转ABP9基因小麦纯合株系的创制与干旱、低氮逆境的抗性鉴定
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摘要
干旱是限制作物产量提升的一个重要胁迫因子,而农田系统氮肥的过量施用则是生态系统的一个重要污染源。小麦是我国的第二大粮食作物,培育耐旱、耐低氮的小麦种质资源有着重要的现实意义。本实验室前期工作从玉米花后17天幼胚的cDNA文库中克隆到一个可以与Cat1基因上游顺式作用元件ABRE2相互作用的bZIP类转录因子——ABP9。拟南芥中的研究结果表明,ABP9基因可以显著提高转基因拟南芥对干旱、盐渍、低温等非生物逆境的耐受性。本论文从创制转ABP9基因小麦株系入手,验证其对干旱、低氮两种非生物逆境胁迫的耐受性,旨在为小麦抗逆育种创造良好的种质资源。研究结果如下:
(1)2006年-2009年通过基因枪共转化的方法将三种不同启动子驱动的ABP9基因(Ubi-ABP9、Ind-ABP9、Pabp9-ABP9)转入两个小麦受体品种石4185和宁春四号中并获得了T0代转化植株。通过PCR方法对T0-T4代的转基因株系进行跟踪检测,在T4代株系中获得Ubi-ABP9和Ind-ABP9石4185各1个纯合株系,Ubi-ABP9宁春四号两个纯合株系。通过Southern进一步验证了PCR结果,通过Northern确定了ABP9基因在受体小麦中的表达。
(2)T2代转Ubi-ABP9和Ind-ABP9基因的石4185小麦株系的耐旱实验表明,转基因小麦株系在干旱逆境胁迫下的生长表型显著优于非转基因株系且存活率显著提高。
(3)2009年和2010年对分别转ABP9基因石4185小麦纯合株系进行了两次盆栽耐旱实验。结果显示,与非转基因小麦株系相比转基因小麦株系在干旱逆境下的生长表型有了显著改善。2009年耐旱实验结果统计分析表明,干旱逆境下转Ubi-ABP9基因的小麦株系较非转基因小麦株系的株高、干物质积累、相对含水量、产量及产量构成要素显著提高;在正常生长条件下,上述指标无显著差异。2010年Ind-ABP9石4185和Ubi-ABP9宁春四号纯合株系耐旱实验初步结果显示两种转基因株系在干旱逆境下的生长表型较非转基因株系也有显著的改善。
(4)利用实时荧光定量PCR的结果显示低氮逆境可以诱导ABP9基因的表达。在此基础之上,于2009年和2010年对Ubi-ABP9石4185株系分别在沙培和蛭石中进行了两次耐低氮胁迫的实验。结果显示,与非转基因小麦株系相比转基因小麦株系在低氮逆境下的长势、叶色等性状显著改善。2009年的耐低氮胁迫实验表明,在低氮逆境条件下转基因小麦株系的生长发育、叶绿素、产量及产量构成要素等指标较非转基因小麦株系有显著的改善。在正常生长条件下,转基因株系与非转基因株系在株高、干物质积累、穗长和穗粒数方面无显著差异,而转基因株系的单株产量、百粒重和穗数较非转基因株系有显著提高。2010年对宁春四号转Ubi-ABP9的株系在蛭石中进行了耐低氮胁迫的实验,初步实验结果显示转基因株系在低氮逆境条件下的生长表型也优于非转基因株系,与Ubi-ABP9石4185低氮胁迫实验结果基本一致。
上述实验结果表明ABP9基因在提高受体小麦对干旱、低氮两种非生物逆境的耐受性方面有重要的作用,且该基因在受体小麦中的表达对其在正常生长条件下的生长发育和产量无负面影响。
Drought is an important factor that restrict the yield improvement of crops and the over application of nitrogen has caused serious eco-system pollution. Our laboratory has cloned a bZIP transcription factor-ABP9 from the cDNA library of maize, which can interact with the cis-acting element of Cat1——ABRE2. According to the function study of ABP9 in Arabidopsis Thaliana, ABP9 can significantly improve its tolerance to abiotic stress such as drought, salinity and cold. Starting from the creation of transgenic wheat expressing ABP9, we further identified the transgenic wheat’s tolerance to the stress of drought and low-nitrogen and created good germplasm resources for the wheat breeding. Results of the study are as following:
(1) From 2006 to 2009 we have successfully transformed ABP9 gene driven by three different promoters into two varieties of SHI4185 and NINGCHUN4 by the method of microprojectile bombardment and obtained the T0 transformed plants. According to the detection of T0-T4 transformed plants by PCR, we obtained one pure line of Ubi-ABP9 SHI4185 and Ind-ABP9 SHI4185 respectively ,two pure lines of Ubi-ABP9 NINGCHUN4 . According to Southern blot and Northern blot, we further confirmed the results of PCR and the expression of ABP9 in the wheat.
(2) We conducted drought tolerance experiment in the two T2 transgenic lines of SHI4185(Ubi-ABP9SHI4185 and Ind-ABP9 SHI 4185). The results showed that the phenotypes of the two transgenic lines under the drought stress and survival rate have been significantly improved.
(3)We conducted drought tolerance experiment for the pure lines of transgenic ABP9 SHI4185 in ports in 2009 and 2010. Results showed that the phenotype of transgenic lines has been greatly improved. The results of 2009 showed that under drought stress the height, biomass, relative water content, yield and yield components have been remarkably improved and statistical analysis showed that the difference of yield and yield components between SHI4185 and Ubi-ABP9 SHI4185 is significant on the level of 1% and the above indexes are not significant between SHI4185 and Ubi-ABP9 SHI4185 under normal conditions.
(4) According to the Real-time Quantitative PCR, we confirmed the induction of ABP9 in maize under the conditions of low nitrogen. We conducted the low-nitrogen tolerance experiment with pure lines of Ubi-ABP9SHI4185 in the sand and vermiculite in 2009 and 2010 respectively. Results of 2009 and 2010 showed that the growth and leaf color of Ubi-ABP9SHI4185 have been greatly improved comparing with SHI4185. Results of 2009 showed that the growth, chlorophyll, yield and yield components have been greatly improved,too. The differences of height,biomass,length of panicle and spikelets per panicle between SHI4185 and Ubi-ABP9SHI4185 are not significant and yield per plant, 100-grain weight and number of panicle of Ubi-ABP9SHI4185 are significantly improved comparing with SHI4185. The results of low-nitrogen tolerance experiment in vermiculite with Ubi-ABP9NINGCHUN4 are basically consistent with that of Ubi-ABP9 SHI4185.
The above results from 2009 to 2010 have showed that ABP9 can significantly improve wheat’s tolerance to drought and low nitrogen stress and has no obvious negative effect on the growth and yield of the wheat under normal conditions.
(1)2006年-2009年通过基因枪共转化的方法将三种不同启动子驱动的ABP9基因(Ubi-ABP9、Ind-ABP9、Pabp9-ABP9)转入两个小麦受体品种石4185和宁春四号中并获得了T0代转化植株。通过PCR方法对T0-T4代的转基因株系进行跟踪检测,在T4代株系中获得Ubi-ABP9和Ind-ABP9石4185各1个纯合株系,Ubi-ABP9宁春四号两个纯合株系。通过Southern进一步验证了PCR结果,通过Northern确定了ABP9基因在受体小麦中的表达。
(2)T2代转Ubi-ABP9和Ind-ABP9基因的石4185小麦株系的耐旱实验表明,转基因小麦株系在干旱逆境胁迫下的生长表型显著优于非转基因株系且存活率显著提高。
(3)2009年和2010年对分别转ABP9基因石4185小麦纯合株系进行了两次盆栽耐旱实验。结果显示,与非转基因小麦株系相比转基因小麦株系在干旱逆境下的生长表型有了显著改善。2009年耐旱实验结果统计分析表明,干旱逆境下转Ubi-ABP9基因的小麦株系较非转基因小麦株系的株高、干物质积累、相对含水量、产量及产量构成要素显著提高;在正常生长条件下,上述指标无显著差异。2010年Ind-ABP9石4185和Ubi-ABP9宁春四号纯合株系耐旱实验初步结果显示两种转基因株系在干旱逆境下的生长表型较非转基因株系也有显著的改善。
(4)利用实时荧光定量PCR的结果显示低氮逆境可以诱导ABP9基因的表达。在此基础之上,于2009年和2010年对Ubi-ABP9石4185株系分别在沙培和蛭石中进行了两次耐低氮胁迫的实验。结果显示,与非转基因小麦株系相比转基因小麦株系在低氮逆境下的长势、叶色等性状显著改善。2009年的耐低氮胁迫实验表明,在低氮逆境条件下转基因小麦株系的生长发育、叶绿素、产量及产量构成要素等指标较非转基因小麦株系有显著的改善。在正常生长条件下,转基因株系与非转基因株系在株高、干物质积累、穗长和穗粒数方面无显著差异,而转基因株系的单株产量、百粒重和穗数较非转基因株系有显著提高。2010年对宁春四号转Ubi-ABP9的株系在蛭石中进行了耐低氮胁迫的实验,初步实验结果显示转基因株系在低氮逆境条件下的生长表型也优于非转基因株系,与Ubi-ABP9石4185低氮胁迫实验结果基本一致。
上述实验结果表明ABP9基因在提高受体小麦对干旱、低氮两种非生物逆境的耐受性方面有重要的作用,且该基因在受体小麦中的表达对其在正常生长条件下的生长发育和产量无负面影响。
Drought is an important factor that restrict the yield improvement of crops and the over application of nitrogen has caused serious eco-system pollution. Our laboratory has cloned a bZIP transcription factor-ABP9 from the cDNA library of maize, which can interact with the cis-acting element of Cat1——ABRE2. According to the function study of ABP9 in Arabidopsis Thaliana, ABP9 can significantly improve its tolerance to abiotic stress such as drought, salinity and cold. Starting from the creation of transgenic wheat expressing ABP9, we further identified the transgenic wheat’s tolerance to the stress of drought and low-nitrogen and created good germplasm resources for the wheat breeding. Results of the study are as following:
(1) From 2006 to 2009 we have successfully transformed ABP9 gene driven by three different promoters into two varieties of SHI4185 and NINGCHUN4 by the method of microprojectile bombardment and obtained the T0 transformed plants. According to the detection of T0-T4 transformed plants by PCR, we obtained one pure line of Ubi-ABP9 SHI4185 and Ind-ABP9 SHI4185 respectively ,two pure lines of Ubi-ABP9 NINGCHUN4 . According to Southern blot and Northern blot, we further confirmed the results of PCR and the expression of ABP9 in the wheat.
(2) We conducted drought tolerance experiment in the two T2 transgenic lines of SHI4185(Ubi-ABP9SHI4185 and Ind-ABP9 SHI 4185). The results showed that the phenotypes of the two transgenic lines under the drought stress and survival rate have been significantly improved.
(3)We conducted drought tolerance experiment for the pure lines of transgenic ABP9 SHI4185 in ports in 2009 and 2010. Results showed that the phenotype of transgenic lines has been greatly improved. The results of 2009 showed that under drought stress the height, biomass, relative water content, yield and yield components have been remarkably improved and statistical analysis showed that the difference of yield and yield components between SHI4185 and Ubi-ABP9 SHI4185 is significant on the level of 1% and the above indexes are not significant between SHI4185 and Ubi-ABP9 SHI4185 under normal conditions.
(4) According to the Real-time Quantitative PCR, we confirmed the induction of ABP9 in maize under the conditions of low nitrogen. We conducted the low-nitrogen tolerance experiment with pure lines of Ubi-ABP9SHI4185 in the sand and vermiculite in 2009 and 2010 respectively. Results of 2009 and 2010 showed that the growth and leaf color of Ubi-ABP9SHI4185 have been greatly improved comparing with SHI4185. Results of 2009 showed that the growth, chlorophyll, yield and yield components have been greatly improved,too. The differences of height,biomass,length of panicle and spikelets per panicle between SHI4185 and Ubi-ABP9SHI4185 are not significant and yield per plant, 100-grain weight and number of panicle of Ubi-ABP9SHI4185 are significantly improved comparing with SHI4185. The results of low-nitrogen tolerance experiment in vermiculite with Ubi-ABP9NINGCHUN4 are basically consistent with that of Ubi-ABP9 SHI4185.
The above results from 2009 to 2010 have showed that ABP9 can significantly improve wheat’s tolerance to drought and low nitrogen stress and has no obvious negative effect on the growth and yield of the wheat under normal conditions.
引文
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