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Abstract Sugarcane smut greatly influences yield and quality of sugarcane. HAS1 gene in biocontrol strain HAS antagonistic to Sporisorium scitanminea was selected for prokaryotic expression vector construction and inducible expression， and the antagonistic effect of HAS1 protein was detected in vitro. The results showed that the expression product of antifungal HAS1 gene obtained through prokaryotic expression vector construction and inducible expression had better antagonistic effect against S. scitanminea. This study provides an experimental basis for further utilization of this protein.
Key words Bacillus subtilis HAS; Sporisorium scitanminea; Prokaryotic expression; Antifungal protein
Received： June 8， 2018 Accepted： September 10， 2018
Supported by College Students Innovation and Enterpreneurship Training Program of Hainan Province （2017116）; College Students Innovation and Enterpreneurship Training Program of Hainan Medical University （HYCX2016073）; Natural Science Foundation of China （31471555）.
Huiqing LIU （1996-）， female， P. R. China， bachelor： biotechnology.
*Corresponding author. Email： 88610653@qq.com; xiongguoru@itbb.org.cn.
Sugarcane smut is one of the main fungal diseases caused by Sporisorium scitanminea， which seriously restricts healthy sustainable development of sugarcane industry and sugar industry. This disease is found for the first time in South Africa， and then in various countries cultivating sugarcane in Asia and Africa[1]. China is also one of the major sugarcaneproducing countries. Sugarcane is mainly cultivated in south central Guangxi， southwestern Yunnan， western Guangdong and northern Hainan in China[2]. Cane sugar produced in China accounts for more than 80% of total sugar output， while sugarcane smut is one of the largest greatest hazards to sugarcane， which would seriously influence yield and quality of sugarcane.
Teliospores of sugarcane smut can spread easily in drought condition， and this disease happens easier under hightemperature highhumidity conditions. With the adjustment of sugarcaneproducing area distribution， more than 90% of sugarcane is planted in dry land or dry sloping land， resulting in increasingly severe sugarcane smut[3]. S. scitanminea would not seriously influence growth and development of sugarcane at a small quantity， but would damage sugarcane in a large scale when it accumulates to a certain quantity. A large quantity of spores would be produced during the process that the fungus infects sugarcane. These spores have certain adsorbability， and could break through cell wall of hosts and enter cell membrane， thereby competitively absorbing nutritive substances in host and multiplying in hosts in a large quantity. As a result， growth of hosts is inhibited， and synthesis of products is interfered in hosts[4]. At present， sugarcane smut is mainly controlled by breeding of resistant varieties， chemical control and biological control[5]. Different physiological races of sugarcane differ in resistance to sugarcane smut， and dominant varieties could be screened through the detection on resistance type of different sugarcane varieties. Breeding of resistant varieties means to screen dominant hybrids by crossing selected good parents through artificial breeding. This method is the most economic and effective method at present， but the hybridization and selection process is timeconsuming[6]. Chemical control is to spray agents or soak sugarcane with agents to endow sugarcane with certain antagonistic effect against sugarcane smut， to thereby inhibit sugarcane smut[7]. However， chemical control has very poor control efficiency， and would cause certain pollution to environment[8]. Biological control is one of the main methods having the highest development potential， which finds corresponding antagonistic strains according to pathogens at first， then finds acting factors in antagonistic strains and finally improves genetic traits of plants according to antagonistic genes. Biological control would not harm environment and human health， and has attracted extensive attention in the world[9].
Bacillus subtilis is a kind of beneficial bacteria with the most extensive application in biological control. Most B. subtilis could produce certain antagonistic substances inhibiting microbial growth[9]. Xiong et al.[10] isolated from strain HAS having an inhibitory effect on sugarcane smut from soil microorganisms in Hainan sugarcaneproducing area[10]. Preliminary analysis on antifungal mechanism of this strain showed that this strain could effectively colonize around rhizosphere and in root， stem and leaf of sugarcane plants[11]， and it is speculated that TasA gene， chitinase gene and HAS1 gene in this strain all have certain antagonistic effect against sugarcane smut. In this study， HAS1 gene in strain HAS was selected for prokaryotic expression vector construction and inducible expression， and the activity of the expressed protein was tested in vitro， so as to provide a basis for understanding of its antagonistic effect.
Materials and Methods
Materials
B. subtilis HAS and pET32a plasmid vector were purchased from Novagen Inc， Malaysia.
Methods
Primers were designed. PCR of HAS1 gene in genomic DNA of B. subtilis HAS was performed. The PCR reaction system is shown in Table 1. The PCR was started at 94 ℃ for 3 min， followed by 28 cycles of 94 ℃ for 30 s， 58 ℃ for 45 s and 72 ℃ for 45 s， and extended at 72 ℃ for 10 min. The product was preserved at 4 ℃.
The PCR product was detected by agarose gel electrophoresis， and the electrophoresis results were observed.
The target gene was recovered. And the target gene and pET32a were subjected to double enzyme digestion （Table 1）. Each of them was placed in a thermostat water bath at 37 ℃ for 4 h， and the digestion product was identified by electrophoresis.
The target gene and vector were recovered after digestion. Then， the target gene was ligated to the vector （Table 1）. The ligation system was placed at 16 ℃ overnight.
The ligation product was transformed into DH5α competent cells. The transformed cells were coated on a bluewhite selection plate and cultured at 37 ℃ overnight. A half of the cultured colony was used for PCR identification. The other half was inoculated on another plate.
Plasmids in the recombinant bacteria containing the target gene were identified by digestion， so as to detect whether target band could be obtained and whether the recombinant bacteria were positive.
The plasmids containing the target gene was transformed to Escherichia coli BL21 （DE3） for induced expression.
Purification of HAS1 protein and renaturation of inclusion body were performed.
Antifungal test of purified protein： Sterilized agar medium was heated to completely melt it and then poured to a culture dish according to 15 ml per dish （lower layer）， followed by cooling to solidification. Then， melted PDA medium was cooled to 50 ℃ （no hot） and mixed with the tested fungus. Then， 5 ml of the medium mixed with the tested fungus was added onto the solidified medium （upper layer）， followed by cooling for solidification. An Oxford cup was vertically placed on the surface of the medium in sterile environment， and slightly pressed to allow sufficient contact with the medium. Tobedetected sample was fully added into the cup without overflowing. The culture dish was covered and cultured at 28 ℃ for 3-5 d and observed during culture.
Results and Analysis
PCR of HAS1 gene
With genomic DNA of B. subtilis HAS as template， a target band （750 bp） was obtained by PCR using specific primers （Fig. 1）.
Doubleenzyme digestion of HAS1 and pET32a vector
HAS1 gene and pET32（a） vector subjected to digestion with BamH I and Xho I were ligated with ligase and transformed into E. coli DH5α， and recombinant plasmids were screened and identified. Fig. 2 is the identification map of recombinant plasmid DH5αpET32（a）HAS1， indicating that the recombinant plasmid HAS1pET32（a） had been constructed successfully. Induced expression of HAS1
In order to verify whether HAS1 protein could be expressed successfully after successfully constructing the vector， SDS polyacrylamide gel electrophoresis was performed. In order to explore the optimal inducer concentration and induction time of HAS1 protein， such six time points as 0， 1， 2， 4， 6 and 8 h were set， and two inducer concentrations were also set according to literature information. Which time and which concentration could achieve the highest protein level were explored at 37 ℃， and the results are shown in Fig. 3.
A： Final IPTG concentration of 0.5 mM; B： final IPTG concentration of 1 mM
Huiqing LIU et al. Identification of Inintro Antagonistic Activity of HAS1 Protein against Sporisorium scitanminea
It could be seen from Fig. 3 that the target protein showed an obvious band at 47 KDa， indicating successful construction and expression of HAS1 protein vector. HAS1 protein could be successfully expressed at both the inducer concentrations of 0.5 and 1 mM under 37 ℃， and the expression effect was better at 2 h.
Purification of HAS1 protein
During the whole purification process， miscellaneous proteins decreased gradually， and the purity of target protein was improved gradually （Fig. 4）.
Antifungal effect of HAS1 protein
HAS protein was added into the Oxford cup of the experimental group， which showed an obvious inhibition zone， while the blank control group showed fungus grown well （Fig. 5）. It was thus demonstrated that HAS1 protein could inhibit sugarcane smut.
Conclusions and Discussion
After transforming antifungal protein HAS1 gene into expression strain through the construction of HAS1pET32（a）， the expression product， HAS1 protein had better antagonistic effect against S. scitanminea.
In the experiment process， the inclusion body was washed with triton X100， and the precipitate was resuspended in cell lysis solution containing EDTA; and after 15 min of lowtemperature
highspeed centrifugation， the supernatant and precipitate were separated and detected by SDSPAGE， to determine which of the two parts did the target protein mainly existed in. It was found by the detection that the inclusion body mainly existed in the precipitate after washing with triton and centrifugation. Therefore， the precipitate was resuspended in inclusion body solublization buffer containing 8 M urea to solubilize the inclusion body. The solublization process required low temperature and longer time， overnight if possible， so as to obtain a large quantity of protein. There have been no effective control measures for sugarcane smut， and biological control has become a hotspot for control of sugarcane smut. B. subtilis HAS isolated from sugarcane soil is exactly a very good biocontrol strains. It could be seen from the experimental results that HAS1 protein expressed by B. subtilis HAS has good antagonistic effect on sugarcane smut. Successful purification of HAS1 protein could provide a new material for breeding of varieties resistant to sugarcane smut. Xiong et al.[12-13] also found that besides HAS1， B. subtilis HAS also contains antagonistic proteins Tas A protein and chitinase， which play an important role in antagonistic effect.
References
[1] JR. SAA， YANG KZ. Sugarcane smut[J]. Sugarcane and Canesugar， 1980（9）： 55-56.
[2] LUO J， PAN YB， XU L， et al. Rational regional distribution of sugarcane cultivars in China[J]. Scientific Reports， 2015（5）： 15721.
[3] SHEN WK， JIANG ZD， DENG HH， et al. Research progress on sugarcane smut disease and Sporisorium scitaminea[J]. Chinese Journal of Tropical Crops， 2013， 34（10）： 2063-2068.
[4] LU WH， LI WF， HUANG YK. Research advances on sugarcane smut disease occurrence and control[J]. Sugar Crops of China， 2008（3）： 64-66.
[5] NING L， PANG XH， WANG Y， et al. Occurrence and control measures of sugarcane smut in Guangxi[J]. Agricultural Research and Application， 2018（2）： 40-42.
[6] XU LP， CHEN RK. Current situation and prospect of sugarcane smut and breeding of resistance sugarcane varieties[J]. Fujian Journal of Agricultural Sciences， 2000（2）： 26-31.
[7] ZHU GN， LI SY， XIAN XY， et al. Field control test of 5 fungicides to sugarcane smut[J]. Guangxi Agricultural Sciences， 2014， 45（8）：1393-1397.
[8] SHEN WK. Sugarcane disease in Guangdong sugarcane regions in recent years and integrated controlling methods[J]. Sugarcane and Canesugar， 2004（1）：1-5.
[9] ZHANG GY. Isolation， screening and identification of antagonistic bacteria against Ustilago scitaminea Sydow and studies on their antagonistic mechanisms[D]. Nanning： Guangxi University， 2002.
[10] XIONG GR， ZHAO GF， WU SR， et al. Screening and identification of a biocontrol strain， HAS antagonistic to Sporisorium scitaminea Syd[J]. Chinese Journal of Tropical Crops， 2013， 34（6）：1149-1154.
[11] XIONG GR， ZHAO GF， WANG WZ， et al. Colonization ability of Bacillus subtilis HAS in sugarcane[J]. Plant Diseases and Pests， 2018， 9（1）： 26-28.
[12] XIONG GR， ZHAO GF， ZHANG SZ， et al. Cloning and analysis of chitinase gene in Bacillus subtilis HAS[J]. Guizhou Agricultural Sciences， 2017， 45（2）： 1-4.
[13] XIONG GR， ZHAO GF， CAO WW， et al. Cloning and analysis of TasA in Bacillus subtilis HAS[J]. Guizhou Agricultural Sciences， 2016， 44（12）：53-57.
Key words Bacillus subtilis HAS; Sporisorium scitanminea; Prokaryotic expression; Antifungal protein
Received： June 8， 2018 Accepted： September 10， 2018
Supported by College Students Innovation and Enterpreneurship Training Program of Hainan Province （2017116）; College Students Innovation and Enterpreneurship Training Program of Hainan Medical University （HYCX2016073）; Natural Science Foundation of China （31471555）.
Huiqing LIU （1996-）， female， P. R. China， bachelor： biotechnology.
*Corresponding author. Email： 88610653@qq.com; xiongguoru@itbb.org.cn.
Sugarcane smut is one of the main fungal diseases caused by Sporisorium scitanminea， which seriously restricts healthy sustainable development of sugarcane industry and sugar industry. This disease is found for the first time in South Africa， and then in various countries cultivating sugarcane in Asia and Africa[1]. China is also one of the major sugarcaneproducing countries. Sugarcane is mainly cultivated in south central Guangxi， southwestern Yunnan， western Guangdong and northern Hainan in China[2]. Cane sugar produced in China accounts for more than 80% of total sugar output， while sugarcane smut is one of the largest greatest hazards to sugarcane， which would seriously influence yield and quality of sugarcane.
Teliospores of sugarcane smut can spread easily in drought condition， and this disease happens easier under hightemperature highhumidity conditions. With the adjustment of sugarcaneproducing area distribution， more than 90% of sugarcane is planted in dry land or dry sloping land， resulting in increasingly severe sugarcane smut[3]. S. scitanminea would not seriously influence growth and development of sugarcane at a small quantity， but would damage sugarcane in a large scale when it accumulates to a certain quantity. A large quantity of spores would be produced during the process that the fungus infects sugarcane. These spores have certain adsorbability， and could break through cell wall of hosts and enter cell membrane， thereby competitively absorbing nutritive substances in host and multiplying in hosts in a large quantity. As a result， growth of hosts is inhibited， and synthesis of products is interfered in hosts[4]. At present， sugarcane smut is mainly controlled by breeding of resistant varieties， chemical control and biological control[5]. Different physiological races of sugarcane differ in resistance to sugarcane smut， and dominant varieties could be screened through the detection on resistance type of different sugarcane varieties. Breeding of resistant varieties means to screen dominant hybrids by crossing selected good parents through artificial breeding. This method is the most economic and effective method at present， but the hybridization and selection process is timeconsuming[6]. Chemical control is to spray agents or soak sugarcane with agents to endow sugarcane with certain antagonistic effect against sugarcane smut， to thereby inhibit sugarcane smut[7]. However， chemical control has very poor control efficiency， and would cause certain pollution to environment[8]. Biological control is one of the main methods having the highest development potential， which finds corresponding antagonistic strains according to pathogens at first， then finds acting factors in antagonistic strains and finally improves genetic traits of plants according to antagonistic genes. Biological control would not harm environment and human health， and has attracted extensive attention in the world[9].
Bacillus subtilis is a kind of beneficial bacteria with the most extensive application in biological control. Most B. subtilis could produce certain antagonistic substances inhibiting microbial growth[9]. Xiong et al.[10] isolated from strain HAS having an inhibitory effect on sugarcane smut from soil microorganisms in Hainan sugarcaneproducing area[10]. Preliminary analysis on antifungal mechanism of this strain showed that this strain could effectively colonize around rhizosphere and in root， stem and leaf of sugarcane plants[11]， and it is speculated that TasA gene， chitinase gene and HAS1 gene in this strain all have certain antagonistic effect against sugarcane smut. In this study， HAS1 gene in strain HAS was selected for prokaryotic expression vector construction and inducible expression， and the activity of the expressed protein was tested in vitro， so as to provide a basis for understanding of its antagonistic effect.
Materials and Methods
Materials
B. subtilis HAS and pET32a plasmid vector were purchased from Novagen Inc， Malaysia.
Methods
Primers were designed. PCR of HAS1 gene in genomic DNA of B. subtilis HAS was performed. The PCR reaction system is shown in Table 1. The PCR was started at 94 ℃ for 3 min， followed by 28 cycles of 94 ℃ for 30 s， 58 ℃ for 45 s and 72 ℃ for 45 s， and extended at 72 ℃ for 10 min. The product was preserved at 4 ℃.
The PCR product was detected by agarose gel electrophoresis， and the electrophoresis results were observed.
The target gene was recovered. And the target gene and pET32a were subjected to double enzyme digestion （Table 1）. Each of them was placed in a thermostat water bath at 37 ℃ for 4 h， and the digestion product was identified by electrophoresis.
The target gene and vector were recovered after digestion. Then， the target gene was ligated to the vector （Table 1）. The ligation system was placed at 16 ℃ overnight.
The ligation product was transformed into DH5α competent cells. The transformed cells were coated on a bluewhite selection plate and cultured at 37 ℃ overnight. A half of the cultured colony was used for PCR identification. The other half was inoculated on another plate.
Plasmids in the recombinant bacteria containing the target gene were identified by digestion， so as to detect whether target band could be obtained and whether the recombinant bacteria were positive.
The plasmids containing the target gene was transformed to Escherichia coli BL21 （DE3） for induced expression.
Purification of HAS1 protein and renaturation of inclusion body were performed.
Antifungal test of purified protein： Sterilized agar medium was heated to completely melt it and then poured to a culture dish according to 15 ml per dish （lower layer）， followed by cooling to solidification. Then， melted PDA medium was cooled to 50 ℃ （no hot） and mixed with the tested fungus. Then， 5 ml of the medium mixed with the tested fungus was added onto the solidified medium （upper layer）， followed by cooling for solidification. An Oxford cup was vertically placed on the surface of the medium in sterile environment， and slightly pressed to allow sufficient contact with the medium. Tobedetected sample was fully added into the cup without overflowing. The culture dish was covered and cultured at 28 ℃ for 3-5 d and observed during culture.
Results and Analysis
PCR of HAS1 gene
With genomic DNA of B. subtilis HAS as template， a target band （750 bp） was obtained by PCR using specific primers （Fig. 1）.
Doubleenzyme digestion of HAS1 and pET32a vector
HAS1 gene and pET32（a） vector subjected to digestion with BamH I and Xho I were ligated with ligase and transformed into E. coli DH5α， and recombinant plasmids were screened and identified. Fig. 2 is the identification map of recombinant plasmid DH5αpET32（a）HAS1， indicating that the recombinant plasmid HAS1pET32（a） had been constructed successfully. Induced expression of HAS1
In order to verify whether HAS1 protein could be expressed successfully after successfully constructing the vector， SDS polyacrylamide gel electrophoresis was performed. In order to explore the optimal inducer concentration and induction time of HAS1 protein， such six time points as 0， 1， 2， 4， 6 and 8 h were set， and two inducer concentrations were also set according to literature information. Which time and which concentration could achieve the highest protein level were explored at 37 ℃， and the results are shown in Fig. 3.
A： Final IPTG concentration of 0.5 mM; B： final IPTG concentration of 1 mM
Huiqing LIU et al. Identification of Inintro Antagonistic Activity of HAS1 Protein against Sporisorium scitanminea
It could be seen from Fig. 3 that the target protein showed an obvious band at 47 KDa， indicating successful construction and expression of HAS1 protein vector. HAS1 protein could be successfully expressed at both the inducer concentrations of 0.5 and 1 mM under 37 ℃， and the expression effect was better at 2 h.
Purification of HAS1 protein
During the whole purification process， miscellaneous proteins decreased gradually， and the purity of target protein was improved gradually （Fig. 4）.
Antifungal effect of HAS1 protein
HAS protein was added into the Oxford cup of the experimental group， which showed an obvious inhibition zone， while the blank control group showed fungus grown well （Fig. 5）. It was thus demonstrated that HAS1 protein could inhibit sugarcane smut.
Conclusions and Discussion
After transforming antifungal protein HAS1 gene into expression strain through the construction of HAS1pET32（a）， the expression product， HAS1 protein had better antagonistic effect against S. scitanminea.
In the experiment process， the inclusion body was washed with triton X100， and the precipitate was resuspended in cell lysis solution containing EDTA; and after 15 min of lowtemperature
highspeed centrifugation， the supernatant and precipitate were separated and detected by SDSPAGE， to determine which of the two parts did the target protein mainly existed in. It was found by the detection that the inclusion body mainly existed in the precipitate after washing with triton and centrifugation. Therefore， the precipitate was resuspended in inclusion body solublization buffer containing 8 M urea to solubilize the inclusion body. The solublization process required low temperature and longer time， overnight if possible， so as to obtain a large quantity of protein. There have been no effective control measures for sugarcane smut， and biological control has become a hotspot for control of sugarcane smut. B. subtilis HAS isolated from sugarcane soil is exactly a very good biocontrol strains. It could be seen from the experimental results that HAS1 protein expressed by B. subtilis HAS has good antagonistic effect on sugarcane smut. Successful purification of HAS1 protein could provide a new material for breeding of varieties resistant to sugarcane smut. Xiong et al.[12-13] also found that besides HAS1， B. subtilis HAS also contains antagonistic proteins Tas A protein and chitinase， which play an important role in antagonistic effect.
References
[1] JR. SAA， YANG KZ. Sugarcane smut[J]. Sugarcane and Canesugar， 1980（9）： 55-56.
[2] LUO J， PAN YB， XU L， et al. Rational regional distribution of sugarcane cultivars in China[J]. Scientific Reports， 2015（5）： 15721.
[3] SHEN WK， JIANG ZD， DENG HH， et al. Research progress on sugarcane smut disease and Sporisorium scitaminea[J]. Chinese Journal of Tropical Crops， 2013， 34（10）： 2063-2068.
[4] LU WH， LI WF， HUANG YK. Research advances on sugarcane smut disease occurrence and control[J]. Sugar Crops of China， 2008（3）： 64-66.
[5] NING L， PANG XH， WANG Y， et al. Occurrence and control measures of sugarcane smut in Guangxi[J]. Agricultural Research and Application， 2018（2）： 40-42.
[6] XU LP， CHEN RK. Current situation and prospect of sugarcane smut and breeding of resistance sugarcane varieties[J]. Fujian Journal of Agricultural Sciences， 2000（2）： 26-31.
[7] ZHU GN， LI SY， XIAN XY， et al. Field control test of 5 fungicides to sugarcane smut[J]. Guangxi Agricultural Sciences， 2014， 45（8）：1393-1397.
[8] SHEN WK. Sugarcane disease in Guangdong sugarcane regions in recent years and integrated controlling methods[J]. Sugarcane and Canesugar， 2004（1）：1-5.
[9] ZHANG GY. Isolation， screening and identification of antagonistic bacteria against Ustilago scitaminea Sydow and studies on their antagonistic mechanisms[D]. Nanning： Guangxi University， 2002.
[10] XIONG GR， ZHAO GF， WU SR， et al. Screening and identification of a biocontrol strain， HAS antagonistic to Sporisorium scitaminea Syd[J]. Chinese Journal of Tropical Crops， 2013， 34（6）：1149-1154.
[11] XIONG GR， ZHAO GF， WANG WZ， et al. Colonization ability of Bacillus subtilis HAS in sugarcane[J]. Plant Diseases and Pests， 2018， 9（1）： 26-28.
[12] XIONG GR， ZHAO GF， ZHANG SZ， et al. Cloning and analysis of chitinase gene in Bacillus subtilis HAS[J]. Guizhou Agricultural Sciences， 2017， 45（2）： 1-4.
[13] XIONG GR， ZHAO GF， CAO WW， et al. Cloning and analysis of TasA in Bacillus subtilis HAS[J]. Guizhou Agricultural Sciences， 2016， 44（12）：53-57.