宁夏粳稻离体再生体系的建立及HAL1基因遗传转化的研究
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摘要
西北粳稻是我国重要的粳稻类型之一,其种植面积小,但地处稻米的优质气候生态带,日照充足,昼夜温差大,有利于养分的积累,具有高产、优质的特点。其中以宁夏尤为突出,该省粳稻单位面积产量在全国粳稻区多年来一直名列前1至2位,在粮食生产中占有重要地位。通过品种改良不断提高宁夏粳稻的产量和品质,是促进当地粮食生产发展的重要途径之一。植物遗传转化技术是在分子生物学基础上发展起来的作物定向改良和分子育种新技术,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,利用HAL1基因转化宁夏粳稻在提高宁夏粳稻耐盐特性上有重要的理论和实践意义。
胚和花药组织是禾谷类作物遗传转化中建立离体再生体系的主要外植体。利用成熟胚和花药组织建立水稻高频再生体系及进行外源基因遗传转化的研究已有一些报道,但利用宁夏粳稻成熟胚和花药培养建立高频再生体系及进一步进行遗传转化研究尚未见报道。本研究以宁夏粳稻为试验材料,通过技术的优化,首先建立了宁夏粳稻高效稳定的成熟胚、花药组织离体培养再生体系;探索并优化了农杆菌介导的宁夏粳稻遗传转化条件及其影响因素,利用农杆菌介导法将HAL1基因转入宁夏粳稻,对获得的宁夏粳稻HAL1基因转化株进行了分子生物学鉴定。主要研究结果如下:
1.建立并优化了宁夏粳稻成熟胚离体培养再生的技术体系,筛选出了离体培养再生特性较好的宁夏粳稻基因型。研究表明,参试的34个宁夏粳稻品种成熟胚的培养特性差异很大,愈伤组织诱导率在16.0%～87.3%之间,绿苗分化率在0%～68.9%之间;其中优育28成熟胚表现出良好的培养特性,愈伤组织诱导率和绿苗分化率分别为87.3%、65.6%;宁粳24、宁粳34成熟胚也表现出较好的培养效果。不同水稻品种成熟胚在N6和NB培养基上培养特性有差异。N6培养基诱导水稻成熟胚组织适宜的2,4-D浓度为2mg/L,且添加0.2-0.5mg/L的6-BA能够改善愈伤组织的质量,提高愈伤组织出愈率和分化率;麦芽糖和蔗糖为糖源时宁夏粳稻均表现出较好的培养效果,高浓度的蔗糖对愈伤组织分化有利,但对愈伤组织的诱导不利,蔗糖浓度为30-40g/L时宁夏粳稻的培养效果较好。添加KT2mg/L、NAA1mg/L、IAA0.5mg/L的Ms培养基有利于宁夏粳稻成熟胚愈伤组织的分化。成熟胚愈伤组织在继代1～2次对分化培养无显著影响,继代3次以上,愈伤组织逐渐变得灰暗、无光泽,生长缓慢,分化效果显著变差。总体而言,宁夏粳稻成熟胚离体培养再生特性最好基因型为优育28,愈伤组织诱导的最佳培养基为N6+2,4-D 2mg/L+6-BA 0.2-0.5mg/L+水解酪蛋白0.5g/L+脯氨酸2.8g/L+蔗糖30g/L+琼脂粉6g/L(pH5.8),最佳分化培养基为MS+KT2mg/L+IAA1mg/L+NAA0.5mg/L+水解酪蛋白0.5g/L+蔗糖30g/L+琼脂粉6g/L(pH5.8)。
2.建立并优化了宁夏粳稻花药离体培养再生的技术体系。参试的22份材料中3279、花107花药具有较好的离体培养特性。以A4为诱导愈伤组织培养基的培养效果优于N6。不同基因型的水稻花药对2,4-D浓度要求不同,N6培养基中,宁夏粳稻适宜的2,4-D的质量浓度在1-3mg/L之间;碳源用蔗糖效果优于葡萄糖和麦芽糖,蔗糖浓度为60g/L时培养效果最好;选取温室种植花药的培养效果优于选取大田种植花药的培养效果;低温预处理8d左右的培养效果最好。故宁夏粳稻花药离体再生技术体系方案应为:选择培养特性较好的材料3279、花107,温室种植,花药在4℃冰箱内低温预处理8d,诱导愈伤组织培养基为N6+2,4-D 1-3mg/L+KT 0.5mg/L+水解酪蛋白0.5g/L+脯氨酸0.5g/L+蔗糖60g/L+琼脂粉6g/L(pH5.8),分化培养基为MS+KT 2.0mg/L+NAA 1mg/L+IAA 0.5mg/L+蔗糖30g/L+琼脂粉6g/L(pH5.8)。
3.建立并优化了农杆菌介导的宁夏粳稻成熟胚愈伤组织遗传转化技术体系。以优育28成熟胚愈伤组织为转化受体,研究了农杆菌介导法转化体系中筛选剂Kan的浓度、Cb对愈伤组织的影响以及共培养时间、洗菌、干燥处理等对遗传转化的影响。结果表明,Kan的浓度为250mg/L时愈伤组织均褐化死亡,可以作为参试材料的筛选浓度。Cb的浓度大于600mg/L时明显抑制了愈伤组织的生长。共培养3d获得的抗性愈伤组织最多。共培养后,利用加入Cb和Cef各500mg/L的无菌水混合液处理共培养的愈伤组织,对抑制农杆菌起到了较好的效果,其抗性愈伤组织获得率最高。在接种筛选培养基之前,干燥处理可以有效的抑制农杆菌的生长和繁殖,提高抗性愈伤组织的获得率。
4.通过对宁夏粳稻成熟胚、花药离体再生体系的建立和HAL1农杆菌法转化水稻的研究,首次建立起了一个有效的宁夏粳稻遗传转化体系。以优育28成熟胚诱导的愈伤组织为受体材料,经农杆菌介导和Kan抗性筛选,共获得了69个转化植株。经PCR分子检测,有3株在879bp处有特异性扩增条带,初步证明HAL1基因已整合到宁夏粳稻优育28的基因组中。
Japonica rice in Northwest is one important type of rice in China with the characteric of high yield and elite quility,because of abundant sunshine and large diurnal temperature amplitude,which was benefit for nutrient accumulation.Inspite of small planting area,it is named as arable ecological zone for high quality rice production.For example,rice production in Ningxia ranked top or second among rice production areas in China according average yield/unit of years.
In Ningxia,improving the varieties was one of important ways to promote local food production through continuously improving yield and quality.Plant genetic transformation, developed from molecular biology,was a new technologies using directional crops modified and breeding on molecular level.HAL1 gene,acting on balance of Na~+/K~+,was a key gen on salt tolerance from Saccharomyces cerevisiae.Despite it was not a kind of gene transferering protein,it can keep lower Na~+/K~+ with other gene and transferering sistem.So HAL1 gene have great potentiality in the gene engineering of salt tolance.In theoretical,it played important role on using HAL1 gene to transform Ningxia Japonica rice for rice salt-tolerant.
Embryo and anther tissues culture are main explants to establish the vitro regeneration system in cereal crops genetic modified.Some reports on using rice mature embryo and anther organizations to establish high frequency rice regeneration system and exogenous genetic transformation can be found on rice,but few on Ningxia Japonica rice.
In this study,experiments were conducted on Ningxia Japonica rice material through technology optimization.Firstly,highly efficient and stable mature embryos and anther organizations vitro regeneration system was established.Secondly,agrobacterium tumefaciens-mediated genetic transformation of conditions and the factors affecting were explored and optimized.Thirdly,using of Agrobacterium-mediated transformed HAL1 into the Ningxia Japonica rice,and finally HAL1 Transgenic rice was identified with molecular biology.The result showed as following.
1.Mature embryos vitro culture regeneration technology system was established and optimized using Ningxia Japonica rice,and genotype of better culture effects were selected. The resulted showed that effects of mature embryo culture was vastly differenence among 34 varieties of Ningxia Japonica rice.Callus induction rate was between 16.0%to 87.3%,green plantlet differentiation rate was between 0%to 68.9%.Mature embryos of Youyu28 performed the best effect culture,with callus induction rate and green plantlet differentiation rate were 87.3%and 65.6%,separately.Effect culture of Ningjing24 and Ningjing34 were also good.Different genotypes had different culture abilities in N6 culture medium and NB culture medium.Using N6 culture,the addition of 2mg/L 2,4-D to induction medium was favorable for effect of culture.Adding 0.2mg/L-0.5mg/L 6-BA can improve mature embryo culture ability.Sucrose and Maltose were higher than Glucose to induce and differentiate callus.And 30-40g/L was the best for proportion of sucrose to induce and differentiate callus.Using Ms culture medium,adding KT2mg/L+NAA1mg/L+IAA0.5mg/L was better for green plantlet differentiation of Ningxia Japonia rice mature embryo callus.Subculture 1-2 times,green plantlet differentiation of callus was no significant impact.Subculture more than 3 times,callus become gloomy,dull,and growing slow down,green plantlet differentiation of callus was significantly deteriorate.Overall,in Ningxia Japonica rice mature embryos vitro regeneration,best genotype was Youyu28,best induction culture medium is N6+2,4-D 2mg/L+6-BA 0.2-0.5mg/L+hydrolyzed casein protein 0.5g/L+Proline 2.8g/L+ sucrose 30g/L+Agarose powder 6g/L(pH5.8),best green plantlet differentiation is Ms+KT 2mg/L+IAA 1mg/L+NAA 0.5mg/L+hydrolyzed casein protein 0.5g/L+sucrose 30g/L+ Agarose powder 6g/L(pH5.8).
2.Established and optimized anther vitro regenetation technology system of Ningxia Japonica rice.The resulted showed that anther of 3279 and Hua107 were high culture ability among 22 Ningxia Japonica rice cultivars.Effects of culture medium showed that A4 culture medium was better than N6 culture medium.Different genotypes had different culture abilities for concentration of 2,4-D,the extent was 1-3mg/L.Sucrose was better to induce callus using anther,and 60 g/L was the best proportion.Effects of anther culture in greenhouse cultivation is better than in field cultivation.Low-temperature pretreated about 8d had the best effect culture.So in anther vitro regeneration system of Ningxia Japonica rice, plant 3279 and Hual07 had better culture effect in greenhouse,and put anther in 4℃refrigerator pretreated 8d.Induction medium is N6+2,4-D 1-3mg/L+KT 0.5mg/L+ hydrolyzed casein protein 0.5g/L+Proline 0.5g/L+sucrose 60g/L+gluose powder 6g/L (pH5.8).Green plantlet differentiation medium was MS+KT 2.0mg/L+NAA 1mg/L+ IAA 0.5mg/L+sucrose 30g/L+gluose powder 6g/L(pH5.8).
3.The Agrobacterium-mediated genetic transformation technology system was established and optimized using mature embryo callus of Ningxiat Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,the effects of agrobacterium-mediated genetic transformation were studied on influence of Kan-resistant concentration,Cb concentration,co-culture time,washing agrobacterium,drying treatment etc.The results showed that 250mg/L was Kan-resistant concentration.Concentration of Cb more than 600mg/L inhibited the growth of the callus significantly.Co-culture 3d can obtain the most resistant callus.Using sterile water added Cb and Cef 500mg/L separately can inhibit agrobacterium perfectly,the resistant callus rate was the highest.Before inoculating filtration medium,drying treatment can inhibit the growth and reproduction of Agrobacterium tumefaciens effectively,and improve resistant rate of callus.
4.Through the establishment of vitro regeneration system and research on HAL1 genetic transformation,genetic transformation system was established firstly using Ningxia Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,by Agrobacterium tumefaciens and Kan resistance filtrating,69 plants resistant to Kan were obtained.The results of PCR amplification showed that 3 transformants obtained with the special bands at 879bp.Initial proof that HAL1 gene had being integrated into the genome of northwest Japonica rice Youyu28.
胚和花药组织是禾谷类作物遗传转化中建立离体再生体系的主要外植体。利用成熟胚和花药组织建立水稻高频再生体系及进行外源基因遗传转化的研究已有一些报道,但利用宁夏粳稻成熟胚和花药培养建立高频再生体系及进一步进行遗传转化研究尚未见报道。本研究以宁夏粳稻为试验材料,通过技术的优化,首先建立了宁夏粳稻高效稳定的成熟胚、花药组织离体培养再生体系;探索并优化了农杆菌介导的宁夏粳稻遗传转化条件及其影响因素,利用农杆菌介导法将HAL1基因转入宁夏粳稻,对获得的宁夏粳稻HAL1基因转化株进行了分子生物学鉴定。主要研究结果如下:
1.建立并优化了宁夏粳稻成熟胚离体培养再生的技术体系,筛选出了离体培养再生特性较好的宁夏粳稻基因型。研究表明,参试的34个宁夏粳稻品种成熟胚的培养特性差异很大,愈伤组织诱导率在16.0%～87.3%之间,绿苗分化率在0%～68.9%之间;其中优育28成熟胚表现出良好的培养特性,愈伤组织诱导率和绿苗分化率分别为87.3%、65.6%;宁粳24、宁粳34成熟胚也表现出较好的培养效果。不同水稻品种成熟胚在N6和NB培养基上培养特性有差异。N6培养基诱导水稻成熟胚组织适宜的2,4-D浓度为2mg/L,且添加0.2-0.5mg/L的6-BA能够改善愈伤组织的质量,提高愈伤组织出愈率和分化率;麦芽糖和蔗糖为糖源时宁夏粳稻均表现出较好的培养效果,高浓度的蔗糖对愈伤组织分化有利,但对愈伤组织的诱导不利,蔗糖浓度为30-40g/L时宁夏粳稻的培养效果较好。添加KT2mg/L、NAA1mg/L、IAA0.5mg/L的Ms培养基有利于宁夏粳稻成熟胚愈伤组织的分化。成熟胚愈伤组织在继代1～2次对分化培养无显著影响,继代3次以上,愈伤组织逐渐变得灰暗、无光泽,生长缓慢,分化效果显著变差。总体而言,宁夏粳稻成熟胚离体培养再生特性最好基因型为优育28,愈伤组织诱导的最佳培养基为N6+2,4-D 2mg/L+6-BA 0.2-0.5mg/L+水解酪蛋白0.5g/L+脯氨酸2.8g/L+蔗糖30g/L+琼脂粉6g/L(pH5.8),最佳分化培养基为MS+KT2mg/L+IAA1mg/L+NAA0.5mg/L+水解酪蛋白0.5g/L+蔗糖30g/L+琼脂粉6g/L(pH5.8)。
2.建立并优化了宁夏粳稻花药离体培养再生的技术体系。参试的22份材料中3279、花107花药具有较好的离体培养特性。以A4为诱导愈伤组织培养基的培养效果优于N6。不同基因型的水稻花药对2,4-D浓度要求不同,N6培养基中,宁夏粳稻适宜的2,4-D的质量浓度在1-3mg/L之间;碳源用蔗糖效果优于葡萄糖和麦芽糖,蔗糖浓度为60g/L时培养效果最好;选取温室种植花药的培养效果优于选取大田种植花药的培养效果;低温预处理8d左右的培养效果最好。故宁夏粳稻花药离体再生技术体系方案应为:选择培养特性较好的材料3279、花107,温室种植,花药在4℃冰箱内低温预处理8d,诱导愈伤组织培养基为N6+2,4-D 1-3mg/L+KT 0.5mg/L+水解酪蛋白0.5g/L+脯氨酸0.5g/L+蔗糖60g/L+琼脂粉6g/L(pH5.8),分化培养基为MS+KT 2.0mg/L+NAA 1mg/L+IAA 0.5mg/L+蔗糖30g/L+琼脂粉6g/L(pH5.8)。
3.建立并优化了农杆菌介导的宁夏粳稻成熟胚愈伤组织遗传转化技术体系。以优育28成熟胚愈伤组织为转化受体,研究了农杆菌介导法转化体系中筛选剂Kan的浓度、Cb对愈伤组织的影响以及共培养时间、洗菌、干燥处理等对遗传转化的影响。结果表明,Kan的浓度为250mg/L时愈伤组织均褐化死亡,可以作为参试材料的筛选浓度。Cb的浓度大于600mg/L时明显抑制了愈伤组织的生长。共培养3d获得的抗性愈伤组织最多。共培养后,利用加入Cb和Cef各500mg/L的无菌水混合液处理共培养的愈伤组织,对抑制农杆菌起到了较好的效果,其抗性愈伤组织获得率最高。在接种筛选培养基之前,干燥处理可以有效的抑制农杆菌的生长和繁殖,提高抗性愈伤组织的获得率。
4.通过对宁夏粳稻成熟胚、花药离体再生体系的建立和HAL1农杆菌法转化水稻的研究,首次建立起了一个有效的宁夏粳稻遗传转化体系。以优育28成熟胚诱导的愈伤组织为受体材料,经农杆菌介导和Kan抗性筛选,共获得了69个转化植株。经PCR分子检测,有3株在879bp处有特异性扩增条带,初步证明HAL1基因已整合到宁夏粳稻优育28的基因组中。
Japonica rice in Northwest is one important type of rice in China with the characteric of high yield and elite quility,because of abundant sunshine and large diurnal temperature amplitude,which was benefit for nutrient accumulation.Inspite of small planting area,it is named as arable ecological zone for high quality rice production.For example,rice production in Ningxia ranked top or second among rice production areas in China according average yield/unit of years.
In Ningxia,improving the varieties was one of important ways to promote local food production through continuously improving yield and quality.Plant genetic transformation, developed from molecular biology,was a new technologies using directional crops modified and breeding on molecular level.HAL1 gene,acting on balance of Na~+/K~+,was a key gen on salt tolerance from Saccharomyces cerevisiae.Despite it was not a kind of gene transferering protein,it can keep lower Na~+/K~+ with other gene and transferering sistem.So HAL1 gene have great potentiality in the gene engineering of salt tolance.In theoretical,it played important role on using HAL1 gene to transform Ningxia Japonica rice for rice salt-tolerant.
Embryo and anther tissues culture are main explants to establish the vitro regeneration system in cereal crops genetic modified.Some reports on using rice mature embryo and anther organizations to establish high frequency rice regeneration system and exogenous genetic transformation can be found on rice,but few on Ningxia Japonica rice.
In this study,experiments were conducted on Ningxia Japonica rice material through technology optimization.Firstly,highly efficient and stable mature embryos and anther organizations vitro regeneration system was established.Secondly,agrobacterium tumefaciens-mediated genetic transformation of conditions and the factors affecting were explored and optimized.Thirdly,using of Agrobacterium-mediated transformed HAL1 into the Ningxia Japonica rice,and finally HAL1 Transgenic rice was identified with molecular biology.The result showed as following.
1.Mature embryos vitro culture regeneration technology system was established and optimized using Ningxia Japonica rice,and genotype of better culture effects were selected. The resulted showed that effects of mature embryo culture was vastly differenence among 34 varieties of Ningxia Japonica rice.Callus induction rate was between 16.0%to 87.3%,green plantlet differentiation rate was between 0%to 68.9%.Mature embryos of Youyu28 performed the best effect culture,with callus induction rate and green plantlet differentiation rate were 87.3%and 65.6%,separately.Effect culture of Ningjing24 and Ningjing34 were also good.Different genotypes had different culture abilities in N6 culture medium and NB culture medium.Using N6 culture,the addition of 2mg/L 2,4-D to induction medium was favorable for effect of culture.Adding 0.2mg/L-0.5mg/L 6-BA can improve mature embryo culture ability.Sucrose and Maltose were higher than Glucose to induce and differentiate callus.And 30-40g/L was the best for proportion of sucrose to induce and differentiate callus.Using Ms culture medium,adding KT2mg/L+NAA1mg/L+IAA0.5mg/L was better for green plantlet differentiation of Ningxia Japonia rice mature embryo callus.Subculture 1-2 times,green plantlet differentiation of callus was no significant impact.Subculture more than 3 times,callus become gloomy,dull,and growing slow down,green plantlet differentiation of callus was significantly deteriorate.Overall,in Ningxia Japonica rice mature embryos vitro regeneration,best genotype was Youyu28,best induction culture medium is N6+2,4-D 2mg/L+6-BA 0.2-0.5mg/L+hydrolyzed casein protein 0.5g/L+Proline 2.8g/L+ sucrose 30g/L+Agarose powder 6g/L(pH5.8),best green plantlet differentiation is Ms+KT 2mg/L+IAA 1mg/L+NAA 0.5mg/L+hydrolyzed casein protein 0.5g/L+sucrose 30g/L+ Agarose powder 6g/L(pH5.8).
2.Established and optimized anther vitro regenetation technology system of Ningxia Japonica rice.The resulted showed that anther of 3279 and Hua107 were high culture ability among 22 Ningxia Japonica rice cultivars.Effects of culture medium showed that A4 culture medium was better than N6 culture medium.Different genotypes had different culture abilities for concentration of 2,4-D,the extent was 1-3mg/L.Sucrose was better to induce callus using anther,and 60 g/L was the best proportion.Effects of anther culture in greenhouse cultivation is better than in field cultivation.Low-temperature pretreated about 8d had the best effect culture.So in anther vitro regeneration system of Ningxia Japonica rice, plant 3279 and Hual07 had better culture effect in greenhouse,and put anther in 4℃refrigerator pretreated 8d.Induction medium is N6+2,4-D 1-3mg/L+KT 0.5mg/L+ hydrolyzed casein protein 0.5g/L+Proline 0.5g/L+sucrose 60g/L+gluose powder 6g/L (pH5.8).Green plantlet differentiation medium was MS+KT 2.0mg/L+NAA 1mg/L+ IAA 0.5mg/L+sucrose 30g/L+gluose powder 6g/L(pH5.8).
3.The Agrobacterium-mediated genetic transformation technology system was established and optimized using mature embryo callus of Ningxiat Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,the effects of agrobacterium-mediated genetic transformation were studied on influence of Kan-resistant concentration,Cb concentration,co-culture time,washing agrobacterium,drying treatment etc.The results showed that 250mg/L was Kan-resistant concentration.Concentration of Cb more than 600mg/L inhibited the growth of the callus significantly.Co-culture 3d can obtain the most resistant callus.Using sterile water added Cb and Cef 500mg/L separately can inhibit agrobacterium perfectly,the resistant callus rate was the highest.Before inoculating filtration medium,drying treatment can inhibit the growth and reproduction of Agrobacterium tumefaciens effectively,and improve resistant rate of callus.
4.Through the establishment of vitro regeneration system and research on HAL1 genetic transformation,genetic transformation system was established firstly using Ningxia Japonica rice.Using Youyu28 mature embryo callus as transformed receptor,by Agrobacterium tumefaciens and Kan resistance filtrating,69 plants resistant to Kan were obtained.The results of PCR amplification showed that 3 transformants obtained with the special bands at 879bp.Initial proof that HAL1 gene had being integrated into the genome of northwest Japonica rice Youyu28.
引文
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