海藻糖合成酶基因转化玉米
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摘要
干旱是玉米生产的主要限制因素。通过品种改良提高玉米品种耐旱能力,是克服干旱危害最为经济有效的方法。然而,玉米的耐旱性为微效多基因控制的数量性状,遗传改良比较困难,多年来进展不大。转基因技术可以打破物种间生殖隔离,转化利用其他物种有益基因。国内外曾用SOD、DREB等多种抗逆耐旱基因转化玉米,试图提高其耐旱性,但因这些基因耐旱能力不够强,耐旱机制与玉米生理代谢不协调等原因,转基因后代的耐旱性不能达到玉米生产要求。探索利用耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因,是转基因耐旱玉米培育的技术关键。利用海藻糖的抗逆耐旱特性,采用基因工程将其相关酶的基因转入玉米等作物,进而培育抗旱耐盐新品种,这对于植物遗传改良具有重要意义。
本研究按Adams介绍的方法提取啤酒酵母AS.1416菌株基因组DNA。依据PCR引物设计原则,采用Premier 5.0软件设计引物,用于扩增酵母基因组DNA上的TPS1片段。该引物在扩增出的TPS1片段两端引入新的酶切位点BamHⅠ和ApaⅠ。琼脂糖凝胶电泳回收扩增的TPS1片段并插入到T载体pMD19-T Vector中,命名为pT-TPS1。
单子叶植物表达载体pC1300UbiDREB含有玉米泛素启动子ubiquitin、由植物组成型3.5S启动子启动的选择标记基因Hyg和大肠杆菌选择标记基因Kan。用限制性内切酶BamHⅠ和ApaⅠ分别消化pC1300UbiDREB质粒和包含TPS1基因的pT-TPS1质粒,琼脂糖凝胶电泳分别回收大片段和小片段,经T_4-DNA连接酶连接后,组成单子叶植物表达载体pC1300UbiTPS1。
根据载体pC1300UbiTPS1多酶切位点,用限制性内切酶BamHⅠ和ApaⅠ消化pC1300UbiTPS1,琼脂糖凝胶电泳显示长1603和11700 bp的两条特异带,分别是TPS1和pC1300UbiTPS1质粒骨架的长度。由此说明,TPS1基因插入到载体pC1300UbiTPS1中。
农杆菌介导法转化“18-599R”和“18-599W”玉米胚性愈伤组织,两轮潮霉素梯度筛选后,得到“18-599R”和“18-599W”抗性愈伤组织各340和607块。愈伤组织的转化率分别为6.4%和10.2%,两者之间没有显著性差异。但是,“18-599R”愈伤组织的再生能力明显高于“18-599W”。抗性愈伤组织分化培养分别得到再生植株54和31株。PCR检测获得1个阳性植株。
研究发现以继代后7-9d的愈伤组织侵染,其抗性愈伤的获得率最高。在共培养时添加400 mg/L半胱氨酸有助于获得稳定的转化水平,半胱氨酸作为一种抗氧化抑制剂有助于T-DNA转移到胚性愈伤组织中去。农杆菌菌液浓度为OD_(600)=0.5,侵染时间为10 min时,杀菌比较容易,愈伤组织褐化率较低,是最适合的侵染浓度和时间。共培养时间以3 d为最佳。
PCR检测阳性的植株,还有待进一步作分子杂交和表达检测,以及后代的分子标记选择和耐旱性鉴定,培育成稳定的转基因株系。除探索转化利用外源海藻糖合成酶基因,提高玉米耐旱性的可能性外,也有助于研究海藻糖代谢的有关机理。
Drought is the main limiting factor in the production of maize.Maize breeding is use to improve the ability of drought tolerance.It is the most economical and efficient way to deduce the loss of drought.But the drought ability of maize is controlled by microeffect multiplegene.Good result is not received because it's difficult to improve the drought ability by breeding.Transgenic technology can break the species isolation and utilize the gene of other species.Trying to improve the ability of drought tolerance,a large number of genes such as SOD and DREB are transferred into maize.But the transgenic plants can not fit the damand of maize production,because the ait-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself.Technically it is very important to make use of hight ait-drought ability of the gene which is more consistant with maize.Trehalose has the trait of adversity tolerance.So it's very significant to transfer the related gene to maize and breed now maize lines which have the strong ability of drought tolerance.
Genomic DNA of Saccharomyces cerevisiae was extracted from strain AS.1416 by the method introduced by Adams et al.(1998).According to the sequence of gene TPS1,a pair of specific PCR primers was designed using primer 5.0.At the 5'ends of the forward and reverse primers,recognition sites of restriction endonucleases BamH I and Apa I.The specific amplified fragment was separated by agarose gel electrophoresis and inserted into cloning vector pMD19-T and named pT-TPS1.
Plasmid pT-TPS1 containing gene TPS1 and plasmid pC1300UbiDREB containing promoter ubiquitin,were digested by restriction endonucleases BamH I and Apa I, respectively.Fragments of gene TPS1 and plasmid pC1300UbiDREB without gene DREB were separated by low melting-point agarose gel eledtrophoresis and ligated by T_4-DNA ligase to form plasmid pC1300UbiTPS1.
According to its multiple cloning sites,the expression structure of pC1300UbiTPS1 was confirmed by restriction digestion of BamH I and Apa I.Two specific fragments of 1603 and 11700 bp were separated by agarose gel electrophoresis.They were the same long as gene TPS1 and the backbone of plasmid pC1300UbiTPS1.This result showed that gene TPS1 had been inserted into vector pC1300UbiTPS1 and the plasmid pC1300UbiTPS1 had the expression structure as designed.
Expression vectors with TPS1 gene promoted by maize ubiquitin promoter and stress inducible promoter(mwcs120)of monocotyledon respectively were used to transfer embyronic calli of "18-599R" and "18-599W" mediated by Agrobacterium.After two times of screening on gradient hygromycin medium,340 and 607 pieces of positive calli were screend from inbred lines "18-599R" and "18-599W",respectively.The rates of positive calli were 6.4 and 10.2%.No significant difference was found between these two rates.However,the regeneration ability of" 18-599R" was higher than "18-599W".The regeneration rate of "18-599R" was more than three times of "18-599W".54 and 31 regeneration plants were obtained.1 positive plant was obtained by PCR amplification.
Reseearch found that the percentage of positive clallus regenerated was the highest when callus subculture for 7～9 days.Supplementary the cysteine(400 mg/L)in co-culture medium was essential to efficient transfer foreign gene to gramineous plants.In addition,the infecting A.tumefaciens concentration was OD_(600)=0.5,10 minutes infection time and co-culture 3 days were the optimal conditions for Agrobacterium tumefaciens mediated maize transformation.
The positive plant which was just tested by PCR need further study such as molecular hybridization,expression test,molecular mark selection and drought tolerance identification until it is breeding to be steady transgenic breed.It's exploration to improve the ait-drought ability on method of transgene and it is also helpful to study the mechanism of trehalose metabolism.
本研究按Adams介绍的方法提取啤酒酵母AS.1416菌株基因组DNA。依据PCR引物设计原则,采用Premier 5.0软件设计引物,用于扩增酵母基因组DNA上的TPS1片段。该引物在扩增出的TPS1片段两端引入新的酶切位点BamHⅠ和ApaⅠ。琼脂糖凝胶电泳回收扩增的TPS1片段并插入到T载体pMD19-T Vector中,命名为pT-TPS1。
单子叶植物表达载体pC1300UbiDREB含有玉米泛素启动子ubiquitin、由植物组成型3.5S启动子启动的选择标记基因Hyg和大肠杆菌选择标记基因Kan。用限制性内切酶BamHⅠ和ApaⅠ分别消化pC1300UbiDREB质粒和包含TPS1基因的pT-TPS1质粒,琼脂糖凝胶电泳分别回收大片段和小片段,经T_4-DNA连接酶连接后,组成单子叶植物表达载体pC1300UbiTPS1。
根据载体pC1300UbiTPS1多酶切位点,用限制性内切酶BamHⅠ和ApaⅠ消化pC1300UbiTPS1,琼脂糖凝胶电泳显示长1603和11700 bp的两条特异带,分别是TPS1和pC1300UbiTPS1质粒骨架的长度。由此说明,TPS1基因插入到载体pC1300UbiTPS1中。
农杆菌介导法转化“18-599R”和“18-599W”玉米胚性愈伤组织,两轮潮霉素梯度筛选后,得到“18-599R”和“18-599W”抗性愈伤组织各340和607块。愈伤组织的转化率分别为6.4%和10.2%,两者之间没有显著性差异。但是,“18-599R”愈伤组织的再生能力明显高于“18-599W”。抗性愈伤组织分化培养分别得到再生植株54和31株。PCR检测获得1个阳性植株。
研究发现以继代后7-9d的愈伤组织侵染,其抗性愈伤的获得率最高。在共培养时添加400 mg/L半胱氨酸有助于获得稳定的转化水平,半胱氨酸作为一种抗氧化抑制剂有助于T-DNA转移到胚性愈伤组织中去。农杆菌菌液浓度为OD_(600)=0.5,侵染时间为10 min时,杀菌比较容易,愈伤组织褐化率较低,是最适合的侵染浓度和时间。共培养时间以3 d为最佳。
PCR检测阳性的植株,还有待进一步作分子杂交和表达检测,以及后代的分子标记选择和耐旱性鉴定,培育成稳定的转基因株系。除探索转化利用外源海藻糖合成酶基因,提高玉米耐旱性的可能性外,也有助于研究海藻糖代谢的有关机理。
Drought is the main limiting factor in the production of maize.Maize breeding is use to improve the ability of drought tolerance.It is the most economical and efficient way to deduce the loss of drought.But the drought ability of maize is controlled by microeffect multiplegene.Good result is not received because it's difficult to improve the drought ability by breeding.Transgenic technology can break the species isolation and utilize the gene of other species.Trying to improve the ability of drought tolerance,a large number of genes such as SOD and DREB are transferred into maize.But the transgenic plants can not fit the damand of maize production,because the ait-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself.Technically it is very important to make use of hight ait-drought ability of the gene which is more consistant with maize.Trehalose has the trait of adversity tolerance.So it's very significant to transfer the related gene to maize and breed now maize lines which have the strong ability of drought tolerance.
Genomic DNA of Saccharomyces cerevisiae was extracted from strain AS.1416 by the method introduced by Adams et al.(1998).According to the sequence of gene TPS1,a pair of specific PCR primers was designed using primer 5.0.At the 5'ends of the forward and reverse primers,recognition sites of restriction endonucleases BamH I and Apa I.The specific amplified fragment was separated by agarose gel electrophoresis and inserted into cloning vector pMD19-T and named pT-TPS1.
Plasmid pT-TPS1 containing gene TPS1 and plasmid pC1300UbiDREB containing promoter ubiquitin,were digested by restriction endonucleases BamH I and Apa I, respectively.Fragments of gene TPS1 and plasmid pC1300UbiDREB without gene DREB were separated by low melting-point agarose gel eledtrophoresis and ligated by T_4-DNA ligase to form plasmid pC1300UbiTPS1.
According to its multiple cloning sites,the expression structure of pC1300UbiTPS1 was confirmed by restriction digestion of BamH I and Apa I.Two specific fragments of 1603 and 11700 bp were separated by agarose gel electrophoresis.They were the same long as gene TPS1 and the backbone of plasmid pC1300UbiTPS1.This result showed that gene TPS1 had been inserted into vector pC1300UbiTPS1 and the plasmid pC1300UbiTPS1 had the expression structure as designed.
Expression vectors with TPS1 gene promoted by maize ubiquitin promoter and stress inducible promoter(mwcs120)of monocotyledon respectively were used to transfer embyronic calli of "18-599R" and "18-599W" mediated by Agrobacterium.After two times of screening on gradient hygromycin medium,340 and 607 pieces of positive calli were screend from inbred lines "18-599R" and "18-599W",respectively.The rates of positive calli were 6.4 and 10.2%.No significant difference was found between these two rates.However,the regeneration ability of" 18-599R" was higher than "18-599W".The regeneration rate of "18-599R" was more than three times of "18-599W".54 and 31 regeneration plants were obtained.1 positive plant was obtained by PCR amplification.
Reseearch found that the percentage of positive clallus regenerated was the highest when callus subculture for 7～9 days.Supplementary the cysteine(400 mg/L)in co-culture medium was essential to efficient transfer foreign gene to gramineous plants.In addition,the infecting A.tumefaciens concentration was OD_(600)=0.5,10 minutes infection time and co-culture 3 days were the optimal conditions for Agrobacterium tumefaciens mediated maize transformation.
The positive plant which was just tested by PCR need further study such as molecular hybridization,expression test,molecular mark selection and drought tolerance identification until it is breeding to be steady transgenic breed.It's exploration to improve the ait-drought ability on method of transgene and it is also helpful to study the mechanism of trehalose metabolism.
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