农杆菌介导高梁转化体系的建立与Bt（Bacillus thuringiensis）抗虫基因的导入
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摘要
在优化高粱再生系统和农杆菌介导的遗传转化体系的基础上，采用农杆菌介导法获得了含密码子优化的杀虫晶体蛋白(Bt)基因cry1Ab转基因高粱。
通过正交试验和单因子试验确定了高梁离体培养的最佳培养基，并对其多种影响因素进行研究。结果表明，高梁茎尖直接再生的培养基为：MS无机盐+B5有机物+KT0.25mg／L+BAP 0.25mg／L+IAA 0.25mg／L+L-Asn 200mg／L+抗坏血酸10mg／L+PVP 100mg／L+蔗糖30g／L+琼脂10g／L；种子(胚)愈伤组织诱导培养基为：MS(C)无机盐+MS有机物+2，4-D 3mg／L+KT 0.05mg／L+L-Pro 50mg／L+水解酪蛋白500mg／L+PVP8mg／L+蔗糖30g／L+琼脂8g／L；种子(胚)愈伤组织继代培养基为：MS(C)无机盐+MS有机物+2，4-D 3mg／L+KT 0.5mg／L+L-Asn 200mg／L+L-Pro 50mg／L+PVP 100mg／L+抗坏血酸500rog／L+蔗糖30g／L+琼脂8e,／L；未成熟胚愈伤组织诱导培养基为：MS无机盐+MS有机物+2，4-D 2mg／L+KT 0.05mg／L+玉米素0.05mg／L+L-Asn 150mg／L+L-Pro700mg／L+水解酪蛋白lg／L+PVP 8mg／L+抗坏血酸10mg／L+NH_4NO_3 3.5g／L+KH_2PO_40.5g／L+蔗糖20g／L+琼脂8g／L；未成熟胚愈伤组织愈伤组织继代培养基为：MS无机盐+MS有机物+2，4-D2mg／L+玉米素0.5mg／L+L-Ash 150mg／L+L-Pro150mg／L+水解酪蛋白500mg／L+PVP 8mg／L+抗坏血酸10mg／L+NH_4NO_3 3.5g／L+KH_2PO_40.5g／L+蔗糖20g／L+琼脂8g／L；幼穗愈伤组织诱导培养基为：YI4培养基(MS+2，4-D2mg／L+玉米素2.2mg／L+PVP 8mg／L+抗坏血酸10mg／L+水解酪蛋白500mg／L+蔗糖30g／L+琼脂7.2g／L)；幼穗愈伤组织继代培养基为YI4+KT0.175mg／L。
对不同外植体和不同基因型诱导产生愈伤组织的再生能力进行比较研究，结果表明，幼穗愈伤组织分化率最高，平均为77.2％；其次是茎尖，平均再生率为39.4％；未成熟胚愈伤组织平均分化率为21.70％；种子(胚)愈伤组织分化能力最差，分化率平均仅为1％。此外，不同基因型间的再生能力差异很大。以幼穗、茎尖和未成熟胚作外植体比较理想，可用于进一步的转化研究。
通过gus基因瞬时表达对农杆菌介导高梁转化体系及其影响因子进行了系统性研究，获得较高转化频率的条件为：(1)茎尖：预培养时间3d，在OD_(600)值为0.5农杆菌菌液中感染10min，共培养时间为3d；(2)未成熟胚和幼穗愈伤组织：预培养时间3d，
最适农杆菌菌液浓度ODm值为0．5—0．7，感染时间5—20Inin，最适共培养基pH值为
5二一5石，最适共培养温度为22—25℃，最佳共培养时间3d；（）在共培养基、农杆菌预
培养基、悬浮培养基中添加＊51001llllOW有助于提高转化频率；N）在共培养基中添
加 PVP 500mg／L，可防止外植体共培养后褐化死亡现象发生，促进转化频率的提高。
首次研究了不同抗生素的抑菌效果和对高梁离体再生培养反应的影响，发现在农杆
菌介导高梁遗传转化时，选用 250—500lug／L．的梭节青霉素抑菌较为合适。不同高梁品
种及外植体对潮霉素和卡那霉素的敏感性不同，但对潮酶素比卡那霉素更敏感。在筛选
转化体时，采用浓度为 256 潮酶素对茎尖和未成熟胚较为适宜，对愈伤组织则以
50－75mg／L为宜。
以幼穗愈伤组织为受体，在世界上第一次通过农杆菌介导法将杀虫晶体蛋白基因
CryIAb导入高梁品种115、ICSZIB和5—27，共获ZI个独立转基因细胞系，52株转基
因植株，平均转化率为1．go／o。经GUS活性、PCR、S。＂them杂交分析表明此基因己整
合进高梁基因组中。Bt蛋白测定表明，Cy1Ab基因能在转基因高粱植株中表达，但不
同转基因植株及不同组织间表达量有所不同。室内饲虫试验表明，转基因高梁对大螟
（＆sanl la帅rens）具有一定抗性。
Transgenic sorghum plants containing a codon-modified insecticidal Bacillus thuringiensic (Bt) crylAb gene were obtained through /Igrofeacrerium-mediated transformation, based on optimizations of regenerative and transformation system.
The cultural medium was optimized by a statistical method and dissecting effect of single factor on in vitro culture. The optimal medium for direct regeneration of shoot apex was MS salts, B5 vitamins, KT 0.25mg/L, BAP 0.5mg/L, IAA 0.25mg/L, L-Asn 200mg/L, ascorbic acid lOmg/L, PVP (polyvinylpyrrolidone) lOOmg/L, sucrose 30g/L and agar lOg/L; for callus induction of seed (mature embryo) was modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.05mg/L, L-Pro 50mg/L, casamino acid 500mg/L, PW 8mg/L, sucrose 30g/L and agar 8g/L; the medium for callus sub-culture contained modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.5mg/L, L-Asn 200mg/L, L-Pro 50mg/L, PVP lOOmg/L, ascorbic acid lOmg/L, casamino acid 500mg/L, sucrose 30g/L and agar 8g/L; for callus induction of immature embryo was MS salts, MS vitamins, 2,4-D 2mg/L, KT 0.05mg/L, zeatin 0.05mg/L, L-Pro 700mg/L, L-Asn 150mg/L, casamino acid Ig/L, PW 8mg/L, ascorbic acid lOmg/L, NH4NO3 3.5g/L, KH2PO4 0.5g/L, sucrose 20g/L and agar 8g/L; for callus sub-culture was MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 0.5mg/L, L-Pro 150mg/L, L-Asn 150mg/L, casamino acid 0.5g/L, PVP 8mg/L, ascorbic acid lOmg/L, Ntt^MOs 3.5g/L, KH2P04 0.5g/L, sucrose 20g/L and agar 8g/L; for callus induction of immature inflorescence is YI4 containing MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 2.2mg/L, PVP 8mg/L, ascorbic acid lOmg/L, casamino acid 0.5g/L, sucrose 30g/L and agar 7.2g/L; for sub-culture was YI4 with KT 0.175mg/L.
The regenerative capability of different explants and genotypes was comparatively investigated. It showed that the regenerative frequency of immature inflorescence-derived callus was higher than that of other types of explant-derived callus and shoot apex. The average regenerative frequencies of callus induced from immature inflorescence, immature embryo and seed (mature embryo), and shoot apex were 77.2%, 21.7%, 1.0% and 39.4%, respectively. The regenerative capability was different among the genotypes. Immature inflorescence, shoot apex and immature embryo were ideal explants suitable for further transformation study.
The genetic transgenic system of sorghum mediated by Agrobacterium and factors were investigated using the transient expression of gus gene and the optimal conditions were determined as follows: for shoot apex, pre-culture 3 days, bacterial concentration ODeoo 0-5, infection time 10 min, and co-culture 3 days; for immature embryo and inflorescence-derived callus, pre-culture 3 days, bacterial concentration ODeoo 0.5 to 0.7, infection time 5 to 20 min, pH value of co-culture medium 5.2 to 5.6, co-culture temperature 22℃ to 25 ℃, and less than 3-day co-culture. To be higher transformation frequency, it was important to add AS to co-culture medium, pre-culture medium of Agrobacterium., and the bacterial cell suspension. To avoid the browning and death of explants after co-culture and improve the transformation frequency, it was necessary to add PVP 500mg/L to co-culture medium.
The effects of antibiotics on bacterial growth and in vitro response were comprehensively investigated. Carbenicillin with 250-500mg/L was optional to remove the bacterial cells after co-cultivation in the process of Agrobacterium-mediated transformation. Sensitivity to kanamycin and hygromycin varied among the varieties and explants, and the higher sensitivity to hygromycin was observed than that to kanamycin. The preferable concentration of hygromycin for transformant screening is 25-50mg/L for shoot apex and immature embryo and 50-75mg/L for callus, respectively.
The insecticidal Bacillus thuringiensis (Bt) crylAb gene were firstly hi the world inserted into the immature inflorescence-derived callus of sorghum varieties, i.e. 115, ICS21B and 5-27 through Agrobacterium-mediated transformation. After gradient selection with hygromycin, a
通过正交试验和单因子试验确定了高梁离体培养的最佳培养基，并对其多种影响因素进行研究。结果表明，高梁茎尖直接再生的培养基为：MS无机盐+B5有机物+KT0.25mg／L+BAP 0.25mg／L+IAA 0.25mg／L+L-Asn 200mg／L+抗坏血酸10mg／L+PVP 100mg／L+蔗糖30g／L+琼脂10g／L；种子(胚)愈伤组织诱导培养基为：MS(C)无机盐+MS有机物+2，4-D 3mg／L+KT 0.05mg／L+L-Pro 50mg／L+水解酪蛋白500mg／L+PVP8mg／L+蔗糖30g／L+琼脂8g／L；种子(胚)愈伤组织继代培养基为：MS(C)无机盐+MS有机物+2，4-D 3mg／L+KT 0.5mg／L+L-Asn 200mg／L+L-Pro 50mg／L+PVP 100mg／L+抗坏血酸500rog／L+蔗糖30g／L+琼脂8e,／L；未成熟胚愈伤组织诱导培养基为：MS无机盐+MS有机物+2，4-D 2mg／L+KT 0.05mg／L+玉米素0.05mg／L+L-Asn 150mg／L+L-Pro700mg／L+水解酪蛋白lg／L+PVP 8mg／L+抗坏血酸10mg／L+NH_4NO_3 3.5g／L+KH_2PO_40.5g／L+蔗糖20g／L+琼脂8g／L；未成熟胚愈伤组织愈伤组织继代培养基为：MS无机盐+MS有机物+2，4-D2mg／L+玉米素0.5mg／L+L-Ash 150mg／L+L-Pro150mg／L+水解酪蛋白500mg／L+PVP 8mg／L+抗坏血酸10mg／L+NH_4NO_3 3.5g／L+KH_2PO_40.5g／L+蔗糖20g／L+琼脂8g／L；幼穗愈伤组织诱导培养基为：YI4培养基(MS+2，4-D2mg／L+玉米素2.2mg／L+PVP 8mg／L+抗坏血酸10mg／L+水解酪蛋白500mg／L+蔗糖30g／L+琼脂7.2g／L)；幼穗愈伤组织继代培养基为YI4+KT0.175mg／L。
对不同外植体和不同基因型诱导产生愈伤组织的再生能力进行比较研究，结果表明，幼穗愈伤组织分化率最高，平均为77.2％；其次是茎尖，平均再生率为39.4％；未成熟胚愈伤组织平均分化率为21.70％；种子(胚)愈伤组织分化能力最差，分化率平均仅为1％。此外，不同基因型间的再生能力差异很大。以幼穗、茎尖和未成熟胚作外植体比较理想，可用于进一步的转化研究。
通过gus基因瞬时表达对农杆菌介导高梁转化体系及其影响因子进行了系统性研究，获得较高转化频率的条件为：(1)茎尖：预培养时间3d，在OD_(600)值为0.5农杆菌菌液中感染10min，共培养时间为3d；(2)未成熟胚和幼穗愈伤组织：预培养时间3d，
最适农杆菌菌液浓度ODm值为0．5—0．7，感染时间5—20Inin，最适共培养基pH值为
5二一5石，最适共培养温度为22—25℃，最佳共培养时间3d；（）在共培养基、农杆菌预
培养基、悬浮培养基中添加＊51001llllOW有助于提高转化频率；N）在共培养基中添
加 PVP 500mg／L，可防止外植体共培养后褐化死亡现象发生，促进转化频率的提高。
首次研究了不同抗生素的抑菌效果和对高梁离体再生培养反应的影响，发现在农杆
菌介导高梁遗传转化时，选用 250—500lug／L．的梭节青霉素抑菌较为合适。不同高梁品
种及外植体对潮霉素和卡那霉素的敏感性不同，但对潮酶素比卡那霉素更敏感。在筛选
转化体时，采用浓度为 256 潮酶素对茎尖和未成熟胚较为适宜，对愈伤组织则以
50－75mg／L为宜。
以幼穗愈伤组织为受体，在世界上第一次通过农杆菌介导法将杀虫晶体蛋白基因
CryIAb导入高梁品种115、ICSZIB和5—27，共获ZI个独立转基因细胞系，52株转基
因植株，平均转化率为1．go／o。经GUS活性、PCR、S。＂them杂交分析表明此基因己整
合进高梁基因组中。Bt蛋白测定表明，Cy1Ab基因能在转基因高粱植株中表达，但不
同转基因植株及不同组织间表达量有所不同。室内饲虫试验表明，转基因高梁对大螟
（＆sanl la帅rens）具有一定抗性。
Transgenic sorghum plants containing a codon-modified insecticidal Bacillus thuringiensic (Bt) crylAb gene were obtained through /Igrofeacrerium-mediated transformation, based on optimizations of regenerative and transformation system.
The cultural medium was optimized by a statistical method and dissecting effect of single factor on in vitro culture. The optimal medium for direct regeneration of shoot apex was MS salts, B5 vitamins, KT 0.25mg/L, BAP 0.5mg/L, IAA 0.25mg/L, L-Asn 200mg/L, ascorbic acid lOmg/L, PVP (polyvinylpyrrolidone) lOOmg/L, sucrose 30g/L and agar lOg/L; for callus induction of seed (mature embryo) was modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.05mg/L, L-Pro 50mg/L, casamino acid 500mg/L, PW 8mg/L, sucrose 30g/L and agar 8g/L; the medium for callus sub-culture contained modified MS salts, MS vitamins, 2,4-D 3mg/L, KT 0.5mg/L, L-Asn 200mg/L, L-Pro 50mg/L, PVP lOOmg/L, ascorbic acid lOmg/L, casamino acid 500mg/L, sucrose 30g/L and agar 8g/L; for callus induction of immature embryo was MS salts, MS vitamins, 2,4-D 2mg/L, KT 0.05mg/L, zeatin 0.05mg/L, L-Pro 700mg/L, L-Asn 150mg/L, casamino acid Ig/L, PW 8mg/L, ascorbic acid lOmg/L, NH4NO3 3.5g/L, KH2PO4 0.5g/L, sucrose 20g/L and agar 8g/L; for callus sub-culture was MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 0.5mg/L, L-Pro 150mg/L, L-Asn 150mg/L, casamino acid 0.5g/L, PVP 8mg/L, ascorbic acid lOmg/L, Ntt^MOs 3.5g/L, KH2P04 0.5g/L, sucrose 20g/L and agar 8g/L; for callus induction of immature inflorescence is YI4 containing MS salts, MS vitamins, 2,4-D 2mg/L, zeatin 2.2mg/L, PVP 8mg/L, ascorbic acid lOmg/L, casamino acid 0.5g/L, sucrose 30g/L and agar 7.2g/L; for sub-culture was YI4 with KT 0.175mg/L.
The regenerative capability of different explants and genotypes was comparatively investigated. It showed that the regenerative frequency of immature inflorescence-derived callus was higher than that of other types of explant-derived callus and shoot apex. The average regenerative frequencies of callus induced from immature inflorescence, immature embryo and seed (mature embryo), and shoot apex were 77.2%, 21.7%, 1.0% and 39.4%, respectively. The regenerative capability was different among the genotypes. Immature inflorescence, shoot apex and immature embryo were ideal explants suitable for further transformation study.
The genetic transgenic system of sorghum mediated by Agrobacterium and factors were investigated using the transient expression of gus gene and the optimal conditions were determined as follows: for shoot apex, pre-culture 3 days, bacterial concentration ODeoo 0-5, infection time 10 min, and co-culture 3 days; for immature embryo and inflorescence-derived callus, pre-culture 3 days, bacterial concentration ODeoo 0.5 to 0.7, infection time 5 to 20 min, pH value of co-culture medium 5.2 to 5.6, co-culture temperature 22℃ to 25 ℃, and less than 3-day co-culture. To be higher transformation frequency, it was important to add AS to co-culture medium, pre-culture medium of Agrobacterium., and the bacterial cell suspension. To avoid the browning and death of explants after co-culture and improve the transformation frequency, it was necessary to add PVP 500mg/L to co-culture medium.
The effects of antibiotics on bacterial growth and in vitro response were comprehensively investigated. Carbenicillin with 250-500mg/L was optional to remove the bacterial cells after co-cultivation in the process of Agrobacterium-mediated transformation. Sensitivity to kanamycin and hygromycin varied among the varieties and explants, and the higher sensitivity to hygromycin was observed than that to kanamycin. The preferable concentration of hygromycin for transformant screening is 25-50mg/L for shoot apex and immature embryo and 50-75mg/L for callus, respectively.
The insecticidal Bacillus thuringiensis (Bt) crylAb gene were firstly hi the world inserted into the immature inflorescence-derived callus of sorghum varieties, i.e. 115, ICS21B and 5-27 through Agrobacterium-mediated transformation. After gradient selection with hygromycin, a
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