SAAT法介导铁皮石斛遗传转化及NAC启动子的克隆与载体构建
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摘要
NSC转录因子是植物特有的、家族成员较多的一类转录因子,其表达受植物发育时期和多种环境因素的诱导,并在植物发育、激素反应以及抗逆性的调控中起重要作用。本实验以铁皮石斛愈伤组织诱导的类原球茎为实验材料,优化超声波处理辅助农杆菌介导铁皮石斛遗传转化体系,使用常规PCR法分离克隆NAC转录因子,再采用染色体步移法获得NAC转录因子5’和3’侧翼序列,通过生物信息学手段对启动子和基因结构进行预测,并构建该启动子的表达载体,从而为进一步研究NAC启动子遗传转化和调控机制提供思路和手段,主要研究结果如下：
(1)以铁皮石斛愈伤组织诱导的类原球茎为材料,通过PLBs对潮霉素敏感性实验确定潮霉素选择压,结果发现,PLBs对潮霉素较为敏感,10mg/L潮霉素能够有效抑制PLBs的增殖和分化并完全死亡,因此确定10mg/L为潮霉素有效选择压浓度。
(2)优化SAAT法转化条件,研究超声波预处理时间、根瘤农杆菌侵染时间和共培养时间对GUS瞬时表达率的影响,实验结果表明,使用40KHZ、90W的超声波对预培养2d的PLBs超声处理3min,再用预培养2hr的OD600为0.8-0.95的农杆菌侵染液侵染30min,转入共培养基暗培养5d,所获得的蓝色斑点数为42个/100mg PLBs,而且其除菌也相对容易,其中预培养基、侵染液和共培养基中均含有100μM AS。
(3)采用常规PCR法和染色体步移法分离克隆出一条长度为6324bp的基因序列,通过分析发现,NAC转录因子在起始密码子和终止密码子之间长度为1611bp,含有3个外显子和2个内含子,在距离终止密码子下游859处和886处分别有一个ployA结构和Stem-loop结构,在TTS上游348处和378处分别为TSS位点和TATA box。5’侧翼序列长度为TSS前3401bp, PlantCare分析表明,该序列不仅含有TATA box、CAAT box等启动子核心顺式作用元件,而且含有光信号反应元件、激发子响应元件、生理节律元件、激素诱导相关的顺式作用元件、生长发育相关顺式元件和逆境肋、迫响应元件,这些顺式元件的存在与NAC转录因子主要生物学功能相符合。
(4)通过软件预测,确定翻译起始位点上游862bp大小的序列为NAC启动子核心区域,分离克隆该段基因,并构建表达载体,采用冻融法转入到农杆菌中,经双酶切验证证明构建和转化成功。
As a kind of plant specific transcriptional factors. NAC plays an important role in plant development and responses to hormone and stresses, whose expression is regulated by plant developmental stages and various environmental factors. In this study, we optimized sonication-assisted Agrobacterium-mediated transformation system of Dendrobium candidum Wall ex Lindl. for PLBs induced from callus, isolated a NAC transcription factor by PCR and its 5' and 3' flanking sequence by genome walking, predicted the promoter and gene structure of NAC gene through bioinformatic websites and softwares, and then constructed the expression vector of the promoter, which provided the ideas and tools to further research the genetic transformation and regulatory mechanism of NAC promote. The main results were described as follows:
1. Confirm that 10mg/L is the concentration of selective agent Hyg for PLBs. Through the sensitive experiment of PLBs to hygromycin, it was found that PLBs were very sensitive to hygromycin, and as high as 10mg/L proliferation and differentiation of PLBs were inhibited and completely dead.
2. An optimal sonication-assisted Agrobacterium-mediated transformation system was obtained by adjusting pre-treatment time of sonication, infection time of Agrobacterium and co-culture time.The results showed that PLBs which pre-cultured 2 days as treated 3 min with sonication of 40KHz,90W were incubated for 30min by Agrobacterium tumefaciens GV3101 which was resuspended in 1/2 MS at OD600 between 0.8-0.95 and pre-cultured 2 hours, and then was placed on the co-culture medium with 5 days co-culture periods. With this transformation system, numbers of blue spots per 100mg PLB was 42, and at the same time A. tumefaciens was sterilized easily.
3. We isolated a 6324bp length gene sequence by convetiona PCR and genome walking. Sequence analysis showed that NAC transcription factor between initiation codon and termination codon was 1611bp in size, including 3 exons and 2 introns. There were ployA and stem-loop structure at a distance of 859bp and 886bp after termination codon respectively. TSS and TATA box were found at a distance of 348bp and 378bp before TTS respectively.5' flanking sequence was 3401bp in size before TSS and not only contained TATA box, CAAT box and other core promoter element but also included light responsive elements, elicitor responsive elements, stress responsive elements, biorhythm element and other cis-elements. which could help us to understand the biological functions of NAC transcription factor.
4. The prediction of softwares showed that the size of core promoter region was 862bp before TTS. This gene fragment was isolated and inserted into expression vector, and then the recombinant plasmid was transferred into Agrobaclerium tutmefaciens using freeze-thaw method, and detected successfully by double enzymes digestion.
(1)以铁皮石斛愈伤组织诱导的类原球茎为材料,通过PLBs对潮霉素敏感性实验确定潮霉素选择压,结果发现,PLBs对潮霉素较为敏感,10mg/L潮霉素能够有效抑制PLBs的增殖和分化并完全死亡,因此确定10mg/L为潮霉素有效选择压浓度。
(2)优化SAAT法转化条件,研究超声波预处理时间、根瘤农杆菌侵染时间和共培养时间对GUS瞬时表达率的影响,实验结果表明,使用40KHZ、90W的超声波对预培养2d的PLBs超声处理3min,再用预培养2hr的OD600为0.8-0.95的农杆菌侵染液侵染30min,转入共培养基暗培养5d,所获得的蓝色斑点数为42个/100mg PLBs,而且其除菌也相对容易,其中预培养基、侵染液和共培养基中均含有100μM AS。
(3)采用常规PCR法和染色体步移法分离克隆出一条长度为6324bp的基因序列,通过分析发现,NAC转录因子在起始密码子和终止密码子之间长度为1611bp,含有3个外显子和2个内含子,在距离终止密码子下游859处和886处分别有一个ployA结构和Stem-loop结构,在TTS上游348处和378处分别为TSS位点和TATA box。5’侧翼序列长度为TSS前3401bp, PlantCare分析表明,该序列不仅含有TATA box、CAAT box等启动子核心顺式作用元件,而且含有光信号反应元件、激发子响应元件、生理节律元件、激素诱导相关的顺式作用元件、生长发育相关顺式元件和逆境肋、迫响应元件,这些顺式元件的存在与NAC转录因子主要生物学功能相符合。
(4)通过软件预测,确定翻译起始位点上游862bp大小的序列为NAC启动子核心区域,分离克隆该段基因,并构建表达载体,采用冻融法转入到农杆菌中,经双酶切验证证明构建和转化成功。
As a kind of plant specific transcriptional factors. NAC plays an important role in plant development and responses to hormone and stresses, whose expression is regulated by plant developmental stages and various environmental factors. In this study, we optimized sonication-assisted Agrobacterium-mediated transformation system of Dendrobium candidum Wall ex Lindl. for PLBs induced from callus, isolated a NAC transcription factor by PCR and its 5' and 3' flanking sequence by genome walking, predicted the promoter and gene structure of NAC gene through bioinformatic websites and softwares, and then constructed the expression vector of the promoter, which provided the ideas and tools to further research the genetic transformation and regulatory mechanism of NAC promote. The main results were described as follows:
1. Confirm that 10mg/L is the concentration of selective agent Hyg for PLBs. Through the sensitive experiment of PLBs to hygromycin, it was found that PLBs were very sensitive to hygromycin, and as high as 10mg/L proliferation and differentiation of PLBs were inhibited and completely dead.
2. An optimal sonication-assisted Agrobacterium-mediated transformation system was obtained by adjusting pre-treatment time of sonication, infection time of Agrobacterium and co-culture time.The results showed that PLBs which pre-cultured 2 days as treated 3 min with sonication of 40KHz,90W were incubated for 30min by Agrobacterium tumefaciens GV3101 which was resuspended in 1/2 MS at OD600 between 0.8-0.95 and pre-cultured 2 hours, and then was placed on the co-culture medium with 5 days co-culture periods. With this transformation system, numbers of blue spots per 100mg PLB was 42, and at the same time A. tumefaciens was sterilized easily.
3. We isolated a 6324bp length gene sequence by convetiona PCR and genome walking. Sequence analysis showed that NAC transcription factor between initiation codon and termination codon was 1611bp in size, including 3 exons and 2 introns. There were ployA and stem-loop structure at a distance of 859bp and 886bp after termination codon respectively. TSS and TATA box were found at a distance of 348bp and 378bp before TTS respectively.5' flanking sequence was 3401bp in size before TSS and not only contained TATA box, CAAT box and other core promoter element but also included light responsive elements, elicitor responsive elements, stress responsive elements, biorhythm element and other cis-elements. which could help us to understand the biological functions of NAC transcription factor.
4. The prediction of softwares showed that the size of core promoter region was 862bp before TTS. This gene fragment was isolated and inserted into expression vector, and then the recombinant plasmid was transferred into Agrobaclerium tutmefaciens using freeze-thaw method, and detected successfully by double enzymes digestion.
引文
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