greatly from the secondary structures of other proteins， as Strands and Coils had larger proportions， and there was a longer segment of Strands.
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