PnKTI基因在转基因拟南芥中的抗虫及功能分析
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摘要
Kunitz型胰蛋白酶抑制剂(Kunitz-type trypsin inhibitor,KTI)属于蛋白酶抑制剂(protease inhibitor,PI)家族中的丝氨酸蛋白酶抑制剂.本研究选择黑杨(Poplusnigra,Pn)中4个可被机械创伤诱导的KTI基因(PnKTIA6、PnKTIB1、PnKTIC1、PnKTID2),在拟南芥中过表达并以转基因拟南芥叶片饲喂小菜蛾,观察其从幼虫到成蛾的整个生长发育周期生理指标,从而检测4种PnKTI基因的抗虫效果.结果发现,4种PnKTI基因的抗虫效果依次为35,S:PnKTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:Pn KTIC1,其中,过表达PnKTIA6基因的拟南芥叶片饲喂的小菜蛾存活率最低,仅为9.8%,.此外,采用酶化学法测定分析了PnKTIs基因编码产物的功能及效果.结果发现,PnKTIs基因的编码产物对胰凝乳蛋白酶没有抑制作用,但对胰蛋白酶有很强的抑制作用,作用效果依次为35,S:PnKTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:Pn KTIC1,与小菜蛾抗性测定结果一致.
The Kunitz-type trypsin inhibitor(KTI) is a serine protease inhibitor that belongs to the protease inhibitor(PI)family. In this study,we selected four KTI genes(PnKTIA6,PnKTIB1,PnKTIC1,and PnKTID2) induced by mechanical trauma in Populus nigra,which were overexpressed in Arabidopsis thaliana that fed on Plutella xylostella to observe the physiological indicators of the entire growth and development cycle from larvae to adult moths and analyze their anti-insect effects. The results showed that the anti-insect effects of the four PnKTI genes were in the order 35,S:Pn KTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:PnKTIC1. Among them,the survival rate of Plutella xylostella fed with Arabidopsis thaliana over-expressing PnKTIA6 was only 9.8%. We also applied enzymatic chemistry to determine the function and effect of the PnKTI gene-encoding product,which showed that the PnKTI encoding product had no inhibitory effect on chymotrypsin,but had a strong inhibitory effect on trypsin. The effects were in the order 35,S:PnKTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:PnKTIC1,which were consistent with the result of Plutella xylostella resistance determination.
The Kunitz-type trypsin inhibitor(KTI) is a serine protease inhibitor that belongs to the protease inhibitor(PI)family. In this study,we selected four KTI genes(PnKTIA6,PnKTIB1,PnKTIC1,and PnKTID2) induced by mechanical trauma in Populus nigra,which were overexpressed in Arabidopsis thaliana that fed on Plutella xylostella to observe the physiological indicators of the entire growth and development cycle from larvae to adult moths and analyze their anti-insect effects. The results showed that the anti-insect effects of the four PnKTI genes were in the order 35,S:Pn KTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:PnKTIC1. Among them,the survival rate of Plutella xylostella fed with Arabidopsis thaliana over-expressing PnKTIA6 was only 9.8%. We also applied enzymatic chemistry to determine the function and effect of the PnKTI gene-encoding product,which showed that the PnKTI encoding product had no inhibitory effect on chymotrypsin,but had a strong inhibitory effect on trypsin. The effects were in the order 35,S:PnKTIA6>35,S:PnKTID2>35,S:PnKTIB1>35,S:PnKTIC1,which were consistent with the result of Plutella xylostella resistance determination.
引文
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[14]王震,乔天磊,霍乃蕊,等.植物叶蛋白提取及应用前景[J].山西农业科学,2015,43(12):1727-1731.Wang Zhen,Qiao Tianlei,Huo Nairui,et al.Extraction and application prospects of plant leaf protein[J].Shanxi Journal of Agricultural Sciences,2015,43(12):1727-1731(in Chinese).
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[16]Wang H L,Swain E W,Wallen L L,et al.Free fatty acids as antitryptic factor in soybeans formulated by Rhizopus oligosporus[J].Journal of Nutrition,1975,105(10):1351-1355.
[17]Wirnt R.Chymotrypsin measurements with N-benzoyl-L-tyrosin methyl ester as substrate[C]//Methods of Enzymatic Analysis.New York:Academic Press,1974:1009-1012.
[18]Yeh K W,Lin M I,Tuan S J,et al.Sweet potato trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera Litura[J].Plant Cell Reports,1997,16(10):696-699.
[19]Broadway R M.Are insects resistant to plant proteinase inhibitors?[J].Journal of Insect Physiology,1995,41(2):107-116.
[20]Yang L,Fang Z,Dicke M,et al.The diamondback moth,Plutella xylostella,specifically inactivates mustard trypsin inhibitor 2(MTI2)to overcome host plant defence[J].Insect Biochem Mol Biol,2009,39(1):55-61.
[2]Herrero S,Yolanda B,Patriciav H M,et al.Susceptibility,mechanisms of response and resistance to Bacillus thuringiensis toxins in Spodoptera spp[J].Current Opinion in Insect Science,2016,15(6):89-96.
[3]Xu L N,Wang Z Y,Zhang J,et al.Cross-resistance of Cry1Ab-selected Asian corn borer to other Cry toxins[J].Journal of Applied Entomoloy,2010,134(5):429-438.
[4]Laskowski K.Protein inhibitors of proteinases[J].Annu Rev Biochem,1980,49(7):593-626.
[5]Lingaraju M H,Gowda L R.A Kunitz trypsin inhibitor of Entada scandens seeds:Another member with single disulfide bridge[J].Biochim Biophys Acta,2008,1784(5):850-855.
[6]Mc Manus M T,Laing W A,Watson L M,et al.Expression of the soybean(Kunitz)trypsin inhibitor in leaves of white clover(Trifolium repens L)[J].Plant Science,2005,168(5):1211-1220.
[7]罗玉娇,李滨,舒衡平,等.Kunitz型胰蛋白酶抑制剂的研究进展[J].中国生化药物杂志,2012,33(3):316-319.Luo Yujiao,Li Bing,Shu Hengping,et al.Research progress of Kunitz trypsin inhibitors[J].Chinese Journal of Biochemical Pharmaceutics,2012,33(3):316-319(in Chinese).
[8]Hartl M,Giri A P,Kaur H,et al.Serine protease inhibitors specifically defend solanum nigrum against generalist herbivores but do not influence plant growth and development[J].Plant Cell,2010,22(12):4158-4175.
[9]江汉民,宋文芹,刘莉莉,等.抗虫相关基因KTI对青花菜的转化及其对小菜蛾抗性的分析[J].园艺学报,2013,40(3):498-504.Jiang Hanmin,Song Wenqin,Liu Lili,et al.Transformation of Brassica oleracea and its resistance to Plutella xylostella with resistance gene KTI[J].Acta Horticulturae Sinica,2013,40(3):498-504(in Chinese).
[10]Ma Y,Zhao Q,Lu M Z,et al.Kunitz-type trypsin inhibitor gene family in Arabidopsis and Populus trichocarpa and its expression response to wounding and herbivore in Populus nigra[J].Genetics&Genomes,2011,7(7):431-441.
[11]畅欣.KTI基因家族在模式植物中的抗虫机制[D].天津:天津大学化工学院,2011.Chang Xin.Insecticidal Mechanism of KTI Gene Family in Model Plants[D].Tianjin:School of Chemical Engineering and Technology,Tianjin University,2011(in Chinese).
[12]Giri A P,Harsulkar A M,Ku M S,et al.Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds[J].Phytochemistry,2003,63(7):523-532.
[13]赵爱平,孙聪,展恩玲,等.蛋白酶抑制剂对梨小食心虫幼虫中肠蛋白酶活性的影响[J].昆虫学报,2016,59(10):1069-1078.Zhao Aiping,Sun Cong,Zhan Enling,et al.Effect of protease inhibitor on midgut protease activity in pear borer Larvae[J].Journal of Entomology,2016,59(10):1069-1078(in Chinese).
[14]王震,乔天磊,霍乃蕊,等.植物叶蛋白提取及应用前景[J].山西农业科学,2015,43(12):1727-1731.Wang Zhen,Qiao Tianlei,Huo Nairui,et al.Extraction and application prospects of plant leaf protein[J].Shanxi Journal of Agricultural Sciences,2015,43(12):1727-1731(in Chinese).
[15]Goldring J.Spectrophotometric methods to determine protein concentration[C]//Methods in Molecular Biology.New York:Humana Press,2015:41-47.
[16]Wang H L,Swain E W,Wallen L L,et al.Free fatty acids as antitryptic factor in soybeans formulated by Rhizopus oligosporus[J].Journal of Nutrition,1975,105(10):1351-1355.
[17]Wirnt R.Chymotrypsin measurements with N-benzoyl-L-tyrosin methyl ester as substrate[C]//Methods of Enzymatic Analysis.New York:Academic Press,1974:1009-1012.
[18]Yeh K W,Lin M I,Tuan S J,et al.Sweet potato trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera Litura[J].Plant Cell Reports,1997,16(10):696-699.
[19]Broadway R M.Are insects resistant to plant proteinase inhibitors?[J].Journal of Insect Physiology,1995,41(2):107-116.
[20]Yang L,Fang Z,Dicke M,et al.The diamondback moth,Plutella xylostella,specifically inactivates mustard trypsin inhibitor 2(MTI2)to overcome host plant defence[J].Insect Biochem Mol Biol,2009,39(1):55-61.