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Abstract SlMC7 prokaryotic expression recombinant protein was used as test materials. SlMC7 polyclonal antiserum was obtained from immunized mice by using purified SlMC7 recombinant protein from Escherichia coli. ELISA and protein dot plot methods were used to detect the antibody titer and expression characteristics and the expression difference at different stages， providing material foundation and reference basis for subsequent SlMC7 functional research in tomato fruit. The results showed that the titer of SlMC7 polyclonal antibodies was more than 1∶256 000 and the sensitivity of SlMC7 polyclonal antibodies was 1.6 ng at the dilutability of 1∶10 000. SlMC7 protein was detected at different stages， and SlMC7 had a higher expression level in color-break fruit， indicating that SlMC7 might be relevant to the fruit ripening of tomato.
Key words Metacaspase； Antibody； Protein expression； Tomato
Cysteine aspartic acid specific caspases are a class of proteases found in animal cells， very conserved evolutionarily[1-3]. With the help of structural analysis of protein sequence and genomic sequencing technology， Uren et al.[4] found a class of proteins sharing sequential structure homology with caspases in plants， fungi and protozoa in 2000， which were designated as metacaspases （MCs）. MCs are a class of cysteine proteases existing in plants[5]， fungi[6] and protozoa[7-8]. MCs are divided into type I and type II according to structural and sequential similarity[9].
In recent years， researches show that MCs play an important role in physiological processes including cell apoptosis， autophagy， cell differentiation and disease resistance. Most studies are concentrated on Arabidopsis thaliana. BOLLHONER et al.[10] found that type II MC AtMC9 of A. thaliana plays an important role in apoptosis of xylem， and it could participate in the degradation of cellular content after the burst of vacuoles in duct wall cells. As to type I MC of A. thaliana， Coll et al.[11] found that AtMC1 has a function similar to autophagy， i.e.， MCs and autophagy both have the function of triggering programmed cell death （PCD）. In seedlings， they regulate the death of hypersensitive reaction （HR） type of cells， and alleviate the aging of mature plants.
Except A. thaliana， studies about corresponding functions of MCs in other species were also conducted. During the resistance of Nicotiana benthamiana against Colletotrichum destructivum， scholars found that type II metacaspase gene NbMCA1-scilenced plant exhibited aggravated lesion after the infection of C. destructivum， but no above change after the infection of P. syringae[12]， indicating that tobacco NbMCA1 plays different roles in different disease reactions. Minina et al.[13] found that Picea abies mcII-Pa is a kind of key protease related to death of suspensor cells， and cell autophagy involving in the death process of suspensor cells of P. abies is closely related to type II MC mcII-Pa. The activation process of autophagy is located downstream of mcII-Pa， and after the knockout of mcII-Pa gene， cell autophagy and body cell development related to it is terminated. There are six kinds of type I and two kinds of type II MCs in tomato. The research on MCs in tomato shows that the infection of tomato leaves with Botrytis cinerea Pers. could induce the up-regulation of the expression level of type II MC （SlMC7）[2]. The studies in recent years also show that in-vitro recombinant tomato type II MC （SlMC7） exhibits self-hydrolysis self-activation phenomenon[14]. The research on MCs as a class of proteins having similar structure to the key factor caspase family of animal PCD is still in its infancy. At present， the research on tomato type II metacaspase is also just started， and there were few studies at home and abroad. Though the expression of MC gene changes in the development and stress responses of plant， MC antibodies should be acquired， so as to investigate whether change happens at protein level. In this study， on the basis of type II tomato MC （SlMC7）， prokaryotically-expressed SlMC7 protein was induced and purified， SlMC7 polyclonal antibodies were prepared， and the expression differences during the development process of SlMC7 in tomato was also preliminarily studied. This study would provide material basis and reference for the study on subsequent SlMC7 functional research in tomato fruits.
Materials and Methods
Experimental materials
The tested tomato variety was "Zhongshu No. 4" （Lycopersicon esculentum cv. zhongshu）； and Escherichia coli DH5α， and prokaryotic expression vector pET28a-LeM of tomato metacaspase gene SlMC7 were provided by Food Biotechnology Laboratory of College of Food Science and Nutritional Engineering， China Agricultural University. Second antibody and Rosetta were purchased from TransGen Biotech. M-MLV reverse transcriptase was purchased from Promega company. Bio-Radreal-time fluorescence quantitative PCR instrument was adopted. The tested mice were provided by Institute of Genetics and Developmental Biology， Chinese Academy of Sciences.
Experimental methods
Prokaryotic expression and purification of SlMC7 protein
With reference to the method of Ma et al.[15]， the prokaryotic expression vector pET28a-LeM （C139） carrying SlMC7 gene with point mutation was transformed into strain Rosetta， and induced with 1 mmol/L IPTG at 37 ℃. The bacteria were subjected to ultrasonic decomposition， and purified protein was obtained by affinity chromatography according to the kit produced by GE company.
Preparation and detection of SlMC7 polyclonal antibodies Preparation of antibodies： The purified recombinant protein was transported to Institute of Genetics and Developmental Biology， Chinese Academy of Science for the preparation of SlMC7 polyclonal antibodies. Seven mice were injected with 200 μg of pure recombinant protein， respectively， and polyclonal antiserum was obtained and detected. Blank control was also set.
Enzyme-linked immunosorbent assay （ELISA）： Known antibodies were diluted with 0.05 mol/L carbonate coating buffer （pH 9.6） to 1， 2， 3， 4， 5， 6， 7， 8， 9 and 10 μg/ml. Then， 0.1 ml of corresponding solution was added into each reaction hole of polystyrene plate， and stood at 4 ℃ overnight. Next day， the solution was discarded， and the holes were closed with 3% bovine serum albumin and washed. Certain diluted to-be-detected sample （unknown antibodies， 0.1 ml） was added into each of above coated reaction holes， and incubated at 37 ℃ for 1 h， and 0.1 ml of second antibody was then added， followed by incubation and washing. A substrate solution was added to perform development， during which 0.1 ml of TMB substrate solution prepared temporarily was added into each reaction hole， and incubation was performed at 37 ℃ for 10-30 min. Into each reaction hole， 0.05 ml of 2 mol/L sulfuric acid was added to terminate the reaction. Finally， on an ELISA detector， at 450 nm， the OD value of each hole was detected after zero setting with blank as control， and if the OD value is higher than 2.1 times of that of the negative control， it is judged as positive.
Protein dot blot： The protein dot blot was carried out with reference to the method of Ma et al.[16]. Nitrocellulose membrane （0.45 μm） was soaked in TBST buffer solution， air-dried and lightly pressed on ELISA plate， exhibiting sample holes. The purified 139 site-mutated SlMC7 fusion protein was directly added to the membrane as antibodies. The sample was diluted with 50 mmol/L （pH 9.6） beforehand， to ensure that that the protein contents in the holes were 51.2， 25.6， 12.8， 6.4， 3.2， 1.6， 0.8， 0.4， 0.2 and 0.1 ng， respectively. According to Western-blot method， closure and detection were performed. In this experiment， the dilution of the used antibodies was 1∶10 000.
Extraction and Western-blot analysis of SlMC7 protein from tomato fruit
Extraction of SlMC7 protein： At first， 0.5 g of powder of fruit sample was mixed with 1.5 ml of 10% trichloroacetic acid uniformly. Centrifugation was performed at 15 000 r/min under low temperature， and the supernatant was discarded. The product was mixed with 0.1 mol/L ammonium acetate 80% methanol solution. Centrifugation was then performed， and the precipitate was washed with 80% acetone. Phenol/SDS solution （volume ratio of tris-phenol to SDS at 1∶1） and 0.1 mol/L ammonium acetate 80% methanol solution was stood at -20 ℃ overnight， and the supernatant was discarded. The product was re-suspended with 100 μl of SDS buffer. The protein concentration was determined by Bradford bovine serum albumin. Western-blot analysis： At first， 5 μg of denatured protein was loaded to perform SDS-PAGE electrophoresis， which was carried out at 80 V for 30 min and 120 V for 80 min. The separated protein was transferred to 0.45 μm nitrocellulose membrane by electrophoretic transfer method. Then， closure was performed with 5% skim milk powder at room temperature for 2 h. Incubation was performed after the addition of primary antibody overnight， and after the addition of second antibody for 2 h. After ECL luminescence， development was performed in a dark room， and protein bands were analyzed. The results of Western-blot were obtained by scanning with Quantity one for optical density values. Analysis of significance of difference was performed on data using SPSS 17.0. P<0.05 indicates a significant difference， and P<0.01 indicates a very significant difference.
RNA extraction from tomato fruit and analysis of expression level of SlMC7
RNA extraction from tomato fruit： Ground sample powder was transferred to a 10 ml centrifuge tube pre-cooled with liquid nitrogen， and 4 ml of Trizol extractant （temporarily added with 2% β-mercaptoethanol） was then added， followed by vortex oscillation and standing at room temperature for 10 min. Then， into each tube， 2 ml of chloroform/isoamylol （49∶1） and 360 μl of anhydrous ethanol was added， and the solution was mixed rapidly and stood at room temperature for 2 min. Centrifugation was performed at low temperature. The supernatant was added with 4 ml of anhydrous ethanol and 200 μl of sodium acetate （3 mol/L， pH 5.2）， and precipitation was allowed for 1 h after mixing well （-20 ℃）. Centrifugation was performed again at low temperature， and the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol， the suspension was centrifugated， and the supernatant was discarded. The product was dissoved in a proper amount of DEPC treatment water， and the quality of the RNA sample was detected by 1% agarose gel electrophoresis. The RNA samples with bright clear sharp 28S and 18S bands （having clear edges） having the brightness of the 28S band about 2 times of that of the 18S band were asserted as having good quality. The qualified RNA samples were subpackaged and stored at -80 ℃， to avoid multigelation.
Analysis of expression level of SlMC7： The RNA of tomato tissue was subjected to reverse transcription， followed by real-time quantificative PCR. The amplification system included 2.5×Real Master Mix/20×SYBR solution 11.25 μl， forward primer （10 μmol/L） 0.5 μl， reverse frimer （10 μmol/L） 0.5 μl， template cDNA 1 μl， and higher purity water 11.75 μl. The primers of refrence gene of tomato （ubi-3 gene， Accession No.X58253） were designed and synthesized by Sangon Biotech （Shanghai） Co.， Ltd. The primers were as follows： ubi3 sense： 5′-GAAGACCTACACCAAGCCAAAGA-3′； ubi3 antisense： 5′-ACTCCTTACGAAGCCTCTGAAC-3′. The real-time quantificative primers of tomato LeMCA1 gene were designed as follows： LeM-Rtsense：5′-CAAGCAAGGTGATGACGATGAAGG-3′； LeM-RTantisense： 5′-ATGAGAGTGGAAAGAGGCAAGGAC-3′. The amplification was started with pre-denaturation at 95 ℃ for 2 min， followed by 40 cyles of 95 ℃ for 15 s， 60 ℃ for 30 s and 68 ℃ for 30 s. The melting curve was analyzed at 60-95 ℃. Each cDNA sample was detected for 3 times in parallel. Agricultural Biotechnology2018
Results and Analysis
Preparation and detection of SlMC7 polyclonal antibodies
Mice were immunized with the recombinant protein， and mice serum was collected. The titer and sensitivity index of antibodies were detected by ELISA and protein dot blot methods. It could be seen from Fig. 1 that with the dilution times of antibodies increasing， the relative times of OD value decreased gradually. When the dilution times of antibodies reached 256 000， the reaction was still detected as positive. It indicated that the titer of the SlMC7 polyclonal antibodies is higher than 1∶256 000.
The sensitivity of polyclonal antibodies was detected under the antibody concentration of 1∶10 000. As shown in Fig. 2， with the decrease of the quantity of pure recombinant protein， the sensitivity signal of the polyclonal antibodies showed a decreasing trend， and the lower limit of the detection signal was reached at 1.6 ng. It was indicated that the sensitivity of the SlMC7 polyclonal antibodies could reach 1.6 ng under the dilution times of 1∶10 000.
Changes in the expression of tomato SlMC7 gene and protein
It could be seen from Fig. 3 that the quantity of the total RNA was very high after extraction and digestion， and the ratio of 28S band to 18S band was not remarkable， but the total RNA was not degraded， and the OD260/OD280 ratio was in the range of 1.8-2.0 （data not shown）. It was indicated that the total RNA suffered from less pollution of protein and other organic reagents， and could be used in followed-up test.
The results of Western-blot analysis （Fig. 4A and Fig. B）， SlMC7 protein had the lowest expression level in peel of tomato fruit at green ripe stage， which was significantly different from those in the tomato fruit at color-break stage， pink stage and red ripe stage （P<0.05）， and the expression level in the tomato fruit at pink stage was the highest. The results of real-time PCR （Fig. 4C） showed that SlMC7 had the lowest expression level in tomato fruit at green ripe stage， and the highest level at color-break stage. The results of Western-blot and real-time PCR showed that the expression level of SlMC7 in the ripening process of tomato had substantially the same trend， and compared with green ripe stage， the expression level of SlMC7 protein significantly increased at color-break stage and pink stage. It is speculated that SlMC7 protein might have certain correlation with the ripening of tomato. Conclusions and Discussion
In recent years， the research on the function of plant metacaspase has become a hot field in plant science， while for tomato as an important model plant， the research on its metacaspase is still in its infancy， and the study on the function of tomato metacaspase will obviously have important significance. In this study， on the basis of type II tomato MC SlMC7 gene， 139 site-mutated tomato SlMC7 protein prokaryotically expressed was induced and purified， SlMC7 polyclonal antibodies were prepared， and titer analysis was performed. The differential expression of SlMC7 during the development process of tomato fruit was preliminarily discussed. A good foundation is laid through the analysis of the expression of tomato SlMC7 protein for the study on the acquisition of protein acting with in-vivo SlMC7 by co-immunoprecipitation using antibodies.
Western-blot and real-time PCR detection of tomato fruit at different stages showed that in the fruit at the four stages， SlMC7 gene was expressed， and the expression level of SlMC7 was relatively lower at green ripe stage， which was significantly different from color-break stage， pink stage and red ripe stages. At transcriptional level， the expression level reached a peak at color-break stage， and decreased at pink stage until red ripe stage. At protein level， the expression of SlMC7 showed a peak at pink stage and decreased at red ripe stage， the expression peak was delayed related to transcription level， but the overall trend did not change. It speculated that SlMC7 might have certain correlation with the ripening of tomato fruit.
MCs are similar to the key protease caspase in apoptosis of animal cells in protein structure， and then， whether MCs play a certain function in PCD needs further study. Hoeberichts et al.[16]showed that the expression level of tomato SlMC7 was up-regulated in PCD induced by B. cinerea Pers.， indicating that SlMC7 might participated in PCD induced by B. cinerea. Suarez et al.[17]reported that during the somatic embryogenesis of P. abies， its MC （mcll-Pa） is transferred from cytoplasm to cell nucleus and finally make cells differentiate， indicating that MCs are the executor of PCD in somatic embryogenesis of P. abies. It could be seen that MCs might play an important role in plant growth and development process or the defense against diseases. In this study， SlMC7 is type II MC， and according to the alignment results with protein sequences， SlMC7 protein shares higher homology with metacaspase 4 in A. thaliana and metacaspase of P. abies， and the function of SlMC7 in the development and stress of tomato fruit will be studied in subsequent experiments. References
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[2] LOS M， STROH C， JANICKE RU， et al. Caspase： more than just killers[J]. Trends Immunol，2001， 22： 31-34.
[3] THORNBERRY NA， LAZEBNIK Y. Caspases： enemies within[J]. Science， 1998， 281： 1312-1316.
[4] UREN AG， O’ROURKE K， ARAVIND L， et al. Identification of paracaspases and metacaspases： Two ancient families of caspase-like proteins， one of which plays a key role in MALT lymphoma[J]. Mol Cell， 2000， 6（4）： 961-967.
[5] VERCAMMEN D， van DE COTTE B， de JAEGER G， et al. Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine[J]. J Biol Chem， 2004， 279： 45329-45336.
[6] MADEO F， HERKER E， MALDENER C， et al. A caspase-related protease regulates apoptosis in yeast[J]. Mol Cell 2002， 9： 911-917.
[7] SZALLIES A， KUBATA B K， DUSZENKO M. A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae[J]. FEBS Lett， 2002， 517： 144-150.
[8] MOTTRAM JC， HELMS MJ， COOMBS GH， et al. Clan CD cysteine peptidases of parasitic protozoa[J]. Trends Parasitol， 2003， 19： 182-187.
[9] TRZYNA WC， LEGRAS XD， CORDINGLEY JS. A type-1 metacaspase from Acanthamoeba castellanii[J]. Microbiol Res， 2008， 163： 414-423.
[10] BOLLHONER B， ZHANG B， STAEL S， et al. Post mortemfunction of AtMC9 in xylem vessel elements[J]. New Phyto， 2013， 200： 498-510.
[11] COLL N S， VERCAMMEN D， SMIDLER A， et al. Arabidopsis type I metacaspases control cell death[J]. Science， 2010， 330： 1393-1397.
[12] BASKETT JA. A type II metacaspase interacts with rps1-k-2 in soybean and analysis of the soybean metacaspase gene family[D]. 2012， Ames， Iowa State University.
[13] MININA E， LADA H， KAZUTAKE F， et al. Autophagy and metacaspase determine the mode of cell death in plants[J]. J Cell Biol， 2013， 203： 917-927.
[14] LIU H， LIU J， WEI YX. Identification and analysis of the metacaspase gene family in tomato[J]. Biochem Bioph Res C， 2016， 479： 523-529.
[15] MA QM， ZHANG YL， LIU JN， et al. Cloning of Lycopersicon esculentum metacaspase gene （Le M） and its effective expression in Escherichia coli Rosetta[J]. Chinese journal of agricultural biotechnology， 2011， 19（2）： 258-262.
[16] HOEBERICHTS F， HAVE A， WOLTERING E. A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea-infected leaves[J].Planta，2003， 217（3）： 517-522.
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Key words Metacaspase； Antibody； Protein expression； Tomato
Cysteine aspartic acid specific caspases are a class of proteases found in animal cells， very conserved evolutionarily[1-3]. With the help of structural analysis of protein sequence and genomic sequencing technology， Uren et al.[4] found a class of proteins sharing sequential structure homology with caspases in plants， fungi and protozoa in 2000， which were designated as metacaspases （MCs）. MCs are a class of cysteine proteases existing in plants[5]， fungi[6] and protozoa[7-8]. MCs are divided into type I and type II according to structural and sequential similarity[9].
In recent years， researches show that MCs play an important role in physiological processes including cell apoptosis， autophagy， cell differentiation and disease resistance. Most studies are concentrated on Arabidopsis thaliana. BOLLHONER et al.[10] found that type II MC AtMC9 of A. thaliana plays an important role in apoptosis of xylem， and it could participate in the degradation of cellular content after the burst of vacuoles in duct wall cells. As to type I MC of A. thaliana， Coll et al.[11] found that AtMC1 has a function similar to autophagy， i.e.， MCs and autophagy both have the function of triggering programmed cell death （PCD）. In seedlings， they regulate the death of hypersensitive reaction （HR） type of cells， and alleviate the aging of mature plants.
Except A. thaliana， studies about corresponding functions of MCs in other species were also conducted. During the resistance of Nicotiana benthamiana against Colletotrichum destructivum， scholars found that type II metacaspase gene NbMCA1-scilenced plant exhibited aggravated lesion after the infection of C. destructivum， but no above change after the infection of P. syringae[12]， indicating that tobacco NbMCA1 plays different roles in different disease reactions. Minina et al.[13] found that Picea abies mcII-Pa is a kind of key protease related to death of suspensor cells， and cell autophagy involving in the death process of suspensor cells of P. abies is closely related to type II MC mcII-Pa. The activation process of autophagy is located downstream of mcII-Pa， and after the knockout of mcII-Pa gene， cell autophagy and body cell development related to it is terminated. There are six kinds of type I and two kinds of type II MCs in tomato. The research on MCs in tomato shows that the infection of tomato leaves with Botrytis cinerea Pers. could induce the up-regulation of the expression level of type II MC （SlMC7）[2]. The studies in recent years also show that in-vitro recombinant tomato type II MC （SlMC7） exhibits self-hydrolysis self-activation phenomenon[14]. The research on MCs as a class of proteins having similar structure to the key factor caspase family of animal PCD is still in its infancy. At present， the research on tomato type II metacaspase is also just started， and there were few studies at home and abroad. Though the expression of MC gene changes in the development and stress responses of plant， MC antibodies should be acquired， so as to investigate whether change happens at protein level. In this study， on the basis of type II tomato MC （SlMC7）， prokaryotically-expressed SlMC7 protein was induced and purified， SlMC7 polyclonal antibodies were prepared， and the expression differences during the development process of SlMC7 in tomato was also preliminarily studied. This study would provide material basis and reference for the study on subsequent SlMC7 functional research in tomato fruits.
Materials and Methods
Experimental materials
The tested tomato variety was "Zhongshu No. 4" （Lycopersicon esculentum cv. zhongshu）； and Escherichia coli DH5α， and prokaryotic expression vector pET28a-LeM of tomato metacaspase gene SlMC7 were provided by Food Biotechnology Laboratory of College of Food Science and Nutritional Engineering， China Agricultural University. Second antibody and Rosetta were purchased from TransGen Biotech. M-MLV reverse transcriptase was purchased from Promega company. Bio-Radreal-time fluorescence quantitative PCR instrument was adopted. The tested mice were provided by Institute of Genetics and Developmental Biology， Chinese Academy of Sciences.
Experimental methods
Prokaryotic expression and purification of SlMC7 protein
With reference to the method of Ma et al.[15]， the prokaryotic expression vector pET28a-LeM （C139） carrying SlMC7 gene with point mutation was transformed into strain Rosetta， and induced with 1 mmol/L IPTG at 37 ℃. The bacteria were subjected to ultrasonic decomposition， and purified protein was obtained by affinity chromatography according to the kit produced by GE company.
Preparation and detection of SlMC7 polyclonal antibodies Preparation of antibodies： The purified recombinant protein was transported to Institute of Genetics and Developmental Biology， Chinese Academy of Science for the preparation of SlMC7 polyclonal antibodies. Seven mice were injected with 200 μg of pure recombinant protein， respectively， and polyclonal antiserum was obtained and detected. Blank control was also set.
Enzyme-linked immunosorbent assay （ELISA）： Known antibodies were diluted with 0.05 mol/L carbonate coating buffer （pH 9.6） to 1， 2， 3， 4， 5， 6， 7， 8， 9 and 10 μg/ml. Then， 0.1 ml of corresponding solution was added into each reaction hole of polystyrene plate， and stood at 4 ℃ overnight. Next day， the solution was discarded， and the holes were closed with 3% bovine serum albumin and washed. Certain diluted to-be-detected sample （unknown antibodies， 0.1 ml） was added into each of above coated reaction holes， and incubated at 37 ℃ for 1 h， and 0.1 ml of second antibody was then added， followed by incubation and washing. A substrate solution was added to perform development， during which 0.1 ml of TMB substrate solution prepared temporarily was added into each reaction hole， and incubation was performed at 37 ℃ for 10-30 min. Into each reaction hole， 0.05 ml of 2 mol/L sulfuric acid was added to terminate the reaction. Finally， on an ELISA detector， at 450 nm， the OD value of each hole was detected after zero setting with blank as control， and if the OD value is higher than 2.1 times of that of the negative control， it is judged as positive.
Protein dot blot： The protein dot blot was carried out with reference to the method of Ma et al.[16]. Nitrocellulose membrane （0.45 μm） was soaked in TBST buffer solution， air-dried and lightly pressed on ELISA plate， exhibiting sample holes. The purified 139 site-mutated SlMC7 fusion protein was directly added to the membrane as antibodies. The sample was diluted with 50 mmol/L （pH 9.6） beforehand， to ensure that that the protein contents in the holes were 51.2， 25.6， 12.8， 6.4， 3.2， 1.6， 0.8， 0.4， 0.2 and 0.1 ng， respectively. According to Western-blot method， closure and detection were performed. In this experiment， the dilution of the used antibodies was 1∶10 000.
Extraction and Western-blot analysis of SlMC7 protein from tomato fruit
Extraction of SlMC7 protein： At first， 0.5 g of powder of fruit sample was mixed with 1.5 ml of 10% trichloroacetic acid uniformly. Centrifugation was performed at 15 000 r/min under low temperature， and the supernatant was discarded. The product was mixed with 0.1 mol/L ammonium acetate 80% methanol solution. Centrifugation was then performed， and the precipitate was washed with 80% acetone. Phenol/SDS solution （volume ratio of tris-phenol to SDS at 1∶1） and 0.1 mol/L ammonium acetate 80% methanol solution was stood at -20 ℃ overnight， and the supernatant was discarded. The product was re-suspended with 100 μl of SDS buffer. The protein concentration was determined by Bradford bovine serum albumin. Western-blot analysis： At first， 5 μg of denatured protein was loaded to perform SDS-PAGE electrophoresis， which was carried out at 80 V for 30 min and 120 V for 80 min. The separated protein was transferred to 0.45 μm nitrocellulose membrane by electrophoretic transfer method. Then， closure was performed with 5% skim milk powder at room temperature for 2 h. Incubation was performed after the addition of primary antibody overnight， and after the addition of second antibody for 2 h. After ECL luminescence， development was performed in a dark room， and protein bands were analyzed. The results of Western-blot were obtained by scanning with Quantity one for optical density values. Analysis of significance of difference was performed on data using SPSS 17.0. P<0.05 indicates a significant difference， and P<0.01 indicates a very significant difference.
RNA extraction from tomato fruit and analysis of expression level of SlMC7
RNA extraction from tomato fruit： Ground sample powder was transferred to a 10 ml centrifuge tube pre-cooled with liquid nitrogen， and 4 ml of Trizol extractant （temporarily added with 2% β-mercaptoethanol） was then added， followed by vortex oscillation and standing at room temperature for 10 min. Then， into each tube， 2 ml of chloroform/isoamylol （49∶1） and 360 μl of anhydrous ethanol was added， and the solution was mixed rapidly and stood at room temperature for 2 min. Centrifugation was performed at low temperature. The supernatant was added with 4 ml of anhydrous ethanol and 200 μl of sodium acetate （3 mol/L， pH 5.2）， and precipitation was allowed for 1 h after mixing well （-20 ℃）. Centrifugation was performed again at low temperature， and the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol， the suspension was centrifugated， and the supernatant was discarded. The product was dissoved in a proper amount of DEPC treatment water， and the quality of the RNA sample was detected by 1% agarose gel electrophoresis. The RNA samples with bright clear sharp 28S and 18S bands （having clear edges） having the brightness of the 28S band about 2 times of that of the 18S band were asserted as having good quality. The qualified RNA samples were subpackaged and stored at -80 ℃， to avoid multigelation.
Analysis of expression level of SlMC7： The RNA of tomato tissue was subjected to reverse transcription， followed by real-time quantificative PCR. The amplification system included 2.5×Real Master Mix/20×SYBR solution 11.25 μl， forward primer （10 μmol/L） 0.5 μl， reverse frimer （10 μmol/L） 0.5 μl， template cDNA 1 μl， and higher purity water 11.75 μl. The primers of refrence gene of tomato （ubi-3 gene， Accession No.X58253） were designed and synthesized by Sangon Biotech （Shanghai） Co.， Ltd. The primers were as follows： ubi3 sense： 5′-GAAGACCTACACCAAGCCAAAGA-3′； ubi3 antisense： 5′-ACTCCTTACGAAGCCTCTGAAC-3′. The real-time quantificative primers of tomato LeMCA1 gene were designed as follows： LeM-Rtsense：5′-CAAGCAAGGTGATGACGATGAAGG-3′； LeM-RTantisense： 5′-ATGAGAGTGGAAAGAGGCAAGGAC-3′. The amplification was started with pre-denaturation at 95 ℃ for 2 min， followed by 40 cyles of 95 ℃ for 15 s， 60 ℃ for 30 s and 68 ℃ for 30 s. The melting curve was analyzed at 60-95 ℃. Each cDNA sample was detected for 3 times in parallel. Agricultural Biotechnology2018
Results and Analysis
Preparation and detection of SlMC7 polyclonal antibodies
Mice were immunized with the recombinant protein， and mice serum was collected. The titer and sensitivity index of antibodies were detected by ELISA and protein dot blot methods. It could be seen from Fig. 1 that with the dilution times of antibodies increasing， the relative times of OD value decreased gradually. When the dilution times of antibodies reached 256 000， the reaction was still detected as positive. It indicated that the titer of the SlMC7 polyclonal antibodies is higher than 1∶256 000.
The sensitivity of polyclonal antibodies was detected under the antibody concentration of 1∶10 000. As shown in Fig. 2， with the decrease of the quantity of pure recombinant protein， the sensitivity signal of the polyclonal antibodies showed a decreasing trend， and the lower limit of the detection signal was reached at 1.6 ng. It was indicated that the sensitivity of the SlMC7 polyclonal antibodies could reach 1.6 ng under the dilution times of 1∶10 000.
Changes in the expression of tomato SlMC7 gene and protein
It could be seen from Fig. 3 that the quantity of the total RNA was very high after extraction and digestion， and the ratio of 28S band to 18S band was not remarkable， but the total RNA was not degraded， and the OD260/OD280 ratio was in the range of 1.8-2.0 （data not shown）. It was indicated that the total RNA suffered from less pollution of protein and other organic reagents， and could be used in followed-up test.
The results of Western-blot analysis （Fig. 4A and Fig. B）， SlMC7 protein had the lowest expression level in peel of tomato fruit at green ripe stage， which was significantly different from those in the tomato fruit at color-break stage， pink stage and red ripe stage （P<0.05）， and the expression level in the tomato fruit at pink stage was the highest. The results of real-time PCR （Fig. 4C） showed that SlMC7 had the lowest expression level in tomato fruit at green ripe stage， and the highest level at color-break stage. The results of Western-blot and real-time PCR showed that the expression level of SlMC7 in the ripening process of tomato had substantially the same trend， and compared with green ripe stage， the expression level of SlMC7 protein significantly increased at color-break stage and pink stage. It is speculated that SlMC7 protein might have certain correlation with the ripening of tomato. Conclusions and Discussion
In recent years， the research on the function of plant metacaspase has become a hot field in plant science， while for tomato as an important model plant， the research on its metacaspase is still in its infancy， and the study on the function of tomato metacaspase will obviously have important significance. In this study， on the basis of type II tomato MC SlMC7 gene， 139 site-mutated tomato SlMC7 protein prokaryotically expressed was induced and purified， SlMC7 polyclonal antibodies were prepared， and titer analysis was performed. The differential expression of SlMC7 during the development process of tomato fruit was preliminarily discussed. A good foundation is laid through the analysis of the expression of tomato SlMC7 protein for the study on the acquisition of protein acting with in-vivo SlMC7 by co-immunoprecipitation using antibodies.
Western-blot and real-time PCR detection of tomato fruit at different stages showed that in the fruit at the four stages， SlMC7 gene was expressed， and the expression level of SlMC7 was relatively lower at green ripe stage， which was significantly different from color-break stage， pink stage and red ripe stages. At transcriptional level， the expression level reached a peak at color-break stage， and decreased at pink stage until red ripe stage. At protein level， the expression of SlMC7 showed a peak at pink stage and decreased at red ripe stage， the expression peak was delayed related to transcription level， but the overall trend did not change. It speculated that SlMC7 might have certain correlation with the ripening of tomato fruit.
MCs are similar to the key protease caspase in apoptosis of animal cells in protein structure， and then， whether MCs play a certain function in PCD needs further study. Hoeberichts et al.[16]showed that the expression level of tomato SlMC7 was up-regulated in PCD induced by B. cinerea Pers.， indicating that SlMC7 might participated in PCD induced by B. cinerea. Suarez et al.[17]reported that during the somatic embryogenesis of P. abies， its MC （mcll-Pa） is transferred from cytoplasm to cell nucleus and finally make cells differentiate， indicating that MCs are the executor of PCD in somatic embryogenesis of P. abies. It could be seen that MCs might play an important role in plant growth and development process or the defense against diseases. In this study， SlMC7 is type II MC， and according to the alignment results with protein sequences， SlMC7 protein shares higher homology with metacaspase 4 in A. thaliana and metacaspase of P. abies， and the function of SlMC7 in the development and stress of tomato fruit will be studied in subsequent experiments. References
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