耐盐相关基因HAL1在烟草、小麦上的遗传转化研究
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摘要
土壤盐渍化是影响农业生产和生态环境的主要非生物逆境之一。随着人口的增加和耕地的减少,如何开发利用盐渍化土壤己成为农业生产和环境生态亟待解决的问题。利用耐盐相关基因遗传转化提高栽培作物的耐盐性,为盐渍化土地的开发与利用,增加粮食产量和维持农业可持续发展提供了一条新的途径和方法。HAL1基因是酿酒酵母中的重要耐盐因子,其表达参与调节细胞内离子浓度。尽管HAL1基因本身不是转运蛋白,但在盐胁迫下能与ENA1基因协同作用促进Na+外排,与其它转运系统协同作用增加K+的吸收,保持细胞内低的Na+/K+比,以减轻Na+毒害,因而在植物耐盐基因工程上具有很大的潜力。本研究通过农杆菌介导和基因枪轰击,进行了耐盐相关基因HAL1转化普通烟草和小麦的研究,探讨了农杆菌介导的烟草及基因枪轰击的小麦遗传转化条件及其影响因素,将HAL1基因转入模式植物烟草和栽培小麦,获得了以下主要研究结果: 1.优化了农杆菌介导的烟草遗传转化体系:研究了秦烟96和秦烟97烟草叶片的耐卡那霉素水平,为转基因株系的选择提供有效依据,卡那霉素浓度梯度筛选试验表明,以50mg/L卡那霉素作为秦烟96和秦烟97在农杆菌转化后抗性芽的筛选浓度为宜;在除菌步骤中,800 mg/L羧苄青霉素(Cb)进行除菌可以获得理想的筛选和除菌效果。
通过对农杆菌介导的烟草转化条件的探索,建立起了一个快速有效的烟草遗传转化体系,优化的农杆菌介导烟草叶片遗传转化条件为:受体烟草品种秦烟96和秦烟97叶圆片未经预培养(预培养0 d)用稀释150倍的农杆菌菌液(OD600 0.5)浸泡1 min,转至MS1培养基上共培养48 h,无菌水冲洗后于体积分数2 % NaClO中浸泡15 min,无菌水冲洗4～5次,置800 mg/L Cb+质量分数0.1 %的甘露醇中浸泡约12 h除菌,后培养于MS1培养基上诱导烟草叶圆片不定芽的发生,50mg/L卡那霉素筛选秦烟96和秦烟97在农杆菌转化后的抗性芽;抗性芽在MS3培养基上生根成苗。
2.获得了烟草转HAL1基因株:经农杆菌介导和抗性筛选共获得了69株抗性苗,经分子检测,有6株在879bp处有特异性扩增条带的烟草植株,初步证明HAL1基因已整合到两个烟草品种的基因组中。
3.优化了基因枪转化小麦幼胚愈伤组织技术体系:在采用基因枪轰击法对小麦幼胚愈伤组织进行转化的体系中,DNA微弹的最佳浓度采用1.0μg/枪;靶材料的被轰击次数为1次;受体愈伤的最佳发育阶段为胚性启动前期;高渗处理可以使基因枪转化频率得到明显提高,用0.4mol/L的蔗糖在轰击前6h和轰击后18h进行高渗处理转化效果最佳,建立起了高效蔗糖高渗基因枪转化受体系统。研究通过小麦愈伤组织对卡那霉素的敏感性试验及其对转化植株的筛选试验,证明了针对小麦上的NPTⅡ抗性标记基因,卡那霉素的适宜用量为100mg/L。基因枪转化技术体系为:以小偃22和213两个品种(系)的幼胚愈伤组织为为受体,基因枪轰击DNA微弹浓度1.0μg/枪;轰击1次;受体愈伤组织的最佳发育阶段为胚性启动前期;轰击前6h和轰击后18h用0.4mol/L的蔗糖于20℃黑暗条件进行高渗处理,筛选,后在含30g/L蔗糖的幼胚愈伤组织诱导培养基上于26℃、3 000Lx光照条件下恢复培养14d,待胚性愈伤组织充分分化后,再用附加100mg/L Kan的再生培养基MS+2,4-D 1.0mg/L+KT 0.5mg/L+NAA 0.01mg/L+Surose 30g/L+Ag 6.0g/L (PH5.8)筛选转化体。
4.获得了小麦抗kan再生株:通过基因枪轰击转化与抗Kan筛选,共获得了26株抗Kan再生株。
Salinization of soil is one of the major abiotic-stresses that influence agricultural production and environment.With the increasing of world’s population and the decreasing of field,how to develop and utilize salinized-soil becomes an urgent problem in agriculture production and ecological environmental protection. It provides a new way to increase food production for the need of population growing and maintain sustainable development of agrieulture;we can use the correlation salt tolerance gene about the transformation technique to improve the salt tolerance of the cultivar and to discover and utilize salinized-soil. HAL1 gene is a key factor of salt tolerance,it’s expression can regulate Na+/K+ balance.Thought HAL1 gene is not a kind of transfererd protein,it can keep lower Na+/K+ with other gene and transfererd system.So HAL1 gene have great potentiality in the gene engineering of salt tolance.HAL1 gene was transformed into tobacco by transfection of Agrobacterium tumefaciens and into wheat by particle Bombardment ,the factors effecting on frequency of gene transfer were examined was analysised.The main results are as follows:
1.The agrobacterium tumefaciens transformation system was be superior : the study did some research on the level of Kan-resistent with the Qin 96 and Qin 97,and provided some efficiency gists for the transgene plants choices .The experiment of Kan-concentration ladder screening show that scanning the explants with 50 mg / L kanamycin (Kan)and degerming with 800 mg / L carboxymethyl benzylpenicillin (Cb), then it got the ideal result. The optimization conditions of Agrobacterium-mediated genetic transformation were: dipping the explants which were not been pre-training (pre-culture 0 d)in the diluted 150 times Agrobacterium bacilli (OD600 0.5)for 1 min, then transferred the explants to the medium MS1. After training 48 h, washing the explants with sterilized distilled water and then dipping them in volume scores 2% NaClO for 15 min ,rinsing with sterilized distilled water four to five times, and dipping the explants in 800 mg / L Cb + quality scores of 0.1 % mannitol about 12 h.And then put the tobacco explants on the MS1 medium to induce the buds,use 50 mg /L kanamycin to screening the resistent bud of the Qin 96 and Qin 97 after agrobacterium tumefaciens transformation;Then the resistent buds breeded their roots on the MS3 medium .
2.Got the HAL1 transgene plant of tobacco: by the agrobacterium tumefaciens-mediated and the Kan-resistent scanning, we totally got 69 resistent plants. The molecule test show that from the PCR amplification we can see six transformants obtained the special bands at 879bp. HAL1 gene had bene integrated into the genome of part of tobacco plants.
3.The particle Bombardment transformation system was be superior : In the system of the transformation of the callus of wheat immature embryos with the particle Bombardment,the best plasmid DNA concentration is 1.0μg per gun;the acceptor was bombed 1 time ; an efficient acceptor system for gene transformation was established in callus of wheat immature embryos by improving the condition of osmotic treatment before bombardment.In this experiment,the acceptors were from 2 wheat cultivars including Xiaoyan 22 and 213.Particle bombardment was carried out using pROKⅡcontaining the NPTⅡgene and HAL1 gene.The factors affecting the Biolistic transformation efficiency like physiological state of acceptor cells and time of osmotic treatment with different osmosis concentration were studies.The result showed that osmotic treatment can enhance the differentiation frequency of bombarded callus.The callus before embryo switching on was optional.Sucrose as osmosis has a less inactive effect than mannitol before bombardment. The callus treated with 0.4mol/L sucrose for 6 hours before bombardment but 18 hours after bombardment could significantly achieved optional transformation efficiency. The antibiotic,which was normally used in plant transformation for the selection gene NPTⅡwas tested for the optional concentrations in wheat transformation. Kanamycin of 100mg/L used for the selection of NPTⅡwas available.The medium of MS with 1.0mg/L 2,4-D,0.5 mg/L KT and 0.01 mg/L NAA has tested to be an optional cultural condition for regeneration of the callus after bombardment and in selection.
4.Got the Kan-resistent rebirth wheat : by the particle Bombardment and the Kan-resistent scanning ,we totally got 26 resistent transplants.
通过对农杆菌介导的烟草转化条件的探索,建立起了一个快速有效的烟草遗传转化体系,优化的农杆菌介导烟草叶片遗传转化条件为:受体烟草品种秦烟96和秦烟97叶圆片未经预培养(预培养0 d)用稀释150倍的农杆菌菌液(OD600 0.5)浸泡1 min,转至MS1培养基上共培养48 h,无菌水冲洗后于体积分数2 % NaClO中浸泡15 min,无菌水冲洗4～5次,置800 mg/L Cb+质量分数0.1 %的甘露醇中浸泡约12 h除菌,后培养于MS1培养基上诱导烟草叶圆片不定芽的发生,50mg/L卡那霉素筛选秦烟96和秦烟97在农杆菌转化后的抗性芽;抗性芽在MS3培养基上生根成苗。
2.获得了烟草转HAL1基因株:经农杆菌介导和抗性筛选共获得了69株抗性苗,经分子检测,有6株在879bp处有特异性扩增条带的烟草植株,初步证明HAL1基因已整合到两个烟草品种的基因组中。
3.优化了基因枪转化小麦幼胚愈伤组织技术体系:在采用基因枪轰击法对小麦幼胚愈伤组织进行转化的体系中,DNA微弹的最佳浓度采用1.0μg/枪;靶材料的被轰击次数为1次;受体愈伤的最佳发育阶段为胚性启动前期;高渗处理可以使基因枪转化频率得到明显提高,用0.4mol/L的蔗糖在轰击前6h和轰击后18h进行高渗处理转化效果最佳,建立起了高效蔗糖高渗基因枪转化受体系统。研究通过小麦愈伤组织对卡那霉素的敏感性试验及其对转化植株的筛选试验,证明了针对小麦上的NPTⅡ抗性标记基因,卡那霉素的适宜用量为100mg/L。基因枪转化技术体系为:以小偃22和213两个品种(系)的幼胚愈伤组织为为受体,基因枪轰击DNA微弹浓度1.0μg/枪;轰击1次;受体愈伤组织的最佳发育阶段为胚性启动前期;轰击前6h和轰击后18h用0.4mol/L的蔗糖于20℃黑暗条件进行高渗处理,筛选,后在含30g/L蔗糖的幼胚愈伤组织诱导培养基上于26℃、3 000Lx光照条件下恢复培养14d,待胚性愈伤组织充分分化后,再用附加100mg/L Kan的再生培养基MS+2,4-D 1.0mg/L+KT 0.5mg/L+NAA 0.01mg/L+Surose 30g/L+Ag 6.0g/L (PH5.8)筛选转化体。
4.获得了小麦抗kan再生株:通过基因枪轰击转化与抗Kan筛选,共获得了26株抗Kan再生株。
Salinization of soil is one of the major abiotic-stresses that influence agricultural production and environment.With the increasing of world’s population and the decreasing of field,how to develop and utilize salinized-soil becomes an urgent problem in agriculture production and ecological environmental protection. It provides a new way to increase food production for the need of population growing and maintain sustainable development of agrieulture;we can use the correlation salt tolerance gene about the transformation technique to improve the salt tolerance of the cultivar and to discover and utilize salinized-soil. HAL1 gene is a key factor of salt tolerance,it’s expression can regulate Na+/K+ balance.Thought HAL1 gene is not a kind of transfererd protein,it can keep lower Na+/K+ with other gene and transfererd system.So HAL1 gene have great potentiality in the gene engineering of salt tolance.HAL1 gene was transformed into tobacco by transfection of Agrobacterium tumefaciens and into wheat by particle Bombardment ,the factors effecting on frequency of gene transfer were examined was analysised.The main results are as follows:
1.The agrobacterium tumefaciens transformation system was be superior : the study did some research on the level of Kan-resistent with the Qin 96 and Qin 97,and provided some efficiency gists for the transgene plants choices .The experiment of Kan-concentration ladder screening show that scanning the explants with 50 mg / L kanamycin (Kan)and degerming with 800 mg / L carboxymethyl benzylpenicillin (Cb), then it got the ideal result. The optimization conditions of Agrobacterium-mediated genetic transformation were: dipping the explants which were not been pre-training (pre-culture 0 d)in the diluted 150 times Agrobacterium bacilli (OD600 0.5)for 1 min, then transferred the explants to the medium MS1. After training 48 h, washing the explants with sterilized distilled water and then dipping them in volume scores 2% NaClO for 15 min ,rinsing with sterilized distilled water four to five times, and dipping the explants in 800 mg / L Cb + quality scores of 0.1 % mannitol about 12 h.And then put the tobacco explants on the MS1 medium to induce the buds,use 50 mg /L kanamycin to screening the resistent bud of the Qin 96 and Qin 97 after agrobacterium tumefaciens transformation;Then the resistent buds breeded their roots on the MS3 medium .
2.Got the HAL1 transgene plant of tobacco: by the agrobacterium tumefaciens-mediated and the Kan-resistent scanning, we totally got 69 resistent plants. The molecule test show that from the PCR amplification we can see six transformants obtained the special bands at 879bp. HAL1 gene had bene integrated into the genome of part of tobacco plants.
3.The particle Bombardment transformation system was be superior : In the system of the transformation of the callus of wheat immature embryos with the particle Bombardment,the best plasmid DNA concentration is 1.0μg per gun;the acceptor was bombed 1 time ; an efficient acceptor system for gene transformation was established in callus of wheat immature embryos by improving the condition of osmotic treatment before bombardment.In this experiment,the acceptors were from 2 wheat cultivars including Xiaoyan 22 and 213.Particle bombardment was carried out using pROKⅡcontaining the NPTⅡgene and HAL1 gene.The factors affecting the Biolistic transformation efficiency like physiological state of acceptor cells and time of osmotic treatment with different osmosis concentration were studies.The result showed that osmotic treatment can enhance the differentiation frequency of bombarded callus.The callus before embryo switching on was optional.Sucrose as osmosis has a less inactive effect than mannitol before bombardment. The callus treated with 0.4mol/L sucrose for 6 hours before bombardment but 18 hours after bombardment could significantly achieved optional transformation efficiency. The antibiotic,which was normally used in plant transformation for the selection gene NPTⅡwas tested for the optional concentrations in wheat transformation. Kanamycin of 100mg/L used for the selection of NPTⅡwas available.The medium of MS with 1.0mg/L 2,4-D,0.5 mg/L KT and 0.01 mg/L NAA has tested to be an optional cultural condition for regeneration of the callus after bombardment and in selection.
4.Got the Kan-resistent rebirth wheat : by the particle Bombardment and the Kan-resistent scanning ,we totally got 26 resistent transplants.
引文
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