光叶楮耐盐无性系的筛选及其对NaCl胁迫的反应
摘要
本论文以光叶楮为材料,建立了组培实验体系,从组织、器官、分子水平系统的研究了NaCl胁迫对光叶楮幼苗生长、部分生理特性、膜脂过氧化及抗氧化保护酶系统、光和特征以及脱水素基因表达的影响,丰富了木本植物对盐胁迫反应及抗性机理的理论基础,为提高构树耐盐性及耐盐构树选育提供实验依据。主要研究结论如下:
1.光叶楮无菌体系建立过程中,以0.1%升汞溶液杀菌10～15min为宜,最优的增殖培养基为MS+6-BA 1.0 mg/1+IBA0.1 mg/1+30g/L蔗糖+卡拉胶6.5g/L,最佳生根培养基为1/2MS+IBA0.5 mg/1+NAA0.3 mg/1+30g/L蔗糖+卡拉胶6.5 g/L。
2.不同浓度NaCl胁迫后,光叶楮组培苗的受害率,盐胁迫指数随着盐浓度的升高而增加,植株株高、鲜重、干重随着盐浓度的升高而降低。
3.NaCl胁迫对可溶性蛋白、脯氨酸含量、抗氧化酶活性和MDA含量均产生一定影响。
4.光叶楮幼苗受到盐胁迫后,叶绿素a呈下降趋势,叶绿素b和类胡萝卜素含量则升高,总叶绿素含量降低,类胡萝卜素/叶绿素显著高于对照。NaCl胁迫具有加速叶片衰老的破坏作用。
5.光叶楮脱水素基因的转录水平在受盐胁迫后呈总体上升趋势。dhn1基因在未受胁迫的植株中也有微量表达,但转录水平较低;当浓度高于75 mmol/L时,脱水素的表达明显增加,100 mmol/L时达到最大。
Using Broussonetia papyrifera(L.) Vent(common name:paper mulberry),an economically important tree species,we established efficient experimental system of tissue culture and studied the effect of NaCl stress on plantlet growth,partial physiological nature, membrane lipid peroxidation,protective enzyme system,photosynthetic characters and Dehydrin gene expression.The major results were summarized as follows:
1.The shoots of Bronssonetia Papyrifera disinfected for 10-15 minutes with 0.1% mercuric chloride could efficiently control contamination in sterile culture.The most suitable proliferation medium was MS+1.0mg/L 6-BA+0.1 mg/L IBA+3.0%sucrose+6.5 g/L carrageenan.Rooting medium is 1/2MS+0.5 mg/L IBA+0.3 mg/L NAA+3.0%sucrose+6.5 g/L carrageenan.
2.The injury rate and salt stress index of Broussonetia papyrifera plantlet was increased with increased NaCl concentration.However,shoot height,fresh weight and dry weight decreased with the increased concentration.The suitable concentration for the selection of salt tolerance plant was 100 mM-125 mM.Meanwhile,the stress time was important to select salt tolerance plantlet.
3.NaCl treatment caused a lot of changes in protein content,antioxidant enzyme activities and the content of proline and MDA.The results demonstrated that soluble protein content increased at low level of NaCl concentration,and when NaCl concentration exceeded 100mM it decreased.Moreover,proline contents markedly increased in response to salt stress.In our study,there was a positive relationship between salt concentration and proline content of tissues.Proline content significantly increased in tissues with increase in salinity.MDA content increased at low levels of salinity and then decreased at 125mM and 150 mM NaCl concentration. The decrease of MDA content probably results from up-regulation of the antioxidative system in response to salt stress.4.Under salt stress chla and total chlorophyl contents in the leaves tended to decline while chlb and carotenoid contents were increased,chla/chlb was decreased while car/chl was higher than control.NaCl stress could accelerate the senescence of leaves.Photosynthetic rate, evaporate and stomatal conductance of Broussonetia papyrifera plantlet was decreased with increased salt concentration.But CO_2 concentration was higher than control.The decrease of net photosynthetic rate of Broussonetia papyrifera plantlet was caused by non-stomatal limitation.The results of correlation analysis showed that: Pn(Photosynthesic rate),Tr(Evaporate) and Gs(Stomatal conductance) had significant negative correlation with treatment concentration,and the correlation between WUE(water use efficiency) and treatment concentration was not significant, Ci(CO_2 concentration) had no correlation with treatment concentration.The effects of 50 mM NaCl on the gas exchange parameters was not significant,which indicated that Broussonetia papyrifera has certain salt tolerance.
5.Salt stress induced the expression of dehydrin gene in transcriptional levels. There was only a little expression for dhnl gene in control plantlet.When treatment concentration was more than 75mM,the expression of dhnl gene was increased obviously.The transcription level reached the highest when stress concentration was 100mM.
1.光叶楮无菌体系建立过程中,以0.1%升汞溶液杀菌10～15min为宜,最优的增殖培养基为MS+6-BA 1.0 mg/1+IBA0.1 mg/1+30g/L蔗糖+卡拉胶6.5g/L,最佳生根培养基为1/2MS+IBA0.5 mg/1+NAA0.3 mg/1+30g/L蔗糖+卡拉胶6.5 g/L。
2.不同浓度NaCl胁迫后,光叶楮组培苗的受害率,盐胁迫指数随着盐浓度的升高而增加,植株株高、鲜重、干重随着盐浓度的升高而降低。
3.NaCl胁迫对可溶性蛋白、脯氨酸含量、抗氧化酶活性和MDA含量均产生一定影响。
4.光叶楮幼苗受到盐胁迫后,叶绿素a呈下降趋势,叶绿素b和类胡萝卜素含量则升高,总叶绿素含量降低,类胡萝卜素/叶绿素显著高于对照。NaCl胁迫具有加速叶片衰老的破坏作用。
5.光叶楮脱水素基因的转录水平在受盐胁迫后呈总体上升趋势。dhn1基因在未受胁迫的植株中也有微量表达,但转录水平较低;当浓度高于75 mmol/L时,脱水素的表达明显增加,100 mmol/L时达到最大。
Using Broussonetia papyrifera(L.) Vent(common name:paper mulberry),an economically important tree species,we established efficient experimental system of tissue culture and studied the effect of NaCl stress on plantlet growth,partial physiological nature, membrane lipid peroxidation,protective enzyme system,photosynthetic characters and Dehydrin gene expression.The major results were summarized as follows:
1.The shoots of Bronssonetia Papyrifera disinfected for 10-15 minutes with 0.1% mercuric chloride could efficiently control contamination in sterile culture.The most suitable proliferation medium was MS+1.0mg/L 6-BA+0.1 mg/L IBA+3.0%sucrose+6.5 g/L carrageenan.Rooting medium is 1/2MS+0.5 mg/L IBA+0.3 mg/L NAA+3.0%sucrose+6.5 g/L carrageenan.
2.The injury rate and salt stress index of Broussonetia papyrifera plantlet was increased with increased NaCl concentration.However,shoot height,fresh weight and dry weight decreased with the increased concentration.The suitable concentration for the selection of salt tolerance plant was 100 mM-125 mM.Meanwhile,the stress time was important to select salt tolerance plantlet.
3.NaCl treatment caused a lot of changes in protein content,antioxidant enzyme activities and the content of proline and MDA.The results demonstrated that soluble protein content increased at low level of NaCl concentration,and when NaCl concentration exceeded 100mM it decreased.Moreover,proline contents markedly increased in response to salt stress.In our study,there was a positive relationship between salt concentration and proline content of tissues.Proline content significantly increased in tissues with increase in salinity.MDA content increased at low levels of salinity and then decreased at 125mM and 150 mM NaCl concentration. The decrease of MDA content probably results from up-regulation of the antioxidative system in response to salt stress.4.Under salt stress chla and total chlorophyl contents in the leaves tended to decline while chlb and carotenoid contents were increased,chla/chlb was decreased while car/chl was higher than control.NaCl stress could accelerate the senescence of leaves.Photosynthetic rate, evaporate and stomatal conductance of Broussonetia papyrifera plantlet was decreased with increased salt concentration.But CO_2 concentration was higher than control.The decrease of net photosynthetic rate of Broussonetia papyrifera plantlet was caused by non-stomatal limitation.The results of correlation analysis showed that: Pn(Photosynthesic rate),Tr(Evaporate) and Gs(Stomatal conductance) had significant negative correlation with treatment concentration,and the correlation between WUE(water use efficiency) and treatment concentration was not significant, Ci(CO_2 concentration) had no correlation with treatment concentration.The effects of 50 mM NaCl on the gas exchange parameters was not significant,which indicated that Broussonetia papyrifera has certain salt tolerance.
5.Salt stress induced the expression of dehydrin gene in transcriptional levels. There was only a little expression for dhnl gene in control plantlet.When treatment concentration was more than 75mM,the expression of dhnl gene was increased obviously.The transcription level reached the highest when stress concentration was 100mM.
引文
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