亚洲棉GaP5CS和GaTPS基因启动子的克隆与功能分析
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摘要
在转基因植物工程中,多数采用的是花椰菜花叶病毒(CaMV)的35S启动子等组成型启动子,它们驱动外源基因在植物的各种组织和各发育时期都表达,因而往往造成物质和能量的浪费,增加了植物的代谢负担,甚至引起植物形态发生改变,影响植物生长发育。因此,采用组织特异启动子或诱导型启动子启动外源基因在转基因植物中特定组织中得到表达,不但可以降低植物耗能、减轻对植物形态和生长发育的影响,还可以提高外源基因在特定部位的表达浓度,增加转基因的效果。
脯氨酸和海藻糖是两种重要的渗透调节物质,吡咯琳-5-羧酸合成酶(pyrroline-5-carboxyate synthetase,P5CS)与海藻糖-6-磷酸合成酶(TPS)分别是脯氨酸和合成途径中的关键酶。本研究从亚洲棉中克隆出P5CS基因启动子和TPS基因启动子,分别命名为GaPROP5CS和GaPROTPS。取得的研究结果如下:
1启动子的克隆和序列分析
利用改良的TAIL-PCR法,从亚洲棉基因组中扩增出2个基因的5’端上游序列,命名为GaPROP5CS和GaPROTPS。GaPROP5CS全长795bp,找到转录起始位点A,TATAbox位于-22处; GaPROTPS全长1269bp,TATAbox距离转录起始位点A有32bp。
2启动子的瞬时表达分析
构建了GaP5CS和GaTPS的启动子和GUS融合基因表达载体,利用基因枪转化法将两种载体转入洋葱表皮细胞,并以含有PEG的培养基进行后期培养。通过GUS染色结果推测2个启动子是受旱胁迫诱导启动子。
3启动子的功能分析
通过农杆菌介导法分别将GaPROP5CS和GaPROTPS转入烟草进行功能验证。在干旱胁迫处理条件下,在转基因植株的叶片和根部都发现GUS基因的表达,从GUS酶活性测定结果表明启动子都具有较强的表达活性,在干旱胁迫诱导后表达活性得到提高。为了进一步研究GaPROP5CS S启动子在棉花中的启动活性,接着将GaPROP5CS启动子通过农杆菌介导法转化陆地棉,继续探讨启动子在转基因棉中的启动活性。
4启动子缺失片段的获得和功能分析
初步探索GaPROP5CS启动子序列中负调控元件,利用PCR法构建了8段启动子5,端缺失序列,通过基因枪转化法转化洋葱表皮细胞, PEG胁迫下检测GUS的表达,GUS活性测定结果表明, -444/+52缺失启动子以及-123/+52响应旱胁迫诱导活性最强,与CaMV-35S活性相当。为了进一步研究缺失启动子片段活性的强弱,将上述表达活性强的缺失启动子构建的表达载体转化农杆菌,利用农杆菌转化法转化烟草,以探讨GaPROP5CS缺失片段中活性最强缺失启动子。
Constitutive promoters, such as CaMV 35S, were mostly used in transgenic plant engineering. They drive foreign gene to express during the whole growth period and in every organizations of transgenic plant, which often causes substances and energy waste, increases the metabolism burden of plant, even leads to changes of plant morphology and affects plant growth and development. Therefore, using tissue specific promoter or inducible promoter can not only improve level of expression of the foreign gene, increase the effect of genetically modified, but also reduce plant energy consumption and the damages of plant.
Proline and trehalose are two important osmolytes. Pyrroline-5-carboxyate synthetase and trehalose 6-phosphate-synthase are the key enzymes in the biosynthesis of proline and trehalose, respectively. The promoters of the two genes were cloned and characterized from Gossypium arboreum(ShixiyaⅠcultivar).The main results were as follows:
1 Promoter cloning and sequence analysis
Using modified TAIL-PCR method, the two genes 5’upstream sequence were amplified from the Asian cotton genome, which named GaPROP5CS and GaPROTPS. Sequence analysis showed that GaPROP5CS was 795bp in length, in which the TATA box was found at position -22 relative to the putative transcriptional star site (TSS). GaPROTPS was 1269bp in length and TATA box was at 32bp away from the TSS.
2 Transient expression analysis of promoter
Two expression vectors were constructed, in which GUS was drove by GaPROP5CS and GaPROTPS, respectively. The vectors were transformed into onion by particle bombardment transformation to test their functions, respectively. The onion were cultivated in the medium with PEG10%.The results suggested that GaPROP5CS and GaPROTPS are the drought stress-induced promoters, and the activity of GaPROP5CS is stronger than that of GaPROTPS.
3 Analysis of promoter function
GaPROP5CS and GaPROTPS were transformed into Nicotiana babacum by Agrobacterium infiltration method to test their functions, respectively. Under drought stress, gene expressions of GUS were found in leaves and roots of transgenic plants, GUS activity showed that the two promoters had a stronger expression of activity after drought stress-induced. To further study the expression activity of GaPROP5CS, we transformed the GaPROP5CS into Gossypium hirsutum L., using Agrobacterium- mediated transformation.
4.Deletion analysis of promoter
To study the negative regulatory element, a series of 5’-deletions were legated to GUS in expression vector. Then, we transformed the deletions into onion by particle bombardment transformation. Lastly, the transgenic onions were cultivated by PEG. The GUS activity results showed that the highest GUS activity were examined at the region of 444/+52 and -123 /+52, which were equal to CaMV-35S activity. Then, we transformed it into Nicotiana babacum by Agrobacterium infiltration method, so as to select the deletion of highest activity.
脯氨酸和海藻糖是两种重要的渗透调节物质,吡咯琳-5-羧酸合成酶(pyrroline-5-carboxyate synthetase,P5CS)与海藻糖-6-磷酸合成酶(TPS)分别是脯氨酸和合成途径中的关键酶。本研究从亚洲棉中克隆出P5CS基因启动子和TPS基因启动子,分别命名为GaPROP5CS和GaPROTPS。取得的研究结果如下:
1启动子的克隆和序列分析
利用改良的TAIL-PCR法,从亚洲棉基因组中扩增出2个基因的5’端上游序列,命名为GaPROP5CS和GaPROTPS。GaPROP5CS全长795bp,找到转录起始位点A,TATAbox位于-22处; GaPROTPS全长1269bp,TATAbox距离转录起始位点A有32bp。
2启动子的瞬时表达分析
构建了GaP5CS和GaTPS的启动子和GUS融合基因表达载体,利用基因枪转化法将两种载体转入洋葱表皮细胞,并以含有PEG的培养基进行后期培养。通过GUS染色结果推测2个启动子是受旱胁迫诱导启动子。
3启动子的功能分析
通过农杆菌介导法分别将GaPROP5CS和GaPROTPS转入烟草进行功能验证。在干旱胁迫处理条件下,在转基因植株的叶片和根部都发现GUS基因的表达,从GUS酶活性测定结果表明启动子都具有较强的表达活性,在干旱胁迫诱导后表达活性得到提高。为了进一步研究GaPROP5CS S启动子在棉花中的启动活性,接着将GaPROP5CS启动子通过农杆菌介导法转化陆地棉,继续探讨启动子在转基因棉中的启动活性。
4启动子缺失片段的获得和功能分析
初步探索GaPROP5CS启动子序列中负调控元件,利用PCR法构建了8段启动子5,端缺失序列,通过基因枪转化法转化洋葱表皮细胞, PEG胁迫下检测GUS的表达,GUS活性测定结果表明, -444/+52缺失启动子以及-123/+52响应旱胁迫诱导活性最强,与CaMV-35S活性相当。为了进一步研究缺失启动子片段活性的强弱,将上述表达活性强的缺失启动子构建的表达载体转化农杆菌,利用农杆菌转化法转化烟草,以探讨GaPROP5CS缺失片段中活性最强缺失启动子。
Constitutive promoters, such as CaMV 35S, were mostly used in transgenic plant engineering. They drive foreign gene to express during the whole growth period and in every organizations of transgenic plant, which often causes substances and energy waste, increases the metabolism burden of plant, even leads to changes of plant morphology and affects plant growth and development. Therefore, using tissue specific promoter or inducible promoter can not only improve level of expression of the foreign gene, increase the effect of genetically modified, but also reduce plant energy consumption and the damages of plant.
Proline and trehalose are two important osmolytes. Pyrroline-5-carboxyate synthetase and trehalose 6-phosphate-synthase are the key enzymes in the biosynthesis of proline and trehalose, respectively. The promoters of the two genes were cloned and characterized from Gossypium arboreum(ShixiyaⅠcultivar).The main results were as follows:
1 Promoter cloning and sequence analysis
Using modified TAIL-PCR method, the two genes 5’upstream sequence were amplified from the Asian cotton genome, which named GaPROP5CS and GaPROTPS. Sequence analysis showed that GaPROP5CS was 795bp in length, in which the TATA box was found at position -22 relative to the putative transcriptional star site (TSS). GaPROTPS was 1269bp in length and TATA box was at 32bp away from the TSS.
2 Transient expression analysis of promoter
Two expression vectors were constructed, in which GUS was drove by GaPROP5CS and GaPROTPS, respectively. The vectors were transformed into onion by particle bombardment transformation to test their functions, respectively. The onion were cultivated in the medium with PEG10%.The results suggested that GaPROP5CS and GaPROTPS are the drought stress-induced promoters, and the activity of GaPROP5CS is stronger than that of GaPROTPS.
3 Analysis of promoter function
GaPROP5CS and GaPROTPS were transformed into Nicotiana babacum by Agrobacterium infiltration method to test their functions, respectively. Under drought stress, gene expressions of GUS were found in leaves and roots of transgenic plants, GUS activity showed that the two promoters had a stronger expression of activity after drought stress-induced. To further study the expression activity of GaPROP5CS, we transformed the GaPROP5CS into Gossypium hirsutum L., using Agrobacterium- mediated transformation.
4.Deletion analysis of promoter
To study the negative regulatory element, a series of 5’-deletions were legated to GUS in expression vector. Then, we transformed the deletions into onion by particle bombardment transformation. Lastly, the transgenic onions were cultivated by PEG. The GUS activity results showed that the highest GUS activity were examined at the region of 444/+52 and -123 /+52, which were equal to CaMV-35S activity. Then, we transformed it into Nicotiana babacum by Agrobacterium infiltration method, so as to select the deletion of highest activity.
引文
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75.夏江东,程在全,吴渝生等.高等植物启动子功能和结构研究进展.云南农业大学学报, 2006, 21(1) :7~14
76.徐琼芳,李连成,陈孝等.基因枪法获得GNA转基因小麦植株的研究.中国农业科学,2001,34(1): 5-8
77.谢迎秋,刘玉乐,朱祯,棉花曲叶病毒互补链基因启动子功能区的缺失分析.中国科学,2000, 10(5):528-535
78.谢迎秋,朱祯,刘玉乐等.一种新型双向启动子-棉花曲叶病毒启动子的分离、序列分析与功能初探.高技术通讯,2002, 4: 13-17
79.袁正强,贾燕涛,吴家和等.三个韧皮部特异性启动子在转基因烟草中表达的比较研究.农业生物技术学报,2002,10(1):6-9
80.于娅,刘传亮,马峙英等.基因枪转化技术在棉花遗传转化上的应用.棉花学报,2003,15(4):243- 247
81.张雪妍,刘传亮,王俊娟,李付广,叶武威.PEG胁迫方法评价棉花幼苗耐旱性研究.棉花学报.2007, 19(3):205-209
82.张震林,刘正銮,周宝良等.转兔角蛋白基因改良棉纤维品质研究.棉花学报,2004,14(2):72-76
83.张春晓,王文棋,蒋湘宁,陈雪梅.植物基因启动子研究进展.遗传学报,2004,31(12):1455-1464.
84.张艳华,季静,王罡.转基因植物与生物安全性,作物杂志, 2003,6: 4-7
85.张晓宁.盐藻耐盐相关基因的克隆、功能分析与高效启动子的分离鉴定:[博士学位论文],复旦大学,2002
86.张秀君,刘俊起,赵倩等.用基因枪将高赖氨酸基因导入玉米及转基因植株的检测.农业生物技术学报,1997 ,7(4):363-367
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81.张雪妍,刘传亮,王俊娟,李付广,叶武威.PEG胁迫方法评价棉花幼苗耐旱性研究.棉花学报.2007, 19(3):205-209
82.张震林,刘正銮,周宝良等.转兔角蛋白基因改良棉纤维品质研究.棉花学报,2004,14(2):72-76
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