内生真菌印度梨形孢对水稻苗期耐盐性的影响
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摘要
【目的】研究印度梨形孢(Piriformospora indica)对盐(NaCl)胁迫后水稻幼苗生长发育的影响,明确印度梨形孢提高水稻幼苗耐盐性的机理。【方法】采用不同浓度(0、50、100、150和200 mmol/L)的盐(NaCl)溶液处理水稻幼苗,72 h后加入5×10~5个/mL的印度梨形孢孢子悬浮液10 mL侵染水稻根部(接种处理),以未侵染为对照(不接种,CK);20 d后测定水稻幼苗叶片的丙二醛(MDA)、脯氨酸(Pro)、叶绿素、可溶性糖和可溶性蛋白含量及抗氧化酶活性等生理指标,并利用实时荧光定量PCR(qPCR)检测耐盐相关基因的表达情况。【结果】盐胁迫处理后,与CK相比,接种处理水稻幼苗的MDA含量降低,叶绿素、可溶性糖和可溶性蛋白含量及抗氧化酶活性升高。其中,盐胁迫浓度为150和200 mmol/L时,CK的MDA含量是接种处理的1.5和2.8倍;盐胁迫浓度为150 mmol/L时,接种处理的Pro含量是CK的2.1倍;盐胁迫浓度为200 mmol/L时,接种处理的可溶性糖含量是CK的1.8倍;差异均达显著水平(P<0.05,下同)。qPCR检测结果表明,经盐胁迫后接种处理水稻幼苗叶片中耐盐相关基因SNAC1、OsNAC6、OsBZIP23和OsDREB2A的表达量均高于CK,各基因表达量均在100 mmol/L处理下达最大值,其中SNAC1和OsNAC6基因表达量分别是CK的1.5和1.1倍,差异达显著水平。【结论】接种印度梨形孢可降低盐胁迫后水稻幼苗叶片的MDA含量,提高抗氧化酶活性及Pro、叶绿素、可溶性糖和可溶性蛋白含量,同时促使水稻叶片中耐盐胁迫相关基因的表达量上调,从而增强水稻幼苗对盐胁迫的耐受性。印度梨形孢作为生物菌肥在农业生产中有潜在的利用价值。
【Objective】Study on the effects of Piriformospora indica on the growth and development of rice seedlings under salt stress(NaCl)was conducted,and the mechanism of P. indica improving salt tolerance of rice seedlings was also researched.【Method】Rice seedlings were treated with different concentrations(0,50,100,150 and 200 mmol/L)of salt(NaCl)solution. After 72 h,5×10~5 spore/mL P. indica spore suspension was added to infect rice roots(inoculation treatment). With the absence of dip-dyeing as control(CK),20 d later,the physiological indicators such as malondialdehyde(MDA),proline(Pro),chlorophyll,soluble sugar,soluble protein contents and antioxidant enzyme activities in rice seedling leaves were determined. Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of salt-tolerant genes.【Result】After salt stress treatment,compared with CK,MDA content decreased,chlorophyll,soluble sugar and soluble protein content and antioxidant enzyme activities increased in infected rice seedlings. Among them,when the salt concentrations were 150 and 200 mmol/L,the MDA contents of CK were as 1.5 and 2.8 times as that of P. indica treatment;when the salt concentration was 150 mmol/L,the Pro content of P. indica inoculated rice was as 2.1 times as that of CK;when the salt concentration was 200 mmol/L,the soluble sugar content of P. indica treatment was as 1.8 times as that of CK;the differences were significant(P<0.05,the same below). The results of qPCR showed that the expression levels of salt-tolerant genes SNAC1,OsNAC6,OsBZIP23 and OsDREB2 A in the leaves of rice seedlings after salt stress were higher than those of CK,and the expression level of each gene reached the maximum at 100 mmol/L.The expression levels of SNAC1 and OsNAC6 genes in P. indica seedlings were as 1.5 and 1.1 times as those of CK,respectively,and the differences were significant.【Conclusion】Inoculation with P. indica can decrease the MDA content and increase the activities of antioxidant enzymes,Pro,chlorophyll,soluble sugar and soluble protein contents in rice seedling leaves under salt stress,and also promote the up-regulation of salt tolerance-related genes in rice leaves,and finally enhance the tolerance of rice seedlings to salt stress. P. indica has potential to be uses as a biological fertilizer in agricultural production.
【Objective】Study on the effects of Piriformospora indica on the growth and development of rice seedlings under salt stress(NaCl)was conducted,and the mechanism of P. indica improving salt tolerance of rice seedlings was also researched.【Method】Rice seedlings were treated with different concentrations(0,50,100,150 and 200 mmol/L)of salt(NaCl)solution. After 72 h,5×10~5 spore/mL P. indica spore suspension was added to infect rice roots(inoculation treatment). With the absence of dip-dyeing as control(CK),20 d later,the physiological indicators such as malondialdehyde(MDA),proline(Pro),chlorophyll,soluble sugar,soluble protein contents and antioxidant enzyme activities in rice seedling leaves were determined. Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of salt-tolerant genes.【Result】After salt stress treatment,compared with CK,MDA content decreased,chlorophyll,soluble sugar and soluble protein content and antioxidant enzyme activities increased in infected rice seedlings. Among them,when the salt concentrations were 150 and 200 mmol/L,the MDA contents of CK were as 1.5 and 2.8 times as that of P. indica treatment;when the salt concentration was 150 mmol/L,the Pro content of P. indica inoculated rice was as 2.1 times as that of CK;when the salt concentration was 200 mmol/L,the soluble sugar content of P. indica treatment was as 1.8 times as that of CK;the differences were significant(P<0.05,the same below). The results of qPCR showed that the expression levels of salt-tolerant genes SNAC1,OsNAC6,OsBZIP23 and OsDREB2 A in the leaves of rice seedlings after salt stress were higher than those of CK,and the expression level of each gene reached the maximum at 100 mmol/L.The expression levels of SNAC1 and OsNAC6 genes in P. indica seedlings were as 1.5 and 1.1 times as those of CK,respectively,and the differences were significant.【Conclusion】Inoculation with P. indica can decrease the MDA content and increase the activities of antioxidant enzymes,Pro,chlorophyll,soluble sugar and soluble protein contents in rice seedling leaves under salt stress,and also promote the up-regulation of salt tolerance-related genes in rice leaves,and finally enhance the tolerance of rice seedlings to salt stress. P. indica has potential to be uses as a biological fertilizer in agricultural production.
引文
陈建勋,王晓峰.2006.植物生理实验指导[M].第2版.广州:华南理工大学出版社.[Chen J X,Wang X F.2006.Plant Physiological Experiment Guide[M].The 2ndEdition.Guangzhou:South China University of Technology Press.]
陈佑源,楼兵干,高其康,林福呈.2013.印度梨形孢诱导油菜抗旱性机理的初步研究[J].农业生物技术学报,21(3):272-281.[Chen Y Y,Lou B G,Gao Q K,Lin F C.2013.Preliminary study on mechanisms of drought resistance in Brassica napus L.conferred by Piriformospora indica[J].Journal of Agricultural Biotechnology,21(3):272-281.]
高俊凤.2006.植物生理学实验指导[M].北京:高等教育出版社.[Gao J F.2006.Plant Physiology Experiment Instruction[M].Beijing:Higher Education Press.]
惠非琼.2014.印度梨形孢对烟草耐盐、抗旱及重金属作用及机理的初步研究[D].杭州:浙江大学.[Hui F Q.2014.Preliminary study on the effect and mechanism of Piriformospora indica on salt tolerance,drought resistance and heavy metals in tobacco[D].Hangzhou:Zhejiang University.]
李合生.2000.植物生理生化实验原理和技术[M].北京:高等教育出版社.[Li H S.2000.Principle and Technology of Plant Physiological and Biochemical Experiments[M].Beijing:Higher Education Press.]
李亮,武洪庆,马朝阳,任亮,王璐欣,齐树亭.2015.印度梨形孢促进蒺藜苜蓿生长及其提高耐盐性研究[J].微生物学通报,42(8):1492-1500.[Li L,Wu H Q,Ma C Y,Ren L,Wang L X,Qi S T.2015.Growth promotion and salt tolerance induction by Piriformospora indica colonization in Medicago truncatul[J].Microbiology China,42(8):1492-1500.]
李亮,陈希,王奋,王晓阳,齐树亭.2016.印度梨形孢通过激活抗氧化物酶活性及诱导P5CS基因表达提高紫花苜蓿耐盐性[J].河北工业大学学报,45(4):29-36.[Li L,Chen X,Wang F,Wang X Y,Qi S T.2016.Piriformospora indica confers salt tolerance in Medicago sativa by stimulating antioxidant enzymes activities and the expression of P5CS genes[J].Journal of Hebei University of Technology,45(4):29-36.]
马杰.2012.印度梨形孢诱导烟草促生、抗病、抗逆作用及其机理的初步研究[D].杭州:浙江大学.[Ma J.2012.Preliminary study on Piriformospora indica induced tobacco growth,disease resistance,stress resistance and its mechanism[D].Hangzhou:Zhejiang University.]
孙超.2010.印度梨形孢诱导小白菜抗病、促生、抗逆的作用及其机理的初步研究[D].杭州:浙江大学.[Sun C.2010.Preliminary study on the effect of Piriformospora indica on disease resistance,growth promotion and stress resistance of Chinese cabbage[D].Hangzhou:Zhejiang University.]
吴金丹,陈乾,刘晓曦,林福呈,高其康,楼兵干.2015.印度梨形孢对水稻的促生作用及其机理的初探[J].中国水稻科学,29(2):200-207.[Wu J D,Chen Q,Liu X X,Lin FC,Gao Q K,Lou B G.2015.Preliminary study on mechanisms of growth promotion in rice colonized by Piriformospora indica[J].Chinese Journal of Rice Science,29(2):200-207.]
韦巧,武美燕,张文英.2017.不同磷水平下印度梨形孢对生菜生长及磷素吸收利用的影响[J].河南农业科学,46(1):100-104.[Wei Q,Wu M Y,Zhang W Y.2017.E-ffects of Piriformospora indica on growth and phosphorus absoption of lettuce under different phosphorus levels[J].Journal of Henan Agricultural Sciences,46(1):100-104.]
杨志敏.2015.生物化学实验[M].北京:高等教育出版社.[Yang Z M.2015.Biochemical Experiment[M].Beijing:Higher Education Press.]
赵世杰.2000.植物生理学实验指导[M].北京:中国农业科学技术出版社.[Zhao S J.2000.Guidance of Plant Physiology Experiments[M].Beijing:China Agricultural Science and Technology Press.]
邹琦.2007.植物生理学实验指导[M].北京:中国农业出版社.[Zou Q.2007.Plant Physiology Experiment Guide[M].Beijing:China Agriculture Press.]
Ashraf M,Foodlad M R.2007.Roles of glycine betain and proline in improving plant abiotic stress resistance[J].Environmental and Experimental Botany,59:206-216.
Boscaiu M,Bautista I,Lidón A,Llinares J,Lull C,Donat P,Mayoral O,Vicente O.2013.Environmental-dependent proline accumulation in plants living on gypsum soils[J].Acta Physiologiae Plantarum,35(7):2193-2204.
Flowers T J,Colmer T D.2008.Salinity tolerance in halophytes[J].The New phytologist,179(4):945-963.
Kumar V,Sahai V,Bisaria V S.2011.High-density spore production of Piriformospora indica,a plant growth-promoting endophyte,by optimization of nutritional and cultural parameters[J].Bioresource Technology,102(3):3169-3175.
Lee D H,Kim Y S,Lee C B.2001.The inductive responses of the antioxidant enzymes by salt stress in the rice(Oryzasativa L.)[J].Journal of Plant Physiology,158(6):737-745.
Singh A,Sharma J,Rexer K H,Varma A.2000.Plant productivity determinants beyond minerals,water and light:Piriformospora indica,a revolutionary plant growth promoting fungus[J].Current Science,79(11):1548-1554.
Szabados L,SavouréA.2010.Proline:A multifunctional amino acid[J].Trends in Plant Science,15(2):89-97.
Varma A,Verma S,Sudha,Sahay N,Butehorn B,Franken P.1999.Piriformospora indica,a cultivable plant-growthpromoting root endophyte[J].Applied&Environmental Microbiology,65(6):2741-2744.
Waller F,Achatz B,Baltruschat H,Fodor J,Becker K,Fischer M,Heier T,Huckelhoven R,Neumann C,Wettstein D,Franken P,Kogel K H.2005.The endophytic fungus Pirifomospora indica reprograms barley to salt-stress tolerance,disease resistance,and higher yield[J].Proceedings of the National Academy of Sciences of the United States of America,102(38):13386-13391.
Xie Q,Frugis G,Colgan D,Chua N H.2000.Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development[J].Genes&Development,14(23):3024-3036.
Yamauchi T,Kamon J,Minokoshi Y,Ito Y,Waki H,Uchida S,Yamashita S,Noda M,Kita S,Ueki K,Eto K,Akanuma Y,Froguel P,Foufelle F,Ferre P,Carling D,Kimura S,Nagai R,Kahn B B,Kadowaki T.2002.Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[J].Nature Medicine,8(11):1288-1295.
陈佑源,楼兵干,高其康,林福呈.2013.印度梨形孢诱导油菜抗旱性机理的初步研究[J].农业生物技术学报,21(3):272-281.[Chen Y Y,Lou B G,Gao Q K,Lin F C.2013.Preliminary study on mechanisms of drought resistance in Brassica napus L.conferred by Piriformospora indica[J].Journal of Agricultural Biotechnology,21(3):272-281.]
高俊凤.2006.植物生理学实验指导[M].北京:高等教育出版社.[Gao J F.2006.Plant Physiology Experiment Instruction[M].Beijing:Higher Education Press.]
惠非琼.2014.印度梨形孢对烟草耐盐、抗旱及重金属作用及机理的初步研究[D].杭州:浙江大学.[Hui F Q.2014.Preliminary study on the effect and mechanism of Piriformospora indica on salt tolerance,drought resistance and heavy metals in tobacco[D].Hangzhou:Zhejiang University.]
李合生.2000.植物生理生化实验原理和技术[M].北京:高等教育出版社.[Li H S.2000.Principle and Technology of Plant Physiological and Biochemical Experiments[M].Beijing:Higher Education Press.]
李亮,武洪庆,马朝阳,任亮,王璐欣,齐树亭.2015.印度梨形孢促进蒺藜苜蓿生长及其提高耐盐性研究[J].微生物学通报,42(8):1492-1500.[Li L,Wu H Q,Ma C Y,Ren L,Wang L X,Qi S T.2015.Growth promotion and salt tolerance induction by Piriformospora indica colonization in Medicago truncatul[J].Microbiology China,42(8):1492-1500.]
李亮,陈希,王奋,王晓阳,齐树亭.2016.印度梨形孢通过激活抗氧化物酶活性及诱导P5CS基因表达提高紫花苜蓿耐盐性[J].河北工业大学学报,45(4):29-36.[Li L,Chen X,Wang F,Wang X Y,Qi S T.2016.Piriformospora indica confers salt tolerance in Medicago sativa by stimulating antioxidant enzymes activities and the expression of P5CS genes[J].Journal of Hebei University of Technology,45(4):29-36.]
马杰.2012.印度梨形孢诱导烟草促生、抗病、抗逆作用及其机理的初步研究[D].杭州:浙江大学.[Ma J.2012.Preliminary study on Piriformospora indica induced tobacco growth,disease resistance,stress resistance and its mechanism[D].Hangzhou:Zhejiang University.]
孙超.2010.印度梨形孢诱导小白菜抗病、促生、抗逆的作用及其机理的初步研究[D].杭州:浙江大学.[Sun C.2010.Preliminary study on the effect of Piriformospora indica on disease resistance,growth promotion and stress resistance of Chinese cabbage[D].Hangzhou:Zhejiang University.]
吴金丹,陈乾,刘晓曦,林福呈,高其康,楼兵干.2015.印度梨形孢对水稻的促生作用及其机理的初探[J].中国水稻科学,29(2):200-207.[Wu J D,Chen Q,Liu X X,Lin FC,Gao Q K,Lou B G.2015.Preliminary study on mechanisms of growth promotion in rice colonized by Piriformospora indica[J].Chinese Journal of Rice Science,29(2):200-207.]
韦巧,武美燕,张文英.2017.不同磷水平下印度梨形孢对生菜生长及磷素吸收利用的影响[J].河南农业科学,46(1):100-104.[Wei Q,Wu M Y,Zhang W Y.2017.E-ffects of Piriformospora indica on growth and phosphorus absoption of lettuce under different phosphorus levels[J].Journal of Henan Agricultural Sciences,46(1):100-104.]
杨志敏.2015.生物化学实验[M].北京:高等教育出版社.[Yang Z M.2015.Biochemical Experiment[M].Beijing:Higher Education Press.]
赵世杰.2000.植物生理学实验指导[M].北京:中国农业科学技术出版社.[Zhao S J.2000.Guidance of Plant Physiology Experiments[M].Beijing:China Agricultural Science and Technology Press.]
邹琦.2007.植物生理学实验指导[M].北京:中国农业出版社.[Zou Q.2007.Plant Physiology Experiment Guide[M].Beijing:China Agriculture Press.]
Ashraf M,Foodlad M R.2007.Roles of glycine betain and proline in improving plant abiotic stress resistance[J].Environmental and Experimental Botany,59:206-216.
Boscaiu M,Bautista I,Lidón A,Llinares J,Lull C,Donat P,Mayoral O,Vicente O.2013.Environmental-dependent proline accumulation in plants living on gypsum soils[J].Acta Physiologiae Plantarum,35(7):2193-2204.
Flowers T J,Colmer T D.2008.Salinity tolerance in halophytes[J].The New phytologist,179(4):945-963.
Kumar V,Sahai V,Bisaria V S.2011.High-density spore production of Piriformospora indica,a plant growth-promoting endophyte,by optimization of nutritional and cultural parameters[J].Bioresource Technology,102(3):3169-3175.
Lee D H,Kim Y S,Lee C B.2001.The inductive responses of the antioxidant enzymes by salt stress in the rice(Oryzasativa L.)[J].Journal of Plant Physiology,158(6):737-745.
Singh A,Sharma J,Rexer K H,Varma A.2000.Plant productivity determinants beyond minerals,water and light:Piriformospora indica,a revolutionary plant growth promoting fungus[J].Current Science,79(11):1548-1554.
Szabados L,SavouréA.2010.Proline:A multifunctional amino acid[J].Trends in Plant Science,15(2):89-97.
Varma A,Verma S,Sudha,Sahay N,Butehorn B,Franken P.1999.Piriformospora indica,a cultivable plant-growthpromoting root endophyte[J].Applied&Environmental Microbiology,65(6):2741-2744.
Waller F,Achatz B,Baltruschat H,Fodor J,Becker K,Fischer M,Heier T,Huckelhoven R,Neumann C,Wettstein D,Franken P,Kogel K H.2005.The endophytic fungus Pirifomospora indica reprograms barley to salt-stress tolerance,disease resistance,and higher yield[J].Proceedings of the National Academy of Sciences of the United States of America,102(38):13386-13391.
Xie Q,Frugis G,Colgan D,Chua N H.2000.Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development[J].Genes&Development,14(23):3024-3036.
Yamauchi T,Kamon J,Minokoshi Y,Ito Y,Waki H,Uchida S,Yamashita S,Noda M,Kita S,Ueki K,Eto K,Akanuma Y,Froguel P,Foufelle F,Ferre P,Carling D,Kimura S,Nagai R,Kahn B B,Kadowaki T.2002.Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[J].Nature Medicine,8(11):1288-1295.