用分割法建立不依赖选择标记的转基因技术
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摘要
21世纪是生命科学的世纪，谁占生物技术的优势地位，谁就会主导未来的生物经济时代。转基因技术是生物技术的重要内容之一，是改良品种的关键技术，在理论研究和实际应用中都具有极其重要的地位，但目前的转基因技术大都依赖于选择标记。本文利用分割法(即通过对转化处理的愈伤组织进行多次分割、培养和直接选择，富集转化细胞的方法)建立了不依赖选择标记的转基因技术，其目的在于解决伴随转基因作物越来越多而日益突出的选择标记问题，它可以消除由于应用选择标记可能在以下五个方面产生的不利影响，1)影响目标基因的表达，2)限制多个基因的转化，3)影响被改良植物的某些性状，4)危及环境安全，5)危害人身健康。近年来许多研究者利用共转化、位点特异性重组、转座子和同源重组等方法，开展了对转基因植物中的选择标记进行剔除的研究。但到目前为止还难以获得令人满意的结果。因此，建立不依赖选择标记的转基因技术比完成转基因后再剔除选择标记具有更重要的意义。
本文先以烟草(Nicotiana tabacum subsp．petit SR1)为受体，通过农杆菌介导转化，以GUS基因为追踪目标摸索了分割法的技术参数。当愈伤组织长至直径0.5cm时就进行均分，每次的分割块数(i)设4个处理，分别为64块、32块、16块和8块，分割次数(n)设4个处理，分别为2次、3次、4次和5次，为了便于将“子愈伤组织”的阳性结果与“母愈伤组织”的转化部位对应起来，均分后的小细胞团按其在“母愈伤组织”中的相邻关系依次摆放在培养基上。通过分割和培养可以削弱转化细胞与非转化细胞之间的竞争强度，给予转化细胞较充分的发展机会，提高有转化愈伤组织中转化细胞所占比例。自第二次分割起，每次分割前，都寻找“转化愈伤组织”集中出现的区域，并选择区域内最靠近中间的愈伤组织进行下次分割，如此重复使转化细胞得到富集，最终得到近似均一的转化愈伤组织，诱导出再生植株，并对再生植株的每个叶片进行检测确定其是否为纯合体(或嵌合体)。然后，通过用PCR代替X-gluc检测，以番茄为受体，以葡聚糖酶基因为供体基因，对该技术进行了验证。
本研究用分割法建立了不依赖选择标记的转基因技术，主要包括三方面内容：优化培养基，建立小细胞团培养技术；优化农杆菌介导的转化技术，提高“起始愈伤组织”中转化细胞的数目；确定分割法的可行性及其必要的参数。
本试验研究表明，较适合培养烟草小细胞团的培养基为：B_5+584mg／l谷氨酰胺+
董文琦中国农业科学院研究生硕士论文
0.6m留1 BA+0.03m留1 IAA十250m留ICarb。外植体为番茄时，将激素调整为BA
1 .2m酬+I AA 0.2 ms/l。较适合农杆菌侵染和共培养的培养基为:BS+谷氨酞胺584m幼十
甜菜碱117.2mg/1+脯氨酸0.12mg/1+肌醇400mg/卜309/1蔗糖，pH 5.5。较合适的侵染和
共培养条件为:将农杆菌稀释至OD(Optical Degree)600=0.05，加入smg/l乙酞T香酮，
侵染外体植30分钟，在附加10 mg/l Agar的培养基上，于避光条件下，共培养3天。
在烟草上，每当转化处理后的愈伤组织长至直径o.scm时就进行分割，前1一2次的
分割块数大于32块、分割递进度(分割块数X分割次数)大于128的处理效果比较理
想，获得的有效转化率都超过了50%。试验数据还表明，在一定的范围内，分割递进度
与有效转化率之间基本上呈现正比关系，因此，我们认为在利用分割法进行基因转化的
早期应尽量增加分割块数，以尽早降低非转化细胞的比率，早日获得转化体。但还应考
虑到不同受体小细胞团培养的难易程度及操作的难易程度，当小细胞团较难培养或转化
细胞在转化愈伤组织中的比率己较高时，应适当减少分割块数。
利用分割法，在不进行药物筛选的前提下，已将p一1，3一葡聚糖酶基因和几丁质酶成
功地转入了SRI烟草外植体，得到了纯合转化植株。并将p一l，3一葡聚糖酶基因转入了
番茄(却e叩ersscon esculentum)的栽培品种(毛T一5番茄)。
The 21th century is the century of life science, and one who keeps ahead in the biotechnology would occupy the predominant position in the bio-economy ages in future. Transgenic technique, an important part of biotechnology, has a great and significant prospective for developing new and improving the existing commercial cultivars. But nowadays the most transgenic techniques depend on the selection markers. We want to establish a new transgenic technique without selection marker by dividing technique in order to eliminate the potential disadvantages of selection markers, such as influencing the gene expression, limiting the further transformation, disturbing the plant characteristics, endangering the environment and jeopardizing the community health. In recent years many researchers have put a lot of efforts to eliminate the selection markers from transgenic plants, but up to now all the results are unsatisfactory. So it is more important to establish a new genetic transformation technique without selection m
arker than to eliminate the selection markers from transgenic plants.
In this study, the explants obtained from tobacco leaves were inoculated by Agrobacterium tumefaciens with GUS as reporter gene. The calli were grown and divided into 8, 16, 32 and 64 pieces for 2, 3, 4 and 5 times, respectively, and placed on media in original parental neighborhood sequence to provide an opportunity to transformed calli pieces to grow by weakening the competition from non-transformed cells. Every time the central calli were selected from the set of calli with positive GUS activity. Seedlings were regenerated from the finally obtained calli and their transformed status was confirmed through GUS or PCR activity.
This research primarily includes three parts: Improving the calli pieces culture technique, improving the DNA transfer technique by Agrobacterium and ascertaining the necessary parameters of dividing technique.
"B5+0.6 mg/L 2ip+0.03 mg/L IAA+584 mg/l Glutamine" was used for small piece of callus culture for tobacco. "B5+ 584 mg/1 Glutamine+117.2mg/l betaine+0.12mg/l proline+400mg/l inositol (pH5.5)" was used for bacterial infection and co-culture. The agro-bacteria was diluted to OD (Optical Degree) 600=0.5 with subsequent addition of
acetosyrimgone at concentration of 5 mg/l, and infected the explants for 30min, followed by 3days co-culture in the dark. When the diameter of callus reached about 0.5 cm, the next division was conducted. That the number of division and division degree(numbre of division X times) at first and second times were more than 32 and 128, respectively, was time saving and cost effective with more than 50% chances to get higher transformed plants. As a whole, the Dividing Degree and percentage of transformed plants are linearly correlated. How many pieces the callus should be divided relies on the difficulty of callus-culturing and operation too. We think, if possible, the more pieces we divide the callus, the better it will be, especially in the earlier stage, In this way we can decrease the ratio of non-transgenic cells and obtain the transgenic plants as early as possible. When there are enough transgenic cells in callus, we can decrease the number of pieces in the latter stage.
Using the transgenic technique by dividing treatment without selection marker, we have transferred chitinase and B-1,3-glucanase genes to SRI tobacco, and transferred B-1,3-glucanase gene to tomato cultivar (Mao T-5) successfully.
本文先以烟草(Nicotiana tabacum subsp．petit SR1)为受体，通过农杆菌介导转化，以GUS基因为追踪目标摸索了分割法的技术参数。当愈伤组织长至直径0.5cm时就进行均分，每次的分割块数(i)设4个处理，分别为64块、32块、16块和8块，分割次数(n)设4个处理，分别为2次、3次、4次和5次，为了便于将“子愈伤组织”的阳性结果与“母愈伤组织”的转化部位对应起来，均分后的小细胞团按其在“母愈伤组织”中的相邻关系依次摆放在培养基上。通过分割和培养可以削弱转化细胞与非转化细胞之间的竞争强度，给予转化细胞较充分的发展机会，提高有转化愈伤组织中转化细胞所占比例。自第二次分割起，每次分割前，都寻找“转化愈伤组织”集中出现的区域，并选择区域内最靠近中间的愈伤组织进行下次分割，如此重复使转化细胞得到富集，最终得到近似均一的转化愈伤组织，诱导出再生植株，并对再生植株的每个叶片进行检测确定其是否为纯合体(或嵌合体)。然后，通过用PCR代替X-gluc检测，以番茄为受体，以葡聚糖酶基因为供体基因，对该技术进行了验证。
本研究用分割法建立了不依赖选择标记的转基因技术，主要包括三方面内容：优化培养基，建立小细胞团培养技术；优化农杆菌介导的转化技术，提高“起始愈伤组织”中转化细胞的数目；确定分割法的可行性及其必要的参数。
本试验研究表明，较适合培养烟草小细胞团的培养基为：B_5+584mg／l谷氨酰胺+
董文琦中国农业科学院研究生硕士论文
0.6m留1 BA+0.03m留1 IAA十250m留ICarb。外植体为番茄时，将激素调整为BA
1 .2m酬+I AA 0.2 ms/l。较适合农杆菌侵染和共培养的培养基为:BS+谷氨酞胺584m幼十
甜菜碱117.2mg/1+脯氨酸0.12mg/1+肌醇400mg/卜309/1蔗糖，pH 5.5。较合适的侵染和
共培养条件为:将农杆菌稀释至OD(Optical Degree)600=0.05，加入smg/l乙酞T香酮，
侵染外体植30分钟，在附加10 mg/l Agar的培养基上，于避光条件下，共培养3天。
在烟草上，每当转化处理后的愈伤组织长至直径o.scm时就进行分割，前1一2次的
分割块数大于32块、分割递进度(分割块数X分割次数)大于128的处理效果比较理
想，获得的有效转化率都超过了50%。试验数据还表明，在一定的范围内，分割递进度
与有效转化率之间基本上呈现正比关系，因此，我们认为在利用分割法进行基因转化的
早期应尽量增加分割块数，以尽早降低非转化细胞的比率，早日获得转化体。但还应考
虑到不同受体小细胞团培养的难易程度及操作的难易程度，当小细胞团较难培养或转化
细胞在转化愈伤组织中的比率己较高时，应适当减少分割块数。
利用分割法，在不进行药物筛选的前提下，已将p一1，3一葡聚糖酶基因和几丁质酶成
功地转入了SRI烟草外植体，得到了纯合转化植株。并将p一l，3一葡聚糖酶基因转入了
番茄(却e叩ersscon esculentum)的栽培品种(毛T一5番茄)。
The 21th century is the century of life science, and one who keeps ahead in the biotechnology would occupy the predominant position in the bio-economy ages in future. Transgenic technique, an important part of biotechnology, has a great and significant prospective for developing new and improving the existing commercial cultivars. But nowadays the most transgenic techniques depend on the selection markers. We want to establish a new transgenic technique without selection marker by dividing technique in order to eliminate the potential disadvantages of selection markers, such as influencing the gene expression, limiting the further transformation, disturbing the plant characteristics, endangering the environment and jeopardizing the community health. In recent years many researchers have put a lot of efforts to eliminate the selection markers from transgenic plants, but up to now all the results are unsatisfactory. So it is more important to establish a new genetic transformation technique without selection m
arker than to eliminate the selection markers from transgenic plants.
In this study, the explants obtained from tobacco leaves were inoculated by Agrobacterium tumefaciens with GUS as reporter gene. The calli were grown and divided into 8, 16, 32 and 64 pieces for 2, 3, 4 and 5 times, respectively, and placed on media in original parental neighborhood sequence to provide an opportunity to transformed calli pieces to grow by weakening the competition from non-transformed cells. Every time the central calli were selected from the set of calli with positive GUS activity. Seedlings were regenerated from the finally obtained calli and their transformed status was confirmed through GUS or PCR activity.
This research primarily includes three parts: Improving the calli pieces culture technique, improving the DNA transfer technique by Agrobacterium and ascertaining the necessary parameters of dividing technique.
"B5+0.6 mg/L 2ip+0.03 mg/L IAA+584 mg/l Glutamine" was used for small piece of callus culture for tobacco. "B5+ 584 mg/1 Glutamine+117.2mg/l betaine+0.12mg/l proline+400mg/l inositol (pH5.5)" was used for bacterial infection and co-culture. The agro-bacteria was diluted to OD (Optical Degree) 600=0.5 with subsequent addition of
acetosyrimgone at concentration of 5 mg/l, and infected the explants for 30min, followed by 3days co-culture in the dark. When the diameter of callus reached about 0.5 cm, the next division was conducted. That the number of division and division degree(numbre of division X times) at first and second times were more than 32 and 128, respectively, was time saving and cost effective with more than 50% chances to get higher transformed plants. As a whole, the Dividing Degree and percentage of transformed plants are linearly correlated. How many pieces the callus should be divided relies on the difficulty of callus-culturing and operation too. We think, if possible, the more pieces we divide the callus, the better it will be, especially in the earlier stage, In this way we can decrease the ratio of non-transgenic cells and obtain the transgenic plants as early as possible. When there are enough transgenic cells in callus, we can decrease the number of pieces in the latter stage.
Using the transgenic technique by dividing treatment without selection marker, we have transferred chitinase and B-1,3-glucanase genes to SRI tobacco, and transferred B-1,3-glucanase gene to tomato cultivar (Mao T-5) successfully.
引文
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