基因枪转化玉米自交系受体系统的建立及转Bt基因的研究
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摘要
玉米是世界上第三大粮食作物，中国是世界上第二大玉米生产国。通过植物基因工程方法将特定基因转入玉米中，从而改善玉米的品质，提高玉米产量具有重要的意义。利用基因枪法获得转基因玉米切实可行。基因枪转化法首先要建立受体系统。本研究以我国北方地区农业生产中较广泛使用的玉米自交系7922为基础材料，全面探讨了影响玉米自交系胚性愈伤组织诱导、Ⅱ型愈伤组织继代培养及分化再生植株的诸多因素，并成功的建立了以7922自交系幼胚为材料，适于基因枪转化法进行遗传转化的受体系统。在此基础上，利用本实验所获得的结果，P1、P2、H99、Mo17等自交系受体系统的建立也取得成功，进一步证明了实验结果的正确性和一定的适用性。
取得的主要研究结果如下：
1 对于玉米自交系幼胚愈伤组织诱导而言：MB培养基是较适宜的诱导培养基；适当的4℃冷藏处理雌果穗(<7天)会提高胚性愈伤组织的诱导率，改善愈伤组织的质量；AgNO_3对于玉米自交系愈伤组织诱导的促进效果不明显：20g／l蔗糖+10g／l的葡萄糖是愈伤组织诱导培养基的适宜碳源。
2 在愈伤组织状态调控的实验中发现DMS培养基可以促进愈伤组织状态由Ⅰ型向Ⅱ型转变。
3 对于玉米Ⅱ型胚性愈伤组织继代培养的研究结果表明：N6和NMB培养基较8114、MS、MB等适于作继代培养的培养基；在继代培养基中附加5g／l的甘露醇有利于愈伤组织胚性状态的保持；继代培养基的适宜碳源为50g／l的蔗糖。同时还发现AgNO_3和活性炭(AC)不利于继代培养过程中Ⅱ型愈伤组织胚性状态的维持，AC会促使愈伤组织分化、生须根等。
4．Basta的有效成分为PPT，愈伤组织对Basta的本底耐性实验表明6mg／l的Basta附加于继代培养基中时，愈伤组织的生长明显受抑，该浓度可以作为转化的愈伤组织和细胞的有效筛选浓度。
5．Ⅱ型胚性愈伤组织的分化主要有体细胞胚途径和器官发生途径两种，随玉米自交系的基因型不同，分化途径也不同，玉米自交系7922的分化途径为体细胞胚途径，其适宜的分化培养基为MS+1.0mg／16-BA+0.4mg／l IBA和N6+1.0mg／l 6-BA，但以前者较好，在其上分化的愈伤组织分化率达到105.26％，分化出的组织培养苗较粗壮，且畸形苗率低。
6 基因枪转化法因受体材料及其生理状态的不同，最适转化条件也有所差异。
以玉米自交系7922的H型胚性愈伤组织作为基因枪转化的受体，对影响转化效果的
回素进行了探讨。实验结果表明：以 11 00psi X gcm的射击压力和射击靶距组合是较适
宜的基因枪轰击参数：轰击2次时的转化效果要好于轰击1次和轰击3次时的转化效
果：300Ug金粉＋03llgDNA为适宜的DNA用量；愈伤组织在轰击前用02M的甘露醇
渗透处理，可以有效地减轻组织受伤害程度，提高转化率。
7 对筛选2个月左右，接到分化培养基上分化培养的愈伤组织分化出的试管苗
移栽后获得的转基因植株提取植物基因组 DNA，进行 PCR检测，结果表明，约 10 5％
的植株呈阳性，在分子水平上初步证明了外源Bt基因己经整合到植物基因组中。
Maize is an kind of importment crop in the world as well as in China. In order to improve the quality of maize and enhance its production, it is important for us to apply the genetic engineering to transfer target gene to maize . It is applicable to use biolistic system to obtain transgenic corn plants. First of all ,if using biolistic system,it is to erect regenable receptor system. In the paper, the method to establish a better receptor system of maize inbred line for biolistic bombarderment was studied.
Many factors which affect callus induction,subculture and ^differentiation were studied. On the basis of this work,the receptor system were successfully built up by using immature embryo of maize inbred line 7922 as model.Subsequently,immature embryos of inbred line Pl,P2,H99,Mol7 also were successfully inoculated, and type-II callus were obtained
The results of the experiment of induction of callus showed that MB medium is the better medium than other media tested,such as 8114,MS,N6,NMB,AG.The frequency of induction of callus was higher and the callus which inducted from MB was better also. Suitable 4"C treatment in a refrigerator(<7 days) prior to immature embryo dissection was an efficient way to improve the frequency of inocution. AgNOsdid not contribute to callus iniation in the study 20g/l sucrose plus 10g/l glucose was suitable carbon source for iniation medium. Immature embryos placed embryo-axis dow(scutellum side up) on callus initiation medium was better. Othenvise,the frequency of iniation was very low.
It is essential for maize callus to transfer from Type-I to Type-II to build up receptor system which is suitable to transfer target gene with biolistics. DMSmedium could significantly expedise the process. The frequency of transformation of callus on DMS medium was about 48.28 per cent while those of MS,N6 were respectively around 15.63 per cent and 12.12 per cent.
Factors influncing the subculture of Type-II callus ,such as kinds of media,concenttrtion of mannitol,carbon source,AgNC>3 and active charcoal were observed. The results showed that N6 and NMBwere two kinds of optimal media for subculture of
ra
maize calus. The callus growed on them was pale yellow or yellow,compact,and friable. Five per cent mannitol which was added to subculture medium was benefitial to enhance the state of callus, at the same time,it could not influence speed of growth. Optimal carbon source in the subculture medium was 50g/l sucrose.At this concentration,sucrose not only provided enough energenic matter to callus,but also maintain a better osmotic condition of medium The results also showed that most callus seemed difficult to be maintained on medium added some active charcoal.Just lg/1 AC could make callus diffrentiation.AgNOs actually did the same effect on the process of subculture.After these factors were analyzed,optimal contition of subculture was established.
Many factors affecting regeneration of Type-II callus, including kinds of medium, sorts of plant growth regulation matters and their concentration were studied.Optimal condition for rediffrentiation of Type-II callus was obtained.MS medium plus l.Omg/1 6-BA and 0.4mg/l IB A or N6 medium plus l.Omg/1 6-BA were two better media, espiecially the former The frequency of rediffrentiation was 105.26 per cent.
The experiment of basic endurance of callus to Basta was done The result showed optimal concentration for screening after bombarderment was 6mg/l which could evidently blocked the growth of non-thansformation callus.
In view of obtaining efficient transformation condition to maize cells by using biolistic system,factors influencing the frequency of transformation, rupture pressure and target distance, bombarderment times,concentration of Au powder and DNA,osmotic treatment for callus,and so on were researched when the Type-II callus of maize inbred line 7922 was taken as receptor system of bombardment.The results showed that optimal biolistic rupture pressureand target distance was HOOpsi X 9cm,optimal bombardment times was twice,suitable co
取得的主要研究结果如下：
1 对于玉米自交系幼胚愈伤组织诱导而言：MB培养基是较适宜的诱导培养基；适当的4℃冷藏处理雌果穗(<7天)会提高胚性愈伤组织的诱导率，改善愈伤组织的质量；AgNO_3对于玉米自交系愈伤组织诱导的促进效果不明显：20g／l蔗糖+10g／l的葡萄糖是愈伤组织诱导培养基的适宜碳源。
2 在愈伤组织状态调控的实验中发现DMS培养基可以促进愈伤组织状态由Ⅰ型向Ⅱ型转变。
3 对于玉米Ⅱ型胚性愈伤组织继代培养的研究结果表明：N6和NMB培养基较8114、MS、MB等适于作继代培养的培养基；在继代培养基中附加5g／l的甘露醇有利于愈伤组织胚性状态的保持；继代培养基的适宜碳源为50g／l的蔗糖。同时还发现AgNO_3和活性炭(AC)不利于继代培养过程中Ⅱ型愈伤组织胚性状态的维持，AC会促使愈伤组织分化、生须根等。
4．Basta的有效成分为PPT，愈伤组织对Basta的本底耐性实验表明6mg／l的Basta附加于继代培养基中时，愈伤组织的生长明显受抑，该浓度可以作为转化的愈伤组织和细胞的有效筛选浓度。
5．Ⅱ型胚性愈伤组织的分化主要有体细胞胚途径和器官发生途径两种，随玉米自交系的基因型不同，分化途径也不同，玉米自交系7922的分化途径为体细胞胚途径，其适宜的分化培养基为MS+1.0mg／16-BA+0.4mg／l IBA和N6+1.0mg／l 6-BA，但以前者较好，在其上分化的愈伤组织分化率达到105.26％，分化出的组织培养苗较粗壮，且畸形苗率低。
6 基因枪转化法因受体材料及其生理状态的不同，最适转化条件也有所差异。
以玉米自交系7922的H型胚性愈伤组织作为基因枪转化的受体，对影响转化效果的
回素进行了探讨。实验结果表明：以 11 00psi X gcm的射击压力和射击靶距组合是较适
宜的基因枪轰击参数：轰击2次时的转化效果要好于轰击1次和轰击3次时的转化效
果：300Ug金粉＋03llgDNA为适宜的DNA用量；愈伤组织在轰击前用02M的甘露醇
渗透处理，可以有效地减轻组织受伤害程度，提高转化率。
7 对筛选2个月左右，接到分化培养基上分化培养的愈伤组织分化出的试管苗
移栽后获得的转基因植株提取植物基因组 DNA，进行 PCR检测，结果表明，约 10 5％
的植株呈阳性，在分子水平上初步证明了外源Bt基因己经整合到植物基因组中。
Maize is an kind of importment crop in the world as well as in China. In order to improve the quality of maize and enhance its production, it is important for us to apply the genetic engineering to transfer target gene to maize . It is applicable to use biolistic system to obtain transgenic corn plants. First of all ,if using biolistic system,it is to erect regenable receptor system. In the paper, the method to establish a better receptor system of maize inbred line for biolistic bombarderment was studied.
Many factors which affect callus induction,subculture and ^differentiation were studied. On the basis of this work,the receptor system were successfully built up by using immature embryo of maize inbred line 7922 as model.Subsequently,immature embryos of inbred line Pl,P2,H99,Mol7 also were successfully inoculated, and type-II callus were obtained
The results of the experiment of induction of callus showed that MB medium is the better medium than other media tested,such as 8114,MS,N6,NMB,AG.The frequency of induction of callus was higher and the callus which inducted from MB was better also. Suitable 4"C treatment in a refrigerator(<7 days) prior to immature embryo dissection was an efficient way to improve the frequency of inocution. AgNOsdid not contribute to callus iniation in the study 20g/l sucrose plus 10g/l glucose was suitable carbon source for iniation medium. Immature embryos placed embryo-axis dow(scutellum side up) on callus initiation medium was better. Othenvise,the frequency of iniation was very low.
It is essential for maize callus to transfer from Type-I to Type-II to build up receptor system which is suitable to transfer target gene with biolistics. DMSmedium could significantly expedise the process. The frequency of transformation of callus on DMS medium was about 48.28 per cent while those of MS,N6 were respectively around 15.63 per cent and 12.12 per cent.
Factors influncing the subculture of Type-II callus ,such as kinds of media,concenttrtion of mannitol,carbon source,AgNC>3 and active charcoal were observed. The results showed that N6 and NMBwere two kinds of optimal media for subculture of
ra
maize calus. The callus growed on them was pale yellow or yellow,compact,and friable. Five per cent mannitol which was added to subculture medium was benefitial to enhance the state of callus, at the same time,it could not influence speed of growth. Optimal carbon source in the subculture medium was 50g/l sucrose.At this concentration,sucrose not only provided enough energenic matter to callus,but also maintain a better osmotic condition of medium The results also showed that most callus seemed difficult to be maintained on medium added some active charcoal.Just lg/1 AC could make callus diffrentiation.AgNOs actually did the same effect on the process of subculture.After these factors were analyzed,optimal contition of subculture was established.
Many factors affecting regeneration of Type-II callus, including kinds of medium, sorts of plant growth regulation matters and their concentration were studied.Optimal condition for rediffrentiation of Type-II callus was obtained.MS medium plus l.Omg/1 6-BA and 0.4mg/l IB A or N6 medium plus l.Omg/1 6-BA were two better media, espiecially the former The frequency of rediffrentiation was 105.26 per cent.
The experiment of basic endurance of callus to Basta was done The result showed optimal concentration for screening after bombarderment was 6mg/l which could evidently blocked the growth of non-thansformation callus.
In view of obtaining efficient transformation condition to maize cells by using biolistic system,factors influencing the frequency of transformation, rupture pressure and target distance, bombarderment times,concentration of Au powder and DNA,osmotic treatment for callus,and so on were researched when the Type-II callus of maize inbred line 7922 was taken as receptor system of bombardment.The results showed that optimal biolistic rupture pressureand target distance was HOOpsi X 9cm,optimal bombardment times was twice,suitable co
引文
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