天山雪莲SikFBP1基因的抗寒性研究
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摘要
在植物体内,FBPase酶是卡尔文循环内一个不可缺少的酶,在干旱、高温、盐碱等非生物胁迫中FBPase同样起着重要的作用。本试验从典型的耐极端低温的植物天山雪莲(Saussurea involucrata Kar.ER Kir.)中克隆了一段FBPase序列,命名为SikFBP1,同时也克隆出了一段SBPase序列命名为SikSBP。实时荧光定量显示2个基因对寒冷胁迫都做出了响应,为验证其抗寒性功能分别转入了烟草中。通过RT-PCR验证获得转化植株,低温胁迫下,通过丙二醛(MDA)含量、相对电子渗透率(REL)、过超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、PSⅡ最大光化学效率(Fv/Fm)及光合效率(Pn)的测量,发现转SikFBP1基因株系与转SikSBP基因株系在光合效率上提高较为微弱,但在耐寒性方面得到了一定的提高,为FBPase的功能研究提供一定参考。
FBPase enzyme is an important enzyme in plant.Recent studies have shown that FBPase also plays an important role against abiotic stress such as high temperature and drought.In this study,aFBPase and a SBPase sequence were cloned fromSaussurea involucrata Kar.ER Kir.,a typical perennial alpine plant resistant to extreme low temperature,and was named as SikFBP1 and SikSBPrespectively.We found that both of them were responsive to low temperature by real-time fluorescence quantitative PCR.In order to verify the function of cold resistance of the sequence,SikFBP1 and SikSBPwere transferred into tobacco respectively.Transformed plants were obtained by RT-PCR verification.We also measured the content of malondialdehyde(MDA),relative electron transport(REL),superoxide dismutase(SOD)activity,catalase(CAT)activity,maximum photochemical efficiency(Fv/Fm)and photochemical efficiency(Pn).The results showed that compared with wild-type,the transgenic SikSBPplants and SikFBP1 plants were obviously improved in cold tolerance,but only slight increased in photosynthetic efficiency.
FBPase enzyme is an important enzyme in plant.Recent studies have shown that FBPase also plays an important role against abiotic stress such as high temperature and drought.In this study,aFBPase and a SBPase sequence were cloned fromSaussurea involucrata Kar.ER Kir.,a typical perennial alpine plant resistant to extreme low temperature,and was named as SikFBP1 and SikSBPrespectively.We found that both of them were responsive to low temperature by real-time fluorescence quantitative PCR.In order to verify the function of cold resistance of the sequence,SikFBP1 and SikSBPwere transferred into tobacco respectively.Transformed plants were obtained by RT-PCR verification.We also measured the content of malondialdehyde(MDA),relative electron transport(REL),superoxide dismutase(SOD)activity,catalase(CAT)activity,maximum photochemical efficiency(Fv/Fm)and photochemical efficiency(Pn).The results showed that compared with wild-type,the transgenic SikSBPplants and SikFBP1 plants were obviously improved in cold tolerance,but only slight increased in photosynthetic efficiency.
引文
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[22]Xiao H D,Chen C S,Xu Y,et al.Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene fromPyropia haitanensis[J].Acta Oceanologica Sinica,2014,33(4):92-100.
[23]Chueca A,Sahrawy M,Pagano E A,et al.Chloroplast fructose-1,6-bisphosphatase:structure and function.[J]Photosynthesis Research,2002,74:235-249.
[24]Asada K.Production and scavenging of reactive oxygen species in chloroplasts and their functions[J].Plant Physiology,2006,141:391-396.
[25]Woodrow I E,Berry J A.Enzymatic regulation of photosynthetic CO2fixation in C3plants[J].Annual Reviev of Plant Physiology and Plant Molecular Biology,1988,39:533-594.
[2]Cséke C,Balogh A,Wong J H,et al.Fructose 2,6-bisphosphate.A regulator of carbon processing in leaves[J].Trends in Biochemical Sciences,1984,9:533-535.
[3]Gibson J L,Chen J H,Tower P A et al.The form-IIfructose-1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides.Primary structure and insertional mutagenesis analysis[J].Biochemistry,1990,29:8085-8093.
[4]Grotjohann N.Fructose 1,6-bisphosphatase in Chlorella kessleri grown in red or blue light[J].Zeitschrift Fur Naturforschung,1993,48c:707-712.
[5]Brown D,Kershaw K A.Seasonal changes in the kinetic parameters of a photosynthetic fructose-1,6-bisphosphatase isolated from Peltigera rufescens[J].Plant Physiology,1986,82:457-461.
[6]Shi H T,Jiang C,Ye T T,et al.Comparative physiological,metabolomic,and transcriptomic analyses revealmechanisms of improved abiotic stress resistance in bermudagrass(Cynodond dctylon(L.)Pers.)by exogenous melatonin[J].Journal of Experimental Botany,2015,56:1064-1079.
[7]Gururani M A,Venkatesh J,Tran L P.Regulation of photosynthesis duringabiotic stress-induced photoinhibition[J].Molecular Plant,2015,8:1304-1320.
[8]Meiling W.Molecular cloning and expression analysis of the gene encoding sedoheptulose-1,7-bisphosphatase from Cucumis sativus[J].Scientia Horticulturae,2011,129:414-420.
[9]Ding F,Wang M L,Zhang S X,et al.Changes in SB-Pase activity influence photosynthetic capacity,growth,and tolerance to chilling stress in transgenic tomato plants[J].Molecular Plant,2016,6:32-41
[10]Feng L L,Han Y J,Liu G,et al.Overexpression of sedoheptulose-1,7-bisphosphate enhances photosynthesis and growth under salt stress in transgenic rice plants[J].Functional Plant Biology,2007,34(9):822-834.
[11]Cheng H Y,Song S Q.Species and organ diversity in the effect of hydrogenperoxide on superoxide dismutase activity in vitro.J.Integr[J].Plant Biology,2006,48:672-678.
[12]Fan J B,Hu Z R,Xie Y,et al.Alleviation of cold damage to photosystem II and metabolisms by melatonin in bermuda grass[J].Frontiers in Plant Science,2015,6:9-25.
[13]陈发菊,杨映根,赵德修,等.我国雪莲植物的种类、生境分布及化学成分的研究进展[J].植物学通报,1999,1(5):561-566.
[14]郭新勇,程晨,王重,等.转天山雪莲冷调节蛋白基因烟草的获得及其抗寒性鉴定[J].西北植物学报,2012,32(1):1-10.
[15]张丽.天山雪莲类黄酮相关基因的克隆及功能分析[D].石河子:石河子大学,2015.
[16]Dhindsa R S,Matowe W.Drought tolerance in two mosses:correlated with enzymatic defence against lipid peroxidation[J].Journal of Experimental Botany,1981,32:79-91.
[17]Lutts S,Kinet J M,Bouharmont J.NaCl-induced senescencein leaves of rice(Oryza sativa L.)cultivars differing in salinity resistance[J].Annals of Botany,1996,78:389-398.
[18]Zhang Y H,Chen L J,He J L,et al.Characteristics of chlorophyll fluorescence and antioxidative system in super-hybrid rice and its parental cultivars under chilling stress[J].Biological Plant,2010,54:164-168.
[19]Giannopolitis C N,Ries S K.Superoxide dismutases.I.Occurrence in higher plants[J].Plant Physiology,1977,59:309-314.
[20]Kooten O,Snel J F H.The use of chlorophyll fluorescence nomenclature in plant stress physiology[J].Photosynthesis Research,1990,25:147-150.
[21]King A I,Joyce D C,Reid M S.Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings[J].Plant Physiol,86,764-768(1998)..
[22]Xiao H D,Chen C S,Xu Y,et al.Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene fromPyropia haitanensis[J].Acta Oceanologica Sinica,2014,33(4):92-100.
[23]Chueca A,Sahrawy M,Pagano E A,et al.Chloroplast fructose-1,6-bisphosphatase:structure and function.[J]Photosynthesis Research,2002,74:235-249.
[24]Asada K.Production and scavenging of reactive oxygen species in chloroplasts and their functions[J].Plant Physiology,2006,141:391-396.
[25]Woodrow I E,Berry J A.Enzymatic regulation of photosynthetic CO2fixation in C3plants[J].Annual Reviev of Plant Physiology and Plant Molecular Biology,1988,39:533-594.