小麦组织培养体系优化及抗冻基因KN2转化小麦的研究
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摘要
与水稻相比,小麦组织培养技术和再生体系还不十分完善,遗传转化效率低得多,研究小麦组织培养条件和植株再生频率的影响因素,优化小麦遗传转化体系对于有效开展小麦转基因育种有重要意义。近年来我国小麦冻害发生频率不断增加,危害程度日趋严重,利用转基因技术引进外源抗(耐)冻基因是增强小麦的抗(耐)冻能力的可行途径之一
本试验以陇春23、科农199、Bobwhite等10个小麦品种为材料,研究了不同诱导培养基、分化培养基、继代方式以及基因枪转化前的预培养等对小麦幼胚离体培养和转化效率的影响；研究了接种方式、诱导培养基等对小麦成熟胚离体培养的影响。结果如下：诱导培养基中添加0.2mg/L的ABA、1.25 mg/L的CuSO4对胚性愈伤组织的形成和再分化有促进作用,分化培养基中含5mg/L的KT、0.25 mg/L CuSO4再生效果较好,愈伤组织整块继代的方式有利于其胚性的维持,幼胚转化前预培养4天而转化后恢复培养15天转化效率较高；半胚法接种方式及添加了CH、VB1、Pro、Gln的诱导培养基有利于成熟胚的组织培养。
在建立了小麦的高频再生和高效遗传转化体系的基础上,我们开展了南极鱼类的超氧化物歧化酶基因(KN2基因)转化小麦的研究,期望获得抗(耐)冻能力增强的小麦种质。用pUBI::Bar和pUBI::KN2表达载体对小麦材料陇春23、科农199和Bobwhite幼胚及其愈伤组织进行了的基因枪法共转化。转化陇春23幼胚共得到耐Bialaphos的再生苗265株,CTAB法提取总DNA进行Bar基因和KN2基因的PCR检测,共检测到转Bar基因的样品92个,转KN2基因的样品98个,转Bar和KN2基因的样品37个,转化率分别为14.91%、15.88%、和6.00%。转化陇春23、科农199和Bobwhite幼胚诱导10周左右的愈伤组织共得到耐Bialaphos的再生苗42株,CTAB法提取总DNA进行Bar基因和KN2基因的PCR检测,共检测到转Bar基因的样品17个,转KN2基因的样品19个,转Bar和KN2基因的样品6个,转化率分别为1.58%、1.77%、和0.56%,其中有4株科农199、7株Bobwhite、6株陇春23获得转基因种子。
Wheat tissue culture and regeneration system is not completely established as in rice, the efficiency of genetic transformation is much lower than that in rice. Optimization of regeneration system of wheat will be significant in promoting the application of transgenic wheat in large scale. In recent years, low-temperature is a common stress that always has adverse effect on the growth and productivity of wheat. In this study, the KN2 gene encoding superoxide dismutase cloned from Antarctic fish was transferred into wheat to improve their capacity of cold-resistance.
Ten wheat genotypes were used to compare the effect of different media on callus induction and differentiation and effect of different ways of inculation, subculture and culture period before transformation on wheat immature and mature embryos culture and genetic transformation efficiency. The result showed that the callus induction media supplemented with 0.2mg/L ABA,1.25mg/L CUSO4 was the optimum media for callus induction and differentiation, the differentiation media supplemented with 5mg/L KT, 0.25mg/L CUSO4 was the optimum media for plantlet regeneration. High frequencies of plantlet regeneration were obtained when whole callus was used to subculture. Pre-incubation of wheat immature embryos for 4 days before transformation seemd to be the optimum pre-incubation period. Half-matured embryos inoculation and callus induction media supplemented with CH, VB1, Pro and Gln were both benefit for culturing mature embryos.
Expression vectors carrying KN2 gene and Bar marker gene were transformed into immature embryos from L23, K199, Bobwhite to improve their capacity of cold-resistance using particle bombardment. Two hundred and sixty-five resistant plantlets from immature embryos of L23 were obtained. PCR analysis identified 92 Bar-positive transgenic plants,98 KN2- positive transgenic plants and 37 double positive transgenic plants. The transformation frequencies were 14.91%,15.88% and 6.00%, respectively. The KN2 gene and Bar marker gene were also transferred into the callus derived from ten-week induction of immature embryos of L23, K199, Bobwhite. Forty two resistant plantlets were obtained. PCR analysis identidfied 17 Bar-positive transgenic plants,19 KN2- positive transgenic plants and 6 double positive transgenic plants. The frequencies of gene transformation were 1.58%,1.77% and 0.56%, respectively. In addition, we also obtained transgenic seeds from 6 L23,4 K199 and 7 Bobwhite regenerated plants.
本试验以陇春23、科农199、Bobwhite等10个小麦品种为材料,研究了不同诱导培养基、分化培养基、继代方式以及基因枪转化前的预培养等对小麦幼胚离体培养和转化效率的影响；研究了接种方式、诱导培养基等对小麦成熟胚离体培养的影响。结果如下：诱导培养基中添加0.2mg/L的ABA、1.25 mg/L的CuSO4对胚性愈伤组织的形成和再分化有促进作用,分化培养基中含5mg/L的KT、0.25 mg/L CuSO4再生效果较好,愈伤组织整块继代的方式有利于其胚性的维持,幼胚转化前预培养4天而转化后恢复培养15天转化效率较高；半胚法接种方式及添加了CH、VB1、Pro、Gln的诱导培养基有利于成熟胚的组织培养。
在建立了小麦的高频再生和高效遗传转化体系的基础上,我们开展了南极鱼类的超氧化物歧化酶基因(KN2基因)转化小麦的研究,期望获得抗(耐)冻能力增强的小麦种质。用pUBI::Bar和pUBI::KN2表达载体对小麦材料陇春23、科农199和Bobwhite幼胚及其愈伤组织进行了的基因枪法共转化。转化陇春23幼胚共得到耐Bialaphos的再生苗265株,CTAB法提取总DNA进行Bar基因和KN2基因的PCR检测,共检测到转Bar基因的样品92个,转KN2基因的样品98个,转Bar和KN2基因的样品37个,转化率分别为14.91%、15.88%、和6.00%。转化陇春23、科农199和Bobwhite幼胚诱导10周左右的愈伤组织共得到耐Bialaphos的再生苗42株,CTAB法提取总DNA进行Bar基因和KN2基因的PCR检测,共检测到转Bar基因的样品17个,转KN2基因的样品19个,转Bar和KN2基因的样品6个,转化率分别为1.58%、1.77%、和0.56%,其中有4株科农199、7株Bobwhite、6株陇春23获得转基因种子。
Wheat tissue culture and regeneration system is not completely established as in rice, the efficiency of genetic transformation is much lower than that in rice. Optimization of regeneration system of wheat will be significant in promoting the application of transgenic wheat in large scale. In recent years, low-temperature is a common stress that always has adverse effect on the growth and productivity of wheat. In this study, the KN2 gene encoding superoxide dismutase cloned from Antarctic fish was transferred into wheat to improve their capacity of cold-resistance.
Ten wheat genotypes were used to compare the effect of different media on callus induction and differentiation and effect of different ways of inculation, subculture and culture period before transformation on wheat immature and mature embryos culture and genetic transformation efficiency. The result showed that the callus induction media supplemented with 0.2mg/L ABA,1.25mg/L CUSO4 was the optimum media for callus induction and differentiation, the differentiation media supplemented with 5mg/L KT, 0.25mg/L CUSO4 was the optimum media for plantlet regeneration. High frequencies of plantlet regeneration were obtained when whole callus was used to subculture. Pre-incubation of wheat immature embryos for 4 days before transformation seemd to be the optimum pre-incubation period. Half-matured embryos inoculation and callus induction media supplemented with CH, VB1, Pro and Gln were both benefit for culturing mature embryos.
Expression vectors carrying KN2 gene and Bar marker gene were transformed into immature embryos from L23, K199, Bobwhite to improve their capacity of cold-resistance using particle bombardment. Two hundred and sixty-five resistant plantlets from immature embryos of L23 were obtained. PCR analysis identified 92 Bar-positive transgenic plants,98 KN2- positive transgenic plants and 37 double positive transgenic plants. The transformation frequencies were 14.91%,15.88% and 6.00%, respectively. The KN2 gene and Bar marker gene were also transferred into the callus derived from ten-week induction of immature embryos of L23, K199, Bobwhite. Forty two resistant plantlets were obtained. PCR analysis identidfied 17 Bar-positive transgenic plants,19 KN2- positive transgenic plants and 6 double positive transgenic plants. The frequencies of gene transformation were 1.58%,1.77% and 0.56%, respectively. In addition, we also obtained transgenic seeds from 6 L23,4 K199 and 7 Bobwhite regenerated plants.
引文
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