以PMI基因为选择标记露地菊转PtDHAR基因体系的建立
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摘要
露地菊(Chrysanthemum morifolium)是园林绿化观赏植物中常用的花卉之一。其显著的特点是植株低矮,覆盖力强,繁殖容易,管理粗放,适应范围广,抗逆性强,花色丰富,花多而密,花期长,观赏价值高。在我国东北盐碱地区以及城市盐渍化严重的地区,大部分栽植的露地菊受盐碱的影响则表现出叶色萎黄、生长不良、花芽分化受阻甚至死亡的现象。本研究旨在利用安全标记的遗传转化方法获得转化植株,培育出抗盐碱能力更强的新品系,丰富盐碱地区花卉种植品种,提高盐碱地区花卉种植业收益水平。
研究利用Gateway技术,以磷酸甘露糖异构酶基因PMI为选择标记,以盐生植物星星草中克隆的耐盐碱相关基因PtDHAR为目的基因,构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入农杆菌菌株EHA105中,通过农杆菌介导的方法,把耐盐基因PtDHAR转入露地菊‘火焰’基因组中。
主要研究结果如下：(1)遗传转化载体的构建。从大肠杆菌中通过PCR克隆PMI基因全长,装入pGEM(?)-T Vector载体中,利用限制性内切酶Xhol单酶切,以PMI-T载体和pCAMBIA1301载体为基础,构建pCAMBIA1301-PMI载体；利用Gateway技术构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入到农杆菌菌株EHA105中,通过PCR验证结果表明pCAMBIA1301-PMI-PtDHAR载体已成功转入农杆菌菌株EHA105中。
(2)露地菊‘火焰’对甘露糖和头孢霉素的敏感性试验。研究结果当甘露糖浓度为10 g/L时即能明显抑制叶片外植体的分化和不定芽的产生,可以作为遗传转化时叶片外植体筛选培养的临界耐受浓度；分化率和不定芽增殖系数随着头孢霉素(Cef)浓度的升高而逐渐下降,300 mg/L的Cef浓度可作为叶片外植体在共培养后抑制农杆菌的抑菌浓度。
(3)露地菊‘火焰’遗传转化体系的建立。以预培养1d的叶片外植体为转化材料,用OD600值为0.4的农杆菌菌液侵染10min,在23-25℃黑暗条件下共培养2d,随后进行2d延迟培养,接下来以300mg/L的Cef浓度为抑菌浓度,采用选择压递增的方式进行筛选,初期筛选培养基的甘露糖浓度为8g/L,后期筛选培养基中甘露糖的浓度增加到10g/L,连续筛选至不定芽产生。把不定芽转入甘露糖浓度为10g/L、Cef浓度为100mg/L的生根筛选培养基中继续筛选,直至长势健壮并生根的抗性植株产生。
(4)转化植株的检测。对获得的9株抗性植株进行PCR检测,结果表明获得了2株转PtDHAR基因植株。
Chrysanthemum morifolium is commonly used in ornamental gardens and flower. The significant features are low power plant, coverage, easy to manage the extensive breeding, wide, and resistant to strong, a great variety, spend more dense, long flowering period, ornamental value. In saline and Northeast China, as well as the city of saline, most intense cultivation of Chrysanthemum morifolium affected by salt of the exhibit leaf chlorosis, bad growth, bud is blocked or even death. This study aims to take advantage of the security token of the transformation method to obtain a conversion plant has produced salt more lines, rich in plants species, improve income in flower growers.
This study draws Gateway technology to phosphomannose isomerase gene PMI is safe for planting in the cloned tenuiflora salt tolerance gene PtDHAR, built for the purpose of gene pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by Agrobacterium-mediated method, the salt tolerance gene PtDHAR turn into Chrysanthemum morifolium'Huoyan'notation genome, alkali more lines, on large daisy in the country, the better for landscaping services.
The major findings are as follows:
(1) Building genetic transformation vector. From the Escheriachia coli PMI by PCR cloning, gene-digestion method to PMI-T carrier and carrier-based, pCAMBIA1301 into pCAMBIA1301-PMI carrier; use Gateway technology to build pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by PCR validation results showed that pCAMBIA1301-PMI-PtDHAR carrier has successfully transferred to Agrobacterium strain EHA105.
(2) Chrysanthemum morifolium'Huoyan'on Mannose and cephalosporin sensitivity. Study results indicate that leaves explants on Mannose is very sensitive, mannose concentration to 10g/L at apparent suppression leaves explants of differentiation and adventitious buds of produce that can be used as genetic transformation when leaves explant culture of critical tolerance filter concentration; differentiation and proliferation factor as adventitious buds cephalosporin Cef concentration but steady decline,300mg/L Cef concentration can be used as leaf explant cultured in total suppression of Agrobacterium tumefaciens inhibitory concentration.
(3) Established in Chrysanthemum morifolium'Huoyan'genetic transformation system. To pre-training 1d of the leaf explants were transformed material, with a value of 0.4 OD600 Agrobacterium infection 10 min,23-25℃in the dark were cultured under the conditions of 2d, for 2d delayed train to 300mg/L of Cef concentration inhibitory concentration, using selective pressure increments were screened, initial screening medium mannose concentration 8g/L, post-screening medium concentration of mannose increased to lOg/L, continuous screening to produce adventitious buds. To shoot into the mannose concentration of 10g/L, Cef concentration of 100mg/L of rooting medium to filter selection, until the growing robust and resistant plants produce root.
(4) Transformed plants testing. To 9 resistant adult plant which obtains carries on the PCR examination, finally indicated that obtained 2 to transfer the PtDHAR gene adult plant.
研究利用Gateway技术,以磷酸甘露糖异构酶基因PMI为选择标记,以盐生植物星星草中克隆的耐盐碱相关基因PtDHAR为目的基因,构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入农杆菌菌株EHA105中,通过农杆菌介导的方法,把耐盐基因PtDHAR转入露地菊‘火焰’基因组中。
主要研究结果如下：(1)遗传转化载体的构建。从大肠杆菌中通过PCR克隆PMI基因全长,装入pGEM(?)-T Vector载体中,利用限制性内切酶Xhol单酶切,以PMI-T载体和pCAMBIA1301载体为基础,构建pCAMBIA1301-PMI载体；利用Gateway技术构建pCAMBIA1301-PMI-PtDHAR双元表达载体,并将此载体转入到农杆菌菌株EHA105中,通过PCR验证结果表明pCAMBIA1301-PMI-PtDHAR载体已成功转入农杆菌菌株EHA105中。
(2)露地菊‘火焰’对甘露糖和头孢霉素的敏感性试验。研究结果当甘露糖浓度为10 g/L时即能明显抑制叶片外植体的分化和不定芽的产生,可以作为遗传转化时叶片外植体筛选培养的临界耐受浓度；分化率和不定芽增殖系数随着头孢霉素(Cef)浓度的升高而逐渐下降,300 mg/L的Cef浓度可作为叶片外植体在共培养后抑制农杆菌的抑菌浓度。
(3)露地菊‘火焰’遗传转化体系的建立。以预培养1d的叶片外植体为转化材料,用OD600值为0.4的农杆菌菌液侵染10min,在23-25℃黑暗条件下共培养2d,随后进行2d延迟培养,接下来以300mg/L的Cef浓度为抑菌浓度,采用选择压递增的方式进行筛选,初期筛选培养基的甘露糖浓度为8g/L,后期筛选培养基中甘露糖的浓度增加到10g/L,连续筛选至不定芽产生。把不定芽转入甘露糖浓度为10g/L、Cef浓度为100mg/L的生根筛选培养基中继续筛选,直至长势健壮并生根的抗性植株产生。
(4)转化植株的检测。对获得的9株抗性植株进行PCR检测,结果表明获得了2株转PtDHAR基因植株。
Chrysanthemum morifolium is commonly used in ornamental gardens and flower. The significant features are low power plant, coverage, easy to manage the extensive breeding, wide, and resistant to strong, a great variety, spend more dense, long flowering period, ornamental value. In saline and Northeast China, as well as the city of saline, most intense cultivation of Chrysanthemum morifolium affected by salt of the exhibit leaf chlorosis, bad growth, bud is blocked or even death. This study aims to take advantage of the security token of the transformation method to obtain a conversion plant has produced salt more lines, rich in plants species, improve income in flower growers.
This study draws Gateway technology to phosphomannose isomerase gene PMI is safe for planting in the cloned tenuiflora salt tolerance gene PtDHAR, built for the purpose of gene pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by Agrobacterium-mediated method, the salt tolerance gene PtDHAR turn into Chrysanthemum morifolium'Huoyan'notation genome, alkali more lines, on large daisy in the country, the better for landscaping services.
The major findings are as follows:
(1) Building genetic transformation vector. From the Escheriachia coli PMI by PCR cloning, gene-digestion method to PMI-T carrier and carrier-based, pCAMBIA1301 into pCAMBIA1301-PMI carrier; use Gateway technology to build pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by PCR validation results showed that pCAMBIA1301-PMI-PtDHAR carrier has successfully transferred to Agrobacterium strain EHA105.
(2) Chrysanthemum morifolium'Huoyan'on Mannose and cephalosporin sensitivity. Study results indicate that leaves explants on Mannose is very sensitive, mannose concentration to 10g/L at apparent suppression leaves explants of differentiation and adventitious buds of produce that can be used as genetic transformation when leaves explant culture of critical tolerance filter concentration; differentiation and proliferation factor as adventitious buds cephalosporin Cef concentration but steady decline,300mg/L Cef concentration can be used as leaf explant cultured in total suppression of Agrobacterium tumefaciens inhibitory concentration.
(3) Established in Chrysanthemum morifolium'Huoyan'genetic transformation system. To pre-training 1d of the leaf explants were transformed material, with a value of 0.4 OD600 Agrobacterium infection 10 min,23-25℃in the dark were cultured under the conditions of 2d, for 2d delayed train to 300mg/L of Cef concentration inhibitory concentration, using selective pressure increments were screened, initial screening medium mannose concentration 8g/L, post-screening medium concentration of mannose increased to lOg/L, continuous screening to produce adventitious buds. To shoot into the mannose concentration of 10g/L, Cef concentration of 100mg/L of rooting medium to filter selection, until the growing robust and resistant plants produce root.
(4) Transformed plants testing. To 9 resistant adult plant which obtains carries on the PCR examination, finally indicated that obtained 2 to transfer the PtDHAR gene adult plant.
引文
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