转耐盐相关基因山新杨的抗逆性分析
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摘要
利用农杆菌(A tumefaciens)介导法将从抗旱、耐盐植物柽柳(Tamarix chinensis)中克隆得到的lea基因(late embryogenesis abundant)转入山新杨,共获得18个卡那霉素抗性芽,对其进行PCR检测,均为阳性;RT-PCR检测,表明外源lea基因在RNA水平上能够表达。
将转3种基因的山新杨进行0.8%的NaCl胁迫,对转基因株系和对照的丙二醛含量,叶绿素含量以及过氧化物歧化酶(SOD),过氧化物酶(POD),抗血酸过氧化物酶(APX),过氧化氢酶(CAT)等抗氧化酶的活性进行了测定。
试验结果表明:在胁迫的第4d和第6d转基因株系的叶绿素平均含量分别高于对照的14.28%和16.42%。
非转基因小黑杨的MDA含量高于胁迫前的7.74%,各转基因山新杨的平均MDA含量均高于胁迫前的1.69%;胁迫第6d非转基因小黑杨的MDA含量最高,高于胁迫前的87.31%,各转基因山新杨的平均MDA含量高于胁迫前的60.63%。
转基因山新杨的4种抗氧化酶活性分析结果如下:
(1)APX活性分析表明,盐胁迫的第2d各转基因株系的APX活性达到最大,其中L15和A11植株内的APX活性明显高于对照,分别高于对照的63.09%和48.85%;
(2)POD活性分析表明,在胁迫的第4d和第6d,转基因株系L10,P6,P8的POD活性分别高于对照的37.34%,28.39%,28.99%,而在胁迫6d时这些转基因株系的POD活性则分别高于对照的38.6%,19.48%,19.34%。
(3)SOD活性分析表明,在胁迫第6d,L15,P1和P8的SOD活性显著的高于对照,分别高于对照的26.36%,27.31%,17.33%。
(4)CAT活性分析表明,在胁迫的第2d,各转基因株系的CAT活性(除了A18)均明显高于对照,但在胁迫第4d和6dL15和L14的CAT活性高于对照。
采用隶属函数法,利用4种抗氧化酶以及叶绿素和丙二醛等指标对转基因株系的耐盐性进行了综合评价。按照耐盐性高低排列为:L10>A11>P1>L14>A18>P8>P6>A16>P7。
This research,using A tumefaciens mediated method,transformed the lea(late embryogenesis abundant) into genome of Populus davidiana×P.bolleana.Eighteen buds that were resistant to kanamycin were obtained.After detecting them with PCR,the results showed that these buds were positive.And then the total RNA was extracted for RT-PCR to confirm that the exogenous gene had expressed on RNA level.
These transgenic plants were treated with 0.8%NaCl.And then the content of malondialdehyde and chloroplast were measured besides activity measurement of other four anti-oxidative enzymes including superoxide dismutase(SOD)、peroxidase(POD)、catalase(CAT)。The results indicated that on the fourth and sixth day of treatment,the average content of chloroplast in transgenic plants were higher than control,14.28%and 16.42% respectively.
Judging from the activity fluctuation of four anti-oxidative enzymes in these transgenic plants:
(1) During the process of fourth and sixth day's stress,the activity of APX reached the peak, especially the activity of APX in the L15 and A11 were remarkably higher than control, 63.09%and 48.85%,respectively.
(2)On the fourth and sixth day of stress,activity of POD in these transgenic plants(L10,P6, P8) were 37.34%,28.39%,28.99%stronger than control while that of POD in those ones were 38.6%,19.48%,19.34%higher than control.
(3)On the sixth day of stress,activity of SOD in L15,P1,P8 and P8 were significantly 26.36%,27.31%,17.33%higher than control respectively.
(4) On the second day of stress,activiy of CAT in all the transgenic plants except for A18 all strikingly higher than control but at the late stage of stress only the activity of CAT in L15 was 65.09%higher than control while that in L14 was higher than control.
After using membership function comprehensively to analyze four anti-oxidative enzymes as well as content of chloroplast and malondialdehyde,the thorough appraise was made about the tolerance to salinity.The sequence of resistant ability from high to low is; L10>A11>P1>L14>A18>P8>P6>A16>P7.
将转3种基因的山新杨进行0.8%的NaCl胁迫,对转基因株系和对照的丙二醛含量,叶绿素含量以及过氧化物歧化酶(SOD),过氧化物酶(POD),抗血酸过氧化物酶(APX),过氧化氢酶(CAT)等抗氧化酶的活性进行了测定。
试验结果表明:在胁迫的第4d和第6d转基因株系的叶绿素平均含量分别高于对照的14.28%和16.42%。
非转基因小黑杨的MDA含量高于胁迫前的7.74%,各转基因山新杨的平均MDA含量均高于胁迫前的1.69%;胁迫第6d非转基因小黑杨的MDA含量最高,高于胁迫前的87.31%,各转基因山新杨的平均MDA含量高于胁迫前的60.63%。
转基因山新杨的4种抗氧化酶活性分析结果如下:
(1)APX活性分析表明,盐胁迫的第2d各转基因株系的APX活性达到最大,其中L15和A11植株内的APX活性明显高于对照,分别高于对照的63.09%和48.85%;
(2)POD活性分析表明,在胁迫的第4d和第6d,转基因株系L10,P6,P8的POD活性分别高于对照的37.34%,28.39%,28.99%,而在胁迫6d时这些转基因株系的POD活性则分别高于对照的38.6%,19.48%,19.34%。
(3)SOD活性分析表明,在胁迫第6d,L15,P1和P8的SOD活性显著的高于对照,分别高于对照的26.36%,27.31%,17.33%。
(4)CAT活性分析表明,在胁迫的第2d,各转基因株系的CAT活性(除了A18)均明显高于对照,但在胁迫第4d和6dL15和L14的CAT活性高于对照。
采用隶属函数法,利用4种抗氧化酶以及叶绿素和丙二醛等指标对转基因株系的耐盐性进行了综合评价。按照耐盐性高低排列为:L10>A11>P1>L14>A18>P8>P6>A16>P7。
This research,using A tumefaciens mediated method,transformed the lea(late embryogenesis abundant) into genome of Populus davidiana×P.bolleana.Eighteen buds that were resistant to kanamycin were obtained.After detecting them with PCR,the results showed that these buds were positive.And then the total RNA was extracted for RT-PCR to confirm that the exogenous gene had expressed on RNA level.
These transgenic plants were treated with 0.8%NaCl.And then the content of malondialdehyde and chloroplast were measured besides activity measurement of other four anti-oxidative enzymes including superoxide dismutase(SOD)、peroxidase(POD)、catalase(CAT)。The results indicated that on the fourth and sixth day of treatment,the average content of chloroplast in transgenic plants were higher than control,14.28%and 16.42% respectively.
Judging from the activity fluctuation of four anti-oxidative enzymes in these transgenic plants:
(1) During the process of fourth and sixth day's stress,the activity of APX reached the peak, especially the activity of APX in the L15 and A11 were remarkably higher than control, 63.09%and 48.85%,respectively.
(2)On the fourth and sixth day of stress,activity of POD in these transgenic plants(L10,P6, P8) were 37.34%,28.39%,28.99%stronger than control while that of POD in those ones were 38.6%,19.48%,19.34%higher than control.
(3)On the sixth day of stress,activity of SOD in L15,P1,P8 and P8 were significantly 26.36%,27.31%,17.33%higher than control respectively.
(4) On the second day of stress,activiy of CAT in all the transgenic plants except for A18 all strikingly higher than control but at the late stage of stress only the activity of CAT in L15 was 65.09%higher than control while that in L14 was higher than control.
After using membership function comprehensively to analyze four anti-oxidative enzymes as well as content of chloroplast and malondialdehyde,the thorough appraise was made about the tolerance to salinity.The sequence of resistant ability from high to low is; L10>A11>P1>L14>A18>P8>P6>A16>P7.
引文
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